t bull f
CHARACTERIZATION OF ANTIBIOTIC-PRODUCING FUNGI FROM UNIMAS RESERVE FOREST AND
THEIR ANTIBIOTICS
Norhafizah Binti Sidek
SF 1S4A NIJ9 Bachelor of Science with Honours lOll (Biotechnology Resource)
2012
Pusat Khidmat Maklumat Akademik bull f UNIVERSm MALAYSIA SARAWAK
PKHIDMAT MAKLUMAT AKADEMIK
111111111 100111111111 1000235680
CHARACTERIZATION OF ANTIBIOTIC-PRODUCING FUNGI FROM UNIMAS RESERVE FOREST AND THEIR ANTIBIOTICS
NORHAFIZAH BINT SIDEK
This project is submitted in partial fulfillment of the requirement for the degree of
Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology
UNIVERSITI MALAYSIA SARA W AK
2012
DECLARATION
I hereby declare that no portion of this dissertation has been submitted in support of an
application for another degree of qualification of this or any other university or institution of
higher learning
(NORHAFIZAH BINTI SIDEK)
Resource Biotechnology Programme
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ACKNOWLEDGEMENT
First of all praise to Allah swt the Mighty One for His bless in giving me the strength and
good health to complete this study
Most appreciation to Prof Dr Ismail bin Ahmad for being such a dedicated and responsible
supervisor Thank you for the knowledge and guidance that has been given throughout
conducting the study This appreciation is also dedicated to Lab Assistant Mr Iskandar for
providing the equipments and laboratory needs and thank you to all MASTER students of
Virology Lab for the guidance and advice
]To my dear colleagues and fiiends especially Olivia Chan Nurulhazwanie Rafiee Mohd
Noorzaifiqrudin and Boo Chie Vi thank you for the support and sharing information To my
dear table partner Rahimah Manan lots of thanks for always helping and sharing knowledge
with me Not to forget to my beloved family thank you for the pray and for always being there
when time needed
1
Pusat Kbidmat Maklumat Akademik UNIVEltSm MALAYSIA SARAWAK
Table of Contents
Acknowledgement i
Table of Contents ii
List of Abbreviations iv
List of Tables and Figures vii
Abstract 1
10 Introduction 2
20 Literature Review 5
21 Fungi 5
22 Soil as Reservoir of Antibiotic-Producing Fungi 5
23 Antibiotic-Resistance 6
24 Screening Method for Antibiotic Activity 7
241 Agar Overlay Technique 7
242 Antimicrobial Susceptibility Test Using Disc Diffusion Method 8
25 Bioautography _ 9
11
f bull t f
30 Materials and Methods 11
31 Preparation of Media 11
311 Preparation ofPDA Media 11
312 Preparation of NA Media 11
32 Sampling Sites and Collections II
33 Isolation of Microorganisms from Soil 12
331 Isolation and Subculturing 12
332 Subculturing of Bacteria and Fungi Colonies 13
34 Identification and Classification ofFungallsolates 13
341 Macroscopic Examination 13
342 Microscopic Examination 13
35 Molecular Identification 14
351 DNA Extraction 14
352 Polymerase Chain Reaction (PCR) 14
353 Purification of PCR Products 000000 00000000 0000 0015
354 Agarose Gel Electrophoresis 15
36 Antimicrobial Screening of Fungal Isolates 16
361 Preparation of Bacteria 00 00 00 00000000 00 000000 00 00 000000 000000 00 0016
111
t f
362 Preliminary Fungal Colony Screening 16
37 Extraction of Antibiotics 17
38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8
381 Test Bacteria Preparation J8
382 Disc Diffusion Test of Antibiotic Extracts 18
39 Antifungal Test 19
310 Fractionation of Extracted Antibiotics Using TLC 20
311 Bioautography 21
40 Result 22
41 Isolation and Subculturing of Microorganism from Soil 22
42 Preliminary Colony Screening of Fungi 22
43 Antibiotic Screening for Hexane and DCM Extraction 25
44 Antifungal Test 27
45 Morphological Characterization 29
46 Thin Layer Chromatography (TLC) 33
47 Bioautography 36
48 Molecular Identification 38
IV
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
Pusat Khidmat Maklumat Akademik bull f UNIVERSm MALAYSIA SARAWAK
PKHIDMAT MAKLUMAT AKADEMIK
111111111 100111111111 1000235680
CHARACTERIZATION OF ANTIBIOTIC-PRODUCING FUNGI FROM UNIMAS RESERVE FOREST AND THEIR ANTIBIOTICS
NORHAFIZAH BINT SIDEK
This project is submitted in partial fulfillment of the requirement for the degree of
Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology
UNIVERSITI MALAYSIA SARA W AK
2012
DECLARATION
I hereby declare that no portion of this dissertation has been submitted in support of an
application for another degree of qualification of this or any other university or institution of
higher learning
(NORHAFIZAH BINTI SIDEK)
Resource Biotechnology Programme
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ACKNOWLEDGEMENT
First of all praise to Allah swt the Mighty One for His bless in giving me the strength and
good health to complete this study
Most appreciation to Prof Dr Ismail bin Ahmad for being such a dedicated and responsible
supervisor Thank you for the knowledge and guidance that has been given throughout
conducting the study This appreciation is also dedicated to Lab Assistant Mr Iskandar for
providing the equipments and laboratory needs and thank you to all MASTER students of
Virology Lab for the guidance and advice
]To my dear colleagues and fiiends especially Olivia Chan Nurulhazwanie Rafiee Mohd
Noorzaifiqrudin and Boo Chie Vi thank you for the support and sharing information To my
dear table partner Rahimah Manan lots of thanks for always helping and sharing knowledge
with me Not to forget to my beloved family thank you for the pray and for always being there
when time needed
1
Pusat Kbidmat Maklumat Akademik UNIVEltSm MALAYSIA SARAWAK
Table of Contents
Acknowledgement i
Table of Contents ii
List of Abbreviations iv
List of Tables and Figures vii
Abstract 1
10 Introduction 2
20 Literature Review 5
21 Fungi 5
22 Soil as Reservoir of Antibiotic-Producing Fungi 5
23 Antibiotic-Resistance 6
24 Screening Method for Antibiotic Activity 7
241 Agar Overlay Technique 7
242 Antimicrobial Susceptibility Test Using Disc Diffusion Method 8
25 Bioautography _ 9
11
f bull t f
30 Materials and Methods 11
31 Preparation of Media 11
311 Preparation ofPDA Media 11
312 Preparation of NA Media 11
32 Sampling Sites and Collections II
33 Isolation of Microorganisms from Soil 12
331 Isolation and Subculturing 12
332 Subculturing of Bacteria and Fungi Colonies 13
34 Identification and Classification ofFungallsolates 13
341 Macroscopic Examination 13
342 Microscopic Examination 13
35 Molecular Identification 14
351 DNA Extraction 14
352 Polymerase Chain Reaction (PCR) 14
353 Purification of PCR Products 000000 00000000 0000 0015
354 Agarose Gel Electrophoresis 15
36 Antimicrobial Screening of Fungal Isolates 16
361 Preparation of Bacteria 00 00 00 00000000 00 000000 00 00 000000 000000 00 0016
111
t f
362 Preliminary Fungal Colony Screening 16
37 Extraction of Antibiotics 17
38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8
381 Test Bacteria Preparation J8
382 Disc Diffusion Test of Antibiotic Extracts 18
39 Antifungal Test 19
310 Fractionation of Extracted Antibiotics Using TLC 20
311 Bioautography 21
40 Result 22
41 Isolation and Subculturing of Microorganism from Soil 22
42 Preliminary Colony Screening of Fungi 22
43 Antibiotic Screening for Hexane and DCM Extraction 25
44 Antifungal Test 27
45 Morphological Characterization 29
46 Thin Layer Chromatography (TLC) 33
47 Bioautography 36
48 Molecular Identification 38
IV
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
DECLARATION
I hereby declare that no portion of this dissertation has been submitted in support of an
application for another degree of qualification of this or any other university or institution of
higher learning
(NORHAFIZAH BINTI SIDEK)
Resource Biotechnology Programme
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ACKNOWLEDGEMENT
First of all praise to Allah swt the Mighty One for His bless in giving me the strength and
good health to complete this study
Most appreciation to Prof Dr Ismail bin Ahmad for being such a dedicated and responsible
supervisor Thank you for the knowledge and guidance that has been given throughout
conducting the study This appreciation is also dedicated to Lab Assistant Mr Iskandar for
providing the equipments and laboratory needs and thank you to all MASTER students of
Virology Lab for the guidance and advice
]To my dear colleagues and fiiends especially Olivia Chan Nurulhazwanie Rafiee Mohd
Noorzaifiqrudin and Boo Chie Vi thank you for the support and sharing information To my
dear table partner Rahimah Manan lots of thanks for always helping and sharing knowledge
with me Not to forget to my beloved family thank you for the pray and for always being there
when time needed
1
Pusat Kbidmat Maklumat Akademik UNIVEltSm MALAYSIA SARAWAK
Table of Contents
Acknowledgement i
Table of Contents ii
List of Abbreviations iv
List of Tables and Figures vii
Abstract 1
10 Introduction 2
20 Literature Review 5
21 Fungi 5
22 Soil as Reservoir of Antibiotic-Producing Fungi 5
23 Antibiotic-Resistance 6
24 Screening Method for Antibiotic Activity 7
241 Agar Overlay Technique 7
242 Antimicrobial Susceptibility Test Using Disc Diffusion Method 8
25 Bioautography _ 9
11
f bull t f
30 Materials and Methods 11
31 Preparation of Media 11
311 Preparation ofPDA Media 11
312 Preparation of NA Media 11
32 Sampling Sites and Collections II
33 Isolation of Microorganisms from Soil 12
331 Isolation and Subculturing 12
332 Subculturing of Bacteria and Fungi Colonies 13
34 Identification and Classification ofFungallsolates 13
341 Macroscopic Examination 13
342 Microscopic Examination 13
35 Molecular Identification 14
351 DNA Extraction 14
352 Polymerase Chain Reaction (PCR) 14
353 Purification of PCR Products 000000 00000000 0000 0015
354 Agarose Gel Electrophoresis 15
36 Antimicrobial Screening of Fungal Isolates 16
361 Preparation of Bacteria 00 00 00 00000000 00 000000 00 00 000000 000000 00 0016
111
t f
362 Preliminary Fungal Colony Screening 16
37 Extraction of Antibiotics 17
38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8
381 Test Bacteria Preparation J8
382 Disc Diffusion Test of Antibiotic Extracts 18
39 Antifungal Test 19
310 Fractionation of Extracted Antibiotics Using TLC 20
311 Bioautography 21
40 Result 22
41 Isolation and Subculturing of Microorganism from Soil 22
42 Preliminary Colony Screening of Fungi 22
43 Antibiotic Screening for Hexane and DCM Extraction 25
44 Antifungal Test 27
45 Morphological Characterization 29
46 Thin Layer Chromatography (TLC) 33
47 Bioautography 36
48 Molecular Identification 38
IV
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
ACKNOWLEDGEMENT
First of all praise to Allah swt the Mighty One for His bless in giving me the strength and
good health to complete this study
Most appreciation to Prof Dr Ismail bin Ahmad for being such a dedicated and responsible
supervisor Thank you for the knowledge and guidance that has been given throughout
conducting the study This appreciation is also dedicated to Lab Assistant Mr Iskandar for
providing the equipments and laboratory needs and thank you to all MASTER students of
Virology Lab for the guidance and advice
]To my dear colleagues and fiiends especially Olivia Chan Nurulhazwanie Rafiee Mohd
Noorzaifiqrudin and Boo Chie Vi thank you for the support and sharing information To my
dear table partner Rahimah Manan lots of thanks for always helping and sharing knowledge
with me Not to forget to my beloved family thank you for the pray and for always being there
when time needed
1
Pusat Kbidmat Maklumat Akademik UNIVEltSm MALAYSIA SARAWAK
Table of Contents
Acknowledgement i
Table of Contents ii
List of Abbreviations iv
List of Tables and Figures vii
Abstract 1
10 Introduction 2
20 Literature Review 5
21 Fungi 5
22 Soil as Reservoir of Antibiotic-Producing Fungi 5
23 Antibiotic-Resistance 6
24 Screening Method for Antibiotic Activity 7
241 Agar Overlay Technique 7
242 Antimicrobial Susceptibility Test Using Disc Diffusion Method 8
25 Bioautography _ 9
11
f bull t f
30 Materials and Methods 11
31 Preparation of Media 11
311 Preparation ofPDA Media 11
312 Preparation of NA Media 11
32 Sampling Sites and Collections II
33 Isolation of Microorganisms from Soil 12
331 Isolation and Subculturing 12
332 Subculturing of Bacteria and Fungi Colonies 13
34 Identification and Classification ofFungallsolates 13
341 Macroscopic Examination 13
342 Microscopic Examination 13
35 Molecular Identification 14
351 DNA Extraction 14
352 Polymerase Chain Reaction (PCR) 14
353 Purification of PCR Products 000000 00000000 0000 0015
354 Agarose Gel Electrophoresis 15
36 Antimicrobial Screening of Fungal Isolates 16
361 Preparation of Bacteria 00 00 00 00000000 00 000000 00 00 000000 000000 00 0016
111
t f
362 Preliminary Fungal Colony Screening 16
37 Extraction of Antibiotics 17
38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8
381 Test Bacteria Preparation J8
382 Disc Diffusion Test of Antibiotic Extracts 18
39 Antifungal Test 19
310 Fractionation of Extracted Antibiotics Using TLC 20
311 Bioautography 21
40 Result 22
41 Isolation and Subculturing of Microorganism from Soil 22
42 Preliminary Colony Screening of Fungi 22
43 Antibiotic Screening for Hexane and DCM Extraction 25
44 Antifungal Test 27
45 Morphological Characterization 29
46 Thin Layer Chromatography (TLC) 33
47 Bioautography 36
48 Molecular Identification 38
IV
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
Pusat Kbidmat Maklumat Akademik UNIVEltSm MALAYSIA SARAWAK
Table of Contents
Acknowledgement i
Table of Contents ii
List of Abbreviations iv
List of Tables and Figures vii
Abstract 1
10 Introduction 2
20 Literature Review 5
21 Fungi 5
22 Soil as Reservoir of Antibiotic-Producing Fungi 5
23 Antibiotic-Resistance 6
24 Screening Method for Antibiotic Activity 7
241 Agar Overlay Technique 7
242 Antimicrobial Susceptibility Test Using Disc Diffusion Method 8
25 Bioautography _ 9
11
f bull t f
30 Materials and Methods 11
31 Preparation of Media 11
311 Preparation ofPDA Media 11
312 Preparation of NA Media 11
32 Sampling Sites and Collections II
33 Isolation of Microorganisms from Soil 12
331 Isolation and Subculturing 12
332 Subculturing of Bacteria and Fungi Colonies 13
34 Identification and Classification ofFungallsolates 13
341 Macroscopic Examination 13
342 Microscopic Examination 13
35 Molecular Identification 14
351 DNA Extraction 14
352 Polymerase Chain Reaction (PCR) 14
353 Purification of PCR Products 000000 00000000 0000 0015
354 Agarose Gel Electrophoresis 15
36 Antimicrobial Screening of Fungal Isolates 16
361 Preparation of Bacteria 00 00 00 00000000 00 000000 00 00 000000 000000 00 0016
111
t f
362 Preliminary Fungal Colony Screening 16
37 Extraction of Antibiotics 17
38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8
381 Test Bacteria Preparation J8
382 Disc Diffusion Test of Antibiotic Extracts 18
39 Antifungal Test 19
310 Fractionation of Extracted Antibiotics Using TLC 20
311 Bioautography 21
40 Result 22
41 Isolation and Subculturing of Microorganism from Soil 22
42 Preliminary Colony Screening of Fungi 22
43 Antibiotic Screening for Hexane and DCM Extraction 25
44 Antifungal Test 27
45 Morphological Characterization 29
46 Thin Layer Chromatography (TLC) 33
47 Bioautography 36
48 Molecular Identification 38
IV
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
f bull t f
30 Materials and Methods 11
31 Preparation of Media 11
311 Preparation ofPDA Media 11
312 Preparation of NA Media 11
32 Sampling Sites and Collections II
33 Isolation of Microorganisms from Soil 12
331 Isolation and Subculturing 12
332 Subculturing of Bacteria and Fungi Colonies 13
34 Identification and Classification ofFungallsolates 13
341 Macroscopic Examination 13
342 Microscopic Examination 13
35 Molecular Identification 14
351 DNA Extraction 14
352 Polymerase Chain Reaction (PCR) 14
353 Purification of PCR Products 000000 00000000 0000 0015
354 Agarose Gel Electrophoresis 15
36 Antimicrobial Screening of Fungal Isolates 16
361 Preparation of Bacteria 00 00 00 00000000 00 000000 00 00 000000 000000 00 0016
111
t f
362 Preliminary Fungal Colony Screening 16
37 Extraction of Antibiotics 17
38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8
381 Test Bacteria Preparation J8
382 Disc Diffusion Test of Antibiotic Extracts 18
39 Antifungal Test 19
310 Fractionation of Extracted Antibiotics Using TLC 20
311 Bioautography 21
40 Result 22
41 Isolation and Subculturing of Microorganism from Soil 22
42 Preliminary Colony Screening of Fungi 22
43 Antibiotic Screening for Hexane and DCM Extraction 25
44 Antifungal Test 27
45 Morphological Characterization 29
46 Thin Layer Chromatography (TLC) 33
47 Bioautography 36
48 Molecular Identification 38
IV
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
t f
362 Preliminary Fungal Colony Screening 16
37 Extraction of Antibiotics 17
38 Minimum Inhibitory Concentration (MIC) of Extracted Antibiotics J8
381 Test Bacteria Preparation J8
382 Disc Diffusion Test of Antibiotic Extracts 18
39 Antifungal Test 19
310 Fractionation of Extracted Antibiotics Using TLC 20
311 Bioautography 21
40 Result 22
41 Isolation and Subculturing of Microorganism from Soil 22
42 Preliminary Colony Screening of Fungi 22
43 Antibiotic Screening for Hexane and DCM Extraction 25
44 Antifungal Test 27
45 Morphological Characterization 29
46 Thin Layer Chromatography (TLC) 33
47 Bioautography 36
48 Molecular Identification 38
IV
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
t
50 Discussion 39
51 Location ofSoiJ Sampling 39
52 Isolation and Subculturing of Microorganism from Soil 39
53 Preliminary Colony Screening 39
54 Antibiotic Screening of Hexane and DCM Extraction 41
55 Antifungal Test 41
56 Morphological Characterization 42
57 Thin Layer Chromatography (TLC) 42
58 Bioautography 43
59 Molecular Identification 44
60 Conclusion 45
References 46
v
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
t t
List of Abbreviation
NA - Nutrient Agar
PDA - Potato Dextrose Agar
DCM - Dichloromethane
MHA - Mueller-Hinton Agar
MHB - Mueller-Hinton Broth
MIC - Minimum Inhibitory Concentration
TLC - Thin Layer Chromatography
PCR - Polymerase Chain Reaction
PBS - Phosphate Buffer Saline
SA Staphylococcuss aureus
ST Salmonella typhi
EC Escherichia coli
EA Enterobacter aerogenes
00 Optical Density
~L - microliter
mm - millimeter
cm - centimeter
mg - milligram
mL - milliliter
vi
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
List of Tables and Figures
TablelFigure Page
Figure 31 Map on location of soil sampling on UNIMAS reserve forest 12
Figure 32 Diagram representing the arrangement of fungal isolates on test 17 bacteria lawn
Figure 33 Diagram represented the arrangement of antibiotic extracts according to 19 concentra tion
Figure 34 Diagram represented the arrangement of selected fungal isolate for 20 antifungal test
Figure 41 Zone of inhibition formed by eight fungi isolates against E aerogene 24
Figure 42 Inhibition zone demonstrated by hexane extract of F5 isolate against EA 25 The MIC obtained was 025 mgml
Figure 43 Formation of concave and non concave inhibition zones between fungal 28 isolates and test fungus isolate Fusarium sp
Figure 44 Pure culture of fungal isolate F5 for the determination of macroscopic 30 characteristics
Figure 45 Fungal isolates F5 and F6 under compound microscope A Fungal isolate F5 30 B Fungal isolate F6
Figure 46 Spots observed under UV light demonstrated by DCM extracts of F7 isolate 36
Figure 47 Activity presented of fractionated hexane extracts of fungal isolates towards SA 38
Table 41 Preliminary colony screening for identifying of antibiotic-producing fungi 23
Table 42 Inhibition zones demonstrated by hexane extracts of F4-F8 isolate against 26 test bacteria
vii
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
I
Table 43 Inhibition zones demonstrated by OCM extracts of F4-F8 isolate against 27 test bacteria
Table 44 Presence of antifungal property in various fungal isolates 30
Table 45a Macroscopic characteristics of isolated antibiotic-producing fungi 31
Table 45b Microscopic characteristics of isolated antibiotic-producing fungi 32
Table 46a Rr value of fungal isolates for hexane extraction 34
Table 46b Rr value of fungal isolates for dichloromethane (OCM) extraction 35
Table 47a Rr value of fractionation hexane extraction on test bacteria ST 37
Table 47b Rr value of fractionation hexane extraction on test bacteria SA 37
VJll
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
t
Characterization of Antibiotic-Producing Fungi from UNIMAS Reserve Forest and Their Antibiotics
Norhafizah Binti Sidek
Resource Biotechnology Programme Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A study was conducted to isolate antibiotic-producing fungal from soil samples collected from UNIMAS forest reserve Kota Samarahan Sarawak The soil was suspended and homogenized in phosphate buffer saline (PBS) and then was inoculated on potato dextrose agar (POA) or nutrient agar (NA) by spread-plate method Antibiotic-producing microorganisms were identified through preliminary screening of the isolated fungi by using agar overlay technique Out of the eight fungi isolated five were selected namely F4 F5 F6 F7 and F8 were selected for extraction with hexane and dichloromethane (OCM) This procedure involved drying 14 days old fungal culture followed by immersion of the dried agar in hexane and OCM The extracts were dried and subjected to disc diffusion assay against StaphylococclIs alreliS (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Result showed that one or both the hexane and OCM extracts of all the five fungi isolates showed inhibitory activity against at least one of the test bacteria but at different concentration The strongest activity was shown by hexane extracts of F5 on EA with the MIC value of 025 mgfmL thin layer chromatography (TLC) and bioautography of all the extracts revealed three to six spots per chromatogram when subjected to Bioautography against the test bacteria only ST and SA were found to be inhibited This showed that further studies to fractionate and purity the antibiotic components could be carried out in the future In addition preliminary colony antifungal assay revealed that six of the eight fungal isolate showed inhibition of test fungus whic h is Fusarillm sp
Keywords Antibiotic antibiotic-producing fungal soil sample
ABSTRAK
Kajian dijalankan IIntllk memencilkan klliat penghasil antibiotik daripada sampel tanah yang diambil dari kawasan hutan sillpan UNMAS Kota Samarahan Sarawak Sampeltanah yang diambil dilarutkan bersama lanttall phosphate buffer saline (PBS) dan diiklti dengan homogenasi Kemlldian sedikit daripada lanttan tanah yang dihomogen diinokulasi pada permllkaan agar potato dextrose (PDA) dan agar nlllrient (NA) melalui kaedah piring sebaran (spread-plale) Penyaringan lInllIk mengenal pasti tindakan penghasilan antibiotic dijalankan menenlsi saringan awal mennggllnakan teknik pelapisan agar Daripada lapan jenis klliat yang diasingkan lima daripadanya iaitll F4 F5 F6 F7 dan F8 dipilih IIntllk diekstrak menggunakan hexane dan dichloromethane (DCM) Ekstrak dikeringkan dall diteruskan dengan penyaringan antibiotic dan diuji ke atas bacteria StaphylococclIs allreus (SA) Salmonella typhi (ST) Escherichia coli (EC) and Enterobacter aerogenes (EA) Hasil kajian IIendapati saw mahllplln kedlla esktrak hexane dan DCM daripada kesemlla lima klliat yang diasingkan menllnjllkkan aktiliti perencatan keatas sekurangnya satll bacteria pengllj i dalam kepekatan yang berbeza Aktiliti perencatan dapat dilihat daripada ekstrak hexane bagi klliat F5 keatas EA dengan nilai kepekatan perencatan minimllm ()25 mgmL kesemlla lima ekstrak kulat yang dillji dengan thin layer chromatography (TLC) dan bioatograji menunjukkan kesemua ekstrak mempunyai tiga hingga lima tlNk per kromatogram tetapi hasil kajian bioalltograji mendapati hanya bacteria SA dan ST yang dapat direncatkan Hasil menllnjllkkan kajian lanjlll IIntllk menyuling komponen antibiotic boleh dijalankan Disamping itll saringan awal koloni anti-klliat menllnjllkkan enam daripada lapan klliat yang diasingkan dapat merencatkan pembesaran klilat pengllji iaitu Fusarium sJl
Kata kunci Antibiotik klliat penghasil antibiotic sampeltanah
1
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
10 INTRODUCTION
Antibiotics are secondary metabolites produced by microbes or chemically synthesized
substances with the ability to kill other microorganism or inhibit their growth (Denyer et
01 2004) The first antibiotic to be commercially manufactured in the world was penicillin
which is produced by the fungus Penicillium nota tum (Silver amp Bostian 1993) Penicillin
became widely used in the late 1940s but its effectiveness was hampered by the
emergence of penicillin-resistant Staphylococuss aurells These Penicillium resistance
Staphylococcus aureus emerged just a few years after the first introduction of penicillin in
1950s (Frisvad et 01 1990)
Since the discovery of penicillin more than 100 antibiotics have been deve10ped by
the pharmaceutical industry and only some are commonly used in the health care
management until now (Borregaard 2001) Newly introduced antibiotics are effective in
the control of bacteria and infection including respiratory infection pneumonia and
bacterial throat infections treatment of typhoid fever and other intestinal infections like
dysentery tuberculosis sexually transmitted diseases like gonorrhoea and syphilis The
introduction of these antibiotics have proven that they are remarkably effective in the
control of bacterial and fungal infections (Eileen et 01 2010)
However antibiotic-resistant strains of bacteria have emerged and this resistance is
rapidly transmitted to other bacterial strains and species (Hughes amp Anderson 2001) In
recent years The Center for Disease Control and Prevention (CDC) USA estimates that
each year nearly 2 million people in the United States have acquired an infection while in
a hospital resulting in 90000 deaths More than 70 percent of the bacteria that cause these
2
(
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
middot
infections are resistant to at least one of the antibiotics commonly used to treat them
(Aureden et al 2010)
Despite the high expectation of finding synthetic molecules with effective
antimicrobial properties this alternative method has not been on the promising Hence
naturally occurring antibiotics are still needed in the programme to curb the problems of
ineffectiveness of existing antibiotics for the control of newly emerging antibiotic-resistant
bacterial strains This need has led to the increased interest in screening for strains of
microorganisms which have the potential of producing novel antibiotic
Soil has been used as a source for screening of antibiotic producing microorganism
for several decades Recent record have shown that screening of soil for antimicrobial
activity are still carried out in many parts of the world (Makut amp Owolewa 2011) One of
the target of soil screening is to find Penicillium sp which are able to produce nonshy
penicillin antibiotics This filamentous fungi of the genus Penicillium that belong to
ascomycetes are known to be easily found in the environment (Peberdy 1987)
Sarawak have been known as the large centre of biodiversity with large amount of
lands still not being used as a source for finding antibiotic-producing microorganism
Previously unexplored environments have their own ecosphere interactions and evolution
that might contain new producer organisms Those facts motivated the development of a
research program which aims to perform the isolation of antimicrobial substances from
UNIMAS soil fungi that might give any prospect to contribute in therapeutic industry
Based on the abundance of penicillin resistance bacteria and the ability of Penicillium sp
3
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
to produce non penicillin antibiotic a research study on discovering non penicillin
antibiotic by Penicillium sp is plausible
OBJECTIVESshy
1 To isolate antibiotic-producing fungi from soil UNIMASs reserve forest with
potential antibiotic
2 To characterize and identifY the isolates using macroscopic and microscopic
examination
3 To fractionate crude extract and identify the presence of antibiotics in the fraction
by using Bioautography
Ii
4
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
usat Khidmat Maldumat Akademik UNIVERSm MALAYSIA SARAWAK
20 LITERATURE REVIEW
21 Fungi
Fungi are microscopic plant-like cells that grow in long threadlike structures or hyphae that
make a mass called mycelium The mycelium absorbs nutrients from the roots it has
colonised surface organic matter or the soil (Tabasso 2006) Fungi is a group of simple
plants that have no chlorophyll There are some species of fungi that are single celled
organisms and there are other kinds of fungi that are multi-cellular organisms It produces
special hyphae that create the reproductive spores Fungi have many different structures
but they can act in similar ways Since the discovery of penicillin a number of antibiotics
have been discovered mostly from soil inhabiting fungi (Vaughan amp Barnes 2009)
A number of metabolites were isolated from fungi which found their way into
medical applications as natural products starting material for pharmaceuticals or as lead
structures for the development of pharmaceutical products (Kavanagh 2011) For example
nowadays research has been improved in order to isolates a novel active metabolites
which produce by fungi This fact together with the knowledge about the great potentiaL of
microscopic fungi for production of bioactive metabolites (eg penicillin from Penicillium
notatum ergotamin from Claviceps purpurea cidosporin from Tolypocladium inflatum)
(Tabasso 2006)
22 Soil as Reservoir of Antibiotic-Producing Fungi
Soil is commonly used as medium of finding a new antibiotic as it rich in diversity of
microorganism including fungi and bacteria Soil has capacity to supply adequate nutrients
5
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
I
to meet the needs of growing microorganism Fungi are an important component of the soil
microbiota typically constituting more of the soil biomass than bacteria depending on soil
depth and nutrient conditions The role of fungi in the soil is an extremely complex and is
fundamental to the soil ecosystem They perform ecological services that strongly impact
the quality of human life and have enormous potential for providing economic benefits
eg the isolation and identification of the soil fungus Penicillium leading to a large
pharmaceutical industry of antibiotics (Takashi et al 2008)
As fungi playa major role in soil ecosystems along with bacteria protists small
invertebrates and plants through complex trophic interactions Most soil fungi are
regarded as saprobes decomposing organic matter and contributing to nutrient cyding
while several species form mycorrhizal associations with plants or are plant pathogens
(Pfenning amp Abreu 2006) Also recognized as prolific secondary metabolite producers
fungi have provided several bioactive compounds and chemical models currently used as
pharmaceuticals and soils are traditionally the main source of fungal genetic resources for
bioprospection programs (Adrio amp Demain 2003) Despite that the biodiversity and
biotechnological potential of the soil mycobiota in many tropical regions is still poorly
studied This environment is thought to select species with adapted metabolism and good
potential for delivering new bioactive metabolites
23 Antibiotic-Resistance
Antibiotic resistance occurs when pathogenic microorganism are capable to inactivate
antibiotics or survive under the selective pressure of antibiotics (Gillespie 200 I)
Resistance is a condition in which the antibiotic fails to harm the pathogen enough to cure
6
shy
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
diseases Emergence of resistance often begins with a large pathogen population in which a
tiny fraction is naturally resistance genes from other microbes (Drlica amp Perlin 2004)The
emergence of antibiotic resistance in bacteria has been mainly attributed to the overuse of
antibiotic and indiscriminate antibiotic treatment are often the main cause that contribute to
the emergence of bacterial resistance to antibiotics (Eileen et aI 20 10)
This emergence of resistance in bacteria towards antibiotics has posed a big
challenge in the treatment of infectious diseases worldwide (Eileen et ai 20 I 0) The
resistance has arisen not only against both natural and semi-synthetc antibiotics but also
towards pure synthetic chemical compounds Some of the bacteria even become multidrug
resistant where resistance has been developed against several drugs
The antibiotic resistance property can be transmitted to the next generation through
vertical transmission of inherited mutations or by horizontally exchange of genetic
materials (Gillespie 200 I) Occasionally spontaneous mutations which occur in receptor
or binding site of the bacteria often leads to the change that renders antibiotics to become
ineffective (FDA 2009) Besides transferring of mutant resistance-causing genes from one
bacterium to another can also cause a bacterium to become resistant rapidly For example
horizontal transfer of mecA gene from Staphylococcus sciuri results in the emergence of
MRSA (Carlos amp Cuevas 1996)
24 Screening Method for Antibiotic Activity
241 Agar Overlay Technique
Agar overlay technique is used for preliminary and secondary antimicrobial activity The
strength of an antimicrobial agent can be detennined by agar overlay technique
7
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
(Frankhauser 20 I 0) In this technique a melted agar with test bacteria is poured evenly on
the top of an agar plate and allowed to solidify Reduction in the turbidity of the test
bacterial lawn around the extracted colonies shows the zone of inhibition Zone of
inhibition produced because of the antibacterial properties of the colony The size of the
zone of inhibition formed around it reflected the strength of the antimicrobial agent A
wider zone of inhibition will be observed for a greater strength of antimicrobial agent
(Hosttetmann 1999)
242 Antimicrobial Susceptibility Using Disc Diffusion Method
For the discovery of new antimicrobial drugs that are effective against an infection it is
crucial to determine the susceptibility of the pathogen Several techniques had been
developed for the test such as Ditch Plate Broth Dilution method standard tube dilution
minimum inhibitory concentrations standardize antibiotic sensitivity testing (AST) method
and others Kirby-Bauer disc diffusion method is commonly used as antibiotic
susceptibility test to determine whether a bacterium is susceptible to a specific antibiotic
(Jacobs et al 1979) The MIC value is determined to evaluate the minimum concentration
ofdrug that will inhibit the bacterial growth
During incubation the antibiotic diffuses outward into the medium and inhibits the
growth of test microorganism at certain concentration The effectiveness of antibiotics
towards test bacteria is analyzed by the formation of inhibition zone around the paper disc
The diameter of the zone of inhibition is proportional to the degree of susceptibility of the
test bacteria toward the antibiotic The organism is characterized as susceptible
8
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
middot
intennediate or resistant based on the diameter of the zone of inhibition formed (Mcdougal
amp Thornsberry 2000)
The paper discs which are impregnated with different concentration of antibiotic
are placed on the surface of an agar plate that has been inoculated with test bacteria Kirby
Bauer disk diffusion method employs the principle of the correlation between the size of
zone of inhibition around the antibiotic disc and sensitivity of bacteria to the antibiotic
(Drew el al 2001) The zone size is directly proportional to the sensitivity of the bacteria
to the antibiotic Antibiotic sensitivity is expressed in the term of MIC in which the
minimum concentration of drug that will inhibit the growth of bacteria was evaluated
25 Bioautography
Bioautography is a technique that combines thin layer chromatography (TLC) with
bioassay in situ (Shittu et al 2006) It can be used for the screening of separated
components of natural product extracts
Bioautography is often the only way to detect antibiotics that have been separated
on paper or thin layer chromatograms It is based on the biological effects of the substances
to be detected In general these effects can be the inhibition or promotion of growth of the
organism that is exposed to the zone that was separated on paper chromatography
(Touchstone 1992) Zones of inhibition in paper chromatography and zones of growth in
thin layer chromatograms make possible the detection of antibiotics Several hundred
antibiotics have been discovered in the past 30 years Due to the wide variations in
chemical properties of these compounds bioautography is the only general method for
their detection on thin layer chromatograms
9
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
In the flrst steps in the investigation of unknown antibiotics bioautography
becomes a method for classiflcation 0 crude antibiotics by TLC when it is not yet available
in pure form The method was developed for identifying the antibiotics contained in a
crude mixture during the early stages of isolation of these compounds The method was
used as a screening method for the present of antimicrobial activities in isolates ( Karen et
al 1996)
10
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
30 MATERIALS AND METHODS
31 Preparation of Media
311 Preparation of PDA Media
A total of 156 g of PDA powder was used to prepare 400 ml media The PDA powder was
transfer into a 400 ml bottle and added with distilled water Then the mixture was stirred
thoroughly until the powder completely dissolved Sterilization of the media was achieved
by autoclaving at 121degC for 2 hours Subsequently the PDA media was poured into petri
dish for preparation of PDA plates use for culturing the sterilize fungi samples About 13
ml of the media was aseptically poured into each plate Then the plates were allowed to
cool and solidify in the laminar flow hood before store at 4degC for future use
312 Preparation of NA Media
Similarly 112 g of NA powder were prepared for 400 ml media After autoclaving the
NA media was dispersed into petri dish to an average depth 4-5 nun After that the plates
were allowed to cool and solidify in the laminar flow hood before store at 4degC for future
use
32 Sampling Sites and Collections
The soil sample was collected in UNIMAS reserve forest areas (Figure 31) The sample
collected from two different sites with depths from 0-10 cm The samples then were
brought into laboratory and let to dry at room temperature
11
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
Figure 31 Map on location of soil sampling on UNIMAS reserve forest
33 Isolation of Microorganisms from Soil
331 Isolation and Subculturing
For each sample 19 of soil sample was transferred into a Falcon tube The soil Was then
suspended with 10 ml of PBS and homogenized with a Vortex mixer The tube was let to
stand at room temperature for 1 hour after which two ten-fold serial dilution with 9 ml PBS
100 11 of each dilution was dispensed into PDA or NA and spread with hockey stick
There were three replicates of NA and three replicates of PDA prepared Then the plates
were incubated at room temperature for 5 days
12
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13
332 Subculturing of Bacteria and Fungi Colonies
After 5 days of incubation fungal colonies that showed inhibition towards the growth of
other microorganism were isolated and subcultured onto new agar media to obtain the pure
culture Then the plates were incubated for another 3 days at room temperature
34 Identification and Classification of Fungal Isolates
The morphologies of the fungal isolates were identified through macroscopic and
microscopic observations
341 Macroscopic Examination
The cultures were observed and physical characteristics were identified such as top and
reverse color parameter growth behavior mycelia mat and changes of medium
342 Microscopic Examination
The slide culture of the fungal isolates was prepared A small sample of fungus and agar
were cut out from the fungal culture and were transferred onto microscope slide The
sample was covered with a cover slip supported by plasticins Subsequently the culture
slide was placed into petri dish which was then sealed with parafilm After 5 days of
incubation at room temperature examination of the slide culture was carried out by
Binocular Compound Microscope (NIKON Corporation Japan) Microscopic
characteristics such as mycelial end branching structure of hypha and presence of spore
were observed using light microscope
13