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Bioassay-guided Isolation of Antibiotics from Selected Marine Fungi
CHONG MENG SHIN
This project is submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARA W AK
2011
Acknowledgement
•
First, I would like to thank to Department of Molecular Biology, Universiti Malaysia
Sarawak for giving me the opportunity to fulfil my Final Year Project (FYP) with the
facilities provided. I really appreciate for all the materials, equipments, instruments, and
other facilities provided which is necessary for the completion of my project.
I would like to take this opportunity to thank my supervisor, Professor Dr. Ismail
bin Ahmad for his guidance, encouragement and concern throughout this project. He is
also the one who constantly keep track on my progress and gave me a lot of precious ideas,
information, knowledge and advices on my project and report writing. He always had time
for questions and discussions. I am very lucky being one of his FYP students.
This study is dealing with marine fungi which, were isolated by Mr Liu Yu Choi,
previous student during his undergraduate study .. I am grateful for being allowed to work
with this resource. A special thanks to all the postgraduates especially Miss Anita for the
guidance, valuable advice and friendly help.
Finally, I would like to thank all my colleagues for their ideas, advices and
collaborations as we shared most of the moment working together at the laboratory. I
appreciate the valuable experience, knowledge and laboratory skills that I gained
throughout this project.
I
Table of Contents
Page
Acknowledgement. . .. . . . . .. . .. . . . . ..... . ..... . . . . .. . . .. .. . . . . .. . . . . .. . .. . . . . ..... . . . . .... ...... I
Declaration...................................................................... ................ II
Table of Contents. ..................................... ........ ......................... ....... III
List of Abbreviations.......................................................................... VI
List of Tables ................................................................................ ". VII
List of Figures. .... ...... ......... ........... ... .... ............... ......... ......... ........ ... VIII
Abstract............................. ...... ..................... .............................. .... 1
1.0 Introduction................................................................................. 2
2.0 Literature Review.......................................................................... 4
2.1 Antimicrobial Compounds ............................. ~ ....................... ". 4
2.2 Antibiotic Resistant Bacteria........................................................ 5
2.3 Marine Environment ...... , .... ..... . ..... . .. . .. . .. . .. . ........ . .. . ..... . .. . .. . .. . .... 6
2.4 Marine Biofilm.................................................................... ..... 7
2.5 Antibiotic Producing Microbes from Marine Biofilm....................... ..... 8
2.6 Antimicrobial Screening Assays.......................... .................. ......... 9
2.6.1 Agar Overlay Technique.................... ................................... 9
2.6.2 Disc Diffusion Assay.......................................................... 9
2.7 Antibiotic Extraction and Fractionation............................................ 10
3.0 Materials and Methods.. ................................. ................................. 11
3.1 Preparation of Sodium Chloride Stock Solution............................. ..... 11
3.2 Preparation of Culture Media.. ...... ......... ...... ...... ......... .................. 11
3.3 Revival and Storage of Selected Fungal Isolates................................. 11
3.4 Identification and Characterization of Fungi.. .................................... 12
III
3.4.1 Macroscopic Examination..................................................... 12
3.4.2 Microscopic Examination..................................................... 12
3.5 Antibacterial Screening of Fungal Cultures............... .................. ....... 12
3.6 Antibiotic Extraction.......................... ........................................ l3
3.7 Minimum Inhibitory Concentration of Extracted Antibiotics ........... ....... 14
3.7.1Test Bacteria Preparation...................................................... 14
3.7.2 Antibacterial Screening of Extracted Antibiotics: Disc Diffusion Assay.. ...... ..................... ..................... ... ..... 14
3.8 Thin Layer Chromatography........ ..... ..................... ....... ................. 15
4.0 R5>Sults............................................................................. ............ 16 /
4.1 Revival and Cultivation of Selected Fungal Isolates........ ............... ....... 16
4.2 Identification and Characterization of Fungi.. ..... ............................... 17
4.2.1 Macroscopic Examination................................... ............. ..... 17
4.2.2 Microscopic Examination..................................................... 17
4.3 Antibacterial Screening of Fungal Cultures...................... ........ .... ....... 20
4.4 Minimum Inhibitory Concentration of Extracted Antibiotics.... ...... .... ..... 21
4.4.1 Preliminary Test.......................................................... ........ 21
4.4.2 Antibacterial Screening of Extracted Antibiotics: Disc Diffusion Assay.......................................................... 22
4.5 Thin Layer Chromatography......................................................... 24
5.0 Discussion......................................... ......................................... 26
5.1 Revival and Cultivation of Selected Fungal Isolates............................. 26 )5.2 Identification and Characterization of Fungi.. ...... ............................... 26
5.3 Antibacterial Screening of Fungal Cultures..................... ................... 27
5.4 Antibacterial Screening of Crude Extracts ofIsolate 4.1.2........................ 29
5.5 Thin Layer Chromatography......................................................... 31
IV
6.0 Conclusion. ................................. ...... ............... ...................... ..... 32
References........ ..................... ...... .................................................... 33
! l.:.1
v
I
DCM
MHA
MHB
MIC
MRSA
NA
NaCI
PDA
TLC
List of Abbreviations
Dichloromethane
Mueller-Hinton Agar
Mueller-Hinton Broth
Minimum Inhibitory Concentration
Methicillins-resistant Staphylococcus au reus
Nutrient Agar
Sodium Chloride
Potato Dextrose Agar
Thin Layer Chromatography
VI
List of Tables
Table Description Page
Table 1 Growth characteristics of the selected fungal isolates..... ................ 17
Table 2 Descriptions for the identitication of fungal isolates...................... 18
Table 3 Antibacterial activities shown by selected fungal isolates against the test bacteria..................................................................... 20
Table 4 Relative strength of extracts in five different concentrations tested against four test bacteria............................... .... ........ ............ 23
Table 5 Thin layer chromatography (TLC) profiling ofDCM extract (6ul) of Bipolaris sp. ...................... ........................ ............ .......... 25
VII
,...
List of Figures
Figure Description Page
Figure 1 PDA with fungal colonies, 4.1.2 (A), P 2.2.1 (B), P 8.1.2 (C)........... 16
Figure 2 Slide-culture of isolate P 2.2.1 (A), 4.1.2 (B) and P 8.1.2 (C)........... 19
Figure 3 Inhibition zones resulting from antimicrobial activities shown by isolate 4.1.2 against S. typhi (A), aerogenes (B), s. alireliS (C) and E.coli (D)................ .... ........... ............................... .......... 21
Figure 4 Disc diffusion for antibiotics screening of methanol fungal extract with different dilution against E.coli (A), S. allreus (B), aerogenes (C), S. typhi (D)..... ......... .................... ........................................ 24
Figure 5 The spots marked with red pencil indicates the UV-spots; the spot marked with green pencil indicates the spot observed with vanillinsulfuric acid ofDCM extract using solvent system DCM-ethyl acetate in ratio of 2: 1.......................................................... 25
VIII
.... •
Bioassay-guided Isolation of Antibiotics from Selected Marine Fungi
Chong Meng Shin
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
The emergence of new life-threatening infectious diseases and the rapid spread of antibiotic-resistant bacteria in nature have raised the awareness of researchers towards screening for novel antibiotics. In response to the marine environment as a potential source of novel secondary metabolites, a study of antibacterial activity in marine fungi isolates from marine biofilm was carried out. Three fungal isolates designated as 4.1.2, P 2.2.1 and P 8.1.2 screened for antibiotic activity using agar overlay technique against one Gram-positive, Staphylococclls aurells and three Gram-negative, Escherichia coli. Salmonella typhi and Enterobacter aerogenes test bacteria. Result showed that only isolate 4.1.2 exerted strong antibacterial activity against Staphylococcus aureus and Enterobacter aerogenes. In contrast, only weak antibacterial activity was obtained against Escherichia coli and Salmonella typhi. Fungal isolates, 4.1.2, P 2.2.1 and P 8.1.2 were putatively identified using slide culture method up to genus level as Bipolaris sp., Endophragmia sp. and Penicillium sp., respectively. Hexane, chloroform, dichloromethane (OCM), ethyl acetate, water and methanol extracts from isolate 4.1.2 (Bipolaris sp.) were subjected to antibacterial screening against the four test bacteria using disc diffusion method. However, none of the extracts showed antibacterial activities. This result suggested that antimicrobial substances produced by Bipolaris sp. could be antimicrobial peptides, and thus failure in obtaining antimicrobial peptide activity could be due to the denaturation of protein-based antimicrobial. The result of thin layer chromatography demonstrated that solvent system OCM-ethyl acetate in ratio of 2: 1 as a better solvent system for fractionation of OCM extract as three spots were visualized under UV light, and with vanillin solution. Further optimization of solvent system could be done for better separation of OCM extract into distinct components. Further studies upon the antifungal activity of Bipolaris sp. can be taken into consideration as its antifungal property was detected against different fungal species.
Keywords: Antibacterial activity, marine fungi, extracted antibiotic, thin layer chromatography
ABSTRAK
Kemunculan penyakit berjangkit yang mengancam nyawa dan penyebaran bakteria rintang-antibiotik telah meningkatkan kesedaran para penyelidik terhadap perlunya penyaringan bagi mendapatkan antibiotik baru. Persekitaran laut yang terkenal dengan potensinya penemuan sumber metabolit sekunder baru, satu kajian aktiviti antibakteria kepada pencilan kulat laut daripada bio-filem laut telah dijalankan. Tiga pencilan kulat yang disebutkan sebagai 4.1.2, P 2.2.1 dan P 8.1.2 telah digunakan bagi penyaringan untuk mengesan aktiviti antibiotik melalui teknik agar overlay terhadap bacteria ujian satu Gram-positif, iaitu Staphylococcus al/reus dan tiga Gram-negatif, iaitu Escherichia coli. Salmonella typlli dan Enterobacter aero genes. Keputusan menjelaskan hanya pencilan kulat 4.1.2 menunjukkan aktiviti antibakteria yang kuat terhadap Staphylococcus fJ1!l1lli§. dan Enterobacter aerorrenes. Aktiviti antibakteria yang lemah terhadap Escherichia coli and Salmonella tyolli. Pencilan kulat iaitu 4.1.2, P 2.2.1 dan P 8.1.2 masing-masing dikenalpastikan dengan kaedah kultur-slid kepada tahap genus sebagai BipOlaris sp., Endophrarrmia sp. dan Penicillium sp. Namun, ekstrak heksana, kloroform, diklorometana(OCM), etil asetat, air dan methanol daripada pencilan kulat 4.1.2 (Bipo/aris sp.) dijalankan penyaringan antibiotik terhadap bakteria ujian dengan kaedah difusi disk. Semua ekstrak tidak menunjukkan aktiviti antibakteria. Keputusan antibakteria yang diperoleh mencadangkan bahawa kemungkinan bahan antibakteria yang dihasilkan oleh Bipolaris sp. adalah antimikrob peptida, dan dengan demikian kegagalan memperoleh aktiviti antimikrob peptida mungkin disebabkan oleh denaturasi antimikrob berasaskan protein. Keputusan kromatografi iapisan nipis menunjukkan sistem pelarut OCM-etil asetat dalam perbandingan 2: 1 sebagai sistem yang lebih baik untuk fraksinasi ekstrak OCM dengan tiga tompok divisualisasikan di bawah sinar UV, dan dengan larutan vanilin. Pengoptimuman sistem pelarut lanjut boleh dilakukan untuk pemisahan ekstrak OCM yang lebih baik kepada bahagian yang berbeza. Kajian lanjut berkaitan aktiviti antikulat Bipolaris sp. boleh dipertimbangkan kerana ciri-ciri antikulat telah dikesan terhadap kulat yang berlainan species.
Kata Kunci: Aktiviti antibakteria, kulat taut, antibiotik ekstrek, kromatografi tapisan nipis
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1.0 Introduction
•
The success of antibiotics as agents for controlling life-threatening infectious diseases is
one of the most profound medical advances in the twentieth century (Simmons et al., 2010).
However, misuse and overuse of antimicrobial drugs have resulted in the emergence of
antibiotic resistant bacteria and new infectious diseases of which a serious threat to global
public health (Simmons et al., 2010). According to the World Health Organization (2001),
more than 95% of Staphylococcus aureus strains worldwide are now resistant to penicillin,
and up to 60% are resistant to its derivative, methicillin. Therefore, a continuing need to
identify novel substances with broadened antimicrobial activity towards highly resistant
pathogens to treat infectious diseases becoming significantly important (Breithaupt, 1999).
Antibiotic-producing microbes such as Actinomycetes are the most widely
distributed group of microorganisms in nature, which primarily inhabit the soil (Parungao
et al., 1997). However, a study in recent has shown the rate of finding of new antimicrobial
substances from terrestrial Actinomycetes has decreased (Lam, 2006). Although the source
from terrestrial environment is abundant, the greatest biodiversity is in the oceans (Lam,
2006). According to Mearns-Spragg et al. (1997), there is high expectation that organisms
from the marine environment that have not been well-investigated will yield a range of
new pharmaceutical compounds with novel activities. Such discovery will provide new
drugs to inhibit microbial pathogens currently developing resistances to conventional
antibiotic therapies. Since ocean has a greater diversity of organisms (Mearns-Spragg et
aI., 1997), many scientists describe marine as a source for possible novel antibiotics to
encounter resistant and new infectious diseases.
While microbes are often thought of as free-floating and rapidly-multiplying single
cells, most microbes actually live in communities (Schachter, 2003). According to
Breithaupt (1997), previous studies in the 1970s found that more than 99.99% of the
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•
resident bacteria in mountain streams were attached to surfaces as biofilm coverings. This
has led to the belief that bacterial biofilms on all surfaces in marine environment possess
expectable enormous biodiversity of marine microorganisms (Schachter, 2003) and such
microorganisms have evolved a plethora of resistance mechanisms in order to survive in
the competition for space. The emergence of new combinations of resistance genes
happens most frequently in compartments with marine biofilms of which bacterial density
is very high (Kummerer, 2004). As a result, an approach for identifying new active
molecules through the screening of new antimicrobial activities of microbes from marine
biofilm is justified.
Accordingly, Liu (2010) had studied the antibiotic production of marine microbes
from marine biofilm. In the present study, three isolates of the fungal isolates were used to
isolate antimicrobial secondary metabolites. First, fungal isolates were subjected to
antibacterial screening using disc diffusion method and subsequently, macroscopic and
microscopic examination via slide culture method. Isolate 4.1.2, which is the most
potential of producing secondary metabolites in the antibacterial screening needed to be
subjected to antibiotic extraction using six solvents of different polarity. Lastly, the
determination of the minimum inhibitory concentration (MIC) of extracts was performed to
allow the selection of potential extract for fractionation. Finally, extract fractionation into
various distinct compounds was carried out using thin layer chromatography (TLC).
The objectives of this study are:
1. To identify and characterize the selected fungal isolates.
2. To screen the fungal isolates for the presence of antimicrobial activities.
3. To extract and fractionate antibiotics from fungal isolate with the most potential of
producing antibiotics.
4. To determine minimum inhibitory concentration (MIC) of extracted antibiotics.
3
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2.0 Literature Review
.,.' •
2.1 Antimicrobial Compounds
The history of antimicrobials begins with the study by Louis Pasteur and Jules Francois
Joubert, in 1877, who found that one type of bacteria could prevent the growth of another
(Walsh, 2003). One bacterium produces antimicrobial substances to inhibit the growth of
other microbes. Subsequent in 1928, Demain and Sanchez (2009) reported that the
discovery of antimicrobials such as penicillin by Alexander Fleming has paved the way for
better health for the world population. Then, efforts for the discovery, development, and
clinical use of antibiotics in treating bacterial infections began in 1930s can be considered
as one of the medical advances (Simmons et al., 2010). With the discovery of penicillin,
most of the outbreaks of infectious diseases were successfully treated and mortalities
caused by bacterial infections have substantially decreased.
An antibiotic or antimicrobial particularly fungi and bacteria (Demain & Sanchez,
2009) is a chemical substance known as secondary metabolites, which mainly produced by
microfungus. Natural antibiotics can be found from terrestrial counterparts, soil and marine
environments. Walsh (2003) claimed that the mode of actions for this natural product is by
killing or inhibiting the growth of other microorganisms, both bacteria and fungi. For
example, one of the major groups of antibiotic-producing bacteria that contributing to
antibiotic production is Actinomycetes (Walsh, 2003).
According to Simmons et al. (2010), antibiotic is one of the most profound medical
advances of the twentieth century that has substantially decreased mortality caused by
bacterial infections. There are almost no therapeutically useful agents that are as effective
as both antibacterial and antifungal because of the different cellular targets and microbial
cell penetration issues (Walsh, 2003). As a result, there is a great demand for novel
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secondary metabolites belonging to the wide range of structural classes which selectively
acting on novel targets with fewer side effects (Wattanadilok et al., 2007).
2.2 Antibiotic Resistant Bacteria
Bacteria have been able to evolve to become antibiotic resistant. Its development has
become one of the greatest concerns with regard to the increased use of antibacterial and
antifungal (Kummerer, 2004). As stated by Asad (2009), the development of antibiotic
resistance mechanism in bacteria most probably is through the transformation of genetic
material from resistant strains to wild strains. Bacteria, especially marine species are able
to produce microbial inhibitory substances results in a serious threat to global public health
as early as 1917 (AI-Raj et aI., 2009).
According to Asad (2009), s. aureus was the first bacteria to resist penicillin. Thus,
results in the occurrence of various antibiotic resistant infections, which mainly caused by
the increasing rate of emerging resistance among pathogenic bacterial populations and
resistance mechanisms to many conventional antibiotics (Breithaupt, 1999). The presence
of antibiotic resistance has greatly reduced the effectiveness of antimicrobial drugs that are
currently available against pathogenic bacteria. Four cases of clinically-important bacteria
such as S. aureus, resistant to vancomycin in overseas countries of which the antibiotic of
last option in treating severe infections (Breithaupt, 1999).
Recently, a team of scientists speaking at the annual meeting of the American
Association for the Advancement of Science, called for new awareness of the potential for
antibiotic resistant infections from the marine environment. Then, a study by Blackburn et
al. (2010) on interspecific comparisons between redfish, Sciaenops ocellata, and sharks
from Louisiana offshore water had been carried out, and eventually found a drastically high
prevalence of antibiotic resistant bacteria in marine predatory fish.
5
In addition, the emergence of the most problematic Gram-positive bacterium,
which is Methicillin-resistant Staphylococcus aureus (MRSA) proved to be highly
prevalent and resistant to almost all available antibiotics except vancomycin and
teicoplanin (Isnansetyo & Kamei, 2003). There is an increased concern in healthcare
institutions worldwide towards the discovery of novel antimicrobial drugs (Sunilson et al.,
2009). To combat the antibiotic resistant pathogen, many efforts have to be done by
researchers to identify novel antimicrobial compounds that function through novel
mechanisms of action from marine sources (Ramesh & Mathivanan, 2009).
2.3 Marine Environment
The ocean covers nearly 71% of the earth's surface rich of marine life (Lam, 2006) such as
marine derived plants, animals and microorganisms, which can produce huge variety of
natural products with great diversity. The exploration of the marine environment began in
1960s as a rich source for the isolation of new compounds. According to Mearns-Spragg et
at. (1997), the ocean has the potential to supply novel source of structurally unique
bioactive agents because it represents a much greater diversity of organisms and
approximately contains up to 200,000 invertebrate and algal species.
The yield of novel metabolites is decreasing although historically, most bioactive
products of microbial origin have come from terrestrial microorganisms belonging to one
taxonomic group, the Actinomycetes (Beman et al., 1997). Lately, marine microorganisms
have been recognized as rich sources of structurally novel and biologically active
secondary metabolites that are different from terrestrial microbes (Cooper, 2004).
Taxonomically diverse fungal species from marine have been found to exhibit highly
specific adaptations within the marine environment (Wattanadilok et al., 2007). Marine
microorganisms are able to survive on their unique habitats with their metabolic and
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physiological capabilities besides providing a great potential for production of metabolites
(Takamatsu et al., 2003). For example, marine derived fungi can produce efficient
antifungal compounds with different modes of action and selective antifungal activity
(Takamatsu et al., 2003).
2.4 Marine Biofilm
Every surface immersed in the sea will rapidly cover with biofilm (Annstrong et aI., 200 I).
The fonnation of biofilms involves attachment of bacteria on the surfaces, which is
mediated by cell-surface organelles (Prub et al., 2010). Highly specific microbial
associations from marine biofilm have lead to nutrient acquisition, metabolic waste
processing, and most importantly production of secondary metabolites (Thomas et al.,
2010). Secondary metabolites production is crucial for the discovery of novel antimicrobial
such as a marine bacterium, Vibrio species, which isolated from the surface of the soft
coral Sinularia polydactyla collected in the Red Sea was found to be a abundant producer
of secondary metabolites with antibacterial activities (Al-Zereini et al., 2010).
Recent findings have implicated that biofilms are taxonomic barrier free
(Kummerer, 2004), which means they contain mix varieties of microbes. It serves as
reservoirs for pathogenic microorganisms and sources of disease outbreaks such as
antibiotic resistant infections (Schachter, 2003). According to Mohamed and Huang (2007),
infections mediated by biofilms are difficult to eradicate because biofilm inhabitants are up
to 1,000 times more resistant to antibiotics than are free-floating bacteria. Although
bacterial biofilms are tough to eradicate and cause problems to living communities on earth
(Prub et al., 2010), they are medically important as a source of new bioactive agents for
use in the treatment of diseases.
7
2.5 Antibiotic Producing Microbes from Marine Biofilm
Microorganisms are capable of making secondary metabolites, which essential for
application in antifungal, antibacterial and antiviral infections (Demain & Sanchez, 2009).
The great diversity of marine environments has exerted a pressure on bacteria selection
leading to the synthesis of new metabolite. Bacteria fonn biofilms when colonizing plants
and sedentary animals. These biofilms have become the source of diverse chemical
compounds producers that have the ability to protect the host against pathogenic
microorganisms (AI-Zereini et aI., 20 I 0).
Marine microorganisms, particularly marine derived fungi, have recently been
highlighted as an important source of biologically active secondary metabolites. Marine
fungi have the potential to produce clinically active compounds is currently even more
vital than that of bacteria (Thomas et al., 20 I 0). According to Zhang et al. (2009), the
marine fungus Pestalotia sp. isolated from the surface of the brown alga Rosenvingea sp.
was able to produce a new chlorinated benzophenone compound, pestalone, which showed
potent antibiotic activity against MRSA with MIC value equals to 37 ng/mL, and VREF
with MIC value equals to 78 ng/mL, revealing its potential as new antibiotic. Besides, a
study by Penesyan et al. (20 I 0), the epibiotic microorganisms that are associated with their
nutrient rich host have also been shown to produce the wide range ofbioactive compounds,
which defends the host against surface colonization. For example, the surfaces of the
lobster Homarus americanlts are covered almost exclusively by a single gram-negative
bacterium that produces an antifungal compound highly effective against the
fungus Lagenidium callinectes, a common pathogen of many crustaceans (Penesyan et al.,
20 lO). Hence, marine microbes that colonized the surface of marine organism provide a
potential source for the discovery of new antimicrobial drugs.
8
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2.6 Antimicrobial Screening Assays
2.6.1 Agar Overlay Technique
Agar overlay technique is used for the screening of antimicrobial activities in microbial
isolates (Saeed et aI., 2004). The technique consists of two layers on assay plates with
lower basal agar layer seeded with bacterial or fungal isolates and being overlaid with
another layer of 0.75% soft agar seeded with test bacteria on top of the basal agar (Waffo,
2004). The resulting inhibition zones formed around the colonies is due to the presence of
antibacterial activities. As commented by Liu (2010), screening for antibacterial activities
in fungal isolates using the agar overlay technique is more successful than for bacteria due
to stronger attachment of fungal colony on solid media without being carried away by the
flow ofoverlaid agar.
2.6.2 Disc Diffusion Assay
A cornmon application of disc diffusion susceptibility test or Kirby-Bauer method is used
to determine whether particular bacteria isolates are susceptible to specific antibiotics
(Wheat, 2001). The principle of this qualitative technique is that antibiotic is placed on
agar the antimicrobial will diffuse from the disc into the agar medium, incubating the plate
overnight, and measuring the presence or absence of a zone of inhibition around the disc.
Zone of inhibition that is observed is a result of the inhibitory effect of antimicrobial
substances preventing growth of the test bacteria around the disc (Drew et aI., 1972). The
extent of the inhibition zone is related to the rate of growth of the indicator organism.
According to Drew et al. (1972), the inhibition zone size is determined by incubation time,
level of incubation popUlation, incubation temperature and the media.
9
2.7 Antibiotic Extraction and Fractionation
In a study by Kellam et al. (1988), solvent extraction method is used to extract antibiotics.
Organic solvents used in solvent extraction to dissolve antibiotics can be classified into two
categories, which are polar and non-polar. Generally, solvents such as methanol and
ethanol are known to polar solvent whereas non~polar solvent, for example chloroform
(Kellam et al., 1988). Various types of extracts can be obtained with different polarity of
solvents.
A method used to separate various components of the extracts is thin layer
chromatography (TLC). TLC is an analytical technique that used for qualitative analysis of
complex mixtures and for the identification of unknown compounds. It is also important
for developing a chromatographic system. TLC is composed of two phases, a mobile and a
solid phase. The solid phase is a thin solid support that usually consists of Alumina or
Silica while the mobile phase is a solvent that moves through capillary action right through
the solid phase (Volland, 2005). TLC is one of the most widely used separation techniques
as it is simple, inexpensive and convenient.
Bioautography on TLC plates is mostly employed to detect the biological activity
of a sample, which has migrated on the plate with appropriate solvent (Marston, 2010). It
can be used for the target-directed isolation of complex mixtures. Chromatoplate spotted
with extract is developed in a solvent system comprising of either one or many different
organic solvents (Chakraborty & Chakraborti, 2010) to separate various components from
the extract, which exhibited antibacterial activity.
10
3.0 Materials and Methods
3.1 Preparation of Sodium Chloride Stock Solution
Sodium Chloride (NaCl) stock solution was prepared by dissolving unprocessed salt (NaCI)
into 200ml of distilled water. Subsequently, the stock solution was filtered through Whatman
No.1 filter paper. Then, the stock solution was stored at 4°C in the cold room.
3.2 Preparation of Culture Media
All media in this study was supplemented with 3.5% NaCI (w/v), which is equivalent to 10%
seawater (Bergman, 2001). Potato Dextrose Agar (PDA) was prepared by adding known
volume of sodium chloride solution into conical flask containing agar media. The media
was boiled with stirring on a magnetic stirrer hotplate and then autoclaved at 121°C, 15 psi
for 20 minutes. Next, the media were cooled down in water bath at 50°C and poured onto
the Petri dishes in the laminar hood to prevent contamination. After the media have
solidified, the plates were stored at 4°C in the cold room.
3.3 Revival and Storage of Selected Fungal Isolates
Three selected fungal isolates were revived by transferring them onto agar plates from
original stock culture, which prepared by Liu (2010). Isolated fungal colonies were
cultured on a fresh plate using the same culture media from which it is derived and
incubated for 2 to 4 days at the room temperature (28°C). Daily examination was carried
out for fungi growth. Subsequently, fungal isolates were sub-cultured to new agar plates by
transferring a small piece of its mycelial growth onto surface of new agar plates where the
mycelial growth were in contact with the agar. Pure isolates were obtained. Then, fungal
isolates were inoculated onto slant agars and incubated at similar incubation temperature
and length. The slant agars were stored at 4°C in the cold room as stock culture.
11
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3.4 Identification and Characterization of Fungi
Selected fungal isolates, which showed the most potential of producing antibiotics in the
antibacterial screening were selected for identification and characterization.
3.4.1 Macroscopic Examination
The growth characteristics of selected fungal isolates were determined by macroscopic
observation after the fungal colony has grown to full plate. Macroscopic observation was
made on the colour of the mycelial mat, reverse colour, margin of the colony, mycelial mat
characteristics, and the colour change of culture media (Maza et al., 1997).
3.4.2 Microscopic Examination
The identification of fungi was carried out through microscopic examination using slide
culture method (Maza et al., 1997). A small sample consisting of agar and fungal growth
was cut out from a fungal culture, and was aseptically transferred onto a sterilized
microscope slide. The sample was then covered with a cover slip, which will be supported
by plastic ins. The culture slides were placed in Petri dish and sealed with parafilm and then
incubated at room temperature for 3 to 5 days to allow sporulation. The slides were then
examined under the microscope for the presence of spores, spore structure and the hyphae
structure. The identification of fungi was based on The Saccardo System of Classification
with the aid of descriptions found in Illustrated Genera of Imperfect Fungi (Barnett &
Hunter, 1972).
3.5 Antibacterial Screening of Fungal Cultures
The fungal isolates were sub-cultured from slant agar onto PDA. After 5 days incubation at
room temperature, the fungal isolates were sub-cultured to new plates with maximum of
four isolates per plate. Subsequently, fungal isolates were incubated at room temperature
12
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for 2 days before screening them against the test bacteria using agar overlay teclmique.
Selected fungal isolates were screened for the presence of inhibition zone against the test
bacteria.
Antibacterial screening was performed against four bacteria species: one Gram
positive bacteria, S. aureus and three Gram-negative bacteria, consisting of E. coli, S typhi
and E. aerogenes. The test bacteria were prepared on NA and incubated at 37°C for
overnight. Single colonies were then inoculated into nutrient broth (NB) and incubated at
the same condition. The final concentration of the test bacterial suspension was adjusted
(Ahmed et al., 2008) to optical density (OD) of 0.168 at 550nm. Test bacterial suspension
were then be added to the soft agar. The soft media were overlaid onto agar plates that
were seeded with the fungal isolates and incubated at room temperature for 24 hours. After
incubation, the plates were checked for the presence of inhibition zone of growth inhibition
around the bacterial spots as a result of antibacterial activities.
3.6 Antibiotics Extraction
As isolate 4.1.2 was the only fungal isolate found to show antibacterial activity against the
test bacteria, this isolate was subjected to a small scale antibiotic extraction. A total of 18
plates were prepared three colonies per plate. These colonies were grown on solid agar
until almost full plate and then let to dry at room temperature to remove most of the water
that is present in the agar. Dried agar were then peeled off from Petri dish and cut into
small pieces.
Subsequently, agar pieces were submerged in six different organic solvents, which
were hexane, chloroform, dichloromethane (DCM), ethyl acetate, water, and methanol of
30 ml using six different conical flasks to dissolve different antimicrobial substances from
marine fungus. Each 250 ml conical flask was specific for one type of organic solvent.
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f'
Each extract was then filtered and poured into 15 ml universal bottles. Initially, the
universal bottles containing extracts were left to dry at room temperature for one month.
Also, organic solvents were let to dry using water bath and incubator because a small scale
antibiotic extraction prohibited us from using the rotary evaporator. Various extracts of
solvents were tested for the presence of antimicrobial activity.
3.7 Minimum Inhibitory Concentration of Extracted Antibiotics
3.7.1 Test Bacteria Preparation
The antibacterial activities of extracted antibiotics were assessed against four bacteria
species: Gram-positive bacteria, S. aureus and Gram-negative bacteria, consisting of E.
coli, S. typhi and E. aerogenes. The test bacteria were prepared on Mueller-Hinton agar
(MHA) and incubated at 37°C for overnight as described by Val gas et al. (2007). Single
colonies were then inoculated into Mueller-Hinton broth (MHB) and incubated at similar
condition. The final concentration of the best bacterial suspension was adjusted (Ahmed et
al., 2008) to OD 0.168 at 550nm.
3.7.2 Antibacterial Screening of Extracted Antibiotics: Disk Diffusion Assay
This step was performed according to method described by Choudhury et al. (2005). 100 ul
of the standardized test bacteria was swabbed onto MHA. When dried, 7 antibiotic free
filter discs of 6 mm diameter were arranged on the petri dish containing agar. 1 mg of
extract was weighted and dissolved in 100 ul of 5 % methanol and 900 ul of sterile distilled
water. The solution was diluted into 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml
and 10 ul of solutions were transferred into each filter disc. 10 ul of sterile distilled water
was used as negative control while 10 ul of 2.5x dilution of Penicillin-streptomycin as
positive control. The petri dish was incubated at 37°C for 18-24 hours. Formation of
14I
J
r,,
inhibition zone was observed and measured. The antibacterial activity was expressed as the
mean of inhibition diameters (mm) produced as described by Choudhury et al. (2005).
3.8 Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out as described by Volland (2005). Initially,
fractionation of extract using TLC was conducted in glass container. The stationary phase
was chromatography plate covered with silica gel while two types of mobile phase
mixtures, which were OCM-hexane and OCM-ethyl acetate solvent mixtures being
employed.
Initially, 6ul of OCM extract (I mg/ml) was dropped at 0.5 centimeter (cm) from the
chromatography plate base. The plate was then allowed to air-dry before inserted into glass
container containing filter paper. Two solvent systems, which were OCM-ethyl acetate
solvent mixture in ratio of 1: 1 and 2: I, and OCM-hexane solvent mixture in ratio of 1.1,
3.1 were used. Each run was stopped when the solvent front reached 0.5 cm from the
chromatography plate end and the solvent end-point was marked with straight line using
pencil. Then, the developed TLC plates were air-dried.
The presence of spots and bands were visualized under UV irradiation at 254 nm
and marked with red colour pencil. After that, the chromatograms were dipped into 10% of
H2S04 in vanillin solution followed with heating at 110°C using hair-dryer until no bands
appeared. The bands were marked with green colour pencil. The Rr value for each spot on
the chromatogram was calculated (Harbone, 1973) using the fonnula:
Distance of sample travelled Rf
Distance of solvent travelled
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4.0 Results
4.1 Revival and Cultivation of Selected Fungal Isolates
All selected marine fungal isolates were successfully revived (Figure 1) from the original
stock culture, which was prepared by Liu (20 1 0). Each revived isolate was cultivated on
PDA supplemented with 3.5% NaCl (w/v), which is equivalent to 10% seawater. In this
study, there was colour change of the culture medium during the culturing of isolate 4.1.2,
whereas isolates P 2.2.1 and P 8.1.2 did not. Culture medium of isolate 4.1.2 that originally
transparent in colour turned into slightly brownish transparent in colour. Also, isolate 4.1.2
was found to be slow in growth as compared to isolate P 2.2.1 and P 8.1.2.
Figure 1: PDA with fungal colonies, 4.1.2 (A), P 2.2.1 (B), P 8.1.2 (C).
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Recommended