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

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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

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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

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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.

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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.

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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.

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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.

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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.

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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

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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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