IDENTIFICATION AND CHARACTERISATION OF GUT MICROFLORA OF INSECTIVOROUS BATS FROM

SARAWAK

Clarice M. Asinti (35812)

QR Bachelor of Science with Honours41.2

(Resource Biotechnology)A832 2015 · 2015

Pusar Khidmat MnkJumat Akad m i VER'm MALAYSIA SARAW,d ~

Identification and Characterisation of Gut Microflora of Insectivorous Bats from Sarawak

"

Clarice M. Asinti (35812)

A Thesis Submitted in Partial Fulfillment of the Requirement of the Degree of Bachelor

Science with Honours (R~source Biotechnology)

p ' Supervisor: Dr. Faisal Ali Anwarali Khan Co-supervisor: Dr. Lesley Mamice Bilung

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2015

DECLARATION

With this, I hereby declare that this thesis entitled "Characterisation and identification of

gut microflora of insectivorous bats from Sarawak" is my original work except for

quotation and citations, all of which have been duly acknowledged. I would also like to

declare that it has not been previously or concurrently submitted for any other degree at

UNIMAS or other institutions.

Clarice M. Asinti

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

University Malaysia Sarawak

ACKNOWLEDGEMENT

First of all, I would like to thank God for His grace that given me strength, determination,

health, and faith to complete my Final Year Project for bachelor degree. I would also like

to take this opportunity to express my deepest appreciation to my supervisor, Dr. Faisal Ali

Anwarali Khan and my co-supervisor, Dr. Lesley Maurice Bilung for their support,

guidance, constructive comment, and continuous help throughout this whole two

semesters.

I would like to thank research grant from Ministry of Education RACE/G(2)/ [ 10712013

(15) and Sarawak Forestry Coperation Sdn. Bhd. (SFCSB) for Permit No:

NCCD.907.4.4(JLD.l 1)-11 and Park Permit No: 12/2015 to conduct research from

biological resources. I would also like to thank postgraduate students especially from

Microbiology Lab, Mdm. Pui Chai Fung and Ms. Cindy, from Department of Zoology,

Mdm Sultanah Habeebur and Ms Farah, fof their kindness helps and assistance throughout

the project. I also owe my gratitude to lab assistances, for both Microbiology lab and

External lab, especially Mr. Azis Ajim and Mr. Huzal Irwan Husin for their help in

supplying all the apparatus needed in the project.

I also like to thank all my Microbiology, Molecular Ecology laboratory and fellow

colleagues for their wonderful friendship, support, and who have helped me a lot during

completion of my project and thesis writing. I wish to thank to, and other mates

I would like to express my gratitude to my beloved family for their loves, advice and

support through my ups and downs that give me strength to finish my project. Lastly,

thanks to all lecturers, course mates and everyone that have contributed either directly or

indirectly toward the completion of this project.

11

P'.sat Khidmat Maklumat Akaden' , rn M....J u~\' ,,, SARAW

TABLE OF CONTENTS

DECLARATION

ACKNOWLEDGEMENTS II

TABLE OF CONTENTS III

LIST OF ABBREVIATIONS v

LIST OF TABLES VI

LIST OF FIGURES VII

ABSTRACT VIII

1.0 INTRODUCTION

2.0 LITERATURE REVIEW

2.1 Insectivorous bat 3

2.2 Microflora 4

2.3 Zoonotic disease 5

2.4 Biochemical tests 6

2.5 16S rRNA sequencing 7

3.0 MATERIALS AND METHOD

3.1 Sample collection of insectivorous bat 8

3.2 Sample colle~tion of bat gut microflora 8

3.3 Enumeration of bacteria 10

3.4 Identification of bacteria using biochemical tests 12

3.4.1 Gram staining 12

3.4.2 Motility test l3

3.4.3 Oxidase test l3

III

3.4.4 Catalase test 13

3.4.5 Citrate test 14

3.4.6 Triple Sugar Iron (TSn test 14

3.4.7 Methyl Red (MR) and Voges Proskauer (VP) test 14

3.4.8 Bile Esculine test 15

3.6 Molecular identification analysis

3.6.1 DNA extraction 16

3.6.2 PCR amplification 16S rRNA gene sequence 16

3.6.3 DNA sequence analysis 17

4.0 RESULTS

4.1 Sampling collection of insectivorous bats 18

4.2 Enumeration of bacteria 18

4.3 Identification of bacteria using biochemical tests 20

4.4 Identification of bacteria using 16S [RNA sequence 22

5.0 DISCUSSION

5.1 Enumeration of bacteria 24

5.2 Identification of bacteria using biochemical tests analysis 25

5.3 Identification of bacteria using 16S rRNA sequence 28

6.0 CONCLUSION AND RECOMMENDATION ' 30 .REFERENCES 31

APPENDICES 34

IV

LIST OF ABBREVIATIONS

% Percentage

°c Degree Celcius

ilL Microliter

bp Base pair

BLAST Basic Local Alignment Search Tool

CFU Colony forming unit

LB Luria-Bertani

mL Millilitre

MR Methyl red

PBS Phosphate-buffered Saline

rRNA Ribosomal ribonucl6ic acid

TSI Triple sugar iron

VP Voges Proskauer

~'

v

LIST OF TABLES

Page

Table I Colony-fonning unit (CFU) per mL and number of isolates for each 19 swab samples from 16 insectivorous bats

Table 2 Occurrence of genus identified isolates from four insectivorous bats 21

Table 3 Identification of isolates based on gene sequence analysis using 23 BLAST

level identity

Table 4 Closest match of isolates from GenBank were compared for their genus 23

.'

vi

LIST OF FIGURES

Page

Figure I A is the region of Southeast Asia with Malaysia highlighted with light

grey colour. B is the enlarge portion of Malaysia to show selected

sampling sites of insectivorous bats

9

Figure 2 Serial dilution-Plate Count agar technique 11

VII

Identification and Characterisation of Gut Microflora of Insectivorous Bats from Sarawak

Clarice M. Asinti

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Bats are among the mammalian species that can serve as the host for zoonotic pathogens, like virus, fungi and bacteria. Therefore, it is important to identify and characterise the microbial diversity that reside within these natural host. The purpose of this project is due to the awareness that the natural microflora of insectivorous bats that could potentially cause zoonotic disease to human. The identification and characterisation of the isolated gut microflora were conducted through biochemical tests analysis and molecular methods using PCR for DNA sequence analysis of 16S rRNA. The estimation of viable bacteria present in the gut of 16 bats through rectal swabbing ranged from 1.03xl04 to 1.49xl09 CFU/mL. A total of II genera of bacteria were identified from 97 isolates by using biochemical tests which were Bacillus, Escherichia, Klebsiella, Enterobacter, Acinetobacter Pseudomonas, Staphylococcus, Salmonella, Shigella, Serratia and Vibrio. A total of II species of bacteria from 14 isolates were identified through comparison of the sequences with BLAST, which were Clavibacter michiganensis, Enterococcus faecalis, Acinetobacter calcoaceticlls, A. nosocomialis, A. baumanii, Bacillus thuringiensis. B. cereus, Enterobacter cloacae, E. hormaechei. Staphylococcus scillri. and S. succinlls.

Keywords: biochemical tests, chiroptera, microorganism, zoonotic disease, 16S rRNA

ABSTRAK

Kelawar adalah antara mamalia yang sesllai menjadi perumah bagi patogen zoonotik, seperti vints, kulat dan bakteria. Tujuan projek ini adalah disebabkan kesedaran ten tang mikroflora kelawar serangga yang boleh menyebabkan penyakit zoonotik kepada manllsia. Oleh itll, adalah penting untuk mengenal pasti dan mencirikan kepelbagaian mikrob yang terdapat dalam hos tersebllt. Pengenalpastian dan pencirian mikroflora IISIIS dijalankan melalui analisis ujian biokimia dan kaedah molekular menggunakan PCR untuk analisis jujukan DNA gen 16S rRNA. Anggaran bakteria yang berada di liSus 16 kelawar melailli teknik mengesat rektum adala" antara I.03xI04 hingga I. 49x109CFU. / mL. Sebanyak II genera bakteria daripada 97 bakteria telah dikenalpasti melalui ujian biokimia iaitu Bacillus, Escherichia, KlebsieIla, Enterobacter, Acinetobacter, Pseudomonas, Staphylococcus, Salmonella, ShigeIla, Serratia dan Vibrio. Sebanyak II spesis daripada 14 bakteria telah dikenalpasti melailli perbandingan jujuran dengan BLAST iaitu Clavibacter michiganensis, Enterococcus faecalis, Acinetobacter ca\coaceticus, A. nosocomialis, A. baumanii, Bacillus thuringiensis, B. cereus, Enterobacter cloacae, E. hormaechei, Staphylococcus sci uri, dan S. succinus.

Kata kunci: IIjian biokimia, khiroptera, mikrorganism, penyakit zoonotik, 16S rRNA

Vlll

1.0 INTRODUCTION

Bats are among mammalian species that can serve as the host for zoonotic pathogens, like

virus, fungi and bacteria (Akobi et aI., 2012; Wood et al., 2012). The food choices of bats,

their colonial or solitary nature and population structure make them serve as natural host

for pathogens. In addition, their ability to fly, seasonal migration and daily movement

patterns, torpor and hibernation, long life span, roosting behaviours, and virus

susceptibility also produce a favourable environment to harbour pathogens (Calisher et ai.,

2006; Veikkolainen et aI., 2014).

In addition, as they live in close proximity to human, and often interact with possible

intermediate hosts for pathogen (livestock and domestic animals) enable them to transmit

disease to human (Wood et al., 2012; Veikkolainen et aI., 2014). Among the recent

reported major outbreak that carried by bats are Ebola, Sudden Acute Respiratory

Syndrome (SARS), Marburg hemorrhagic fever, and rabies (Wood et al., 2012; O'Shea et

al., 2014; Chomel et al., 2015). Similarly, bats are also reservoir hosts for bacterial

pathogens such as Bartonella mayotimomensis that is responsible to cause endocarditis in

human (Veikkolairien et aI., 2014), Kluyvera species that cause several human clinical

infection (Han et aI., 2010), Staphylococcus aureus that cause staph infection in human

(Akobi et aI., 2012), and hemotropic mycoplasma species can cause infectious anaemia in

mammalian species (Mascarel1i et al., 2014). However, most of the studies only focus on

the frugivorous (fruit) bats, instead of insectivorous bats, in studying the emerging

zoonotic disease that caused by bats.

1

The study was conducted due to the awareness that the natural microflora of insectivorous

bats in Sarawak could potentially include those of medical concerns. Besides that it is also .

important to learn about the microflora that is important for their diet. Therefore, it is

important to identify and characterise the microbial diversity that reside within these

natural host.

The aims of the project are:

i) to identify and characterise the gut microflora genus of insectivorous bats using

biochemical analysis, and

ii) to provide genetic identification for the isolates based on GenBank sequence

comparison

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2

.~

2.0 LITERATURE REVIEW

2.1 Insectivorous bat

Bats are from the order Chiroptera, and are divided into two suborders called

Megachiroptera and Microchiroptera. Megachiroptera mostly consist of frugivorous bats

that often referred to as flying foxes. This suborder only consists of one family of bats,

which is family Pteropodidae. Insectivorous bats belong to the suborder Microchiroptera,

which consume insects as their primary diet (Francis & Payne, 1985; Findley, 1993;

Neuweiler, 2000). Most of the insects they consume are from the order Lepidoptera

(moths), Orthoptera (crickets), Coleoptera (beetles), Diptera (mosquitoes) and

Hymenoptera (winged ants) (Neuweiler, 2000; Jayaraj et at., 2011) .

The family of insectivorous Qats that are found in Sarawak are family

Emballonuridae, Megadermatidae, Nycteridae, Rhinolophidae, Hipposideridae, and

Vespertilionidae. The insectivorous bats from family Vespertilionidae are the most widely

distributed groups of bats in Borneo but are rarely found or occurred only in small number.

Examples of bats from this family are Kerivouta and Myotis species. The bats from family

Hipposideridae such as Hipposideros cervinus are commonly found and are among the , . .

high abundant species in Sarawak (Jayaraj et at., 2D 11; Rahman et at., 2011).

The insectivorous bat is small with 5 g to 20 g body weights (Francis & Payne,

1985; Neuweiler, 2000). These species own a highly specialized echolocation system that

enables them to recognize their foods. According to Jayaraj et al. (2011), species diversity

of insectivorous bats in Sarawak are determined by the type of forest, presence of roosting

sites, species distribution and abundance.

2.2 Microflora

Nonnal microfloras are the microorganisms that live in or on some part of the host such as

human and animals. For example, gut microflora of bats are the microorganisms that are

commonly found along the gastrointestinal tracts of bats. The micro flora consists of three

types of microorganisms, which are symbionts, commensals and opportunists. The

symbionts are beneficial to their host. Commensals are mostly neither beneficial nor

hannful to their host, while opportunists are the microflora that might become pathogenic

as the opportunity arises (Cheesbrough, 2006). These microflora are nonnally hannless or

rarely cause disease to their host, but might cause disease to other organisms.

According to Daniel et al. (2013) study, the bats microflora that were successfully isolated

in that study were from genus Bacillus, Enterobacter, Enterococcus, Pantoea,

Pseudomonas, Klebsiella, Escherichia, and Serratia. That study also mentioned that

bacteria from genus Actinomycetes, Aerobacter, Acaligenes, Citrobacter, Bacteriodes,

Clostridium, HaJnia, Micrococcus, Morganella, Proteus, Stapylococcus, and Streptococcus

also the microflora of the bats .

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\4 1

" usnt KI idm. t l\Iaklumat -ad m:' 1J " T1 M Y SARAW

2.3 Zoonotic disease

Zoonotic diseases are also called zoonoses. They are diseases that transferred from animal

to human and other animals. They are caused by bacteria, parasites, fungi and viruses

(CDC, 2013; WHO, 2014). According to WHO (2014), over 200 zoonoses have been

described_ People are infected to zoonoses due to the close interaction with animals such as

in a county fair, petting zoo and through encountering wildlife in a clear wooded land for

new construction (CDC, 20l3).

Bats are among animals that commonly associated with zoonotic viral and bacterial

pathogens. As revised in Chomel et al. (2015), the viral pathogens that have been found in

bats were Rabies, Hendra, Nipah, SARS-coronaviruses, Marburg, and Ebola viruses.

While, the bacterial pathogens that have been found were enteropathogenic bacteria (such

as Salmonella, Shigella, Yersinia, and Camphylobacter) and vector-borne bacteria (such as

Borrelia, Bartonella, and Neorickettsia) . Leptospira also has been found in bats.

5

2.4 Biochemical tests

Biochemical tests are available to identify the genus level of bacteria, but are critical in

identifying the species level of the bacteria. This tests help visually by the presence or

ab ence of substrate or end-products of the enzymatic reaction. The bacteria can be

identified by their biochemical characteristics. Example of the biochemical tests are

Catalase test, Citrate Test, Indole test, Voger-Proskauer test, Triple Sugar Iron (TSI) test,

Bile Eskuline test, Urease test, and Oxidase test (Cheesbrough, 2006).

Catalase test is used to distinguish catalase producing bacteria, like staphylococci and non­

catalase producing bacteria, such as streptococci. Catalase serves as a catalyst to

breakdown the hydrogen peroxide into oxygen and water. The microorganism is a catalase

producer if oxygen bubbles are release as it tested by bringing into contact with hydrogen

peroxide. Citrate test is used for identification of the Enterobacteriaceae, Gram-negative

and non-fermentative bacteria. It is based on the ability of the microorganism tested to

consume citrate, which is the only carbon source in the medium. The Voges-Proskauer can

aid in differentiation between genera and species, and it helps in identification of Listeria

monocytogenes. Bile Eskuline test is used to differentiate Streptococcus pneumoniae with

streptococci. Urease test helps to differentiate enterobacteria based on their urease activity,

while Oxidase test is used to identify bacteria that able to produce cytochrome oxidase,

such as Pseudomonas, Neisseria, and Vibrio. In Oxidase test, if the microorganism tested

is oxidase-producing, phenylenediamine of the reagent strip used in the test will be

oxidised to form deep blue colour (MacFaddin, 1999; Cheesbrough, 2006).

2.5 168 rRNA sequencing

168 rRNA gene is found universally in all bacteria and archaebacteria. According to

Rajendhran et al. (2011), 168 rRNA sequencing is widely used in identification and

classification of pure culture, and estimation of bacterial diversity in environmental

sample. The 165 rRNA gene is used in identification of microorganism due to the presence

of species-specific variable region, it is highly conserved within the bacterial species, and

distributes in all bacteria (Drancourt et aI., 2000).

According to Claridge (2004), sequence analysis using 165 rRNA gene can identify

microorganism that non-cultured, poorly described, rarely isolated and difficult to identify

by conventional bacteriological identification techniques. This sequencing also can lead to

the discovery of novel pathogen, such as Helicobacter winghamensis.

7.

3.0 MATERIALS AND METHODS

3.1 Sample collection of insectivorous bat

Bat samples were collected from Bako National Park and Samunsam Wildlife Sanctuary of

Sarawak (Figure I). The bats were trapped using ten mist nets and four harp traps at the

suitable areas of the field sites. The mist nets and harp traps were set up at 1600 hrs and

monitored continuously every five minutes for the first to second hour (1800-1900 hrs),

then IS minutes for 1900 to 2000 hrs, and 30 minutes for 2000 to 2300 hrs. Trapped bats

were removed from the traps and placed in the cloth bags. The external characters of the

collected bats were examined and identified following species identification by Payne and

Francis (1985).

3.2 Sample collection of bat gut microflora

The rectal or anal swab technique as described in Tse et at. (2012) was used to isolate the

gut microflora of the bats. The sterile cotton bud was used to swab the bat rectum and

directly suspended in sterile microtube that contained PBS solution under sterile condition

to prevent the cross-contamination with environmental microorganism. The tube was

inverted for a few times for proper mixing. The tube was stored in the polystrene box

containing ice before transportation. Tubes were immediately stored at 4 °C after reaching

the laboratory until further processing.

8

B

o

Legend

L Sal11lllSllmWS

... BakoNP 1.......___....

100 200 300 km

Figure I. A is the region of Southeast Asia with Malaysia highlighted with light grey colour. B is the enlarge portion of Malaysia to show selected sampling sites of insectivorous bats.

3.3 Enumeration of bacteria

Enumeration of bacteria was using serial dilution-Plate Count Agar technique with three

replicates of 1 to 7 ranges of 10-fold dilutions of the samples as shown in Figure 2. This

technique was done following Reynold (2013) with some modifications. Seven microtubes

containing 900 ilL PBS each, were used in the serial dilution for every replicate. The

microtubes were labelled based on its dilution. The 100 ilL of sample was transferred to

the first dilution and mixed it. Then, 100 ilL of first dilution mixture was transferred to

second dilution. The step was repeated until seventh dilution. Control was also prepared,

where it only contains PBS without any swab sample. Next, the 100 ilL diluted sample for

every dilution and Control were pipetted and spread on Plate Count Agar plate using

spread plate technique.

The plates were incubated at 37°C for 24 hours. Then, the colony-fonning on the plates

were recorded and the plates having 30-300 bacterial colonies will be calculated with

colony forming unit (CFU) per mL. The colonies with different morphologies then were

picked and inoculated in LB broth. The LB broth were placed on a shaker and incubated at

37°C for 24 hours. The next day, the LB broth were inoculated and streaked on the slant

(LB) agar using inoculum loop, and 750 ilL of the LB broth were transferred to cryo tube

containing 750 ilL glycerol mixtures. Then, the slant agar was incubated at 37°C for 24 ,

hours and glycerol stocks were stored in -20°C (Reynolds, 2013).

100 ilL sample

100 ilL

900 ilL 10-2 10-3 10-6 10-7

PBS

Transfer 100 ilL diluted sample into

Petri dishes

[ ]e ][ JC J 10-1 10-2 10-3 10-4

C JC JC J 10-5 10-6 10-7

Figure 2: Serial dilution-Plate Count agar technique

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3.4 Identification of bacteria using biochemical tests

After the pure culture was obtained, the Gram staining and the observation of cell

morphology under compound light microscope were perfonned to classify them through

their Gram reaction. They were then furthered tested with motility test (Daniel et a/. ,

20 13). The isolates were further tested with several biochemical tests, which were Oxidase

test Catalase test, Citrate test, Triple Sugar Iron (TSI) test, Methyl Red - Voger-Proskauer

(MR-VP) test, and bile esculine test. The biochemical tests were conducted mainly based

on protocol as described in MacFaddin (1999) and Cheesbrough (2006).

3.4.1 Gram Staining

The Gram staining technique was perfonned as described in Chessbrough (2006) and

Kumari (2000). The smear was prepared b~fore staining. The pure colony was emulsified

in sterile distilled water on a microscopy slide with thin preparation. The smear was then

air-dried and fixed. All the slides used were labelled. The fixed smear was covered with

crystal violet stain for 30 to 60 seconds, and then washed with clean water. All the water

must be tip off. Next, the smear was covered again with Gram iodine for 30 to 60 seconds.

The iodine was washed with clean water. The acetone-alcohol was used rapidly to

decolorize the smear and immediately washed with. clean water. The safranin was covered

the smear for 2 minutes and washed with clean water. The smear then was left to air-dry.

Then, the smear was examined under light microscope and recorded.

I.

1

3.4.2 Motility test

The culture was inoculated in the motility medium using sterile needle about two thirds

down and the needle must pulled up through the same path. The medium then incubated

for 24 to 48 hours.

3.4.3 Oxidase test

Strip of oxidase detection was prepared on a petri dish. Then, a colony of pure culture was

transferred to the strip using sterile cotton swab. The strip was observed in five seconds for

the fonnation of deep blue or violet colour as a positive result. No change of colour was

detected as negative result.

3.4.4 Catalase test

A colony of pure culture was transferred to a clean and dry glass slide using inoculum

loop. Then, a drop of 3% hydrogen peroxide, H20 2, was placed on the colony. The

production of bubbles was detected as positive result, while a few or no bubble was

detected as negative result.

3.4.5 Citrate test

The Simmon's citrate agar was prepared in the universal bottles according to manufacturer

protocol. The slant agar was prepared prior to cool at room temperature after autoclaved. A

colony of pure culture was picked by inoculum loop and streaked on the slant citrate agar.

Then, the agar was incubated for 18 to 24 hr to 7 days at 37°C. After incubation, the

changes of green colour of Simmon's citrate agar to blue colour was indicated as positive

result and no change ofcolour (maintained in green) was detected as negative result.

3.4.6 Triple Sugar Iron (TSI) test

The Triple sugar iron (TSI) or Kligler iron agar was prepared according to manufacturer's

protocol and slanted in the tubes. The culture was picked with inoculum needle,

aseptically. The needle containing the culture was stabbed into the TSI agar up to the butt

of the tube and streaked the surface of the slant agar. Then, the tube was incubated for 18

to 24 hr at 37°C. After incubation, the condition of agar was recorded.

3.4.7 Methyl Red (MR) and Voges-Proskauer (VP) test

Prior to both tests, MR-VP broth was prepared in the tubes according to manufacturer's

protocol. For Methyl red test, methyl red solution was required. While for VP test, Baritt's

A and Barrit's B reagents were required. Baritt's A reagent was prepared by dissolving 6.0

g of a-naptholin in 100 mL of 95% ethyl ethanol, and Barrit's B reagent by dissolving 16.0

g of potassium hydroxide in 100 mL of distilled water.