Faculty of Resource Science and Technology

Prevalence of Listeria monocytogenes in wild birds, rodents and bats from Nanga Merit,

Kapit

Annastasia anak Mathew (18051)

Bachelor of Science with Honors

Resource Biotechnology

2010

I

ACKNOWLEDGEMENT

Thank you to God that I had completed my final year project. I would like to express my

sincere thank you to those who were helping much during the progress of my project

especially my supervisor, Dr. Lesley Maurice Bilung and co-supervisor, Assoc. Prof. Dr.

Kasing Apun who gave precious advice, support and very good guidance. For postgraduate

students from Microbiology Laboratory: Ms. Khoo Kai Ling, Ms. Chen Yik Ming and Mr.

Adom Benjamin, thank you so much for your care and guidance. For my friends: Evlyn,

Audrey, Ngah Kiat and the others in Microbiology Lab, thank you for your wise opinion,

advice and support. Last but not least are my parents, brothers, sisters and the others, thank

you for your prayer, physically and mentally support and advice.

II

TABLE OF CONTENTS

ACKNOWLEGDEMENT I

TABLE OF CONTENTS II

LIST OF ABBREVIATIONS IV

LIST OF TABLES AND FIGURES V

ABSTRACT 1

CHAPTER 1 INTRODUCTION

1.1 Introduction

1.2 Objectives

2

2

4

CHAPTER 2 LITERATURE REVIEW

2.1 Description of Genus Listeria and Listeria monocytogenes

2.2 Sources, transmission and pathogenicity

2.3 Isolation and identification of Listeria monocytogenes

2.4 Chromogenic and selective media

2.5 Biochemical tests

5

5

6

7

8

9

CHAPTER 3 MATERIALS AND METHODS

3.1 Source of Listeria monocytogenes strains

3.2 Enrichment, Isolation and Identification

10

10

10

III

CHAPTER 4 RESULTS

4.1 Sources and Nomenclature of Listeria monocytogenes Isolates

4.1.1 Bacterial identification on selective culture media and Gram staining

4.2 Biochemical testing

CHAPTER 5 DISCUSSION

5.1 Bacterial identification on selective culture media

5.2 Biochemical testing

12

12

13

15

23

23

25

CHAPTER 6 CONCLUSION 28

CHAPTER 7 REFERENCES 29

IV

LIST OF ABBREVIATIONS

H2O2 Hydrogen peroxide

L. grayi Listeria grayi

L. inocua Listeria inocua

L. ivanovii Listeria ivanovii

L. monocytogenes Listeria monocytogenes

L. seeligeri Listeria seeligeri

L. welshimeri Listeria welshimeri

µl Microliter

µm Micrometer

MR-VP Methyl red- Voges Proskaeur

SIM Sulphide Indole Motility

TSI Triple Sugar Iron

V

LIST OF TABLES AND FIGURES

Table 1 Nomenclature of Listeria monocytogenes isolates used in the study

Table 2 Results of 12 isolates for biochemical testing

Table 3 Overall number and percentages showing the presence of Listeria

monocytogenes strains isolated from different sources

Table 4 Overall number and percentages showing the presence of Listeria

monocytogenes strain isolated from different sites

Figure 1 The grey-green colonies of Listeria monocytogenes isolates on PALCAM

Listeria Selective Agar

Figure 2 The blue-green colonies of L. monocytogenes on CHROMagar Listeria

Figure 3 The bacterial appearance under the light microscope

Figure 4 Simmon’s citrate test result

Figure 5 Triple Sugar Iron (TSI) test result

Figure 6 Sulphide Indole Motility test result

Figure 7 Methyl red test result

Figure 8 Voges Proskauer test result

Figure 9 Pie chart showing the prevalence of L. monocytogenes isolates from

different sources

Figure 10 Pie chart showing the percentages of L. monocytogenes isolates from

different sites

1

Prevalence of Listeria monocytogenes in wild birds, rodents and bats from Nanga Merit,

Kapit

Annastasia anak Mathew

Resource Biotechnology Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Listeria monocytogenes is gram-positive and facultatively anaerobic bacteria. It is an

important pathogen which caused listeriosis in both human and animals. This study is focusing

on the prevalence of L. monocytogenes in three selected animals namely wild birds, rodents

and bats from Nanga Merit, Kapit. Seventy four samples were tested. Seven samples had

positive growth on CHROMagar Listeria and five samples had positive growth on PALCAM

Listeria Selective Agar. Fraser Broth was used to enrich the samples. There were two types of

medium used to culture the bacteria which were CHROMagar Listeria and PALCAM Listeria

Selective Agar. Positive growths of L.monocytogenes on CHROMagar Listeria were blue-

green colonies with white haloes. Meanwhile, the appearance of positive colonies on

PALCAM Listeria Selective Agar is grey-green colonies with black haloes. The positive

growths were further tested with biochemical tests. In this study, the biochemical tests which

had been conducted were oxidase test, catalase test, Simmon’s Citrate test, triple sugar ion test

and sulphide indole motility test. This study showed two samples had positive results for all

biochemical tests while the others had atypical results.

Key words: Listeria monocytogenes, prevalence, biochemical tests

ABSTRAK

Listeria monocytogenes ialah bakteria gram-positif dan mampu untuk hidup dalam keadaan

kekuranagn udara. Bakteria ini adalah patogen yang menyebabkan listeriosis dalam manusia

dan haiwan. Kajian ini memberi fokus kepada kehadiran L.monocytogenes di dalam tiga

haiwan yang terpilih iaitu burung liar, roden dan kelawar dari Nanga Merit, Kapit. Tujuh

puluh empat sampel telah diuji. Tujuh sampel menunjukkan pertumbuhan positif di atas

CHROMagar Listeria dan lima sampel menunjukkan pertimbuhan positif di atas Agar

Terpilih PALCAM Listeria. Cecair Fraser telah digunakan untuk meningkatkan kualiti

sampel. Dua jenis media telah digunakan untuk mengkulturkan bakteria ini iaitu CHROMagar

Listeria dan Agar Terpilih PALCAM Listeria. Pertumbuhan positif L.monocytogenes di atas

CHROMagar Listeria ialah koloni yang berwarna biru-hijau serta halo putih. Manakala,

pertumbuhan positif di atas Agar Terpilih PALCAM Listeria ialah koloni yang berwarna

kelabu-hijau serta halo hitam. Pertumbuhan positif ini telah diuji selanjutnya dengan ujian

biokimia. Dalam kajian ini, ujian biokimia yang telah dijalankan ialah ujian oksides, ujian

katales, ujian Simmon’s sitrat, ujian ion tiga gula dan ujian pergerakan indol sulfida. Kajian

ini menunjukkan dua sampel mempunyai keputusan positif untuk semua ujian biokimia

manakala yang lain mempunyai keputusan yang berlainan.

Kata kunci: Listeria monocytogenes, kehadiran, ujian biokimia

2

CHAPTER 1

INTRODUCTION

1.1 Introduction

Listeria monocytogenes is gram positive, non-spore forming and facultatively

anaerobic bacteria which is mainly foodborne pathogen. Listeria monocytogenes is one of two

pathogenic species in the genus Listeria while another one is Listeria ivanovii (Romich, 2008).

These bacteria are closely related to a disease known as listeriosis, a febrile, infectious,

endemic disease in both animal and human (Dijkstra, 1981). Jones (1991) stated that listeriosis

primarily occurs in newborn infants, elderly patients and immunocompromised people. This

type of disease had a number of syndromes which can be classified as acute to hyperacute,

subacute, chronic and abortive. Listeriosis presents in three disease forms which are

meningoencephalitis, abortion and stillbirth and septicemia (McGavin and Zachary, 2007).

Dijkstra (1981) reported that L. monocytogenes is a significant cause of encephalitis in

domestic ruminants, of septicemia in monogastric animals and birds and of meningitis in

human.

According to Ryser and Marth (2007), L. monocytogenes is mainly foodborne

pathogen because outbreaks of infections are related with the consumption of ready-to-eat

food and food that is well-cooked. This bacterium can withstand the drying, freezing and heat.

Because of the ability, the bacteria can grow and multiply on contaminated food even at the

refrigeration temperature. In addition, Romich (2008) reported that L.monocytogenes also

survive at pH range 3.6 to 9.5 and rapidly grow at a pH greater than five.

3

Apart from foodborne pathogen, Listeria monocytogenes is a zoonotic agent. Wild

animals are the spreading agent as the infected animals can infect human through direct

contact. The organism had been found in at least thirty seven mammalian species and at least

seventeen species of birds and some species of fish, shellfish and insects (Romich, 2008).

Animals’ intestinal tracts are the main reservoirs of Listeria monocytogenes. Animals which

carry the bacteria do not appear ill. This can cause the contamination of foods from animal

origin. Hellstrom et al. (2008) reported that birds commonly carry L. monocytogenes and

strains are frequently similar with those detected in foods.

Listeria monocytogenes is found widely around the globe. The recent outbreak of

listeriosis was reported in Texas, United States in the year 2003 which caused by the

consumption of unpasteurized food but no death had been reported (Ryser and Marth, 2007).

The greatest outbreak happened in the year 1992 in France with two hundred and seventy nine

cases and eighty five deaths had been reported (Ryser and Marth, 2007). This illness is greatly

affecting the public concern all around the world. The source of Listeria monocytogenes is not

only from food but is also found in wild animals. This study was conducted to detect the

prevalence of Listeria monocytogenes in wild animal namely wild birds, rodents and bats from

Nanga Merit, Kapit.

4

1.2 Objectives

This study was carried out with the objectives:

i. To detect the prevalence of Listeria monocytogenes in wild birds, rodents and bats

from disturbed and pristine area at Nanga Merit, Kapit.

ii. To isolate and detect the presence of Listeria monocytogenes using two selective

media, CHROMagar Listeria and PALCAM Listeria Selective Agar.

iii. To identify and confirm the isolates of Listeria monocytogenes via series of

biochemical tests.

5

CHAPTER 2

LITERATURE REVIEW

2.1 Description of Genus Listeria and Listeria monocytogenes

Listeria had diameter of 0.5 µm and length of 2 µm, Gram-positive rod with rounded

ends and found as single units or in short chains (Ryser and Marth, 2007). These bacteria are

non-capsulate and non-spore forming. The presence of few peritrichious flagella helped in the

bacterial motility. The bacteria were first cultured by Murray, Webb and Swann in 1926 from

Guinea pigs and rabbits with hepatic necrosis and named for Joseph Lister, an English surgeon

(Romich, 2008). Currently, genus Listeria contains six species which are L. monocytogenes, L.

ivanovii, L. innocua, L. welshimeri, L. seeligeri and L. grayi (Ryser and Marth, 2007). Among

these, L. monocytogenes and L. ivanovii are pathogenic (Romich, 2008). Jones (1991) stated

that the important human and animal pathogen is L. monocytogenes.

According to Romich (2008), Listeria monocytogenes are gram-positive, non-

endospore-forming, motile, pleomorphic bacilli and arranged in short chains. L.

monocytogenes had been used by Joseph Lister, an English surgeon, to study the bacteria’s

effect on monocytes (Romich, 2008). These bacteria are closely related to foodborne pathogen

and can survive in wide temperature and pH range. Ryser and Marth (2007) reported that L.

monocytogenes can tolerate up to 20% of salt concentration, can multiply in the temperature

from 1 to 45oC and can survive acid stress. Jones (1991) stated that the outbreak of listeriosis

in both human and animal is commonly caused by L. monocytogenes.

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2.2 Sources, transmission and pathogenicity

Listeria are found in a wide range in the environment. These bacteria are the inhabitant

of soil and animals’ intestinal tract. These bacteria are also found in contaminated processed

food and raw food. The finding done by Ben Embarek (1994) showed that Listeria

monocytogenes and other Listeria species had been isolated from seafood and able to grow

well at refrigeration temperature on contaminated seafood. Meanwhile, Skovgaard and

Morgen (1988) detected the presence of L. monocytogenes in faeces from farm animals.

According to Kalorey et al. (2006), Listeria monocytogenes were not only found in water, soil

and plant but these bacteria could be isolated from both domestic and wild animals. At least

forty two species of wild and domestic animals and seventeen avian species were the carrier of

the bacteria and the microbe had been detected in the faeces of apparently health and diseased

animal (Jones, 1991).

Listeria can be spread by ingestion, by inhalation and direct contact. But, Listeria are

mainly spread through ingestion according to Ryser and Marth (2007). Listeria transmitted in

human and animal through consumption and ingestion of contaminated food and direct contact

with infected animals. Animal can be infected by L. monocytogenes when they are fed on the

contaminated feed (Jones, 1991). Animals which are the carrier of these bacteria do not show

any symptoms of listeriosis, however mainly shed the bacteria in their faeces (Kalorey et al.,

2006). There are two types of L. monocytogenes transmission had been detected which are

venereal and vertical transmission (Romich, 2008). Both of the transmission occurs in human

and animals. Venereal transmission is through the consumption of contaminated food and

silage in human and animal respectively whereas vertical transmission is the route of infection

for newborn human infants and ruminants via placenta or infected birth canal (Romich, 2008).

7

According to Ryser and Marth (2007), there are four virulence and resistance factors

associated with Listeria monocytogenes which are the ability to grow at low temperature,

resistance to drying, motality, adherence and invasion. Exposure to the bacteria does not

always results in illness as the systemic infection depends on host susceptibility, infectious

dose and virulence factors (Jones, 1991). Listeria monocytogenes entered the human or animal

body through the ingestion of contaminated food. In the digestive canal, the binding of L.

monocytogenes to epithelial cells of gastrointestinal tract triggers phagocytosis. Listeria

bacteria can survive and replicate in the host cell’s cytoplasm (Romich, 2008) in order to

avoid to be identified by the host’s immune system.

2.3 Isolation and identification of Listeria monocytogenes

Kalorey et al. (2006) had done a study on the occurrence of Listeria species from wild

animals in captivity. Listeria monocytogenes often found in the faeces of healthy animals. The

prevalence of L.monocytogenes in zoo animals was low compared to farm animals probably

due to better hygienic and care.

In the other study of prevalence of L.monocytogenes in common wild birds had been

carried out by Hellstrom et al. (2008). They were comparing the genotypes of

L.monocytogenes is commonly found in wild birds with the one that was isolated from food

and food processing environment. The samples were birds’ feces which were collected from

urban areas in Helsinki. After being analyzed, it was proven that L. monocytogenes strains

found in birds were similar with those found in foods and foods processing environment. It

was concluded that birds were the agent of spreading the Listeria bacteria in nature and

contributed to food contamination when they came to the food processing environments.

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Apart from the above investigations, L. monocytogenes also had been found in farm

ruminant. Nightingale et al. (2004) had investigated the existence of L. monocytogenes in

ruminant and farm environment by comparing the farms with recent case of listeriosis and

control farms where no listeriosis cases had been reported. The result of the investigation

showed that the prevalence of Listeria was higher in small ruminant farms. Human are rarely

infected directly from infected animals or from agricultural environment that had been

contaminated with Listeria species. It was animal-derived food products that are not processed

before consumption (e.g., raw milk) and raw foods of plant origin that have been contaminated

by manure from infected or shedding animals represent direct links between human infections

and L . monocytogenes in farm animals and farm environments (Nightingale et al., 2004).

2.4 Chromogenic and selective media

L. monocytogenes is exist in low numbers and mix with high number of other

competing microorganism, makes it often difficult to isolate and identify. Hedge et al. (2007)

reported that cultural methods for the detection of L.monocytogenes in food and environmental

samples are based on two-stage enrichment procedures, isolation using selective agars and

confirmation by biochemical means. PALCAM, LPM, Oxford and Modified Oxford agar were

commonly used for Listeria isolation. Brooke (2005) stated that the combination of

chromogenic differential selective agar, CHROMagar Listeria and prescribed selective agar,

the PALCAM agar will improve the isolation and the identification of Listeria

monocytogenes. The bacterium appeared as visible blue/green colonies with distinctive opaque

white halo on CHROMagar Listeria (Brooke, 2005).

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2.5 Biochemical tests

The isolates were identified by conducting biochemical testing. Listeria species are

catalase positive, oxidase negative, methyl red and Voges-Proskauer test positive (Ryser and

Marth, 2007). Hydrogen peroxide (H2O2) is used in catalase test to detect the presence of

bacteria. H2O2 is an oxidizing agent which can destroy the bacterial cells. The aerobic

microbes are using enzyme catalase to get rid of the peroxide. Catalase enzyme will

breakdown H2O2 into water and oxygen. Oxidase test is carried out to identify the aerobic

microbes. Cytochrome oxidase is an enzyme found in some bacteria that transfer electron to

oxygen, the final electron acceptor in electron transport chain (Cappuccino and Sherman,

1992). Beta hemolysis is one of the confirmation tests to differentiate Listeria monocytogenes

from other Listeria species because L. monocytogenes is hemolytic for blood of various

origins (Ryser and Marth, 2007).

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CHAPTER 3

MATERIALS AND METHODS

3.1 Source of Listeria monocytogenes strains

In this study, the bacteria strains were collected from different species of wild birds,

rodents and bats from disturbed and pristine area at Nanga Merit, Kapit by a team of

researchers from Microbiology Laboratory, Faculty of Resource Science and Technology.

Anal, cloacal, intestine and feces swabs were collected using sterile cotton buds and kept in

phosphate buffer saline. The samples were brought back to the laboratory for further

processing.

3.2 Enrichment, Isolation and Identification

The samples were enriched in Fraser broth with the ratio 1:9 (one part of sample

enriched in nine part of Fraser broth) according to CHROMagar Listeria method. 100 µl of

sample was enriched in 900 µl of Fraser Broth. Then, it was incubated at 37oC for 24 hours.

Listeria monocytogenes were isolated using two different types of selective media

which were CHROMagar Listeria (CHROMagar Microbiology, France) and PALCAM-

Listeria Selective Agar (MERCK, Germany). The media were well-dried before being used

and divided into two portions per plate. Each portion was labeled with the code number of the

samples. Approximately a loop-full of 24 hours incubated Fraser broth containing the bacterial

strains was streaked on the well-dried chromogenic agar. After that, the chromogenic agar was

incubated at 37oC for 20-24 hours. The growth of Listeria monocytogenes was observed

around 20-24 hours. A well-prepared PALCAM agar was red in colour.

11

Similar to the method and streaking pattern used in CHROMagar Listeria, the agar

plates were divided into two portions and each portion was labeled with the samples number.

After streaking process, the samples were incubated at 37oC for 20-24 hours. The

incubation period could be prolonged up to 48 hours depending on the bacterial growths

observed after 24 hours. The positive growths were inoculated on Tryptone Soy Agar and

incubated at 37oC overnight for further testing.

The bacterial growths on both medium were further tested using series of biochemical

tests for identification and confirmation of isolates. The series of biochemical tests carried out

in this study included oxidase test, catalse test, Simmon’s citrate test, Triple Sugar Iron (TSI)

test, Sulphide Indole Motility (SIM) test and Methyl Red-Voges Proskauer (MR-VP) reaction.

12

CHAPTER 4

RESULTS

4.1 Sources and Nomenclature of Listeria monocytogenes Isolates

The geographical origin of Listeria monocytogenes isolates used in the study was

Nanga Merit, Kapit.

Table 1: Nomenclature of Listeria monocytogenes isolates used in the study

Sources Date of processing Samples and swabs

Wild birds October 2009

January 2010

February 2010

A2712 [NR], A2715 [NR], A2737 [A], A2740 [C]

A2732 [C], A2936 [A], A2737 [A], A2740 [C], A2773 [C],

A2779 [C], A2781 [C], A2782 [C], A2783 [C]

A2732 [C], A2936 [A], A2737 [A], A2740 [C], A2773 [C],

A2779 [C], A2781 [C], A2782 [C], A2783 [C], A2788 [F],

A2791[F]

Rodents October 2009

January 2010

February 2010

U2043 [NR], U2049 [NR], U2102 [I], U2103 [F]

U2021 [A], U2031 [A], U2078 [A], U2078 [F], U2082 [F],

U2083 [F], U2102 [I], U2102 [A], U2103 [F]

U2114 [I], U2115 [I], U2117 [F], U2118 [I], U2118 [I2], U2119

[I]

Bats October 2009

January 2010

U2009 [A], U2012 [A], U2016 [A], U2023 [I], U2034 [F]

U2009 [A], U2026 [A], U2028 [A], U2028 [F], U2029 [F],

U2030 [A], U2032 [A], U2033[A], U2036 [F], U2070 [A],

U2077 [I],U2087 [A]

NR February 2010 FNM151 [I], DO2602 [I], AO9053 [C], FNM091[A], FNM074

[A], AO9049 [C], FNM068 [I], CO5301 [C], FNM056 [I],

AO9063 [F], CO3365 [A], FNM073 [A], BO9711 [I], AO9059

[C], FNM063 [I], AO9075 [C], AO9069 [C], CO3365 [F],

AO9075 [C], FNM057 [F], AO9046 [C], BO9710 [C], FNM057

[I], FNM069 [I], BO9709 [C],CO3360 [A],CO3377[A]

Notes; [A], anal swab; [C], cloacal swab; [F], feces swab; [I], intestinal swab; [NR], not

recorded

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4.1.1 Bacterial identification on selective culture media and Gram staining

There were two selective media used for the isolation of Listeria monocytogenes in this

study namely CHROMagar Listeria and PALCAM Listeria Selective Agar. Positive growths

of Listeria monocytogenes on CHROMagar Listeria were blue-green colonies with white

haloes. On the PALCAM Listeria Selective Agar, the positive growths of the bacteria were

grey-green with black centre and haloes. There were 12 samples which showed the positive

growth on both agars.

The bacteria were observed blue in colour, rod-shaped bacteria, arranged in short chain

or single under the microscope after gram staining.

Figure 1: The grey-green colonies of Listeria monocytogenes isolates on PALCAM Listeria

Selective Agar

14

Figure 2: The blue-green colonies of L. monocytogenes on CHROMagar Listeria

Figure 3: The bacterial appearance under the light microscope

15

4.2 Biochemical Testing

The bacterial growths on both media were further tested using series of biochemical

tests for identification and confirmation of isolates. There were seven types of biochemical

tests were carried out in this study namely oxidase test, catalase test, Simmon’s citrate test,

Triple Sugar Iron (TSI) test, Sulphide Indole Motility (SIM) test and methyl red-Voges

Proskauer (MR-VP) reaction. All Listeria monocytogenes isolates showed positive results for

oxidase test, catalase test and SIM test. However, atypical results were shown by some of the

bacteria isolates for Simmon’s citrate test, TSI test and MR-VP reaction as shown in Table 2.

Table 2: Results of 12 isolates for biochemical testing

Isolates Oxidase Catalase Simmon’citrate TSI SIM MR VP

AO9069 + + - A/G + - +

BO9709 + + + A/A + - -

FNM057 + + + A/A + + -

FNM063 + + + A/A + + -

FNM151 + + - A/G + - +

U2016 + + + N/A + + -

U2030 + + + A/A + - -

U2070 + + + A/A + + +

U2077 + + + A/A + - +

U2078 + + + A/A + - -

U2083 + + + A/A + + +

U2102 + + + A/A + + -

Note: Slant/Butt: A, acid (yellow); N, alkaline (pink); G, gas present (crack or air gap); +,

positive result; -, negative result

16

Figure 4: Simmon’s citrate test result

Note: Green slant for positive result and blue slant for negative result

Figure 5: Triple Sugar Iron (TSI) test result

Note: on the left, uninoculated slant; on the right, inoculated

Slant/butt: A, acid (yellow), A/A for the positive result

17

Figure 6: Sulphide Indole Motility test result

The absence of cherry ring (indole) on the agar for positive result

Figure 7: Methyl red test result

On the left, red colour for positive result; on the right, yellow colour for negative

result

18

Figure 8: Voges Proskauer test result

On the left, copper layer on top for negative result; on the right, red and yellow layer

for positive result

Copper layer

Yellow layer

Red layer