Characterization of Escherichia Coli and Listeria Isolated from Milk at Different Levels of Collection and Processing in Goa

CHARACTERIZATION OF ESCHERICHIA COLI AND LISTERIA ISOLATED FROM MILK AT DIFFERENT LEVELS

OF COLLECTION AND PROCESSING IN GOA

A Thesis Submitted To

Goa University

For The Award Of Degree Of

DOCTOR OF PHILOSOPHY IN

MICROBIOLOGY By

DILECTA COLAÇO

GOA UNIVERSITY, TALEIGAO, GOA

2011

CHARACTERIZATION OF ESCHERICHIA COLI AND LISTERIA ISOLATED FROM MILK AT DIFFERENT LEVELS

OF COLLECTION AND PROCESSING IN GOA

A Thesis Submitted To

Goa University

For The Award Of Degree Of

DOCTOR OF PHILOSOPHY IN

MICROBIOLOGY By

DILECTA COLAÇO

Research Guide Prof. Saroj Bhosle

GOA UNIVERSITY, TALEIGAO, GOA

2011

ACKNOWLEDGEMENTS

First and above all, I praise God, the almighty for providing me this opportunity and granting me the capability to proceed successfully.

It would not have been possible to write this doctoral thesis without the help and support of the kind people around me, to only some of whom it is possible to give particular mention here.

The good advice, support and friendship of my guide, Dr. Saroj Bhosle, Professor and Head, Department of Microbiology has been invaluable on both an academic and a personal level, for which I am extremely grateful. Her patience, generous spirit and positive disposition only match her impressive professional competence.

This thesis would not have been possible without the help, support and patience of my co-guide Dr. S. B. Barbuddhe, Principal Scientist not to mention his advice and unsurpassed knowledge of Listeria. I am heartily thankful to Barbuddhe Sir, whose encouragement, guidance and support from the initial to the final level enabled me to develop an understanding of the subject. His humility, perseverance and willingness to share knowledge speak of his intellectual avatar.

I thank Prof. G.N. Nayak, Dean, Faculty of Life and Environmental Sciences for his timely advice.

I also thank Dr. D. J. Bhat, Professor, Department of Botany, Goa University and member of my Faculty Research Council for his constructive suggestions during the course of my study.

I am indebted to Dr. V.S. Korikanthimath, then Director, ICAR Research Complex for Goa, Old Goa and Dr. N. P. Singh, Director, ICAR Research Complex for Goa, Old Goa for providing me the necessary facilities to carry out my research and for the steadfast support of the ICAR staff.

I profoundly thank Prof.(Dr.) Trinad Chakraborty, Director, Institute of Medical Microbiology, Justus-Liebig University, Giessen, Germany for his valuable suggestions during the course of this study.

My special thanks to my principal Dr. M. Sangodkar for motivating me to pursue research.

I am most grateful to Dr. R. B. Dhuri, Manager (Animal Health) and Dr. N. S.

Sawant of Goa Dairy for their help and unconditional support at each turn of the road.

I owe my deepest gratitude to my brother-in-law Dr. Ernest D’Costa who paved a way for my quest for research by introducing me to my co-guide.

I would like to express my gratitude to the personnel of Goa Dairy, dairy farmers all over Goa, members of Dairy Co-operative Societies and Mr. Sudin Jambhale for enabling me to procure samples.

My special thanks goes out to my colleagues Sushanta, Jenney, Swapnil and Krupali who supported me right through my research and provided valuable feedback at both the initial and closing moments of this thesis.

I would like to acknowledge the financial, academic and technical support of the University Grants Commission, New Delhi particularly in the award of a Postgraduate Research Studentship that provided the necessary financial support for this research and also for a travel grant to present my paper at ISOPOL 2010, Portugal.

It is a pleasure to thank those who made this thesis possible like Dr. Archana Verma of NDRI, Karnal, Dr. P.M. Korgaonkar (Vet. Officer), Dr. Seema Rath (Officiating Principal of Government College, Khandola), Dr. Sunita Borkar and Fr. Fredrick Rodrigues (FIP committee members), my niece Stephanie, my friends Marina, Charlotte, Nisha, Vineeti, Chandrika especially Manasi and Aditya, my ex-students Nimali, Zico, Diana and Milgen, my ever- cooperative laboratory staff Sylvia, Vimal and Kunda and the administrative staff at Government College, Khandola, and the staff of Microbiology Dept. Goa University.

I would like to thank my husband William for his solicitude, personal support and infinite patience at all times and above all for believing in my potential. His high regard for my ambition gave me the strength to carry on. This thesis is a small tribute to my dearest children Warren and Waluscha from a mother still anxious to learn from them. A special thanks to my mother-in-law for appreciating my endeavour towards research and for her care and concern. My father, brothers, sisters and in-laws have given me their unequivocal support

and admiration throughout, as always, for which my mere expression of thanks likewise does not suffice. I am deeply indebted to my beloved family.

I offer my regards and blessings to all of those who supported me in any respect during the completion of the thesis.

Last but not the least, I wish to dedicate this thesis to my late parents who have always been with me as a source of inspiration in my chosen path.

DILECTA COLAÇO

This thesis is dedicated to my late beloved parents

Mr. Honoristo J. G. Colaço and

Mrs. Maria Antonieta Colaço.

CONTENTS

Page

List of Abbreviations i

List of Tables v

List of Figures vi

Chapter

I Introduction, Aim and Scope of Work 1

II Review of Literature 12

III Microbiological analysis of milk, milking equipments and milk processing 72 environment

IV Isolation and Characterization of Escherichia coli 93 V Isolation and Characterization of Listeria 124

VI Summary and conclusions 158

Future Prospects 163

Bibliography 167

Appendices

Appendix A – Media Composition 233

Appendix B – List of Primers 239

i

List of Abbreviations

µg Microgram

µl Microliter

µM Micro molar

AFLP Amplified Fragment Length Polymorphism AOAC Association of Analytical Chemists

API Analytical Profile Index

ARS-MMA ARS - Modified McBride Agar

ASFBC Aerobic Spore Forming Bacteria Count ATCC American Type Culture Collection BAM Bacteriological and Analytical Method

BCM Biosynth Chromogenic Medium

BHI Brain Heart Infusion

BIMPs Beacon Immuno Magnetic Nanoparticles bp Base pairs

BTM Bulk Tank Milk

CAM Chorio Allantoic Membrane

CAMP Christie Atkins Munch Peterson

CC Coliform Count

CCEA Corn Cob Extract agar

CDC Centre for Disease Control and Prevention CDSC Communicable Disease Surveillance Centre CECM Chromogenic E. coli Coliform Medium

cfu Colony forming units

CHEF- DR II Contour Clamped Homogeneous Electric Field – Dynamic Regulation II

CHROM Chromogenic

CMT California Mastitis Test

CNS Coagulase Negative Staphylococci

CO Chrom agar Orientation

CTAB Hexadecylcetyl Trimethyl Ammonium Bromide DCS Dairy Co-operative Society

DIM Differentiation of innocua and monocytogenes DIS Draft International Standards

DLABN D,L-Alanine β-Naphthylamide

DMC Direct Microscopic Count

DNA Deoxy Ribo Nucleic acid

dNTP Deoxyribose Nucleoside Tri Phosphates DRIA Dominguez- Rodriguez Isolation Agar EDTA Ethylene Diamine Tetra Acetic acid EHEC Entero Hemorrhagic Escherichia coli ELISA Enzyme Linked Immuno Sorbent Assay

ii EMB Eosine Methylene Blue EPEC Entero Pathogenic E. coli

ETEC Entero Toxigenic E. coli

FAO Food and Agriculture Organisation

FDA Food and Drug Administration

FOOD Food borne Outbreak Database

g Gram

GDP Gross Domestic Product

HC Haemorrhagic colitis

HECM Harlequin E. coli Coliform Medium HUS Hemolytic Uremic Syndrome

ICMSF International Commission on Microbiological Specifications for Food

IDF International Dairy Federation

ILCC In-Line milk Coliform Count

IMI Intra Mammary Infection

IMS Immuno Magnetic Separation

IMTECH Institute of Microbial Technology

IMViC Indole Methyl red Voges Proskauer Citrate ISO International Organization for Standardization

km Kilometre

L Litre

LABC Lactic Acid Bacteria Count

LCAM Lithium Chloride- Ceftazidime Agar Modified

LEB Listeria Enrichment Broth

LES Lawrence Experimental Station

LIPI Listeria Pathogenicity Island

LLO Listeriolysin o

LMBA Listeria monocytogenes Blood Agar

LPC Laboratory Pasteurization Count

LSAM Listeria Selective Agar Medium Modified M Molar

MA Mesophilic Aerobes

MALDI-TOF MS Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectroscopy

mg Milligrams

min Minute ml Millilitre

MLA McBride Listeria Agar

MLEE Multi Locus Enzyme Electrophoresis MLST Multi Locus Sequence Typing

MLVA Multiple Locus Variable no. tandem repeats Analysis

mM Milli Molar

iii mm Millimetre

MMLA Modified McBride Listeria Agar

mPCR Multiplex Polymerase Chain Reaction

MPN Most Probable Number

MR-VP Methyl Red-Voges Proskauer

MTCC Microbial Type Culture Collection MUG – 7 4-methyl umbelliferyl-β -d-glucuronide

MVJ Modified Vogel Johnson

MVJM Modified Vogel Johnson Modified further NAHMS National Animal Health Monitoring System

NARMS National Antimicrobial Resistance Monitoring System

NC Non Clean

NCCLS National Committee for Clinical Laboratory Standards NCTC National Collection of Type Cultures

ng Nanogram

NM Non Motile

PALCAM Polymyxin Acriflavine Lithium chloride Ceftizidime Aesculin Mannitol

PALCAMY Polymyxin Acriflavine Lithium chloride Ceftizidime Aesculin Mannitol Egg Yolk

PBC Psychrotrophic Bacteria count

PBS Phosphate Buffered Saline

PCR Polymerase chain reaction

PCR-ELISA Polymerase Chain Reaction-Enzyme Linked Immuno Sorbent Assay

PCR-RFLP Polymerase Chain Reaction Random Fragment Length Polymorphism

PFGE Pulsed Field Gel Electrophoresis PHLS Public Health Laboratory Service PI-PLC Phophatidyl Inositol Phospho Lipase C

PLC Phospho Lipase C

QCM Quartz Crystal Microbalance

qPCR Quantitative Polymerase Chain Reaction

RAPD Random Amplified Polymorphic DNA

RAPD-PCR Random Amplified Polymorphic DNA- Polymerase Chain Reaction

rpm Revolutions per minute RTE Ready To Eat

RTi PCR Real Time Polymerase Chain Reaction

SBA Sheep Blood Agar

SCC Somatic Cell Count

SCM Sub Clinical Mastitis

SPC Standard plate Count

iv

STEC Shiga Toxigenic Escherichia coli

Stx Shiga toxin

TC Total Count

TE Tris EDTA

TPC Total Plate Count

TSB Tryptic Soy Broth

UPB Universal Pre-enrichment Broth

USDA United States Department of Agriculture USFDA United States Food and Drug Administration

UTI Urinary Tract Infection

UV Ultra Violet

UVM University of Vermont Medium

VRBA Violet Red Bile agar

VT Vero Toxin

WASP 2 Web Agri Statistical Programme 2

X-Gal Indoxyl Galactose

YETSB Yeast Extract Tryptic Soy Broth

v

List of Tables

Table 3.1 – Details of samples collected from different sources for analysis of microbiological parameters - 76

Table 3.2 – Average total plate counts of milk samples at different levels (season wise) - 83 Table 3.3 – Average total plate counts at different levels of collection - 85

Table 3.4 – Analysis of swab samples and bulk coolers - 87

Table 4.1 - The details of the primer sequences for virulence genes of E. coli - 101 Table 4.2 - Detection of virulence associated genes in Escherichia coli isolates - 106 Table 4.3 - No of isolates showing frequency of virulence marker genes in E. coli - 111 Table 4.4 - Percent sensitivity/resistance of E. coli isolates - 120

Table 5.1 - Primer sequences for L. monocytogenes used in Multiplex-PCR serotyping - 135 Table 5.2 - Biochemical and pathogenicity profiles of pathogenic Listeria isolates - 142 Table 5.3 - Isolation of Listeria at different levels of collection and processing - 145 Table 5.4 - Serotypes of L. monocytogenes isolates recovered from milk - 149 Table 5.5 - Percent sensitivity/resistance of L. monocytogenes isolates - 156

vi

List of Figures

Fig. 3.1 - Cleaning of udder before milking - 76

Fig. 3.2 - Filtering of milk after receiving at dairy co-operative society - 78 Fig. 3.3 - Average total plate counts of different levels of collection - 85 Fig. 4.1 - Amplification of the stx1 gene by PCR - 109

Fig. 4.2 - Amplification of the stx2 gene by PCR - 109

Fig. 4.3 - Amplification of virulence associated genes in Escherichia coli - 111 Fig. 4.4 - Dendrogram showing diversity of Escherichia coli strains isolated at udder

level - 113

Fig. 4.5 - Dendrogram showing diversity of Escherichia coli strains isolated from milking utensils - 114

Fig. 4.6 - Dendrogram showing diversity of Escherichia coli strains isolated at dairy cooperative level - 114

Fig. 4.7 - Dendrogram showing diversity of Escherichia coli strains isolated form samples collected at receiving dock - 115

Fig. 4.8 - Dendrogram showing diversity of Escherichia coli strains isolated from market milk - 115

Fig. 4.9 - Dendrogram of the 74 E. coli strains based on PFGE patterns after digestion with enzyme XbaI - 116

Fig 4.10 - PFGE comparing restriction profiles for Escherichia coli isolated from milk samples - 117

Fig. 4.11 - Antibiotic susceptibility testing of Escherichia coli isolates from milk samples - 120

vii

Fig. 4.12 - Antibiotic susceptibility testing of Escherichia coli isolates from milk samples - 121

Fig. 5.1 - The grey green colonies with black sunken centers from PALCAM agar - 141 Fig. 5.2 - L. monocytogenes isolates showing haemolysis on sheep blood agar - 141 Fig. 5.3 - L. monocytogenes showing positive CAMP test - 141

Fig. 5.4 - L. monocytogenes showing positive PI-PLC activity on ALOA - 141 Fig 5.5 - Amplification of the hlyA gene in Listeria monocyotgenes isolates - 147 Fig 5.6 - Serotype profile of Listeria species by multiplex-PCR serotyping - 149

Fig. 5.7 - Dendrogram of Listeria monocytogenes isolates obtained from milk samples using ApaI - 151

Fig. 5.8 - Dendrogram of PFGE of Listeria monocytogenes using AscI - 152 Fig. 5.9 - AscI and ApaI profiles of selected Listeria monocytogenes isolates - 153 Fig. 5.10 - PFGE analysis of Listeria monocytogenes strains isolated from milk and milk products at different locations in India - 153

“The cow is the foster mother of the human race. From the time of the ancient Hindu to this time have the thoughts of men turned to this kindly

and beneficent creature as one of the chief sustaining forces of the human race.” –

W. D. Hoard.

1

Chapter I.

General Introduction

2

Agriculture is the base of Indian economy. Agriculture forms 26% of the national GDP and approximately 75% of India’s population live in villages and depend on crop and livestock farming for their livelihood. Livestock production including dairying plays a multipurpose role in the agriculture systems of India. (FAO, 2011; NIC, 2011). Dairying plays a dynamic role in India’s agro-based economy. The dairy sector in India has shown remarkable development in the past decade. Today, India ranks first in the world in terms of milk production with 112.5 million tons per year during 2009-10 (Anon, 2011).

Milk may be defined as the normal secretion of the mammary gland of mammals. Milk contains many essential nutrients, such as carbohydrates, proteins, lipids, minerals and vitamins. According to Hindu mythology as well as the Indian traditional medical practices, cow’s milk has rejuvenator, health protecting and health promoting properties and hence can be referred to as one of the best vitalisers. Most of the changes which take place in the flavour and appearance of milk, after it is drawn from the udder are the results of the activities of microbes. Milk as it is secreted by the gland of the mammals is free of microorganisms. However, microorganisms associated with the teat move up the teat canal and into the interior of the udder. This causes even aseptically drawn milk to contain microorganisms, mostly bacteria. Bacteria in aseptically drawn milk are usually limited in number and include mostly Micrococci, Lactococci, Staphylococci, Streptococci and Bacillus (LCT, 2011).

Milk may become contaminated with bacteria during or after milking.

The mammary glands of cows can become inflamed due to a bacterial

3

infection called mastitis. During mastitis, very high numbers of bacteria are present in the udder and are excreted through the milk. Some disease causing organisms (pathogens) can be shed through cow feces and may contaminate the outside of the udder and teats, the farm environment (bedding, for example) and the milking equipment. Although optimal growth conditions for bacteria/pathogens are varying, milk contains important nutritional components for growth, and, therefore, it is also an ideal medium for the growth of most of the bacteria/pathogens. Temperature plays an important role in bacterial growth. Many bacteria prefer to grow at body temperature (86- 98⁰F, 30-37⁰C), but may grow at lower temperatures (such as refrigerator temperature) at slower rates.

The area of dairy microbiology is large and diverse. Clean milk is generally defined as “Milk drawn from the udder of healthy animals, which is collected in clean dry milking pails and free from extraneous matter like dust, dirt, flies, hay, manure, etc. Clean milk has a normal composition, possesses a natural milk flavour with low bacterial count and is safe for human consumption”. Some bacteria may be specifically added to milk for fermentation to produce products like yogurt and cheese. The spoilage and pathogenic bacteria present in milk and dairy products may cause spoilage of milk or disease in consumers, respectively. Besides being a health hazard, contamination of milk and milk products can lead to huge economic losses.

The employment of hygienic practices at the time of milking is therefore one of the first and most important steps in clean milk production.

Clean milk production results in milk that is safe for human consumption, free from disease producing microorganisms, has a high keeping

4

quality, can be transported over long distances, has a high commercial value and is a high quality base suitable for processing resulting in high quality finished products. Milk needs to be protected from all possible sources of microbial contamination. Potential sources of contamination of milk are dung, water, utensils, soil, feed, air, milking equipment, animal and the milker.

Contamination of milk can occur during storage and transport (FAO, 2011).

Human illness from milkborne pathogens is usually associated with consumption of raw milk or products made from raw milk. In the past 20 years, foodborne illnesses from dairy product consumption have been predominantly associated with Salmonella enterica, Listeria monocytogenes, Campylobacter jejuni, and Escherichia coli O157:H7. These organisms have been isolated from bulk tank samples (Jayarao et al., 2001; 2006; Van Kessel et al., 2004). Because there is a risk of pathogen contamination in milk produced from healthy cows under sanitary milk conditions, pasteurization of milk prior to consumption will destroy pathogens and provide protection for illness associated with milk borne pathogens. Occasionally, human illness has been linked to pasteurized milk products but these cases usually have been a result of contamination of the product after pasteurization or improper pasteurization (Oliver et al., 2005).

Mastitis is recognized as the most costly disease in dairy cattle. Losses to the USA dairy industry is about $2 billion per year. Mastitis is one of the economically most important diseases affecting the Indian dairy industry causing an annual economic loss of Rs.7165.51 crores (US$ 1492.8 millions) (Bansal and Gupta, 2009). Mastitis reduces milk production and alters milk composition.

The magnitude of these changes in individual cows varies with the severity and

5

duration of the infection and the causative microorganisms. Mastitis is commonly caused by bacteria like Staphylococcus aureus, Streptococcus agalactiae, E. coli, Klebsiella, Listeria monocytogenes etc. These microorganisms produce toxins that can directly damage milk producing tissue of the udder and the presence of bacteria initiates inflammation within the udder tissue in an attempt to eliminate the invading microorganisms. The inflammation contributes to decreased milk production and is primarily responsible for the compositional changes observed in milk fat and protein content. Mastitis not only reduces dairy producers’ profits but also results in important and costly losses to processors due to poor quality milk. Awareness of the economic losses associated with mastitis is resulting in a desire for mastitis control programs (Hurley, 2011).

Dairy cattle are considered as the primary reservoir of Shiga toxin- producing Escherichia coli (STEC) and the main route of STEC infections in humans is via consumption of contaminated food (Hussein and Sakuma, 2005;

Meng et al., 2007). STEC strains have been isolated from a large variety of different foods including raw-milk cheeses (Caro and Garcia-Armesto, 2007;

Stephan et al., 2008) and although the first STEC outbreaks were associated with consumption of contaminated and undercooked hamburgers, subsequent outbreaks have incriminated both animal and plant origin foods (Erickson and Doyle, 2007). Clinically, STEC infection is characterized by non-bloody-to- bloody diarrhoea, haemorrhagic colitis, thrombocytopenia and fatal haemolytic uremic syndrome (HUS) (Bertholet-Thomas et al., 2011). Fecal specimens examined from healthy cattle during the investigations of two sporadic cases of hemolytic uremic syndrome (HUS) associated with raw milk consumption and

6

an outbreak of gastroenteritis and HUS caused by E. coli 0157:H7 demonstrated that dairy cattle are a reservoir of E. coli 0157:H7 and other STEC (Wells et al., 1991). Enterohemorrhagic E. coli (EHEC) infection can occur through ingestion of improperly cooked food and raw milk contaminated with bovine feces containing E. coli 0157:H7. There is a growing concern over the emergence of highly virulent non-O157 STEC serotypes that are globally distributed. The recent outbreak of non O157 E.coli i.e. O104:H4 in more than 14 European countries has caused 42 deaths and more than 3792 people were ill (WHO, 2011). Studies in India have confirmed that cattle are the principal reservoir of non-O157 serotypes (Khan et al. 2002). In India, limited information is available regarding the STEC in animals including cattle (Pal et al., 1999), sheep (Bhat et al., 2008), fish (Sanath Kumar et al., 2001), beef (Khan et al., 2002) and human faeces (Khan et al., 2002).

Listeria monocytogenes is a foodborne pathogen of great concern for the food industry and food producing companies. Due to its physiological characteristics, such as resistance to acidic and sodium chloride stress, ability to grow at low temperature and possibility to form biofilms (Harvey et al., 2007), it can persist and/or re-contaminate food products, thereby representing an important risk for the safety of the consumers (Olesen et al., 2009; Gardan et al., 2003; Liu et al., 2002; Pan et al., 2006). The term “Listeria hysteria”

was coined towards the end of 1980s following a series of listeriosis outbreaks due to the consumption of soft-cheese and ready-to-eat (RTE) meats in the UK.

Recently, this emerged again in the large outbreaks in Canada caused by deli meats (Warriner and Namvar, 2009) and in USA (Anon, 2011). The increase in the occurrence of listeriosis, reported in the community summary report on

7

foodborne outbreaks in the European Union in 2007 (Anonymous, 2009), warns for the need of special attention to this foodborne pathogen in order to combat its presence in foodstuffs. Listeria spp. are ubiquitous bacteria widely distributed in the environment (Liu et al., 2006). Among the eight species of Listeria, only Listeria monocytogenes is commonly pathogenic for humans.

Although human listeriosisoccurs only sporadically (Farber and Peterkin, 1991 and Schuchat et al., 1991) several outbreakshave been observed during the last two decades (McLauchlin et al., 2004). It is established that food-borne transmission constitutesthe main route of acquisition of listeriosis (Farber and Peterkin, 1991; Pinner et al., 1992). Although the incidence of the first human case of listeriosis was reported by Nyfeldt (1929), it is only since 1981, after the three well investigated listeriosis epidemics, first caused by coleslaw (Schlech et al., 1983), second caused by whole and 2% fat milk (Fleming et al., 1985) and third caused by consumption of soft Mexican-style cheese (Linnan et al., 1988), that this organism came to be considered as a foodborne pathogen. Multinational outbreak from dairy products was reported recently by Fretz et al. (2010). Large majority of patients with listeriosis have an underlying condition which predisposes to infection by interfering with T cell- mediated immunity. It can cause serious infections such as meningitis or septicemiain newborns, immunocompromised patients, and the elderly or lead to abortion (Vazquez-Boland et al., 2001). At great risk are pregnant women and the unborn child, alcoholics, drug abusers, diabetics, patients receiving treatments which alter their natural immunity such as AIDS patients, patients with malignancies and the elderly (WHO, 1988). Infection acquired early in pregnancy may lead to abortion, stillbirths or premature delivery. When the

8

infection is acquired late in pregnancy, it can be transmitted transplacentally and lead to neonatal listeriosis which may be manifested at birth or late in the neonatal period. Non-perinatal listeriosis is seen mainly in immuno- compromised adults and children. Typical overt listeriosis presents as sepsis and meningitis. Although the presence of L. monocytogenes has been reported from a wide variety of foods, the incidence in tropical foods is very low (Karunasagar and Karunasagar, 2000). The epidemiology and risk management of listeriosis in India has been reviewed (Barbuddhe et al., 2011).

Hence, in the present study, isolation of Listeria from milk samples was undertaken.

The availability of subtyping procedures to track individual strains involved in outbreaks, and to examine the epidemiology and population genetics of bacteria, is integral to control and prevention programs aimed at limiting infections. Application of subtyping methods also provides insight into the population genetics, epidemiology, ecology, and evolution of bacteria.

A variety of conventional, phenotypic, and DNA-based subtyping methods have been described for differentiation of L. monocytogenes and E. coli beyond the species and subspecies levels (Graves et al., 1999). While phenotype-based methods have been used for many years to subtype L. monocytogenes and other food-borne pathogens, DNA-based subtyping methods are generally more discriminatory and amenable to inter-laboratory standardization and are thus increasingly replacing phenotype-based subtyping methods (Wiedmann, 2002; Karama and Gyles, 2009; Foley et al., 2009). Commonly used phenotype-based subtyping methods for L. monocytogenes and other food- borne pathogens include serotyping, phage typing, and multilocus enzyme

9

electrophoresis (MLEE) (Seeliger and Hohne, 1979; Weintraub, 2007). The genetic subtyping approach encompasses PCR-based approaches (e.g., random amplified polymorphic DNA and amplified fragment length polymorphism), PCR-restriction fragment length polymorphism (PCR-RFLP), ribotyping, pulsed-field gel electrophoresis, and DNA sequencing-based subtyping techniques (e.g., multilocus sequence typing (MLST)) (Wiedmann, 2002;

Karama and Gyles, 2009; Hyytiä-Trees et al., 2007).

Raw (unpasteurized) milk can be a source of food borne pathogens.

Raw milk consumption results in sporadic disease outbreaks. Pasteurization is designed to destroy all bacterial pathogens common to raw milk, excluding spore-forming bacteria and possibly Mycobacterium paratuberculosis, but some people continue to drink raw milk, believing it to be safe.

The territory of Goa with the high rainfall (3000 mm), 100 km. long coastal boundary with the available water resources and moderate temperature ranging from 24⁰C to 32⁰C, provides ample scope for income generation through agriculture and livestock production. The territory has about 100,000 cattle and 45,000 buffaloes. The human population of the state is around 14 lakhs in addition to the visiting tourists and migrant labourers which accounts for about one third of the local population (Anon, 1997).

Food borne illnesses continue to pose a threat to human health. Foods of animal origin are usually implicated as a vehicle for such illnesses.

Production of milk and its products involves a long sequence of operations from harvesting to final consumption during which it is exposed to various microorganisms. The climate in Goa is very congenial with high humidity and relatively constant temperature throughout the year which in turn favours the

10

rapid multiplication of the microbes in foods of animal origin. The microbial growth is undesirable as it may cause spoilage as well as food-borne illnesses.

Microbiological examination of milk is essential to find the degree of contamination. The assessment of microbial load at various stages of manufacture or processing may serve as a useful tool for quality assessment and improvement which will result in longer shelf life which is a desirable market requirement. The detection of coliform bacteria and pathogens in milk indicates a possible contamination of bacteria either from the udder, faecal sample, milk utensils or water supply used (Olson and Mocquot, 1980; Bonfoh et al., 2003). However, keeping milk in clean containers at refrigerated temperatures immediately after milking process may delay the increase of initial microbial load and prevent the multiplication of micro-organisms in milk between milking at the farm and transportation to the processing plant (Bonfoh et al., 2003). Contamination of mastitis milk with fresh clean milk may be one of the reasons for the high microbial load of bulk milk (Jeffery and Wilson, 1987).

Guaranteeing a greater food safety level for consumer products warrants an integrated approach to controlling food safety throughout the entire food chain (Stefan, 1997; Valeeva et al., 2005). A number of regulations have been developed and introduced to assure food safety at different stages of the food production chain. Given the many potential and emerging hazards along the chain, it is of practical importance to prioritize attributes. Spoilage and contamination may occur in the milk chain as a result of poor hygiene, long periods of transportation and lack of proper storage facilities. Deficient hygiene has often been considered to be one of the major causes of spoilage of

11

products resulting in a loss of income, both for farmer and smallholder dairies.

Although many studies are reported on analysis of milk collected at different stages of processing, data is lacking particularly on the analysis of milk in production chain i.e from farm to table. Hence, the proposed study was envisaged with the following objectives.

a) To assess the microflora of milk at different points of collection and processing.

b) To standardize the conventional methods of isolation of microorganisms.

c) To isolate the specific pathogens viz. Escherichia coli and Listeria monocytogenes from milk and milk products.

d) To analyse the sources of contamination of milk.

e) To characterize the specific pathogens by molecular techniques.

12

Chapter II.

Review of Literature

13

Food-borne or waterborne microbial pathogens are leading causes of illness and death in less developed countries, killing an estimated 1.9 million people annually at the global level. Even in developed countries, it is estimated that up to one third of the population are affected by microbiological food- borne diseases each year (Schlundt et al., 2004). The factors involved in significant increase are generally agreed to include changes in animal production systems and in the food production chain. Both types of changes can cause corresponding changes in patterns of exposure to the pathogens and the susceptibility pattern of the human population (Schlundt et al., 2004).

Mastitis, on account of its causing serious wastage and undesirable milk quality, is emerging as a major challenge among the others (like breeding improvement, nutrition management, control of infectious, tick-borne, blood and internal parasitic diseases) in dairy development of tropics. Subclinical mastitis was found more important in India (varying from 10-50% in cows and 5-20% in buffaloes) than clinical mastitis (1-10%) (Joshi and Gokhale., 2006).

2.1 Microbiological quality of milk

The total count of bacteria in milk has a decisive effect on the quality and safety of dairy products (Szteyn et al., 2005). Contamination of milk with high levels of spoilage bacteria is usually unsuitable for further processing since it does not meet the consumer's expectations in terms of health (nutritional value), safety (hygienic quality) and satisfaction (sensory attributes) (Nanu et al., 2007). As a result, total viable bacterial counting has become one of the accepted criteria for grading milk intended for consumption and processing for dairy products. The importance of various etiological agents

14

in milk-borne diseases has changed dramatically over time. However, more than 90% of all reported cases of dairy related illness continued to be of bacterial origin, with at least 21 milkborne or potentially milkborne diseases currently being recognized (Bean et al., 1996). Pathogens that have been frequently involved in foodborne outbreaks associated with the consumption of milk include Listeria monocytogenes, Salmonella, Campylobacter, Staphylococcus aureus, Bacillus cereus, Escherichia coli and Clostridium botulinum. The presence of these pathogenic bacteria in milk emerged as major public health concerns, especially for those individuals who still drink raw milk (Ryser, 1998). Keeping fresh milk at an elevated temperature together with unhygienic practices in the milking process may result in microbiologically inferior quality (Chye et al., 1994).

Fresh milk drawn from a healthy cow normally contains a low microbial load (less than 1000 CFU/ml), but the loads may increase up to 100 fold or more once it is stored for some time at normal temperatures (Richter et al., 1992). Contamination of mastitis milk with fresh clean milk may be one of the reasons for the high microbial load of bulk milk (Jeffery and Wilson, 1987). The detection of coliform bacteria and pathogens in milk indicates a possible contamination of bacteria either from the udder, milk utensils or water supply used (Olson and Mocquot, 1980; Bonfoh et al., 2003).

Of the 507 milk samples collected from 16 milk collection centres in Trinidad, 454 (89.5%) were California mastitis test (CMT)-positive. The total aerobic plate count per ml was generally high for all samples ranging from 5.8 x 10⁵ +/- 3.1 x 10⁵ to 5.7 x 10⁸ +/- 1.5 x 10⁹ (Adesiyun, 1994).

15

Evaluation of bacteriological quality of raw cow's milk taken from udder, bucket, storage container before and after cooling and upon arrival at the processing plant from four dairy farms and a milk collection centre in and around Addis Ababa (Godefay and Molla, 2000) indicated a high increase in the mean total aerobic plate count in milk samples taken from the bucket (1.1 x 10⁵ CFU/ml), storage container before cooling (4 x 10⁶ CFU/ml) and upon arrival at the processing plant (1.9 x 10⁸ CFU/ml). The mean coliform counts ranged from 1.3 x 10⁴ CFU/ml (storage container before cooling) to 7.1 x 10⁴ CFU/ml (upon arrival at the processing plant). Lack of knowledge about clean milk production, use of unclean milking equipment and lack of potable water for cleaning purposes were some of the factors which contributed to the poor hygienic quality of raw milk in the study farms (Godefay and Molla, 2000).

Analysis of the microbiological quality of raw cow’s milk taken at different intervals from the udder to the selling point in Bamako, Mali revealed a strong increase in the total count (TC) of bacteria during transport from the farm to the market (10⁷ CFU/ml). The milk containers of the farmer and the milk vendor played a major role in the increase in the milk flora that occurred during transport from the farm to the selling points (Bonfoh et al., 2003).

Quantification of viable cells is a critical step in almost all biological experiments. A Methylene Blue dye Reduction Test (MBRT) to quantify viable cells based on reduction of methylene blue dye in cell cultures has correlated well with colony forming units (cfu) up to an 800 live cells as established by plating. The utility of the developed assay to monitor cfu

16

rapidly and accurately for E. coli, Bacillus subtilis and a mixed culture of E.

coli and B. subtilis has been demonstrated (Bapat et al., 2006).

In a study to improve the microbiological quality of the milk, from .cow’s udder to the selling point by container washing and disinfecting, a significant decrease of the total counts and Enterobacteriaceae counts in milk at the selling point as compared to the cow’s udder was reported (Bonfoh et al., 2006). The study suggested that in milk production area, besides udder infection and water quality, hygiene behaviour with respect to hand washing, containers cleaning and disinfection were the key areas of relevance to milk hygiene intervention (Bonfoh et al., 2006).

The bacterial composition of bulk tank milk from 13 farms was examined over a 2-week period to characterize sudden elevations in the total bacterial count referred to as "spikes." Twenty standard plate count spikes were observed: 12 associated with streptococci, 4 associated with gram- negative organisms, 2 associated with streptococci and gram-negative organisms, and 2 that were not definitively characterized. Spikes ranged from 14,000 to 600,000 CFU/ml (Hayes et al., 2001). Microbiological enumeration of 112 samples of raw buffalo milk collected at four locations in China revealed total mesophilic aerobic bacteria counts of 5.59 log CFU/ml (Bei- Zhong et al., 2007).

Analysis of bulk-tank milk samples in Estonia for lactic acid bacteria count (LABC), psychrotrophic bacteria count (PBC), aerobic spore-forming bacteria count (ASFBC) and total bacterial counts revealed LABC below 10⁴ CFU/ml in most samples, while psychrotrophic micro-organisms dominated in 60% of farms. PBC ranged from 4.2 × 10² to 6.4 × 10⁴ CFU/ml, and ASFBC

17

varied from 5 to 836 CFU/ml. The microbiological quality of the farm bulk- tank milk was good - more than 91% of samples contained <50,000 CFU/ml, and SCC in the majority of samples did not exceed the internationally recommended limits (Stulova et al., 2010).

During an evaluation of on-farm pasteurization systems, milk samples were examined for standard plate count (SPC), coagulase-negative staphylococci count, environmental streptococci count, coliform count, gram- negative non-coliform count, and Staphylococcus aureus count.

Bacteria counts were significantly reduced by pasteurization, and pasteurized milk contained acceptable numbers of bacteria in >90% of samples indicating pasteurization to be effective in lowering bacterial contamination of milk. However, bacteria numbers significantly increased after pasteurization and, in some cases, bacteria counts in milk fed to calves were similar to pre- pasteurization levels. Milk handling after pasteurization was identified as an important issue on the farms studied (Elizondo-Salazar et al., 2010). While determining the total plate counts and total coliform counts in 250 samples of kraals and indigenous milk products in the coastal savannah zone of Ghana, total plate counts exceeded 10⁵ CFU/ml in 45.2% of the samples while coliforms exceeded 10³ CFU/ml in 66.0%. E. coli was detected in 11.2%

samples (Addo et al., 2011).

In an investigation, the effects of season, cow cleanliness and milking routine on bacterial and somatic cell counts of bulk tank milk was studied on a total of 22 dairy farms in Lombardy, (Italy) (Zucali et al., 2011). Season had effect on cow cleanliness with a significantly higher percentage of non-clean (NC) cows during cold compared with mild season. Standard plate count

18

(SPC), laboratory pasteurization count (LPC), coliform count (CC) in milk significantly increased in hot compared with cold season. The effect of cow cleanliness was significant for SPC, PBC, CC and Escherichia coli in bulk tank milk. Milking operation routine strongly affected bacterial counts: farms that accomplished a comprehensive milking scheme including two or more operations among fore stripping, pre-dipping and post-dipping had lower teat contamination and lower milk SPC, PBC, LPC, CC and LS than farms that did not carry out any operation (Zucali et al., 2011).

Relationships of cleaning procedures for milking equipment applied in intensive dairy farms in Lombardy, (Italy) with bacterial count of bulk milk and hygienic condition of milking machine components was studied on a group of 22 dairy farms. The results showed that farms classified as high and low milk total bacteria count significantly differed both in terms of liners and receiver bacterial contamination and in terms of water temperature reached during the detergent phase of cleaning milking equipment. Significant positive correlations were found among total bacterial counts in milk and bacterial contamination of the liners. Routine check and regulation of water temperature during the washing phase of the milking machine can be a simple and effective way to control one of the main risk factors for bacteriological quality of bulk tank milk (Bava et al., 2011).

In a nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows on dairy farms in Sweden among 583 quarter milk samples collected from 583 dairy cows, the most common bacteria isolated were S. aureus - 31%, CNS - 27%, Str. Dysgalactiae - 15%, Str. Uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1% (Persson et al., 2011).

19 2.2 Coliforms in Milk

The presence of total coliforms in foods of animal origin indicates environmental sources of contamination (Mhone et al., 2011). Amongst the coliforms, Escherichia coli is the most common contaminant of raw and processed milk (Quinn et al., 2002). It is a reliable indicator of faecal contamination of water and food such as milk and dairy products (Todar, 2008). Coliforms were detected in 62.3% of 131 bulk tank milk samples in eastern South Dakota and western Minnesota (Jayarao and Wang., 1999).

Counts ranged from 0 to 4.7 log10 CFU/ml. The mean count was 3.4 log10 CFU/ml. Gram-negative non-coliform bacteria were observed in 76.3% of bulk tank milk. Counts ranged from 0 to 6.2 log10 CFU/ml. The mean count was 4.8 log10 CFU/ml. Coliforms and gram-negative non-coliform bacteria accounted for 32.9 and 67.1% of the total isolates, respectively. Examination of bulk tank milk for coliforms and non-coliform bacteria could provide an indication of current and potential problems associated with bacterial counts and milk quality (Jayarao and Wang., 1999). Investigation of the rate of contamination with coliforms and incidence of E. coli in raw milk supplied by farmers to dairy cooperative societies for marketing revealed about forty two (42.2%) percent of the milk samples from farmers’ cans and 10.3% of samples from cooperative cans to be free of coliforms (Ombui et al., 1994), while 89.5% of the samples from farmers cans and 50% samples from cooperative cans could be considered to be of good quality with no more than 50,000 coliforms/ml of milk. A good number of farmers were drawing milk under satisfactory conditions, but awareness campaigns on clean milking, milk

20

handling and storage practices should be stepped up in order to reach farmers who may not be informed (Ombui et al., 1994).

As part of the NAHMS Dairy 2002 survey, 861 bulk tank milk samples from farms in 21 states, coliforms were detected in 95% (818 of 860) of the samples, and the average SCC was 295,000 cells/ml (Van kessel et al., 2004).

Total plate counts, total coliform counts and the presence of Escherichia coli and E. coli O157:H7 were determined in 250 samples of kraals and indigenous milk products in the coastal savannah zone of Ghana. Total plate counts exceeded 10⁵ CFU/ml in 45.2% of the samples while coliforms exceeded 10³ CFU/ml in 66.0% and E. coli was detected in 11.2%. Antibiotic residues were detected in 3.1% of raw cow milk samples (Addo et al., 2011).

2.3 Escherichia coli

Shiga toxigenic Escherichia coli, including E. coli O157:H7, produce a family of toxins known as Shiga toxins, or verotoxins, related to the toxin produced by Shigella dysenteriae. This bacterium is one of the major bacterial pathogens causing food-borne illnesses, ranging from mild diarrhea to a life threatening complication known as hemolytic uremic syndrome (Friedrich et al., 2002). The large number of cases of human illness caused by Shiga toxin- producing Escherichia coli (STEC) worldwide has raised safety concerns for foods of bovine origin. These human illnesses include diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Severe cases end with chronic renal failure, chronic nervous system deficiencies, and death (Hussein and Sakuma, 2005a). The cattle have been shown to be a major reservoir of STEC and raw foods such as ground beef and

21

milk are the most common vehicles of infection (Gomez et al., 2002). A large number of STEC strains (e.g., members of the serogroups O26, O91, O103, O111, O118, O145, and O166) have caused major outbreaks and sporadic cases of human illnesses that have ranged from mild diarrhea to the life- threatening hemolytic uremic syndrome. The global nature of food supply suggests that safety concerns with beef and dairy foods will continue and the challenges facing the dairy industry will increase at the production and processing levels (Hussein and Sakuma, 2005a).

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains have been linked to outbreaks and sporadic cases of illness worldwide. Illnesses linked to STEC serotypes other than O157:H7 appear to be on the rise in the United States and worldwide, indicating that some of these organisms may be emerging pathogens (Mathusa et al., 2010). Various virulence factors are involved in non-O157 STEC pathogenicity; the combined presence of both eae and stx genes has been associated with enhanced virulence. Worldwide, foods associated with non-O157 STEC illness include sausage, ice cream, milk, and lettuce, among others. Subtilase cytotoxin SubAB is an additional STEC virulence factor which contributes to HUS. SubAB has a strong preference for a sialic acid that humans are unable to synthesize but which is derived from meat and dairy products. Thus, a two-hit process is seen in the pathogenesis of milk-borne SubAB-producing STEC strains, thereby causing HUS (Lofling et al., 2009). Results from several studies suggest that control measures for O157 may be effective for non-O157 STEC (Mathusa et al., 2010). Non-O157 STEC contributes to the burden of illness but has been under recognized as a result of diagnostic limitations and inadequate surveillance (Brooks et al.,

22

2005). Of the 940 human non-O157 STEC isolates from persons with sporadic illnesses submitted between 1983 and 2002, by 43 state public health laboratories to the Centers for Diseases Control and Prevention reference laboratory for confirmation and serotyping, the most common serogroups were O26 (22%), O111 (16%), O103 (12%), O121 (8%), O45 (7%), and O145 (5%). Non-O157 STEC infections were most frequent during the summer and among young persons. Virulence gene profiling revealed 61% stx1 but not stx2; 22% stx2 but not stx1; 17% both stx1 and stx2. STEC O111 accounted for most cases of HUS and was also the cause of 3 of 7 non-O157 STEC outbreaks reported in the United States. Strains that produce Shiga toxin 2 are much more likely to cause HUS than are those that produce Shiga toxin 1 alone. Improving surveillance will more fully elucidate the incidence and pathological spectrum of these emerging agents (Brooks et al., 2005).

In US, 27 milkborne general outbreaks of infectious intestinal disease characterized by significant morbidity were reported to the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC). Unpasteurized milk (52%) was the most commonly reported vehicle of infection in milkborne outbreaks, with milk sold as pasteurized accounting for the majority of the rest (37%) and most outbreaks were linked to farms (67%) (Gillespie et al., 2003).

Ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases (Blanco et al., 2005).

23 2.3.1 Isolation of E. coli

Universal pre enrichment broth (UPB) can be effectively used for the isolation of E. coli from dairy farm environmental samples (Nam et al., 2004).

A sensitive procedure based on ISO/DIS 16654:1999 (later ISO 16654:2001), which includes an immunomagnetic separation step can be used for the isolation of E. coli (Conedera et al., 2004).

A rapid fluorogenic medium (4-methylumbelliferyl-beta-D-glucuronide (MUG-7) capable of detecting E. coli after 7.5 h incubation at 41.5^OC was evaluated for the detection of E. coli in dairy products in comparison with Violet red bile agar. There were no significant differences between the numbers of E. coli detected on the two media (Sarhan et al., 1991).

Immunomagnetic separation (IMS) has been found to be a sensitive method for the recovery of E. coli O157:H7 in milk creams and recommended for isolation of the pathogen, the enrichment in tryptone soya broth with vancomycin, cefixime and tellurite, application of IMS, and plating of immunobeads onto nonselective agar (sorbitol MacConkey agar) and selective agar (sorbitol MacConkey agar with cefixime and tellurite or chromogenic agar with tellurite and cefixime) (Rojas et al., 2006).

Alternative media such as LES Endo agar medium (LES Endo), Colilert-18 with 51-well Quanti-tray (Colilert), Chromocult Coliform agar (CC), Harlequin E. coli/ Coliform medium (HECM) and Chromogenic E. coli/

Coliform medium (CECM) for detection and enumeration of E. coli and coliform bacteria were compared to the reference method ISO 9308-1 (LTTC) using non-disinfected water samples with background flora. Results suggested that Colilert, CC and CECM are potential alternative media for detection of

24

coliform bacteria and E. coli from non-disinfected water (Pitkanen et al., 2007).

Enrichment in TSB added with yeast extract (YETSB) resulted in higher detection rates of E. coli O157 and O26 than in TSB. When YETSB was used as enrichment broth, the analysis of the confluent growth from the media gave more positive results than that from E. coli O157:H7-ID medium (Caro et al., 2011).

Chromogenic agars have been developed to recognize frequently occurring microorganisms directly on primary cultures, thus reducing the daily workload in a clinical microbiology laboratory. A new chromogenic medium CCEA was compared with a classical medium of violet red bile agar (VRBA), and other two Chromogenic media Agar I and Agar II. The accordant rates were 90%, 71.88%, 86.25% and 81.25% respectively, showing CCEA > Agar I

> Agar II > VRBA. The CCEA might be more advantageous than the VRBA, having the same efficacy as with Agar I and Agar II (Lu et al., 2007).

Two chromogenic agars, CHROM agar Orientation (CO) and CPS ID 3 (CPS3) compared with routine media (biplate technique using trypticase soy blood agar and eosin methylene blue agar) for the isolation, enumeration and identification of organisms in urinary tract infection (UTI). Approximately 91.9% of E. coli could be identified directly on CO media, while 97.5% of E. coli could be identified on CPS3 media. The use of CO and CPS3 as single media is promising for clinical urine culture (Chang et al., 2008).

The Sanita-kun E. coli and coliform sheet medium, a chromogenic medium containing X-Gal, consisting of a transparent cover film, an adhesive sheet, a layer of nonwoven fabric, and a water-soluble

25

compound film, including a culture medium formula has been devised for the enumeration of total coliforms and differentiation of E. coli (Ushiyama and Iwasaki., 2010). The X-Gal is hydrolyzed by beta-galactosidase from coliforms to produce a visible blue dye and Salmon-glucuronic acid, which is hydrolyzed by beta-glucuronidase from E. coli to produce a red-purple dye. It is easy to distinguish the difference between E. coli and coliform (other than E. coli) colonies. The Sanita-kun E. coli and Coliform sheet medium has been granted performance tested method status (Ushiyama and Iwasaki., 2010).

2.3.2 Prevalence of E. coli in foods

Shiga toxin-producing E. coli can potentially enter the human food chain from a number of animal sources, most commonly by contamination of meat with feces or intestinal contents after slaughter or cross-contamination of unpasteurized milk products (Alexandre and Prado., 2003). E. coli particularly O157:H7 have emerged as significant foodborne pathogens that pose major public health concern in the world, with milk and milk products constituting the high-risk foods category (Manoj et al., 2004). In cheese, E. coli is used as an indicator to assess post-pasteurisation contamination and its presence may indicate inadequate pasteurisation, poor hygiene conditions during processing or post-processing contamination (Martina et al., 2009). Bulk tank milk samples from the 248 dairy herds were examined for foodborne pathogens and Shiga toxin-producing Escherichia coli were detected in 2.4% samples (Jayarao et al., 2006). The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted

26

in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk (Solomakos et al., 2009).

All STEC strains from frozen hamburgers and soft cheeses were characterized as eaeA-/EHEC-hlyA+. The stx2 genotype was highly prevalent (77.8%) (Gomez et al., 2002). Raw poultry samples were highly contaminated with E. coli (45%) and classified as high-risk food in Vietnam. E. coli was also detected in raw meat, fish, and vegetables with the rate of 21.3%, 6.6%, and 18.5%, respectively (Ha and Pham., 2006).

STEC were detected in 12% of the 785 minced beef samples collected from 30 local stores in Lugo city, Spain. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. The highly virulent seropathotypes O26:H11 stx1 eae-beta1, O157:H7 stx1stx2 eae-gamma1 and O157:H7 stx2eae-gamma1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates (Mora et al., 2007).

Escherichia coli is a common contaminant of seafood in the tropics and is often encountered in high numbers. A survey of E. coli was conducted in 644 molluscan shellfish samples marketed in the Apulia region of southern Italy. Levels of E. coli and fecal coliforms were above the Italian legal limit in 27 and 34 samples (4 and 5%), respectively (Parisi et al., 2004). Shellfish collected in coastal environments can serve as a vehicle for STEC transmission (Gourmelon et al., 2006). In a study carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments, stx genes were detected in 40 of

27

144 (27.8%) sample enrichments from mussels, oysters or cockles. Five strains carrying stx1 or stx(1d) genes and one stx negative, eae and ehxA positive E.

coli O157:H7 were isolated from six of 40 stx-positive enrichments (Gourmelon et al., 2006). In another study, all the fish (Rastrineobola argentea) samples (60) analysed from in Kisumu town, Kenya were found to be contaminated with E. coli. The occurrence of multiple drug resistant (MDR) E. coli was identified as some of the possible health risks that may be associated with R. argentea (Sifuna et al., 2008). Pao et al. (2008) evaluated the microbial quality of raw fillets (n=272) of aquacultured catfish, salmon, tilapia, and trout. Escherichia coli was detected in 1.4, 1.5, and 5.9% of trout, salmon, and tilapia, respectively. E. coli was also found in 13.2% of catfish, with an average of 1.7 log MPN/g. Sushi is a traditional Japanese food, mostly consisting of rice and raw fish. Sushi samples (250) were analyzed for their microbiological status and the prevalence of pathogenic bacteria. The prevalence of Escherichia coli was higher in the fresh samples (Atanassova et al., 2008).

In India, four shiga toxin-producing Escherichia coli (STEC) strains were isolated from seafood, six from beef and one from a clinical case of bloody diarrhoea in Mangalore, the isolates were positive for Shiga toxins stx1 and stx2 and also for stx1 and stx2 genes (Kumar et al., 2004). While, in a study in Cochin a total of 484 presumptive E. coli were isolated from 414 finfish samples composed of 23 species of fresh fish from retail markets and frozen fish from cold storage outlets. Results indicated 81.4% of the E. coli isolates to be sorbitol positive (Thampuran et al., 2005). Microbiological quality of fish and shellfish from Kolkata with special emphasis on E. coli was determined

28

and indicated poor hygiene and sanitary conditions. Although E. coli O157 could not be detected, a few samples were contaminated with non-O157 serotypes of enterohaemolysin- and Shiga toxin-producing E. coli, raising public health concern (Manna et al., 2008). In another study, screening of fish and shrimp samples obtained from different retail fish markets in Cochin, India, by direct PCR assays targeting the eaeA gene, hlyA gene and stx gene revealed one shrimp sample to be positive for all these virulence markers, and recovery of seven typical E. coli O157:H7 isolates from the marker-positive shrimp sample indicated the need for strict adherence to hygienic handling methods and proper cooking or processing before consumption of these products (Surendraraj et al., 2010).

E. coli O157:H7 is capable of survival but not growth on the surface of fresh strawberries throughout the expected shelf life of the fruit and can survive in frozen strawberries for periods of greater than 1 month (Knudsen et al., 2001).

2.3.4 Milkborne E. coli outbreaks

Haemolytic uremic syndrome (HUS) is characterized by thrombotic microangiopathy with acute renal failure, haemolytic anaemia with schizocytes and thrombocytopenia. It is caused by gastrointestinal infection with Escherichia coli species producing verotoxins (or Shiga toxins, STEC) (Bertholet-Thomas et al., 2011). It is estimated that 5-8 % of infected individuals will develop HUS following STEC infection. Vehicles of STEC transmission are contaminated food (ground beef, unpasteurised dairy products, unwashed and uncooked fruit and vegetables), person-to-person

29

transmission and contact with farm animals with STEC. After an average incubation period of 3 to 8 days, patients develop painful bloody diarrhoea followed by systemic toxemia. This may lead to thrombotic microangiopathy with endothelial damage and activation of local thrombosis (Bertholet-Thomas et al., 2011).

Raw milk has been implicated as source of foodborne outbreaks. In Connecticut, two children experienced Escherichia coli O157-associated hemolytic uremic syndrome (HUS) after consuming raw milk purchased at a retail market and a farm (Guh et al., 2010). E. coli O157: NM outbreak strains were isolated from stool specimens of 6 case patients and 1 milking cow. The total estimated outbreak cost was $413,402. The outbreak resulted in substantial costs and proposed legislation to prohibit non-farm retail sale, strengthen advisory labels, and increase raw milk testing for pathogens (Guh et al., 2010). Annual Listings of Disease Outbreaks and the Foodborne Outbreak Database (FOOD) to establish epidemiologic baseline characteristics for disease outbreaks associated with fluid milk during 1990-2006 data by the Centers for Disease Control and Prevention reported eighty-three fluid milkborne outbreaks between 1990 and 2006, resulting in 3621 illnesses (Newkirk et al., 2011). The mean number of illnesses per outbreak was 43.6 (illness range: 2-1644). Consumption of unpasteurized milk was associated with 55.4% of reported outbreaks and Escherichia coli in 10.8% of reported outbreaks. Private homes accounted for 41.0% of outbreak locations (Newkirk et al., 2011).

A study was carried out on hospitalized patients with hemorrhagic colitis in Georgia who have consumed not washed raw fruits or vegetables,

30

non pasteurized dairy products, food from street vendors, soft cheeses made from raw milk and untreated water in areas lacking adequate chlorination.

Increased rate of patients indicated circulation of shiga-toxin producing E.

coli (Vashakidze et al., 2010). Another mixed-serotype outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145:H28 and O26:H11 was reported in the province of Antwerp, Belgium in September-October 2007 (Buvens et al., 2011). The epidemiological and laboratory investigations revealed ice cream as the most likely source of the outbreak. The ice cream was produced at a local dairy farm using pasteurized milk. VTEC of both serotypes with indistinguishable pulsed-field gel electrophoresis patterns were isolated from patients, ice cream, and environmental samples. The data suggested that O145:H28 played the most important role in the outbreak (Buvens et al., 2011).

2.3.5 Detection of virulence genes

The prevalence of Shiga toxigenic group of E. coli (STEC) in food products of bovine origin was 16% in France (Madic et al., 2009). In USA, samples of bulk tank milk from dairies suggested that 4.2% were positive for one or both Shiga toxin genes (stx1 and stx2) (Karns et al., 2007). Baseline data on the prevalence and characteristics of Vero cytotoxin-producing E. coli (VTEC) organisms in lactating animals in Ireland suggested ~3% of milk samples contained E. coli O157 (Murphy et al., 2007). In France, the prevalence of STEC-positive samples in raw milk as determined by PCR- ELISA was 21%, of these strains, ~72% were confirmed positive for stx (Perelle et al., 2007).