ISOLATION AND PHENOTYPTIC CHARACTERISATION OF E. COLI O157 AND SALMONELLA SPP IN A SLAUGHTER HOUSE AND MEAT SALE TABLES AT NSUKKA MARKETENUGU STATE

ABSTRACT:

Escherichia coli O157 and Salmonella spp. are major food borne pathogens and the emergence of these pathogens has been reported in many countries. The objectives of this study were to examine the meat contact surfaces in Nsukka slaughterhouse and meat sales environment for E. coli O157 and Salmonella species, determine the enterotoxigenic potential and antibacterial resistance profile of the isolated bacteria. For this study, a total of 448 swab samples randomly collected from slaughter slab (112), slaughter floor (112), meat holding drums (112) and meat

sales tables (112) were pre-enriched in nutrient broth at 37oC for 24hours. For E. coli O157

isolation, a loopful of  each pre-enrichment broth was streaked on Cefixime Tellurite Sorbitol MaConkey (CT-SMAC) agar (Oxoid®). After 24 hours of incubation, colourless colonies were subcultured on plain MacConkey, incubated at 37oC for 24 hours and thereafter a rose pink colony was subcultured on Eosin Methylene Blue (EMB) agar and observed for colonies with greenish metallic sheen appearance. E.coli O157 was further confirmed using Oxoid O157 latex

agglutination kit. For Salmonella isolation, a loopful of the pre-enrichment broth was inoculated into Selenith F broth and incubated at 42oC for 24hours after which  loopful of the broth was streaked on Brilliance Salmonella agar (Oxoid®). After incubation at 37oC for 18-24hrs, purple colonies (presumptive Salmonella organism) were further subjected to biochemical tests for confirmation. The saccharytic activity of the organisms were verified using 13 sugars – sorbitol, glucose, cellobiose, sucrose, saccharose, arabinose, maltose mannitol, mannose, dulcitol, xylose, Inositol  and  lactose.  The  ability  to  produce  heat-labile  and  heat-stable  enterotoxin  was determined using infant mouse assay. Antibiogram of the test isolates was determined using disc

diffusion technique with the following 11 antimicrobial agents – chloramphenicol, ciprofloxacin, ceftazidime, gentamicin, cefotaxime, ampicillin, aztreonam, ceftriaxone, sulphamethoxazole/trimethoprim, enrofloxacine and streptomycin. The results of this study have shown that Escherichia coli O157 was isolated from 3(0.68%) of the 448 samples analysed while Salmonella species were recovered from 10(8.93%) of the samples. The E.coli O157 strains were isolated from the slaughter floor 1(0.9), slaughter slabs 1(0.9), and sales tables 1(0.9). Four (3.57%) of the salmonellae were obtained from meat holding drums, and 4 (3.57%) from sales tables while 2 (1.79%) were isolated from slaughter floor. All the E.coli O157 isolated fermented glucose, sucrose, lactose, mannitol, dulcitol, saccharose, arabinose, maltose, cellobiose, xylose and mannose and none fermented inositol and sorbitol.  All the Salmonella isolated  fermented glucose, mannitol, dulcitol, saccharose, cellobiose and mannose, while 80% fermented xylose and none fermented sucrose, inositol, lactose, arabinose and maltose. The three  E.coli O157 and

Salmonella isolates produced heat-labile enterotoxins as evidenced by death of all infected mice none of the E.coli O157 and Salmonella isolates produced heat-stable enterotoxins since the gut to carcass ratio produced ranged from 0.043-0.062. The three E.coli O157 isolates were sensitive to ceftriaxone, enrofloxacin, streptomycin, cefotaxime and gentamicin, but resistant to ampicillin and  ceftazidime.  All  the Salmonella  isolates  were sensitive to  enrofloxacin  but  resistant  to ampicillin and ceftazidime. This study has shown that E.coli O157 and Salmonella are present on meat contact surfaces in Nsukka Slaughterhouse. This suggests that meat from these surfaces is likely to be contaminated by these organisms and may therefore predispose the consumer to bacterial food poisoning. The multiple antibiotic resistances displayed by zoonotic bacteria is of serious public health significance because infections in humans caused by these bacteria will be difficult to treat with the commonly available antibiotics.

CHAPTER ONE

INTRODUCTION

1.1        Background of Study

Abattoir is a special facility designed and licensed for receiving, holding, slaughtering and inspecting meat animals and meat products before release to the public (Alonge, 2005). Abattoir environment and slaughtering process play a very important role in the safety and wholesomeness of meat. Meat contamination by pathogens such as Listeria monocytogenes, Salmonella spp. and enterohaemorrhagic E. coli (Oboegbulem and Muogbo, 1981; Tabukun et al., 1996, Tompkin, 2004; Abiade-Paul et al., 2006) and chemicals in abattoir can persist in meat processing environment, contaminate meat as they are being processed and lead to food borne illnesses and intoxications (Okolocha et al., 2002). Thus, meat borne diseases and intoxications are major public health concerns. The types and incidence of meat related illnesses are becoming increasingly widespread and consumers becoming more cognizant now than ever of the dangers associated with raw meat contamination.

Escherichia  coli  O157  and  Salmonella  species  are  two  most  important  food  borne bacterial pathogens, about 95% of the emerging diseases that have affected humans in the past years are caused by pathogens originating from these organisms (WHO, 2007).  Diseases caused by these organisms range from bloody diarrhea, enteritis, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (Griffin and Tauxe,

1991; Nataro and Kaper, 1998). The growth of these organisms in the human intestine is known to produce large quantity of toxins, which can cause severe damage to the lining of the intestine and other organs of the body (Nataro and Kaper, 1998). The potentially high mortality associated with  E.coli  O157  and  Salmonella  infections,  makes  their  presence  in  any  food  material worrisome and of serious public health concern as most of the outbreaks recorded has been traced to consumption of beef and food products contaminated with these organisms (Hussein,

2007., Mead et al., 1999).

Domestic animals like cattle, sheep and goats are reported as sources and reservoirs of these organisms (Beutin et al., 1993), these animals are being slaughtered and the meat sold in the slaughterhouse, probably contaminates the environment, and act as a vehicle for transmission of E.coli O157 and salmonella species to the public (Doyle and Cliver 1990; Griffin and Tauxe

1991). These pose serious public and environmental health risk.

Meat contact surfaces in the abattoir such as equipment (e.g. meat holding drums, knives, axes), walls and floors may serve as sources of infectious agents and are therefore considered important factors in the evaluation of the level of contamination in such establishment (Okolocha et al., 2002., Bello and Son., 2009). Contamination occurs during slaughtering, handling, cutting, processing and storage (Madder, 1994).   Salmonella species are primarily located in the gastro- intestinal tract of the sub-clinically infected animals and the epidemiology of Salmonella at the slaughter house level is first of all a question of direct and indirect fecal contamination of carcasses (Oosteron, et al., 1985). Carcasses may be contaminated or cross-contaminated by manual or mechanical handling. During slaughtering, transfer of microorganisms continues from carcasses to hands of workers and equipment surface and from them to other carcasses.

Although slaughter equipment is often the immediate source of contamination, the carrier animals play an important role in the dissemination of Salmonella and E. coli O157. As a result of the technical  difficulties  in  detecting carrier  animals  before slaughtering or during meat inspection, carrier animals are continually serve as sources of contamination in slaughter houses and ultimately in food products (Haddock, 1970). Slaughter of cattle under poor hygienic conditions leads to the contamination of carcasses by Salmonella sp and E. coli O157 thus facilitating their transmission and increasing the risk of food borne diseases in humans (Mafu, et al., 1989).

1.2     Statement of Problem

Escherichia coli O157 and Salmonella species are among the major food borne pathogens causing high mortality in animals and man (WHO, 2007; Bello and Son, 2009). Reports have shown that the intestinal tracts of humans and animals are major sources of   these organisms in nature and approx 1 to 3% of all domestic animals are infected with Salmonella and these can be shed in their feaces (Litchfield, 1980).

Abattoir is a major link in the transmission of E. coli O157 and Salmonella spp to the food  chain  (Richard  et  al.,  1998);  reports  have  shown  that  abattoir  waste  with  all  its contaminants are found littered on the slaughter floors and environment (Nwanta and Achi,

2002; Adeyemo 2002; Abiade-Paul, et al., 2006). In many abattoirs, in Nigeria, meat is often dipped into water in drums instead of a chilling room and this bad practice contaminates the meat (Ezenduka et al., 2010).  Increasing numbers of human E.coli O157:H7 induced illnesses have been related to contact with animals or to water supply contaminated with runoff water from cattle farms or abattoir/slaughter houses (Adetosoye et al., 1976., Adesiyun et al., 1983., Oboegbulem and Muogbo 1981,  Abiade-Paul et al., 2006; Tabukun. 1996, Nwanta et al., 2010).

Studies have been carried out on the prevalence of E.coli O157 and Salmonella species in Nsukka slaughter house (Abiade-Paul et al., 2006; Oboegbulem et al., 1981; Nwanta, et al., 2010). Inspite of the wide knowledge of these organisms, data concerning isolation and characterization of these microorganisms from meat contact surfaces in Nsukka slaughter house and sales environment is lacking, it is therefore necessary that a study be conducted in Nsukka slaughter house to ascertain the presence of these organisms in Nsukka slaughter house meat contact surfaces and sales tables.

Antibiotic resistance is also a growing public health problem and has been attributed to overuse of antibiotics in humans, and its use as growth promoters in food animals (Johnson et al., 2006). Antibiotic resistant E.coli may also pass on the genes responsible for antibiotic resistance to other species of bacteria, such as Staphylococcus aureus.  E. coli O157 often carries multidrug resistant plasmids and under stress readily transfers these plasmids to other species (Salyers et al., 2004). Indeed, E. coli O157 is a frequent member of biofilms where many species of bacteria exist in close proximity to each other. This mixing of species allows E. coli strains that are piliated to accept and transfer plasmids from and to other bacteria. Thus E. coli and other enterobacteria are important reservoirs of transferable antibiotic resistant (Salyers et al., 2004; Enumeration of Escherichia coli and the Coliform Bacteria, 2002).

The wide spread occurrence of drug resistance E.coli and other pathogens in our environment  has necessitate the need for regular monitoring of antibiotics susceptibility trends to provide the basis for developing rational prescription  programs, making policy decisions and assessing the effectiveness of both (Omigie et al., 2006).

1.3   Objectives of the Study

The  overall  objective  of  the  study  is  to  examine  the  meat  contact  surfaces  in  Nsukka slaughterhouse and meat sales environment for E. coli O157 and Salmonella species.

Specifically the study seeks to:

1.   Determine the prevalence of E. coli O157 and Salmonella species on slaughter floor, slabs, sales tables and water in meat holding drums in Nsukka slaughter house.

2.   Determine the saccharolytic activity of the E.coli O157 and Salmonella isolates.

3.   Evaluate the E.coli O157 and Salmonella isolates for enterotoxin production.

4.   Determine the antibiotic sensitivity profile of the E.coli O157 and Salmonella isolates.

1.4    SIGNIFICANCE OF THE STUDY

The study will provide baseline data on the occurrence of E. coli O157 and Salmonella species on meat contact surfaces which are essential for the establishment of a Hazard Analysis Critical Control Point (HACCP) plan in any meat processing chain.

It will provide the information on the deleterious effect of location of abattoirs and discharge of its effluents near markets and residential areas.

Rational antimicrobial therapy requires the determination of the antimicrobial resistance or susceptibility pattern of any clinical isolates prior to drug administration. Unfortunately, in Nigeria, there are not enough veterinary diagnostic laboratories to achieve this purpose. The results of this study will also provide information on the antibiotic resistance profile and enterotoxin production profiles of the zoonotic bacterial isolates to the relevant public health authorities, abattior workers, livestock farmers, veterinary authorities and the  Ministry of Health for the prevention, control and management of health problems.

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