The Prevalence and Antibiogram of Salmonella and Shigella Isolated from abattoir, Jimma town, South West Ethiopia.

The Prevalence and Antibiogram of Salmonella and Shigella Isolated from abattoir, Jimma town, South West Ethiopia. 1Anbessa Dabassa and 2Ketema Bacha Department of Biology, Jimma University, P.O. Box 378, Jimma, Ethiopia E-mail address: adabassa@gmail.com Abstract A disease due to Contamination of food by Salmonella and Shigella are among the major challenges worldwide. Evaluation of the existing safety status of foods, including meat and meat products, is a pro-active measure to reduce the possible risk due to associated foodborne pathogens. To this effect, the study was done over a 5- month period between December 2009 and May 2010. In total 180 animal samples composed from cattle, goat, and sheep, meat and feces were analyzed for microbial load determination using conventional culture method. The aerobic mesophilic counts varied from 3.0 to 9.0 log10 CFU/g. Coliforms were encountered in most samples. Enterobacteriacea were present at 2-4 log10 CFU/g in 19 %( 45) meat, and 21% (22) faeces samples. Salmonella species were present in 4.4 % (8.0) of all the samples. Among the samples only Goat faeces sample was not contaminated with Salmonella whereas Shigella was not isolated from any samples. The prevalent Salmonella species were further characterized using API 20E kit. Isolated Salmonella strain was displayed multidrug resistance to several antibiotics including ampicillin, Naldixic Acid, Streptomycin; Tetracycline, and Chloramphenicol. The present study revealed that despite low contamination rate, foodstuffs particularly beef, chevon and mutton parts could be a potential vehicle for foodborne infections and implementation of preventive measures and consumer food safety education efforts are needed. Key words/phrases: Prevalence, Antibiogram, Salmonella, Shigella, Meat, MDR 1. Introduction Microbial food safety is an increasing public health concern worldwide (Kabir, 2010),food borne diseases often follow the consumption of contaminated food-stuffs especially from animal products such as meat from infected animals or carcasses contaminated with pathogenic bacteria as Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, Campylobacter spp., and Escherichia coli O157: H7(Nouichi and Hamdi, 2009). Meat is a highly perishable food. The rich source of nutrients of fermented meat provides both pathogenic and nonpathogenic microbes a suitable environment to grow during preparatory stage and storage (Zakpaa et al., 2009). Increase in demand for meat without the infrastructure for proper sanitary handling may lead to transfer of pathogenic organisms from the animals to the consumer (Mrema et al., 2006). Furthermore, Food safety issues have recently gained considerable attention from the public and represent an important concern for the veterinary profession. Problems related to food contamination, however, are not unique to humans, as dogs are also susceptible to a large number of food-borne infections (LeJeune and Hancock, 2001). Salmonella is one of the most important pathogenic genera implicated in foodborne bacterial outbreaks and diseases (Cetinkaya et al., 2008). Teklu, A. and Nigussie, H. (2011), points on the reason for Ethiopian abattoir meat contamination as, Salmonella may occur at abattoir from the excretion of symptomless animals, contaminated abattoir equipments, floor, and personal and the pathogen can get an access to the meat at any stage during butchering. Cross contamination of carcasses and meat products could continue during subsequent handling, processing, preparation and distribution. Shigella dysentery usually indicates improper sanitary conditions and poor personal hygiene and is principally a disease shigellosis of humans, as well as other primates (Asghar et al., 2002). The species involved are mostly Shigella sonnei (about 70%) and S. flexneri (approximately 25%) while other species are seldomly implicated (Mead et al., 1982). Understanding the prevalence and distribution of Salmonella and Shigella species in food animals and determining management strategies associated with lower prevalence is key to decreasing the risk of high pathogen loads at harvest (Foley and Lynne, 2008). In recent years, the health concern all over the world is the occurrence of antibiotic resistant strains of a number of pathogenic bacteria including Salmonella in foods (Cetinkaya et al., 2008). The presence of antimicrobial resistance determinants have the potential to adversely affect human health by causing illness that is more difficult to treat because of the resistance profile of the organism (Foley and Lynne,2008). ISSN : 0976-285X Vol 3 Issue 4 Aug-Sep 2012 143

This study was conducted to determine the prevalence of Salmonella and Shigella in cow, sheep, goat meat and feces in abattoir at jimma (Ethiopia), and to evaluate the isolates for their antibiotic resistance. 2. Materials and methods 2.1. Study Design and Sampling Experimental study undertaken on apparently health slaughtered sheep and goat, apparently health abattoir personnel and the abattoir environment at an export abattoir at Jimma Ethiopia from December 2009 and May 2010. The variable of interest considered as an output variable at the slaughterhouse was Salmonella and Shigella status. The explanatory variables considered were Salmonella and Shigella status of Sheep, Goat and Cattle meat and feces. The sample size used for this study was a total of 180 animal samples, were collected from Jimma town abattoir (Ethiopia). The Samples included were: (a) beef (n = 30), (b) chevon (n = 30), (c) mutton (n = 30), (d) cattle feces (n=30), sheep feces (n=30), and goat feces (n = 30). All samples were taken by using sterilized utensils and placed in separate sterile plastic bags. Samples were transported to the laboratory immediately after sampling. Bacteriological analyses were carried out immediately after the sample arrival at laboratory. 2.2. Sampling procedure Individual animals were systematically sampled depending on the number of animals slaughtered on each day Samples were collected on each day visit 8 animal meat and feces sample were collected. From each selected slaughtered sheep, goat and cattle, the carcass and caecal content were collected in separate sterile containers. The samples were then transported on ice to the Jimma University, Faculty of Natural Science Microbiology laboratory, Jimma Ethiopia for processing and analysis upon arrival. 2.3. Microbiological analysis 2.3.1. Determination Microbial Spectrum For carcass and caecal content total Aerobic Mesophilic counts (TAMC), Counts of Staphylococci, Count of Enterobacteriacea, Bacillus, Counts of yeast and molds and Counts of Coliforms were determined according to the criteria specified in Bacteriological analytical manual, and by standard plate count methods on PCA (Plat Count Agar)(Oxoid) for TAMC at 32 C during 72h, Mannitol Salt agar (Oxoid) for staphylococci count, Chloramphenicol Bromophenol Blue Agar for yeast and mold and VRBGA(Violet Red Bile Glucose Agar) (Oxoid) for Enterobacteriacea and VRBA (Violet Red Bile Agar) (Oxoid) for total coliform count (TCC) during 24 hrs at 32 C for TCC, and 44 C for FCC. 2.3.2. Salmonella Isolation For isolation of Salmonella and Shigella spp., 25 g of each sample was homogenized in 225 ml of Peptone Water (PW) and incubated at 37 oc overnight for the metabolic recovery and proliferation of cells which could be injured during processing or to bring the number of target organisms to a detectable level. One milliliter of this pre-enrichment culture was inoculated into tubes containing 10 ml tetrathionate (TT) broth (Oxoid), Selenite Broth (SB) (Oxoid), Rappaport vassiliadis (RVS) (merck) broth for secondary enrichment, and both incubated for 24 h at 37 oc except tetrathionate was incubated at 43oC for 48 hours in water bath. Bacteria growing in TT, SB, RVS, broth were streaked on xylose lysine desoxycholate (XLD) agar (Oxoid), MacConkey agar (Oxoid), Bismuth sulfate (BS),and Salmonella Shigella (SS) Agar and incubated at 37 oc overnight. The plates were examined for the presence of typical colonies of Salmonella, i.e. pink colonies with or without black centres on XLD agar and colourless colonies on MacConkey agar, Black, surrounded by a black or brownish zone with a metallic sheen on BS and colorless colonies on SS agar and for Shigella convex colorless colonies on MacConkey agar and colorless colonies on XLD. In addition to gram staining and hanging drop observation of cell shape, cell arrangement and motility using microscope Presumptive Salmonella and Shigella colonies were then subjected to initial screening tests using KOH test, Oxidation fermentation test( O/F), Catalase test, Cytochrome oxidase test, and triple sugar iron agar (oxoid), lysine iron agar (oxoid), urea agar (oxoid) and Simmon citrate Agar (Oxoid),Methyl red Voges Proskauer reagent, Mannitol and Glucose/Sucrose fermentation and SIM Medium (Oxoid). All biochemical tests were performed at 37 oc for 18 24 h. For confirmation of results of the conventional biochemical tests, additional test of the presumptive Salmonella isolates were characterized using the more sensitive API 20E (biomérieux, Marcy I Etoile, France) identification system as described by the manufacturer. 2.4. Antimicrobial susceptibility testing Antimicrobial susceptibility tests were carried out by the disc diffusion technique described according to the recommendation of the Clinical and Laboratory Standards Institute (CLSI) (NCCLS, 2002) on Mueller Hinton agar (Oxoid). Antimicrobials used by using commercial antibiotic Bio- discs (Oxoid) (μg): Amikacin (Amk)(30 ) ampicillin (Amp) (10), chloramphenicol (Chl) (30), ciprofloxacin (Cip) (5), gentamycin (Gen) (500 ), Kanamicin (Kan) (30), norfloxacine (Nor) (10),nalidixic acid (Nal) (30), streptomycin (Str) (10 ) and ISSN : 0976-285X Vol 3 Issue 4 Aug-Sep 2012 144

tetracycline (Tet) (30). Results were recorded by measuring the inhibition zones and scored as susceptible or resistant according to Manual of antimicrobial susceptibility test of American society of microbiology (ASM), (2005). Standard reference strains, which include E. coli (25922), Staphylococcus aureous (ATCC 25923), Psudomonas aruginosa (ATCC 27853), and Salmonella typhi (NCTC 8553) were used as quality control. The reference strains were kindly obtained from Ethiopian Health and Nutrition Research Institute (EHNRI), Ethiopia. 3. Results and discussion The major sources of contamination are multiple contacts with contaminated tools and Operators hands, and the Cattle carcasses, which are initially sterile, become contaminated with bacteria pathogens via transmission of organisms from the exterior of the live animal, and/or from the environment, to the meat surface. Extensive contamination, or abusive conditions that allow bacteria to reproduce, increase risk for presence of pathogenic bacteria and formation of toxins in food. Microbial population that comes in contact with fresh meat during slaughtering, dressing and processing presents a challenging problem to the abattoir. Table 1. Frequency distribution (%) of dominant bacteria in meat samples collected from Abattoir, Jimma town, 2009/2010. Sample Number of Bacillus Micro Entero- Stphylo Coli Other Gram Type Isolate coccus bacteriacea cocci Forms Positive Beef 116 72(62%) 5(4% ) 21(18%) 9(8%) 8(7%) 1(1%) Mutton 78 37(47%) 8(10%) 17(22%) 6(8%) 8(10%) 2(3%) Chevon 47 19(40%) 9(19%) 7(15%) 6(13%) 4(9%) 2(4%) Total 241 128(52%) 22(8%) 45(19%) 21(9%) 20(10%) 5(2%) Generally, 54 %( 128) of the meat samples were dominated by gram positive organisms. Bacillus spp. is dominated the bacterial flora (52%) in meat samples followed by gram negative enterobacteriacea families (19%) (Table 1). Similarly the aerobic mesophilic bacterial flora of the caecal content of the sample were also dominated by Bacillus spp. (42%), followed by enterobacteriacea families (20%) and other gram positive road bacteria (16%). Micrococcus, Staphylococcus and coli forms were also encountered in both meat and caecal content. Even though, 7% of the beef, 10% of the mutton, and 9% of the Chevon, were contaminated with E. coli and had populations of log 1-3.47 CFU/g. fecal Coliforms contamination in this work was much lower than the 2.55 log cfu /cm2 reported by (Nouchi, 2009). 3.1. Salmonella Spectrum of meat and feces sample From a total of 180 samples including beef, chevon, mutton, cattle feces, goat feces and sheep feces were examined for the presence of Shigella and Salmonella. Shigella was not isolated from any of the analysed samples. Meanwhile, out of 60 cattle, 60 goats and 60 sheep part 4(13.3%) beef, 1(3.3%) cattle feces,1(3.3%) chevon, 1(3.3%) mutton, 1(3.3%) sheep feces were contaminated with Salmonella. only one goat feces sample were not contaminated with Salmonella (Table 1). Table 1. Prevalence of Salmonella and Shigella from Cattle, Goat and sheep carcasses and faeces. Sample Sample No Shigella Positive Salmonella Positive Beef 30 0/30 4/30 13.3 Cattle faeces 30 0/30 1/30 3.3 Chevon 30 0/30 1/30 3.3 Goat faeces 30 0/30 0/30 0 Mutton 30 0/30 1/30 3.3 Sheep faeces 30 0/30 1/30 3.3 Total 180 0/180 8/180 4.4 % Salmonella positive ISSN : 0976-285X Vol 3 Issue 4 Aug-Sep 2012 145

Fresh raw meat like Mutton, Chevon and Beef has been implicated for many meat borne diseases in several countries.salmonella species were isolated from 5 of the total samples type obtained from Abattoir. Salmonella spp. was relatively isolated from the beef than from mutton, chevon, cattle faeces, and sheep faeces. Salmonella were not isolated from Goat faeces. In the rest of each sample type 3.3% of the samples contained Salmonella and beef which was the least clean had 13.3% positive for Salmonella, while a far cleaner samples, like Mutton, Chevon, Cattle faeces, and sheep faeces, each had 3.3% positive. The frequency of isolation, however, did correspond with the exposure of the sampled organisms with environmental contact, as the result of the size of the organism during dressing. The prevalence for Salmonella in the different types of beef, sheep and goat samples varied from 3.3% (1/30) samples each from sheep and goat to 13.3% (4/30) samples from cattle meat. This is much lower than that reported from raw meat (White et al, 2001). Overall, the prevalence of Salmonella in meats and faeces samples collected from the investigated abattoir was only 4.4% (8/180) indicating the relatively better hygienic practice during processing and handling the meat at abattoirs. According to Ejeta et al., (2004), high numbers of Salmonella were isolated from minced beef, mutton and pork meat samples obtained from retail supermarkets in Addis Ababa. The presence of even small numbers of Salmonella in carcass meat and edible offals may lead to heavy contamination of minced meat and sausage. When meat is cut into pieces, more microorganisms are added to the surfaces of exposed tissue (Ejeta et al., 2004). The contamination of equipment, material, and workers hands can spread pathogenic bacteria to noncontaminated carcasses (Nouichi and Hamdi, 2009). In this study no Shigella was isolated from all the analyzed meat and faeces samples. Similarly, the absence of Shigella was reported from related study on different meat products (Cetinkaya et al., 2008, Dhanashree and Mallya, 2008). Furthermore, Heredya et al, (2004) reported the absence of Shigella spp. in ground meat samples collected from retail stores. Similarly, an investigation by White et al, (2001), retail market poultry meats of south-east India revealed the occurrence of the pathogen in the poultry meat sample. FDA recommends zero tolerance to potential pathogens in food samples. 3.2. Antimicrobial Susceptibility patterns of Salmonella isolate 3.2.1. Susceptibility Patterns Salmonella is a leading cause of foodborne illness. The emergence of antimicrobial-resistant salmonella is associated with supplement of antibiotics to animal feed and for their treatments. Resistant bacteria can be transmitted to humans through foods, particularly those of animal origin (White et al, 2001). Almost all Salmonella isolates were sensitive to Amk, Cip, Kan, Nor and Gen. Among Salmonella isolates the most frequent resistance was noted for ampicillin (100%), followed by Naldixic Acid and streptomycin (87.5%), (87.5%) respectively and tetracycline (50%) Chloramphenicol (12.5%) (Table 2). Antimicrobial Drug Table 2. Salmonella Isolates resistance to Antimicrobial type Total No. (%) of isolates resistant Total Beef Resistant (n=4) Isolate(n=8) Mutton (n=1) Chevon (n=1) Beef faeces (n=1) Sheep faeces(n=1) AMK 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) AMP 8(100) 4(50) 1(12.5) 1(12.5) 1(12.5) 1(12.5) CIP 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) CHL 1(0) 0(0) 1(12.5) 0(0) 0(0) 0(0) GEN 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) KAN 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) NOR 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) NAL 7(87.5) 3(37.5) 1(12.5) 1(12.5) 1(12.5) 1(12.5) STR 7(87.5) 1(12.5) 1(12.5) 1(12.5) 1(12.5) 1(12.5) TET 4(50) 2(25) 1(12.5) 0(0) 1(12.5) 0(0) Were, AMK=Amikacine, AMP=Ampicillin, CHL=Chloramphenicol, CIP=Ciprofloxacin, GEN=Gentamicine, KAN=Kanamycin NOR=Norfloxacine, NAL=Naldixic Acid, STR=Streptomycin, TET=Tetracycline. The Salmonella species isolates in this study showed marked resistance to five of the ten antibiotics tested. A high proportion of Salmonella isolates were resistant to ampicillin, Naldixic acid, streptomycin, tetracycline and Chloramphenicol, i.e., 100% (8/8), 87.5% (7/8), 87.5% (7/8), 50% (4/8), 12.5 %( 1/8), respectively. All isolates of Salmonella were susceptible to, amikacine, ciprofloxacin, norfloxacine, Kanamycin and gentamicine. No isolates were susceptible to all of the antibiotics, and 3 different multidrug resistance patterns were identified among the 8 isolates. Of the total eight isolates of Salmonella, four (50%) of the isolates were resistant to four antibiotics, three (37.5%) to three antibiotics, and one isolate to two antibiotic. The most common multidrug resistance profile was Amp, Chl, Nal, Tet and Str (multidrug resistance to ampicillin, Chloramphenicol, ISSN : 0976-285X Vol 3 Issue 4 Aug-Sep 2012 146

Naldixic acid, tetracycline and streptomycin). Resistance to the antibiotics could be the consequence of the frequent use of ampicillin and tetracycline both in animal husbandry and in human medicine. Despite that, the use of antimicrobial agents in any environment creates selection pressures that favor the survival of antibioticresistant pathogens (White et al, 2001). 3.2.2. Multi Drug Resistance Patterns A total of 3 multiple drug resistance (MDR) patterns were detected among Salmonella isolates. About 37.5% (3.0) of the isolates showed resistance to three drugs, 50% (4.0) to four drugs and 25% (2) to two drugs. The most frequent MDR pattern was Amp/ Str/ Nal /Tet and was seen in 50%of the isolates followed by Amp/Nal/Str pattern observed in 37.5% (Table 3). Table 3. Multi-drug Resistance Pattern of Salmonella Isolated from Meat and Faeces samples. Number of Resistant Pattern No of Isolate (%) Resistance One Amp, Chl 12.5 (1.0) Three Amp, Nal, Str 37.5 (4.0) Four Amp, Str, Nal,Tet 50.0 (4.0) Where, Amp= ampicillin; Nal=Naldixic Acid, Str= Streptomycin; Tet= tetracycline, Chl=Chloramphenicol In conclusion, Shigella was not detected from analysed foods whereas S. typhi was isolated from beef, chevon and mutton which could be a potential source of human salmonellosis. The study suggests that despite the low prevalence rate, Salmonella continues to contaminate raw meat parts at abattoir level and constitute a threat for public health. The results also indicated that food-related infections could be acquired from ingestion of meat harboring Salmonella resistance to multiple antibiotics, and that these infections would be more difficult to treat. Consequently, careful usage of antibiotics in food animals plays an important role in the prevention of antibiotic resistance and implementation of increased hygienic measures on farm and both at slaughter and processing line is necessary to ensure food safety. 4. Conclusion Microbiological analysis of meat from abattoir for foodborne organisms like Salmonella is of principal importance in ensuring the supply of safe food for the consumers. The information collected in this study coupled with other similar studies at different critical points earlier meat distribution to the consumer can be used as a basis to undertake qualitative microbiological risk assessment and provide essential information in designing monitoring and surveillance programs. Microflora of meat samples were dominated by Gram-positive bacteria in which Bacillus species were the dominant groups followed by Staphylococcus and Micrococcus spp. Although these genera are abundant in livestock environment, appropriate hygienic practice and storage conditions could reduce their load. The detection of Salmonella in some samples (13.3 %) signals the need for further strengthening of the management system in the existing abattoir. The evisceration process was also found to be a contributing factor towards the contamination of sampled carcasses, especially when analyzed for coliform, Salmonella spp. and Enterobacteriacea were the predominant bacteria. This also would imply that there is a need for improving the evisceration process, in order to eliminate microbial contamination as this would contribute towards not only improving the quality but also the safety of meat produced in the abattoir studied. References [1] Asghar, U., Abdus, N., Samad, A., Qazilbash, A., (2002). Identification, Characterization and Antibiotic Susceptibility of Salmonella and Shigella species Isolated from Blood and Stool Samples of Patients Visiting N. I. H, Islamabad. J. Med. Sci., 2: 85-88 [2] Cetinkaya,F., Cibik,R., Ece Soyutemiz,G., Ozakin, C., Kayali, R., Levent, B., (2008) Shigella and Salmonella contamination in various foodstuffs in Turkey. Food Control, 19:1059 1063 [3] Dhanashree, B. and Mallya, P. S. (2008) Detection of shiga-toxigenic Escherichia coli (STEC) in diarrhoeagenic stool & meat samples in Mangalore, India. Indian.J.Med. Resea. 128:271-277 [4] Ejeta, G., Molla, B., Alemayehu, D. and Muckle, A. (2004). Salmonella serotypes isolated from minced meat beef, mutton and pork in Addis Ababa, Ethiopia. Revue Méd. Vét., 155, 547-551 [5] Foley, S. L. and Lynne, A. M. (2008).Food animal-associated Salmonella challenges: Pathogencity and antimicrobial resistance. J Anim Sci.86:E173-E187. [6] Hardia, A. (2004).Salmonella: a continuing problem. Postgard Med.J, 80:541-545 ISSN : 0976-285X Vol 3 Issue 4 Aug-Sep 2012 147

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