resistance of Escherichia

Aim: The aim of this study was to assess the hygienic quality of foods of animal origin. Thus samples from foods of animal origin, viz. mutton, chicken meat, milk and milk products were processed. 
 
Materials and Methods: Two hundred samples from foods of animal origin viz., mutton, chicken meat, milk and milk products were processed for isolation of Escherichia coli. The isolates were got serotyped and also subjected to detection of virulence genes viz., stx1, stx2, eaeA and hlyA by PCR.The isolates were also tested against commonly used antibiotics. 
 
Results: The prevalence of E. coli was 30% in mutton, 40% in chicken meat, 33.96% in milk and14.89% in milk products samples. All the 60 isolates of E. coli were grouped into 24 serogroups with O60 and O123dominant strains (8.33%) followed by O22 (6.66%). The PCR detected 21(10.5%) of samples possessing stx1, 14(7%) stx2, 3(1.5%) both stx1 and stx2, 16(8%), eaeA and 4(2%) EHEC-hlyA gene. However, the prevalence of Shiga toxin producing E. coli (STEC) was 20% in mutton, 30% in chicken meat, 16.98% in milk and 8.51% in milk products. Whereas the prevalence of enteropathogenic E. coli (EPEC) was 2%, in mutton, 4% in chicken meat, 7.54% in milk and 2.12% in milk products samples. The 4 isolates O60, O101, O131 and one untypeable strain possessed the EHEC-hlyA gene. 22 of 50 (44%) of isolates from meat, milk and milk products showed multidrug resistance to four or more antimicrobial comprising ten of 25 (40%) isolates from chicken meat samples and 12 of 25(48%) from milk and milk products were multidrug resistance to four or more antimicrobial. 
 
Conclusions: It is concluded that partial cooked or raw milk, meat and their products prepared under unhygienic conditions may not be directly consumed as they may be carrying the pathogenic microbes.


Introduction
Thus the aim of this study was to elucidate the presence of E.coli with emphasis on STEC and their Among emerging foodborne bacterial pathogens, virulence determinants in food of animal origin of Shiga toxin producing Escherichia coli (STEC) is a Jammu region.pathogen of concern associated with the change in the livestock practices, food processing techniques along

Materials and Methods
with change in food habits of people.The pathogen A total of 200 samples from foods of animal origin whose reservoirs include the gastrointestinal tract of viz.mutton, chicken meat, milk, and milk products animals especially ruminants viz., cattle, sheep and (ice-cream, kulfi, paneer, milk cake, rasmalai, cream goats is mainly transmitted to humans through oral roll) were collected (Table-1) from Jammu region and route.The oral route of transmission is of significance processed as per the standard microbiological as various food products viz., meat, milk and their techniques [6].The isolation of E.coli was achieved by products derived from animals can be contaminated by enrichment in selective E. coli broth and plating on intestinal contents of animals during production and MacConkey agar (MA).3-4 lactose fermenting ingestion of inappropriate processed foods and could colonies from MA were selected and streaked on EMB lead to serious complications including haemorrhagic agar.The colonies producing metallic sheen were colitis (HC) or the haemolytic uraemic syndrome selected for biochemical identification.(HUS) in children [1,2].
In general, the pathogenicity of STEC is governed illuminator (Biometra, Germany).in brain heart infusion broth at 37 C for 4 hours.One ml of the broth culture was centrifuged at 8000 rpm for 5 Antibiotic sensitivity/Resistance pattern of E.coli minutes followed by washing of pellet with NSS at isolates: A total of 50 isolates (25 from mutton and 8000 rpm for 5 minutes.The pellet was mixed with 0.5 chicken meat and 25 from milk and milk products) ml of nuclease free water and subjected to heat lysis in were subjected to disc diffusion antibiotic sensitivity boiling water bath for10 minutes followed by testagainst 15 commonly used antibiotics (

Serogroups of E. coli isolates:
The 60 E. coli isolates PCR was performed in eppendorf gradient belonged to 24 different serogroups with 5 rough and thermo-cycler with heated lid using cycling conditions Screening of samples for the presence of stx1, stx2, 72 C for 1 minute followed by a final extension at 72 C eaeA and EHEC-hlyA gene was done by PCR (Fig. 1 & for 7 minutes.The PCR product was analyzed byagarose 2).SixtyE.coli isolates comprising of 15from mutton, gel electrophoresis.The PCR for eaeA and EHEC-hlyA 20 from chicken meat, 18 from milk, and 7 from milk genes was performed as per the method of [3] with products were examined for the presence of these minor modifications.For both genes, the cyclic genes.38(19%) of samples possessed stx1and/or stx2 conditions were different while the reaction mixture genes and were designated as STEC.PCR revealed that components were same.The components for reaction 21 (10.5%)samples harboured only stx1 gene, 14 (7%) mixture were: 10X Taq buffer (with 1.5mM MgCl ) = 2.5µl, 2 Deoxynucleotide triphosphate (dNTP) 200µM each= 2.5µl, MgCl 2 only stx2 and 3 (1.5%)both stx1and stx2 genes; (25mM) = 2.5µl, Primers 10 pmol each= 1.0µl, Taq DNA polymerase whereas 8(4%) possessed the eaeA geneonly but not stx 1U= 0.5µl, Template DNA= 2.5µl, Nuclease free water up to= 25µl designated as Enteropathogenic E. coli (EPEC) (Table -The cycling parameters used were initial is presented in table 5. 20% mutton samples yielded The amplified products were analysed by electro-STEC belonging to serogroup O60(4), O80, O22(2), phoresis in 1.5% agarose gel containing ethidium O102, with one rough, and one untypeable whereas 2% bromide @ 0.5µg/ml along with 100 bp molecular belonging to serogroup O123were EPEC.30% weight DNA marker (Bangalore Genei)in horizontal samples carrying E. coli strains belonging to seven electrophoresis unit (Biometra, Germany) for 2 hours sergroups (O5, O8, O20, O22(2), O102, O147, O162, at 7 v/cm.The gel was visualized under UV trans-  with two rough, and 5 untypeable from chicken meat isolates from milk and milk products: Twelve of were STEC and 4% were EPEC from chicken meat 25(48%) isolates from milk and milk products showed carrying E. coli isolates, one rough and one untypeable.
multidrug resistance to four and more antibiotics.But The prevalence of the STEC and EPEC was 16.98% 80% were sensitive to Norfloxacin and Cephoxitin.and 7.54% from milk, 4.25% and 2.12% that of milk 76% to Tetracycline and Chloramphenicol, 72% to products samples respectively.Amikacin, Gentamicin, Tobramycin, and Streptomycin, 60% to Ciprofloxacin, Co-trimoxazole, Colistin, and

0 0
18 untypeable strains.The detailed results of E. coli as follows: initial denaturation at 94 C for 4 minutes, 35 serogroups of each category of food are shown in Table-4.amplification cycles each of 1 minute denaturation at 0 Detection of stx1,stx2, eaeA and EHEC-hlyA genes: 94 C, annealing at 55 C for 1 minute and extension at 0 0 o 5).Only 4(2%) samples possessed EHEC-hlyA gene denaturation at 94 C for 5 min followed by 34 cycles of o o belonging to serogroup O60, O101, O131 and one denaturation at 94 C for 1 minute, annealing at 62 C for 1 o untypeable strain.minute and extension at 72 C for 1 minute followed by o Prevalence of (STEC) and enteropathogenic E. coli final extension at 72 C for 1 minute; while for eaeA (EPEC) among mutton, chicken meat, milk and milk gene amplification, the thermocycler conditions were product samples: Prevalence of STEC and EPEC from same as those of hlyA except that the annealing o mutton, chicken meat, milk and milk products samples temperature used for eaeA was 63 C for 1 minute.
, R = Resistant, I = Intermediate, n=number of the isolates tested, figure in parenthesis indicates number of isolates

Fig.- 1 .
Fig.-1.Agarose gel showing the amplification product of PCR performed on E. coli strains for stx1and stx2genes.Lane M: 100 bp molecular weight marker.Lane 1: Positive control of 350 bp of stx1 and 478 bp of stx2gene, Lane 2: Negative control, Lane 3: Negative sample, Lane 4: Amplified product of 350 bp of stx1 and478 bp of stx2gene, Lane 5: Amplified product of 350 bp of stx1 gene Fig.-2.Agarose gel showing the amplification product of PCR Performed on E. coli strains for eaeA and EHEC-hly genes, Lane M: 100 bp molecular weight marker, Lane 1, 2, 3, 4: Amplified product of 384 bp of eaeA gene, Lane 5: Amplified product of 534 bp of EHEC-hlyA gene All the E. coli isolates were serotyped by two phage -encoded cytotoxins called shiga toxins from National Salmonella and Escherichia Centre, Central viz., Stx1 and Stx2,produced by stx1 and stx2 genes, Serogrouping: Research Institute, Kasauli-173204 (H.P) India.respectively[3].In addition to these toxins,the presence Polymerase Chain Reaction (PCR) for detection of of eaeA gene encoding 'intimin' protein enhances the stx1, stx2, eaeA and EHEC-hlyAgenes: Primers used in virulence of STEC causing intimate attachment to the the study are listed in Table 2.The template DNA was intestinal epithelial cells [4].Also, EHEC-hlyA gene prepared as per the method of Blanco et al. [

Table - 5
. Genetic profile and prevalence of Shiga toxin-producing E. coli (STEC) and Enteropathogenic E. coli (EPEC) from foods of animal origin* * figure in parenthesis indicates number of isolates

Table - 6
. Antimicrobial sensitivity/resistance pattern of E. coli isolates from meat, milk and milk products