Genetic Variation of E . coli Strains Isolated from Poultry Slaughterhouses at Ismailia Governorate , Egypt

| Shiga toxin producing E. coli is represented as one of the main source of foodborne infectious disease distributed all over the world. This area of study not fully explained before at Ismailia governorate. So this study aimed to make genetic survey of all isolated E. coli strains with attention to shiga toxins isolated from different sections of poultry slaughterhouses and workers at Ismailia governorate. One hundred and fifty swab samples were collected from baskets, workers hands, machines, processing tools and food contact surfaces and subjected to bacteriological examination. Ten samples were positive for E. coli with a percentage 6.6%. Different serogroups of E.coli isolated from poultry slaughterhouses were O63:H7 (2), O125:H5, O63:H5, O119:H6, O125:H2, O112ac:H2, O136:H2, O127:H2, O1:H2. Nine E.coli strains were subjected to PCR for genetic detection of Shiga-like toxins genes (stx 1 and stx 2), attaching and effacing (eae A) and enterohaemolysin gene (hly) gene in isolates. 5 E.coli isolates showed positivity for the stx1 gene (55.5%), 2 isolates for stx2 gene (22.2%) and 4 isolates for eaeA gene (44.4%). While, all isolates showed negativity for hly gene. In conclusion, genotypic survey proved the presence of shiga toxins producing E. coli and other virulence E.coli strains at examined slaughterhouses.


INTRODUCTION
P oultry meat and its products are very popular food in Egypt as well as throughout the world.The source of contamination may cross to the poultry meat during slaughtering and processing leading to introduction of pathogens into the meat.The source of these pathogens may be coming from the gastrointestinal tract or from the surrounding environment in farm and/ or slaughterhouse.Poultry is the most common food source of human infection byenteric pathogens throughout the world (Samaha et al., 2012) Because of the relatively high frequency of contamination of poultry with potential pathogens, raw poultry meats are often known to be sources of infections in man (Soares et al., 2018).So there are certain measures must be followed in poultry meat to prevent possible contamination from digestive tracts, during packaging, from utensils and from handlers (Althaus et al., 2017).
The poor hygienic practices lead to contamination in meat by E.coli.Detection of E.coli in food means that there are other harmful bacteria could be detected in the same food (Beutin et al., 2007).Also, detection of E. coli virulence strains in poultry meat lead to infection for slaughterhouse workers and consumers (Xia et al., 2010).
The virulent E.coli strains especially Stx1 and stx2 toxins producing E.coli, have the ability to attach with intestinal epithelium making hemorrhagic colitis and hemolytic -uremic syndrome.Other important virulence factors of STEC are intimin (encoded by eae A gene), a plasmid-encoded enterohaemolysin (ERFAN).

gEnEtic dEtEction oF isolatEd E.coli VirulEncE gEnEs
Nine E. coli strains were subjected to PCR for detection of shiga toxins (stx 1 and stx 2), eae A and hly genes.QIAamp DNA Mini Kit used for extraction of DNA .Primers used were shown in Table 1.Temperature and time conditions of the primers during PCR are shown in Table 2. Amplified PCR product was analyzed by electrophoresis on a 1.5% agarose gel stained with 0.5 µg of ethedium bromide/ mL.The gels were photographed by a gel documentation system.

RESULTS
The bacteriological examination of 150 Swab samples (50 swab samples from different stations of each poultry slaughterhouse) revealed in 10 samples (6.6%) positive reaction to E.coli as shown in the Table 3.All isolates showed the typical colonies characteristic of E.coli where it appeared a smooth, shiny and strong lactose fermenting (pink) colonies on MacConkey agarand metallic sheen green colonies on Eosin Methylene Blue.On blood agar, colonies were non hemolytic, gray, white, moist, glistening, opaque, circular and convex with entire edges.

DISCUSSION
The bacteriological examination of 150 Swab samples (50 swab samples from different stations of each poultry slaughterhouse) revealed in 10 samples (6.6%) positive reaction to E. coli as shown in the Table 3.Seven samples (70%) from dirty area including machines, workers hands and carcasses and 3 samples (30%) from clean area including machines, workers hands and product.As in dirty area workers hand, evisceration machine, scalding tank, act as a source of E.coli contamination (Van den Bogaard et al., 2001;Farah et al., 2007) Who found the E. coli counts were around 3.0 log CFU/cm 2 at the scalding machine.
Generally, the presence of E. coli in poultry slaughterhouse considered as an indicator for improper handling or unhygienic conditions, which agreed with (Samaha et al., 2012).
The negativity of all the isolates to hemolysis on 5% sheep blood agar was in accordance with (Peer et al., 2013) who attributed heavy mortality in chicks due to non -hemolytic strains indicating that pathogenic E.coli to be independent of hemolytic activity and (Literak et al., 2013) who reported that none of their isolates was positive for hemolysis on 5% sheep blood agar.However, the hlyA gene was not detected in strains tested negative for enterohemolysin that was differ with (Farhan et al., 2014) who detect hlyA in seven strains tested negative for enterohemolysin.
In the present study, 5 E.coli isolates showed positivity for the stx1 gene, with an incidence of 55.5%, 2 isolates showed positivity for stx2 gene, with incidence of 22.2%, 4 isolates showed positivity for eaeA gene, with incidence of 44.4%, all isolates showed negativity for hly gene.Regarding to the occurrence of stx1 gene in 9 E.coli strains, only 5 E.coli strains were detected, containing stx1 gene by percent of 55.5%.This result was different from finding which was recorded by (Momtaz and Jamshidi, 2013) who showed that 100% of EHEC serogroups were positive for all stx1.
Regarding to the occurrence of stx2 gene in 9 E.coli strains, only 2 E.coli strains were detected, containing stx2 gene by percent of 22.2%.This result was nearly similar to finding which was recorded by (El-Jakee et al., 2012) who detected stx2 by percentage 41.67% in 5 E.coli isolates out of 12 one.
Other studies, documented in the absence of both types of Shiga toxin type 1 and type 2 genes in E.coli isolates from various types of samples (Fereidouni et al., 2009;Murthy and Rao 2014).
Concerning to examination of E.coli strains for the presence of Intimin gene, results detected 4 out of 9 E.coli strains, by percent of 44.4%.These findings were nearly agreed with those obtained from (Dutta et al., 2011) who detected eaeA in 4 E.coli strains out of 10 samples by percent of 40% and (El-Jakee et al., 2012) who detected eaeA in 5 E.coli strains out of 12 by percentage 41.67%.
In this study all 9 E.coli isolates showed negativity for hly gene, the present findings do not coincide with what was reported by (Dutta et al., 2011) who detected hly in 7 E.coli strains out of 10 samples by percentage 70%.Meanwhile, the shiga toxin positive isolates were negative by PCR for the eaeA and hly genes were reported by (Parreira and Gyles, 2002;Zakeri and Kashefi, 2012).Our results con- firmed the presence of virulence genes including stx, eaeA.Therefore, the environment, tools, machines and handlers of slaughterhouses may be a reservoir of the organism that can cause serious diseases in humans.These results occurred by (Sirotkin et al., 2006) who reported that Shiga toxin-producing E.coli (STEC) has been demonstrated to exist in E.coli samples which is considered as a threat for human health.

Advances in
PCR is a powerful molecular biology technique for the detection of virulence genes.It is not only highly sensitive and specific, but it also provides rapid and reliable results.Detection of virulence genes was determined in the present study as a molecular tool for identification of E.coli isolates using the PCR technique.

CONCLUSION
Protection of poultry meat from exposure to different types of bacteria during the manufacturing stages is to protect the national economy as well as the protection of human health.stx1, stx2 and eaeA virulence genes were detected examined slaughterhouses at Ismailia, Egypt.So applying of strict hygienic measures inside poultry slaughterhouses urgent needs to decrease the opportunity for access of food poisoning microorganisms.A comprehensive educational program about the potential hazards associated with bacterial infection in foods of animal origin should be in place.

Table 1 :
Oligonucleotide primers of E.

Table 3 :
Prevalence of E.coli isolates from different stations of poultry slaughterhouses swab samples SamplesNumber of Swab samples E.coli isolates

Table 4 :
Prevalence of E.coli serotypes from different poultry slaughter-houses

Table 5 :
Detection of virulence genes in E.coli isolates by PCR