ESCHERICHIA COLI PATHOTYPES ASSOCIATED WITH DIARRHEA IN HUMAN AND DOMESTIC ANIMALS

Ruminants are important reservoirs for zoonotic pat hogenic E. coli. The objective of this study was to characterize pathogenic E. coli isolates from cattle and sheep linked to human ill ness with respect to their path types, serotypes and genotypes. E. coli O157:H7 isolated from cattle, sheep and human pati ents were compared for their genomic similarity by Pulsed-Fie ld Gel-Electrophoresis (PFGE). PCR detection of virulence factors associated with different E. coli path types (VTEC, ETEC, EPEC, EAEC and EIEC) revea led that VTEC was the most prevalent path type (22/45; 48.9%), followed by EAEC (3/45; 6.7%), EPEC (1/45; 2.2%), a EPEC (3/45; 6.7%), ETEC (1/45; 2.2%) and E IEC (1/45; 2.2%). E. coli O157:H7 represented the most prevalent VTEC serotypes (11/22; 50%). Pulsed fiel gel electrophoresis typing revealed exact mat ches between E. coli O157:H7 isolates from the human patients, sheep an d c ttle in the same municipality. VTEC play an important cause of diarrhea in human, sheep and cattle. The molecular relatedness between PFGE profiles of E. coli O157:H7 isolates from human, sheep and cattle supp orted the hypothesis that ruminants especially cattle and sheep act as reservoirs of E. coli O157:H7 for human infection.


INTRODUCTION
Diarrheal diseases are a leading cause of morbidity and mortality in developing countries. Escherichia coli is recognized as an important cause of both sporadic cases and outbreaks of diarrhea all over the world. Nguyen et al. (2005). Diarrheagenic Escherichia Coli (DEC) strains can be classified into five major categories on the basis of the nature of their infection and pathogenic mechanisms: They are Enter Pathogenic E. Coli (EPEC), Enter Hemorrhagic E. Coli (EHEC), Enterotoxigenic E. Coli (ETEC), enter invasive E. Coli (EIEC) and Enter Aggregative E. Coli (EAEC) (Tamaki et al., 2005). O157:H7 and related non-O157 VTEC strains are pathogens of public health concern worldwide. They may cause severe outbreaks of gastrointestinal illness with clinical symptoms ranging from diarrhea and hemorrhagic colitis to the life-threatening Hemolytic Uraemic Syndrome (HUS) (Lynn et al., 2005). VTEC strains have been isolated from a variety of animals and cattle are considered to be the major reservoir for VTEC strains (Ferens and Hovde, 2011). However, recent evidence has indicated that small domestic ruminants, including sheep and goats, are also key reservoirs of VTEC (La Ragione et al., 2009). In particular, sheep and their products have been documented as reservoirs for VTECs that belong to a diverse set of non-O157 Serogroup that harbor genes encoding key virulence factors that have been implicated in human disease (Djordjevic et al., 2001). Transmission of VTEC to humans occurs through direct or indirect contact with animals or their environment, consumption of contaminated food or water and through person-toperson contact (Gillespie et al., 2005).
In order to prevent and control DEC epidemics, a reliable procedure should be followed to identify and categorize DEC isolates. Since several virulence factors have been identified in DEC strains, modern molecular detection methods, including PCR have been developed (Teng et al., 2004). They are performed at the genetic Science Publications AJAVS level and directly detect genes for specific virulence factors, which themselves determine the pathogenicity. The presence of these genes is the evidence that renders the virulence and that can be used to categorize DEC strains (Yang et al., 2007).
This study aimed to document the association of pathogenic Escherichia coli isolates with diarrhea in human and animals, based on the PCR-based identification of their intrinsic virulence. And try to find the genetic relatedness among VTEC strains from human, cattle and sheep to identify the zoonotic possibility.

Bacterial Strains
Stool samples were collected from patients with diarrhea and rectal swabs were collected from cow and sheep with diarrhea. Typical E. coli isolates selected were selected from Eosin Methylene Blue (EMB) agar, which discourage the growth of gram positive bacteria and give a distinctive metallic green sheen, these isolates were then subjected to the IMViC test for the further selection of E. coli strains.

Somatic and Flagella Serotyping
Isolates were grown on nutrient agar plates at 37 8C overnight, then the cells were collected and suspended in 0.9% sterile normal saline (154 mEq/L sodium and 154 mEq/L chloride, pH adjusted to 7) and autoclaved at 121 8C for 15 min and then the cells were collected by centrifugation and resuspended in an appropriate volume of sterile normal saline. Detection of O-serogroup was performed using a commercially available Oserogrouping Kit (Denka Seiken, Tokyo, Japan) (Takahashi et al., 2008). The Flagellar phase inversion was carried out using the standard Craigie tube technique by passage through semi-solid agar containing the appropriate flagellar antis era (Davies and Wray, 1997).

Template DNA Preparation
Single colonies obtained from nutrient agar were inoculated into Luria Bertani (LB) broth (Merck) and were grown at 37°C for 12 h. Cells were harvested from 2 mL of the cell suspension by centrifuging at 16 000×g for10 min and discarding the supernatant. Genomic DNA was then isolated by boiling method (Usein et al., 2009).

Detection of Virulence Factors
Detection of virulence factors was performed by PCR. Primer sequences and PCR conditions used for the study listed in Table 1, (Moyo et al., 2007;Ochoa et al., 2008;Vu-Khac et al., 2007;Mendez-Arancibia et al., 2008: Zweifel et al., 2006Osek, 2003;Shabana et al., 2013). PCR performed in the Takara thermal cycler (Takara, Tokyo, Japan). PCR products were separated and visualized by gel electrophoresis in 1.5% agarose (Wako, Japan) in Tris-Acetate-EDTA (TAE) buffer at 100 V. And a 100 or 500 bp DNA ladder (one-step ladder, Wako) was included in each agarose run, accordingly the amplified product.

Pulsed Field Gel Electrophoresis (PFGE)
PFGE was performed according to CDC (the Pulse Net protocol of the (CDCP, 1998). The agaroseembedded bacterial genomic DNA was digested with restriction enzyme XbaI at 37 8C for 4 h. Electrophoresis was performed on 1% agarose gel with 0.5-Tris-borate-EDTA buffer. The electrophoretic conditions were optimized for the separation of the 24-to 600-kB XbaI-digested macro restriction fragments. The following PFGE parameters were applied: Voltage of 6 v/cm, initial switch time of 2.2 s, final switch time of 54.2 s and run time of 19 h. Electrophoresis was conducted by using a CHEF-DRII (Bio-Rad Laboratories, Tokyo, Japan). The gel was stained with ethidium bromide and imaged with the Gel Doc 2000 and Multi-Analyst program (Bio-Rad). Dendrograms were created with a Molecular Analyst (Bio-Rad) by using the Dice coefficient, Un weighted Pair Group Method with Arithmetic means (UPGMA) and a position tolerance of 1.3%.

Virulence Genes Detection and Categorization of DEC:
From the total of 45 isolates, 31 isolates were positive for at least one of the targeted virulence genes. The minimal criteria for the determination of DEC were as follows: The presence of V1 and/or V2 for VTEC, the presence of LT and /or ST for ETEC, the presence of aggR and or astA for EAEC, the presence of bfpA and eae for typical EPEC and the presence of eae only for atypical EPEC, the presence of ipaH for EIEC. Based on these criteria, the 31 isolates were classified as VTEC (22 isolates) expressed either V1 or V2 or both, ETEC (1 isolate) from calf, two isolates were EAEC positive for aggR and astA and one isolate EAEC (astA) was heterogenous among the putative DEC, typical EPEC represented by only one strain, 3 a EPEC isolates and 1 isolate EIEC, respectively ( Table 2).

PFGE
A PFGE analysis was conducted to identify the genetic relatedness in the recovered O157 isolates from the diarrheic human (n = 6), calf (n = 3) and sheep (n = 2), Fig.  1. the strains had more than 80% similarity in the PFGE pattern, the results indicated five PFTs, A: (n = 1) and B: (n = 1), C (n = 1), D (n = 7) and E (n = 1); briefly pulsotype A represented by single human strain expressing V2, pulsotype B represented by single calf strain expressing V1 and pulsotype C represented by single human strain expressing V2. Strains with the pulsotype D were recovered from the three types of feces and all shared expression of both V1 and V2, Pulsotype E also represented by single isolate from sheep expressing both V1 and V2.

DISCUSSION
The reference diagnostic approach in the present study was The PCR-based detection of specific genes that render virulence to E. coli. In addition, the traditional method of Serotyping, which still occupies a central place in the history of E. coli. Specific E. coli

AJAVS
Serogroup can be associated reproducibly with certain clinical syndromes, the serotypes and Serogroup serve as readily identifiable chromosomal markers that correlate with specific virulent clones (Whittam et al., 1993).
Almost half of the isolates carried virulence genes common to Verotoxin-producing Escherichia Coli (VTEC), which appeared to be the most prevalent DEC category in this study (48.9%), VTEC distributed among human, calf and sheep isolates and assigned to serotypes O157:H7, O26:H11, O121: HNM, O165: HUT and O8:H4 harbored either V1 or V2 or both in addition to Ehly and eaeA. This is in concordance with other report highlighting the involvement of VTEC in human, calf and sheep illness (Blanco et al., 2003).
EPEC represented by four strains restricted to human isolates; three were atypical EPEC possessed only the virulence marker eae, indicative for the presence of the Locus of Enterocyte Effacement (LEE) pathogenicity island and single typical EPEC strain possessed an inducible bundle-forming pilus (bfpA) associated with the presence of the EPEC Adherence Factor (EAF) plasmid and Localized Adherence (LA) on HEp-2 cells in addition to LEE-encoded virulence factors. These findings agreed with previous report about the prevalence of EPEC and how atypical EPEC had more implication than typical EPEC (Afset et al., 2003). The majority of EPEC isolates belong to classic serotypes derived from 12 classical O serogroups (O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142 andO158) (Scaletsky et al., 2010). This study reported O115: HUT as EPEC which agreed with (Saito et al., 2005).
ETEC was detected in only one strain from cow assigned for serotype O8:H12, the classical serotype of ETEC (Huasai et al., 2012). It possessed heatlabile, heat-stable enterotoxins and plasmid-borne astA gene encoding the EAST1. Although the lack of references, which monitors the presence of Enteroinvasive Escherichia coli (EIEC) in cow, the current study recorded one strain assigned for O74:H9 and possessed Invasion plasmid antigens (Ipa) proteins which encoded in the ipa operon.
Theoretical digestion of sequenced genomes of E. coli O157 strains should generate 41 fragments with XbaI. Some smaller bands run off the gel, while others are too similar in size and co migrate. As a consequence only half to three quarters of these bands can usually be distinguished by PFGE analysis. (Kudva et al., 2002) demonstrated that PFGE diversity in E. coli O157 is primarily attributed to insertions and deletions, not to point mutations. PFGE was used to genetically discriminate VTEC O157:H7 isolates from different hosts, similar patterns were found among calf, sheep and human strains in Lineage D, which accounted for 63.3% of the tested strains. The strains had expressed the same virulence genes, both V1 and V2, in addition to Ehly and eaeA, while other lineages were distinctly different due to the variation in expression of virulence genes.
Enterohaemorrhagic Escherichia Coli (EHEC) O157:H7 is a zoonotic enteric pathogen of worldwide importance; ruminants cited as its primary reservoirs. Cattle considered to be the sole source of E. coli O157:H7 outbreaks in humans; the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle and with cattle considered to be the primary reservoir (La Ragione et al., 2009).
Transmission of E. coli O157:H7 to humans is principally via contamination of food by animal feces (Jaros et al., 2013). Food products of animal origin have been confirmed as vehicles of disease transmission in case-control studies of VTEC outbreaks and sporadic VTEC infections; these included raw milk (Guh et al., 2010) and undercooked meat products (Chapman et al., 2001).
World Health Organization (WHO) listed Escherichia coli spp. as one of the most important Pathogenic bacteria which transmitted by food especially Enterotoxigenic E. coli (ETEC), enteropathogenic E. Coli (EPEC), Enterohaemorrhagic E. Coli (EHEC), enteroinvasive E. coli (EIEC), however the restricted number of the strains in the present study; EHEC, ETEC and EIEC strains were reported in cattle,. The findings refer to the importance of cattle and sheep as sources for zoonotic Escherichia coli strains.

CONCLUSION
High prevalence of VTEC among diarrheic human, sheep and cattle and highly similar genotypes of VTEC O157:H7 are found in both cattle and sheep and cause human illness suggest that pathogenic strains can circulate freely between them. This information will be of importance for future efforts to trace sources of infection and reduce the burden of disease caused by O157:H7.

ACKNOWLEDGEMENT
I'd like to thank Dr. Hiroshi Sato, Niigata Prefectural Institute of Public Health and Environmental Sciences, Department Of Bacteriology, Niigata, Japan; for his valuable help and contributions.