Phylogenetic of Shiga Toxin-Producing Escherichia coli and a typical Enteropathogenic Escherichia coli Strains Isolated From Human and Cattle in Kerman , Iran

Background: Shiga toxin-producing Escherichia coli (STEC) have emerged as the important zoonotic food-borne pathogens and confirming the risk to public health. Enteropathogenic Escherichia coli (EPEC) is a major cause of children diarrhoea in developing countries. E. coli strains can be assigned to four main phylogenetic groups, A, B1, B2 and D. Objectives: The aim of the current study was to analyze the distribution of phylogenetic groups and presence of STEC and atypical EPEC pathotypes in E. coli isolated from human diarrhea and fecal samples of healthy cattle in Kerman, Iran by PCR. Materials and Methods: A total of 188 E. coli isolates were isolated from human diarrheic (94 isolates) and fecal healthy cattle (94 isolates) samples. The isolates were identified by standard bacteriological tests. The confirmed isolates were examined to detect the phylogenetic groups and a selection of virulence genes including stx1, stx2 and eae by PCR. Results: Phylotyping of isolates from diarrheic human showed that 38.29% belonged to A, 20.21% to B1, 14.89% to B2 and 26.59% to D phylo groups. The isolates of healthy cattle distributed in A (34.04%), B1 (47.88%), B2 (7.44 %) and D (10.64%) phylo-groups. Prevalence of eae gene in human diarrheic isolates was 5.32% (5 isolates), whereas none of the human diarrheic isolates were positive for stx1 and stx2 genes. Among cattle isolates 7.44% (7 isolates) were positive for stx1 gene and 5.32% (5 isolates) possessed eae gene. Of the all isolates examined, none were positive for the stx2 gene. The eae gene were positive for isolates of human diarrhea distributed in A and B2 phylo-groups and isolates possessed stx1 and eae genes from healthy cattle fell into A (4 isolates), B1 (7) and B2 (one isolate). Conclusions: The isolates of human diarrhea samples and fecal healthy cattle were distributed into different phylogenetic groups, which mostly distributed in A and B1 phylo-groups. In addition, results of this study revealed the lower prevalence of SETC and aEPEC in isolates.


Background
Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) produce the characteristic attaching and effacing (A/E) lesions in the gut mucosa of humans and animals (1).Based on the molecular studies, currently EPEC is responsible, on average, for 5-10% of pediatric diarrheal episodes in the developing world.Diarrheagenic E. coli have been classified into six categories based on epidemiologic, clinical, and molecular criteria: enteropathogenic E. coli (EPEC); enterotoxigenic E. coli (ETEC); Shiga toxin-producing E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC) or verotoxin-producing E. coli (VTEC); enteroinvasive E. coli (EIEC); enteroaggregative E. coli (EAEC or EAggEC); and diffusely adherent E. coli (DAEC) (2).Domestic ruminants, especially cattle and sheep harboring STEC may constitute an important reservoir for STEC infection of humans (3).STEC can cause severe diseases in humans, such as hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombocytopenic purpura (TP), which may prove fatal in immunodeficiency patients (4).Typical EPEC strains express eae gene, which encodes intimin, and the bundle-forming pili (BFP) responsible for enterocyte attaching and effacing lesions, whereas strains with the A/E genotype which do not possess bfpA2 gene are classified as atypical EPEC (aEPEC) (5).STEC with and without the eaeA genotype, may expresses one or two shiga-like toxin encoding genes, stx1 and stx2 (6).E. coli strains can be classified to one of the main phylogenetic groups: A, B1, B2 or D. The diarrheagenic E. coli strains belong to groups A, B1 and D, the commensal strains to groups A and B1, whilst the extra-intestinal pathogenic strains usually belong to groups B2 and D (7,8).

Objectives
Healthy asymptomatic bovine are the best-recognized animal reservoir for STEC strains.Sources of human infection include primarily foods of cattle origin; mainly undercooked beef products, unpasteurized milk, and direct contact with bovine and person to person transmission.The aim of this study was to analyze the distribution of phylogenetic groups (A, B1, B2 and D) and occurrence of STEC and atypical EPEC pathotypes encoding genes stx1, stx2 and eaeA in E. coli isolated from patients with diarrhea and fecal samples of healthy cattle in Kerman, Iran by PCR.

Source of the E. coli Isolates
A total of 94 E. coli isolates were obtained from diarrheic samples of human and 94 isolates were taken from rectal swabs of the healthy cattle.The human samples were related to both male (n=51) and female (n=43).The isolates were collected during 2010 to 2011 in Kerman province, Iran.In the laboratory, samples were cultured on Mac Conkey agar and EMB (Biolife Laboratories, Milano, Italy).E. coli isolates were isolated and identified by standard biochemical and bacteriological methods.Isolates were stored in Luria-Bertani broth (Invitrogen, Paisley, Scotland) with 30% sterile glycerol at -20°C.

Reference Strains
In this study two E. coli strains were used as positive controls: E. coli Sakai for EHEC and atypical EPEC (stx1+, stx2+ and eaeA+) and E. coli ECOR62 for (chuA+, yjaA+ and TspE4.C2+).E. coli strain MG1655 was used as a negative control for virulence genes and as a positive control for phylogenetic ECOR group A (chuA, yjaA-and TspE4.C2).All the reference strains were from the bacterial culture collection of Microbiology Department of Ecole Nationale Veterinaire Toulouse, France.

Pathotype and Phylotype Determination by PCR Assay
DNA of E. coli isolates and reference strains was extracted by lysis method (9).The phylogenetic analyses of the isolates were carried out by combinations of three genetic markers chuA, yjaA and DNA fragment TspE4.C2 by a triplex PCR method (10).All isolates were tested by multiplex PCR assay for the presence of the genes encoding intimin, stx1 and stx2 described by China et al. (11).The primers used for amplification of the virulence genes to determine STEC and atypical EPEC pathotypes and phylogenetic groups are presented in Table 1.

Discussion
STEC strains, the cause of human infections, occur after consumption of contaminated food or contact with an infected animal.Cattle are thought to be the main reservoir of STEC and often carry this pathotype in their intestinal flora and serve as source of food contamination (12).
Prevalence of EPEC varies in human related to differences in study population, age group, diagnostic criteria and methods used for diagnosis.Recent studies suggest that atypical EPEC are more prevalent than typical EPEC in both developed and developing countries (2).In the present study, STEC and atypical pathotypes occurred at lower frequencies in human with diarrhea and fecal of healthy cattle in the studied region of Kerman (Iran).
In studies on the capital of Iran (Tehran) 808 isolates which obtained from patients with acute diarrhea 7.92% isolates were positive for STEC and 1.48% for aEPEC, which stx1 and stx2 genes were detected in 34.37% and 43.75% of SETC strains, respectively (13).In the study of Aktan et al. (14) on faeces of healthy cattle, sheep and pigs entering abattoirs 8.06% isolates were eae probe positive that presumptively identified A/E.All isolates were positive for eae gene, five of these isolates possessed stx1 gene and none of them were positive for stx2 gene.The results from the present study are in accordance with the mentioned study by Aktan et al. (14), which showed none of the faeces of healthy cattle isolates were positive for stx2 gene.In a study on Iran, 29 STEC strains were isolated in northern (Mazandaran province) and southwest (Ilam province), which 28 of them revealed the presence of stx1 and one strain possessed stx2 gene.None of the strains carried the eae gene (15).Reports revealed discrepancies in prevalence of STEC in different countries because this pathogen has not been isolated from diarrhea specimen in human (13,16).Food-borne outbreaks caused by STEC can affect large numbers of people.Since there is currently no specific treatment for infections of this pathotype, an understanding of the epidemiology of STEC infections is urgently needed.In the current study frequency of aEPEC in human was 5.32%, as was found in study conducted in Thailand (5.5%) (17); however, in Brazil (34%) and Korea (56%) EPEC were detected with high frequency (18).Alikhani et al. (6) reported that aEPEC strains possess the eaeA gene are a common cause of children with diarrhea in three Iranian provinces, Tehran, Ilam and Mazandaran of Iran.In Spain, distribution of the types of the eae gene among a collection of AEEC strains isolated from healthy cattle and healthy sheep was investigated, which healthy sheep isolates were high percentage types of eae gene than healthy cattle isolates (18).
In the current study indicated the distribution of the main phylogenetic groups among E. coli strains isolated from human diarrhea and healthy cattle.Carlos et al. (19) concluded that geographic variation of the E. coli population structure related to different phylogenetic groups.
One of the major forces that shape the genetic structure of E. coli populations among the hosts is domestication.Escobar-Paramo et al. (20) analyzed fecal strains isolated from humans and animals indicated the prevalence of groups A and B2 in humans and A and B1 in animals.In this study phylogenetic groups of human diarrhea isolates mostly fell into group A, followed by D and B1, and isolates from healthy cattle mostly distributed in phylogroups B1, A and D. Phylogenetic analysis showed that EPEC strains of human are clustered mostly in groups B1 and B2 (21), whereas in the current study mostly aEPEC positive isolates of human diarrheic samples distributed in B2 and A phylo-groups.Phylogenetic analysis of STEC positive isolates of human showed that the isolates belonged to A (3 isolates), B1 (3 isolates) and B2 (one isolate) groups.Phylogenetic study on human diarrheagenic E. coli isolates indicated that STEC strains were segregated in A and B1 phylo-groups (22), whereas Phylotyping of STEC strains from animals mostly distributed in B1 (38.7%) and A (35.5%) phylogenetic groups (1).
In conclusion, the E. coli isolates of human diarrhea samples and fecal healthy cattle were distributed into different phylogenetic groups, which A and B1 phylogenetic groups were represented the majority of isolates.In addition, results of this study revealed the lower prevalence of SETC and aEPEC in isolates.In order to determine detailed genetic studies of other diarrheagenic E. coli pathotypes in human and also typical and atypical EPEC in animal's future research will still be needed.

Figure 1 .
Figure 1.Positive Multiplex PCR Results for the Detection of E. coli Phylogenetic Groups Among the Patients with Diarrhea and Fecal Samples of Healthy Cattle.

Figure 2 .
Figure 2. The Multiplex PCR Results for stx1 and eaeA Genes.

Table 1 .
Oligonucleotide Primers Used in This Study