The overall occurrence of virulence genes observed in this study is significantly high compared to 22 isolates (30%) that carried no virulence genes. The result is comparable with what is reported by , occurrence of virulence genes in 76.45% of fecal samples collected from 824 diarrheic calves in Iran. But it is comparatively high compared to 30.1 % occurrence of isolates carrying at least one of the eight virulence genes from 620 calves investigated by Piccoa et al.  in Cordoba province, Argentina and 15.2% prevalence of virulence genes reported in Australia . This indicates high level of virulence factor gene carrying E. coli in the sample area, indicating high level of pathogenic potential in the sample population.
The high frequency of virulence genes observed in the current study is mainly attributed to high occurrence of eaeA (59%), that cods for an adhesin protein intimin in both EPEC and EHEC. This result is in agreement with previously reported occurrence of this gene in 38% of diarrheic calve fecal samples in Iran by Shahrani et al.  and 57.1% from Australian calves by Luna et al. . But it is by far more than 6% prevalence of eae in Mediterranean water buffalo calves in Italy Borriello . Intimin is a 94-kDa protein encoded by the eae gene on locus of enterocyte effacement and it is found in all strains capable of inducing A/E histopathology .
The locus of enterocyte effacement also harbors additional virulence factors such as T3SS which provide the pathogen leverage over the normal intestinal flora in the competition for intestinal site by intimate adhesion and biofilm formation, favoring overgrowth of pathogen at low infection dose [15, 40]. Thus, the relative abundance of A/E E. coli; EHEC (28%) and EPEC (30 %) in isolates of the current study can be explained by the presence of LEE in this isolates and combined effect of different virulence factors encoded on it.
In the current study comparably, higher frequency of stx1 harboring STEC strain were observed in sample area. As shiga like toxin I is reported to be more often detected than shiga like toxin II in STEC strains, the relative abundance stx1 in current investigation is in agreement with most reports of studies conducted around the world. For instance, Seyda et al. , showed that all STEC isolates from cow with mastitis harbor stx1 gene and identified stx2 only in 20% of the STEC isolates. Similarly, high frequency of E. coli isolates carrying the stx1 was observed in 86.67%, 40.7%, 12.2% E. coli isolates from diarrheic calves compared to 26.67%, 28.3%, 7.8% of stx2 in Egypt, Iran and Australia respectively [19, 43, 29]. In contrast higher prevalence of 33% stx2 gene compared to 17% stx1gene in cattle has been reported in Iran .
Epidemiological studies revealed that stx2 is more associated with severe human disease such as; HC and HUS than stx1 . In one study for instance, stx2 and a variant, stx2c, were the only subtypes found from HUS cases . Thus, even though stx2 is less frequently detected than stx1, the overall occurrence of stx2 among sample isolates is 25%, indicating a potential risk for severe illness following DEC infection in the areas.
In addition to Shiga-like toxin, virulence potential of STEC strains is also determined by additional virulence factors. Among others, virulence factors encoded on LEE had been well characterized so far and it is associated with to A/E lesion in the host resulting sever illnesses such as HUS and HC [39, 15]. In this regard considerable amount of STEC strains 22 of the 28 STEC isolates (78.57%) were found to contain eae and additional virulence factors that are harbored in locus of enterocyte effacement, a pathogenic island that harbored the gene, indicating the associated potential risk following DEC infection in the area. This result is in agreement with results obtained by Seyda et al.  who identified eae in 40% of STEC isolates.
LEE harboring STEC strains stem as distinct subtype EHEC Nataro and Kaper , 22 of the isolates in the current investigation can be regarded as EHEC. Among the EHEC isolates 18 (50%) harbored only stx1 and 2 (5.6%) isolates carried stx2 as a single Stx gene. While the remaining 16 isolates (44.4%) contained both stx1/stx2 Virulence genes. This result is inconsistent with the findings of Badouei et al.  who identified 15.4% isolates to harbored stx1 and 56.4% carried stx2 as a single Stx gene. While the remaining contain both stx1and stx2 from 39 EHEC isolates collected from cattle in Iran. Similarly, Borriello  identified STEC isolates from fecal samples of water buffalo calves, were all Stx and intimin-positive, with Stx1 (80%) more frequent than Stx2 (27%).
In Addition to shiga-like toxins, EHEC strains may carry the EHEC hemolysin toxin encoded by hlyA or ehx. In the current investigation ehlyA was detected in 23 percent of the total isolates. In similar study, Luna et al.  identified Ehly genes in 56% of isolates from diarrheic calves in Australia. This is a pore-forming toxin that lyses erythrocytes and found to be cytotoxic to endothelial cells contributing to the development of HC and HUS  indicating a great potential risk posed by these isolates.
The other virulence gene, EAEC transcriptional regulator gene (aatA) were detected in 5.5% of isolates. In contrast the previously mentioned study in Australia, showed the occurrence of this VG in 29% of isolates . This result would be an evidence for the presence the said pathotype in the study area.
On the basis of combinations of Virulence genes, the E. coli isolates characterized in this study were further placed into one of the three pathotypes of DEC; STEC, EPEC, EAEC and two subtypes of STEC and EPEC each.
Approximately 70% of E. coli investigated in this study were classified as distinct pathotype based on combinations of virulence genes detected. The recognized pathotypes, 6% STEC, 30% EHEC, 12% tEPEC, 16% aEPEC, 6% EAEC were found to be comparable with similar studies such as; 9%STEC, 26% EHEC, 12% EPEC in Iran  and 2.2% tEPEC, 6.7% aEPEC, 48.9% STEC, 6.7% EAEC and 2.2 % of both ETEC and EIEC in Egypt . Similarly, studies made in Egypt that tried to characterize diarrheagenic E. coli virulence genes in newborn calves, identified different set of virulence genes; ST; 33.3%, LT; 30%, Stx1; 86.67%, Stx2; 26.67% and two ETEC adhesions (F5; 13.3%) and (F41; 16.67%) .
The overall occurrence of the different virulence factors in E. coli isolates of the current investigation show the potential risk posed by these organisms not only to humans and domestic animal but also for wild animals. The potential transmission among different sources has been demonstrated by Miko et al.  who isolated 140 STEC strains from game animals in Germany between 1998 and 2006 and compared with 101 STEC isolates from farm animals, their feed products and human patients. The result showed genes linked to high-level virulence for humans (stx2, stx2, and eae) belong to the same genotypes and other virulence attributes, regardless of origin.
Moreover, the current study tried to investigate plasmid content of study isolates as plasmids within DEC have been shown to carry both antibiotic resistance genes as well as virulence factor genes among others . The study has identified ten different kinds of plasmids, with size ranging from 2.6 + 0.14 Kbp to 98.2 + 4.17 Kbp. In agreement with the current study, Nsofor and Iroegbu  isolated E. coli plasmids from domestic animals, that harbor one or more plasmids with molecular size in the range of 1 to 120 Kbp. Similarly, 7 different plasmids 2.4 kbp to 65 kbp in size were isolated from 1,266 fecal specimens derived from environmental, human, animal and food sources .
Among the various plasmids identified, a large plasmid with slight variation in size has been observed in all isolates examined for their plasmid content. DECs are known to harbor large conserved plasmids associated with virulence potential as well as resistance trait [28, 7]. For instance, the presence of large conserved plasmid is established in DEC pathotypes EAEC, EHEC and EPEC which are pAA, pO157 and pEAF respectively . The size variation in the large conserved plasmids is also likely. For instance, the large plasmids of STEC were shown to vary between 115Kbp and 65Kbp in different studies [47, 35, 48].
The smaller plasmids identified in the current investigation were regarded as putative due to their inconsistent distribution, even among isolates with similar characteristics in terms of virulence factors as well as antibiotic resistance profile. However, the study identified high level of antibiotic resistance in isolates containing these plasmids. Though results cannot be directly compared, as different methods employed, Bello et al.  (2013) and Kalantar et al.  detected plasmids more frequently in resistant strains than susceptible strains. Bello et al.  for instance isolated a total of 48 different plasmids occurring in various combinations from 50 E. coli isolates that showed MDR. Similarly, resistance genes for MDR were reported in E. coli plasmids by Kalantar et al. .
Even though plasmids were found to be strongly correlated with resistance phenotype, no particular plasmid was found to be significantly associated with a particular drug resistance phenotype. This result confirmed that there was no a linear correlation between the plasmids content and the antibiogram studies of the isolates, which suggests resistance genes might be located in various plasmids.
Antibiotic resistance is not only conferred by plasmids, studies also show inherent resistance to antibiotics by chromosomally encoded resistance genes . Moreover, the large conserved plasmid of STEC strains has been shown to harbor genes resistance for antibiotics . These facts potentially explain the observed antibiotic resistance despite the absence of putative plasmids in PP-I isolates.
Studies made in understanding the immense antibiotic resistance in E. coli indicated plasticity of E. coli genome and the role of HGT by mobile genetic elements such as plasmids in conferring MDR and the possibility for the transmission of these plasmids among distantly related microbial population . Thus, the results of the current study indicate the need for early intervention to prevent further spread of DEC in the sample areas in order to, as the occurrence of such high level of mobile genetic elements in highly resistant bacteria presents a serious challenge in transmission of antibiotic resistance among other pathogens.
Similarly, the significant positive correlation between antimicrobial resistance and virulence gene content showed in this study agrees with what is reported by Zhang et al.  who showed the prevalence of resistance to single and multiple antibiotics to be significantly higher for pathogenic isolates than for commensal isolates. In contrast, Seyda et al. , identified no significant differences in virulence genes between antibiotic-resistant and antibiotic-susceptible strains. However, the observed significant correlation between virulence and antibiotic resistance in this study can be explained by the effects of selection pressure and genetic association between these factors according to Zhang et al.  and Schroeder et. al. . This study can also be taken as additional evidence for the positive correlation between antibiotic resistance and virulence factor carriage similar to the results obtained by Zhang et al. .
Furthermore, this study showed significant correlation between plasmid carriage and virulence factor, even though all the virulence genes used in the study are not plasmid encoded, where 80% of the putative plasmids were detected in intimin containing isolates. This finding may explain the abundance of EHEC among test samples, as extra virulence factors like additional adhesins can be conferred by plasmid encoded Virulence genes. DEC plasmids are known to be associated with virulence potential [28, 7]. Such strong association between plasmids and virulence factors may be explained based on pathogenomics and concurrent evolution according to Johnson and Nolan .
Based on all the study variables i.e. each of virulence factors, plasmids and antibiotic resistance traits, clear clustering of isolates across sample sites was observed indicating significant phenotypic difference across sampling sites. Such clustering of isolates in spatial and seasonal difference has also been reported on the incidence of E. coli strains in diarrheic calves by Shahrani et al.  in Egypt and Sidhu et al.  in Australia. This clustering of isolates among sites may be explained by similar sources of infection in the area as a result of environmental contamination by contaminated litters and asymptomatic carriers or as a result of difference in geography, climate and antibiotic consumption trend among sample sites.
Despite the valuable information generated in this study, the study had limitations in showing all the relevant information to get the clear picture of the different DEC pathotypes, their biology and distribution as a single host can be infected by multiple pathotypes of DEC. This is mainly the result of limited resources available to conduct the research.