Molecular based prevalence of shigatoxigenic Escherichia coli in rectal swab of apparently healthy cattle in Mymensingh district, Bangladesh

Objective: Cattle are regarded as the principle reservoir of O157 and non-O157 shigatoxigenic Escherichia coli (STEC). Spreading of the STEC to human is primarily happens through contaminated meat, milk and their byproducts. The present study was aimed to explore the occurrence of STEC in the rectal swab of apparently healthy cattle. 
Materials and methods: A total of 60 E. coli isolates that were previously isolated from the rectal swab of cattle were used in this study. DNA were extracted from the isolates and screened by PCR to detect E. coli stx (stx1, stx2), ehxA and rfbO157 genes. Representative amplicons of the PCR products were sequenced. The prevalence of the STEC was determined based on the detection of STEC specific stx genes. The prevalence data were further analyzed by SPSS to elucidate any difference among different demographic groups of the study population. 
Results: Overall, 43.33% (n=26/60) of the isolates were found carrying stx genes. Based on the presence of stx and ehxA genes, 6 different types of STEC were identified, of which 20% (n=12/26) were carrying both stx1 and stx2 genes. None of the isolates was positive for rfbO157. The PCR amplicons were sequenced, and the nucleotide sequences were deposited in GenBank (accession: KM596779-KM596784). 
Conclusion: In this study, non-O157 STEC were found highly prevalent in the local cattle. This study suggests that the apparently healthy cattle may act as a potential source of STEC infection for humans.


INTRODUCTION
Shiga toxigenic Escherichia coli (STEC), has become an increasing public health concern since its first identification in 1982 (Mainil and Daube, 2005). STEC becoming major concern for their association with hemolytic uremic syndrome and hemorrhagic colitis In human. Along with O157:H7 STEC infection, outbreaks and isolation of no-O157:H7 are increasing from different sources with time, and from 1983 to 2002 STEC infection with non-O157:H7 was recorded as approximately 70% (Brooks et al., 2005).
Stx is the major virulence property of STEC resulting host cell death by inhibiting protein synthesis. STEC produces one or more heterogeneous and immunologically non cross reactive Stxs (stx1, stx2 or variants). Though stx1 is identical to shigatoxin of Shigella dysenteriae, stx2 shares only ~56% identity with stx1 (Islam et al., 2008). In addition, some potential virulence genes viz., ehxA, katP, espP and and type II secreting system (etpD) has been reported in a ~90kb plasmid present in certain STEC strains (Farooq et al., 2009). Vast majority of enterohaemorrhagic E. coli (EHEC) associated with HUS harbors EHEC-Hly, a cytolysin, belongs to the RTX family (Schmidt et al., 1996;Bielaszewska et al., 2007). EHEC-Hly has the ability to injure microvascular endothelial cells (Aldick et al., 2007). STEC (O157 and non-O157) has been reported in the intestinal tract and dropping of different animal and birds including its major reservoir as the cattle and sheep (Griffin and Tauxe, 1991;Hazarika et al., 2007;Jomezaden et al., 2009). Although ruminants harbor STEC in their intestine, they are not affected by shigatoxins, due to the lack of specific receptors for shigatoxins on their cell surface. There are reports suggesting that ruminants could shed and spread STEC to humans through fecal contamination of meat and milk (Elder et al., 2000;Asakura et al., 2001;Naidu et al., 2007). Additionally, person to person contact is also well documented as a mode of transmission of STEC (Rodolpho and Marin, 2007

Ethical statement: Not applicable.
Bacterial strains and cultural conditions: Previousl we isolated E. coli from rectal swab of apparenty healthy cattle in Mymensingh (Hassan et al., 2014). Sixty E. coli strains (n=60) from that previous study were selected randomly and used in this study for detection of STEC.

DNA extraction and detection of virulence genes:
DNA from the pure isolates was extracted by boiling (Hassan et al., 2014). The presence of virulent genes (e.g., stx1, stx2, ehxA and rfbO157) was detected using specific primers listed in Table 1. PCR reaction mixtures were adjusted to 25 µL with PCR master mix (Promega, USA) and 10 pmol of each primer. PCR was performed with an initial denaturation at 94°C for 5 min, followed by 30 cycles of amplification [denaturation: 94°C for 1 min; annealing: 1 min at varying temperature depending on the target genes (i.e., 58°C for E. coli 16S rRNA gene, 61°C for stx1, 59°C for stx2, 49°C for ehxA, and 48°C for rfbO157); extension: 72°C for 2 min] with a final extension for 5 min at 72°C. PCR products were separated in 2.0% agarose and DNA was visualized in UVsolo TS Imaging System (Biometra, Germany).

Prevalence of STEC and their virulence genes
The prevalence of the STEC was determined based on the PCR amplification of stx (stx1, stx2) genes. Among the 60 isolates, 43.33% (n=26/60) were found to be positive for stx genes (Figure 1-2). The primers targeting hemolysin i.e., ehxA and E. coli O157 specific O antigen i.e., rfbO157 were also used. Among the 60 isolates, 10% (n=6/60) were found to be positive for ehxA (Figure 3). None of the isolates was positive to rfbO157. Prevalence analysis based on the demographic factors revealed higher prevalence of non-O157 STEC in local cattle aged above 3 years that were maintained under unorganized farming (management) systems ( Table 2).

Distribution of stx1, stx2 and ehxA among the STEC
Among the 60 isolates screened, STEC harboring 6 different combinations of target genes were identified among which isolates bearing both stx1 and stx2 are predominant ( Table 3). The PCR amplicons of the target genes were sequenced. Upon alignment, all the stx1 gene sequences were found to be identical and 3 stx2 and 2 ehxA gene sequences were found dissimilar at different level. One (1) stx1 gene sequence, 3 of the stx2 gene sequences and 2 of the ehxA gene sequences were deposited to GenBank (accession: KM596779, KM596780, KM596781, KM596782, KM596783, KM596784).      several factors such as the sample size and demographic characteristics of the study population. The prevalence of O157 E. coli as revealed in this study is inclined with the previous report of Hussein and Sakuma (2005).

DISCUSSION
In the present study, we found STEC positive for either stx1 or stx2 singly, while some were both stx1 and stx2 positive. In addition, ehxA was also identified in some of these stx1 and/or stx2 positive SETC. However, it was not surprising to observe these kind of findings since occurrence of either stx1 or stx2 or both in a single strain of STEC has earlier been identified from cattle (Renter et al., 2007).
This study also revealed 6 sub-groups of STEC based on the presence of different genes screened, where the prevalence of stx1 or stx2 alone was lower than the finding of Kesava et al. (2011). The prevalence of stx1 alone was lower but occurrence of stx2 alone or in combination with stx1 was higher than that of Cookson et al. (2006). This variation might be due to differences in study population and location.
Prevalence of non-O157 STEC was found significantly higher in local cattle over 3 years of age maintained under unorganized farming system compared to organized farm which might be resulted from recurrent exposure of the animals to STEC contaminated feed materials. However, it's difficult to make certain inference on the potential risk group of cattle to STEC based on this small number of samples analyzed in this study. Further studies covering more areas need to be focused to identify the potential risk group of animals and minimize the spread of STEC to human being.

CONCLUSION
Cattle are regarded as the major reservoir of STEC for human infection. About 43.33% of the rectal swab collected from the apparently healthy cattle was found to be positive for STEC in Mymensingh, Bangladesh. All the STEC isolates revealed in this study belongs to non-O157. In addition to the presence of stx encoding genes, hemolysin encoding genes have also been detected in thee STEC isolates. The occurrence of STEC in the rectal swab of apparently healthy cattle signifies that these cattle could be the potential source for pathogenic E. coli to human.