Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes

Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously. It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.


Introduction
Shiga toxin-producing E. coli (STEC) are considered important foodborne pathogens of zoonotic importance which causing mild to severe bloody diarrhea with the emergence of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) which is a life-threatening disease [1,2]. There are nearly more than 200 serotypes of STEC are recognized and the most frequent outbreaks of STEC are documented to be related to serotype O157: H7 strain throughout the globe [3]. E. coli O157: H7 serotype is the most important strain in hundreds of the other E. coli serogroups which live inside healthy humans and animal's digestive organs it delivers an intense toxin that can cause serious public complications [4]. The toxin produced by STEC is in similarity with Shigella dysentery producing toxin is also called Shiga-like toxins, or verodoxins [5]. In 1982 the pathogen STEC O157: H7 was recognized for the first time during an outbreak in the United States (US) [6,7]. Since then, for public health importance nowadays E. coli O157: H7 is widely recognized as a foodborne pathogen [8,9]. STEC primary transmission occurs through fecal-oral route by either indirectly use of a broad preparation of unhygienic foods, through contaminated water ingestion or directly through animals contact and their condition as well as from individual to individual straightforwardly [5,10]. Ruminant animals especially cattle, goats and sheep serve as a natural reservoir for STEC, which exist in the guts of these animals and appear to be the supportive hosts for STEC O157: H7. Thus, when animals are butchered, bacteria from animal intestines may contaminate their meat [11,12]. In most human cases cattle have been considered the suspected domestic ruminant of the source of infection [13]. Sheep have also been suggested as a source of human infection and a major cause of contamination to the food industry [14][15][16]. Like cattle and sheep, goats have also been considered the sub-clinical carrier of STEC O157, as they are the asymptomatic shedder of these bacterial pathogens [17]. In 2010, there are 1.78 STEC infection cases for each 1 lac populace are reported in the United States (US) [18]. In the European Union, STEC infection rate in 2011, is 1.93 cases per 1 lac populace [19]. Similarly, in New Zealand in 2011, the documented rate of STEC is 3.5 cases for each 1 lac populace (154 cases) [20]. In Argentina, between 2002 and 2015 only 4 cases of HUS were reported which are connected with food intake and these all cases were linked with STEC O157: H7 ehxA, eae and stx 2 [21][22][23]. STEC contamination rate in 2012, reported in Australia is 0.5 cases for every 1 lac populace [24]. In Pakistan, surveillance data regarding this organism is very sparse. Although demonstrated by several studies reported by [25][26][27][28][29][30].
Therefore, the present study was designed to estimate the prevalence rate and recognize the zoonotice potential of E. coli O157: H7 disseminating from adult sheep, goats, cows and buffaloes slaughtered in the slaughterhouses of District Rawalpindi and islamabad, Pakistan.

Study area
The present study was carried out at the Bacteriology laboratory of Animal Health Program, Animal Sciences Institute, National Agricultural Research Centre (NARC), Islamabad, Pakistan in a duration of 8-months from May 2017 to December 2017. Two local government slaughterhouses located at District Rawalpindi and Islamabad, Punjab Pakistan, included slaughterhouse of Rawalpindi which distributed the products of healthy slaughtered sheep and goats, while slaughterhouse of Islamabad which distributed the products of healthy slaughtered cows and buffalos to the other parts of the country.

Recto-anal samples collection
A total of 400 Recto-anal junctions (RAJ) samples were collected with the help of sterile labeled cotton-tipped swab sticks from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included healthy slaughtered adult sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included healthy slaughtered adult cows (n = 120) and buffalos (n = 80). The complete data history such as age, sex, weight, and species with each sample was recorded. In the studied animals, the output variable was the status of Escherichia coli O157. Slaughterhouses were visited seven and six times respectively, during the hot months from May to July 2017, because STEC O157 can easily survive in warm temperatures. On each visit, Twenty-five samples were randomly collected from both regions which included adult sheep, goats, cows and buffaloes. These RAJ samples were placed into modified Stuart's transport medium (Bacti Swab NPB, Thermo Scientific, Lenexa, KS), and maintained approximately at 4 °C until processed in the Laboratory.

RAJ swab samples processing
RAJ swab samples were initially processed in 20 ml of buffered peptone water (BPW) taken in the sterilized universal bottle. Each RAJ swab sample was enriched in the enrichment broth (BPW) by cutting the swab sample inside each universal bottle using a sterilized scissor and incubated for 24 h at 37 °C.

DNA extraction from enriched broth (BPW)
After enrichment, DNA extraction was carried out from enriched broth (BPW; Difco™, Becton, USA) by boil cell lysate method [31,32]. A 1-ml aliquot of enriched broth was taken and centrifuged at 13,000 rpm for 3 min. The supernatant was discarded after centrifugation and the pellet was re-suspended in 500 µl double distilled water (ddH 2 O). Vertexing was done at high speed for 10 s. The aliquot was heated at 95 °C for 10 min. Suspension of the lysed bacterial cell was then cooled at 4 °C for 5 min and was re-centrifuged again at 13,000 rpm for 3 min. The supernatant containing the DNA was then collected and transferred to another eppendorf tube. It was then subjected towards conventional PCR to detect rfbE O157 gene [33].

Initial screening for rfbE O157 serotype by conventional PCR
The rfbE gene is responsible for the production of the lipopolysaccharide (LPS) O side chain of the STEC O157: H7 cell surface and is a highly preserved gene specific to the serotype E. coli O157: H7 [34]. Conventional PCR was performed in the Gene Amp PCR system 9700 (Applied Biosystems, Melbourne, Australia). Already standardized Oligonucleotide specific sequence of rfbE O157 primers along with the amplified product size were specifically used for the synthesis of the rfbE O157 gene (Table 1)

E. coli O157 isolation and confirmation by conventional PCR
Agar media is one of the important sources for desire colonies isolation and for the use of further confirmation purposes [36]. STEC O157 can typically and also be effectively recognized by its capability to ferment Sorbitol in 24 h on Sorbitol MacConkey agar media as compared to other E. coli strains. The RAJ swab samples suspected positive for E. coli O157 gene by conventional PCR were then streaked onto Sorbitol MacConkey Agar media plates (SMAC) and incubated at 37 °C for 24 h. After the incubation period, only Sorbitol fermented pink-colored colonies were grown on SMAC media plates. About 6 different isolated colonies were selected from each plate for further analysis. DNA extraction was carried out from each colony using the simple boiling method [31,32]. The extracted DNA was then analyzed via conventional PCR under similar conditions to conform the presence of rfbE (O157) gene.

Multiplex PCR for STEC virulent genes (stx1, stx2, eae and hlyA)
Isolates positive for rfbE O157 serogroup by conventional PCR were then briefly subjected towards multiplex PCR (Gene Amp PCR system 9700; Applied Biosystems, Melbourne, Australia) to detect the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. Already standardized (Oligonucleotide) specific sequence of primers and its desirable base-pair sizes were used by multiplex PCR assay for the amplification of STEC virulent genes ( Table 2)  for 8 min at 72 °C, followed by maintenance at 4 °C, after which the PCR products were electrophoresed through an agarose (2% w/v) gel (Invitrogen, NZ) and visualized using ethidium bromide under Gel documentation system. The isolates were then sub-cultured onto the SMAC media plates to confirm pure growth and stored at − 80 °C in nutrient broth containing 15% (v/v) glycerol.

Results and discussion
In the present study, of 400 RAJ swab samples, only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) showed negative results for E. coli O157 (Table 3). From each positive sample (sheep and buffalo), 6 different Sorbitol fermented pink-colored colonies were isolated onto two SMAC agar media plates (Fig. 1). DNA was extracted from each colony using simple boil cell lysate method and analyzed via conventional PCR to confirm the presence of rfbE O157 gene. Results indicated that only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes (Figs. 2 and 3), While, rest of the five isolates showed negative results.
To concern the observed variation of our findings with these studies could be attributed to the differences in a wide range of sample collection, culture and molecular-based methods being applied for screening, detection and characterization of STEC. In a recent study, samples collected from ovine and bovine hosts via fecal palpitation and recto-anal junction swabs were reported most appropriate for the identification of STEC [45,46]. For E. coli O 157 colonization the RAJ site is a good indicator to collect a sample [47].
According to these reports, we acquired the same appropriate methodology for a sample collection from the studied animals.
During the enrichment process, the recovery of high STEC and other E. coli O157 strains may be difficult to isolate because of the presence of other competing flora in the medium. Hence, this could be one of the obvious reasons behind the recovery of a low number of STEC while testing samples [48,49]. Additionally, the temperature required for STEC detection during the enrichment process may be preferred to particular serotypes to show enough growth [50,51].
Similarly, culture-based methods (selective and differential media) is almost difficult to differentiate STEC from other E. coli strains, as STEC can only be recognized by its capability to produce Shiga toxins, however, it cannot be used as a phenotypic marker for the identification of STEC when there is an availability of mixed culture [52]. Besides, there is no assurance for the accuracy, specificity and safe use of these cultural methodologies [53]. As in the O serogroup of STEC a huge variability has also been observed [54,55]. As compare to immunomagnetic separation techniques, Sorbitol MacConkey agar has also been recommended for direct STEC O157: H7 isolation. However, its less sensitive factor was confirmed [56].
While some appropriate molecular techniques have been applied in recent studies for the quantification of STEC in bovine feces [36,57,58], in ovine feces [59] and in agricultural food matrices [60]. However, these molecular techniques are more costly to apply in less facilitative areas as compare to culture-based methods. The misidentification of a culture-positive sample, giving false-negative and falsepositive results, the targeted genes need to analyze may be present in different viable cells and the detection of a gene does not indicate if it may be expressed or not are the certain limitations and apparently main reasons behind the detection of a limited number of targeted samples [52]. It is also stated that polymerase chain reaction (PCR) may sometimes incapable to differentiate between live and dead cells, as the amplified DNA from dead STEC cells and the presence of background flora in the sample sometimes makes the PCR more vulnerable to give the exact prevalence ratio of STEC [61].
The DNA extracted from each single rfbE O157 isolate of sheep and buffalo RAJ samples were then briefly subjected towards multiplex PCR to detect the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously (Fig. 4). Results revealed that the single rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes (Fig. 5). It only harbored rfbE O157 gene and thus possessed rear chances of dissemination in the region of Rawalpindi. On the other hand, the single rfbE O157 isolate (01) of buffalo sample carried four STEC clinical virulent genes (sxt1, In consistence with our findings, a total of 6 (4%) E. coli O157: H7 was separated from fecal samples and all isolates were found that contained ehxA, stx 2 c and eaeA-γ1. The non-O157 STEC observed in 2 (1.5%) fecal samples contain one isolate which carried ehxA, stx 2 c; stx 2 a and stx 1 and the other isolates containing stx 1 a only [62]. STEC O157 was also reported as (81-87%) in Irish cattle and beef-derived isolates. Among them, the most predominant virulent strain was stx2 and eae [63,64]. Followed by similar results were noted among E. coli O157 isolates from cattle in France [65]. Likewise, a total of 55/1317 (4.18%) STEC O157 was also recovered from RAMS samples of cattle in Ireland. Amongst 50/55 E. coli O157 isolates harbored stx2 genes and all were eae positive [66].
In the current study, none of the E. coli O157: H7 was detected in RAJ samples of goats and cows. The reason may be due to very fewer chances of E. coli O157 colonization in intestinal hosts of these animals. As goat cannot be colonized exclusively with E. coli O157 and they have been considered the sub-clinical carrier of STEC [67,68] The present study revealed a very low prevalence ratio of rfbE O157 recovered from healthy slaughtered adult sheep and buffalo in the region of Rawalpindi and Islamabad, Punjab Pakistan. One of the possible reasons for the low prevalence rate 2 (0.5%) reported in our study is the inclusion of healthy adult ruminant animals (sheep, goats, cows and buffaloes). The animals bring to the slaughterhouses for buttering in these regions are majority of the adult age.
Studies indicated that when sheep and cattle get older so changes in the composition of gut microbiota (gastrointestinal tract and recto-anal site) of these animals occur consequently the prevalence rate of STEC decreases [69,70]. In the United States, STEC prevalence rate was reported higher in fecal samples of younger sheep (22.7%) at slaughter than older animals (0-1.9%) at pasture [71]. Similarly, in New Zealand, a higher prevalence rate of STEC was reported in slaughtered lamb (3.8%) than ewes (0.9%) at pasture [72]. Another study was reported on a group of older Scottish beef cattle potentially related with a lower risk of E. coli O157 shedding [73].
Even though several factors such as study design, sample collection and isolation methods used have a profound impact on the prevalence rate of E. coli O157. Despite this, the intrinsic factors (age, sex etc.) and extrinsic factors (season, diet, and climate etc.) have also a significant impact on the prevalence rate of E. coli O157 [74]. Keeping in view these differences thus limits the application of our study which could be one of the noticeable reasons behind the low prevalence rate 2/400 (0.5%) of E. coli O157 reported in our findings.

Conclusion
The present study revealed a low prevalence rate of 2/400 (0.5%) reported in sheep 1/75 (1.33%) and buffalo 1/80 (1.25%) RAJ samples at District Rawalpindi and Islamabad, Pakistan. However, it cannot be underestimated as healthy adult sheep and buffalo was possibly essential carriers of STEC O157 in these regions. But, as compared to Rawalpindi, the Islamabad region was at high risk because STEC O157 with 4 clinical relevant virulent genes (stx1, stx2, eae and hlyA) were detected in positive RAJ sample of buffalo

Recommendations
Data about the study of disease transmission of STEC O157 in these specific areas Rawalpindi and Islamabad as well in other parts of Pakistan are rare. Subsequently, more data is required for the study of disease transmission of STEC O157, distribution of virulence genes and their subtypes in E. coli separates from small and large ruminants and transmission of STEC O157 from these living organisms is to devise proper control systems. These control procedures would ultimately help in diminishing the increasing number of human STEC cases in Pakistan and further keep away from possible losses to Pakistan's economy.