Characterization of Diarrheagenic Escherichia Coli Strains Isolated from Slaughtered Sheep in México

Sheep represent one of the main reservoirs of diarrheagenic Escherichia coli; this microorganism is an etiological agent of food-borne diseases, therefore, this work aimed to identify and characterize the principal pathotypes of diarrheagenic E. coli obtained through rectal swabs and samples from sheep carcasses slaughtered in an abattoir at the central region of Mexico. The isolates were subjected to bacteriological identication, serotyping; phylogenetic classication; detection for virulence factors, and antimicrobial sensibility. A total of 90 E. coli isolates were obtained, diarrheagenic serotypes with health public relevance were found: O76:H19 (5), O146:H21 (3), O91:H10 (2), O6:NM (1), and O8:NM (1). According to pathotype, 47.7% of total isolates were Shiga toxin-producing E. coli, while 3.3% were enteropathogenic, 2.2% enterotoxigenic, and 1.1% enteroinvasive E. coli; the remaining isolates did not express the genes used to assign them to some pathotype. Regarding the Shiga toxin subtypes, 31/43 (72.09%) were cataloged as stx1c, 11/43 (25.5%), stx1a- stx1c and 1/43 (2.3%) stx1a- stx1d; while for stx2 it was possible identify stx2g 4/7(57.14%), stx2b 1/7 (14.7%) and stx2b-stx2g 2/7 (28.5%). Almost all pathotypes (91– 100%) belonged to phylogroup B1. Furthermore, it was observed that the 90 isolates showed an antimicrobial resistance of 100% to nitrofurantoin, followed by ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. These results highlight the importance of diarrheagenic E. coli as a potential risk for public health during the slaughtering process.


Introduction
Sheep and other ruminants are regular carriers of commensal Escherichia coli; however, they may harbor some pathogenic E. coli and cause either, diarrhea or extraintestinal illness (Bettelheim 2000). The relevance of these diarrheagenic E. coli (DEC) isolates as causative agents of food-borne diseases (FBD) has been recently studied in Latin America, although there is a lack of information in some countries regarding the main reservoirs and infection routes (Torres 2017). Animal products, like sheep and beef meat, are at risk of contamination by poor hygiene practices during the slaughtering process in the abattoirs, hence, the implementation of good production practices (GPP) and good manufacture practices (GMP) are essential to prevent bacterial contamination of carcasses and ensure food safety (FAO 2005). Sheep without diarrhea usually are asymptomatic carriers of zoonotic pathogens and reservoirs of DEC, which could enter the production line, especially in the critical control points (Reyes- Rodríguez et al. 2015). The animals that arrive at the slaughterhouse are the principal focus of contamination towards drinking water and animal products, allowing the direct transmission of zoonotic microorganisms to the human population (Blanco et al. 2003).
At least 5 E. coli pathotypes have been related to gastrointestinal illness in humans: Shiga-toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) (Nataro and Kaper 1998). These pathotypes are classi ed according to their virulence factors. The principal virulence factor of STEC is the production of a toxin that inhibits protein synthesis coded by stx 1 and stx 2 genes and their variants, moreover, other virulence factors like the intimin (encoded by eae gene) or autoagglutinating adhesins can be found (Paton et al, 2001).
Shiga toxins are classi ed as stx1 and stx2. The stx1 toxins are a homogeneous group with 3 subtypes: stx1a, stx1c, and stx1d. On the other hand, stx2 toxins are more heterogeneous with a greater number of subtypes that include stx2a, stx2b stx2c stx2d stx2e stx2f, and stx2g, being stx2c and stx2d strongly associated with HUS. Other relevant virulence factors include the intimin (encoded by eae gene), a plasmid-carried enterohaemolysin (encoded by ehxA gene), putative adhesins genes like Tox B, saa, espC, and espP (Prager et al., 2011, Beutin 2007. The presence or absence of eae gene in STEC strains allows classifying them into the typical virulent (t-STEC) or atypical strains of low virulence (a-STEC). STEC strains induce gastroenteritis and further complications as hemolytic uremic syndrome (HUS) or hemorrhagic colitis (HC) which can lead to chronic kidney dysfunction, especially in infants and the elderly (Nataro and Kaper 1998).
EPEC produces the attachment and effacing (A/E) lesions onto intestinal mucosa. This pathotype is divided into two categories based on the presence or absence of the bundle-forming pilus (bfp) gene; strains that contain this gene are classi ed as typical (t-EPEC), while the ones that lack this gene are atypical (a-EPEC). Curiously, the a-EPEC strains are more common in developing countries, in contrast, t-EPEC causes diarrhea in children from developed countries (Trabulsi et al. 2002). The main feature of ETEC is the production of two enterotoxins: the heat labile-toxin (LT) and the heat-stable toxin (ST), The ETEC strains are the leading cause of traveler´s diarrhea and is also related to children´s diarrhea (Sjöling et al. 2007).
The EIEC group and Shigella spp. are biochemically and genetically related. The pathogenicity mechanism is through the invasion of the colon´s epithelium; several involved proteins like Ipa and others are encoded in the 140 MDa plasmid pInv, generally, watery diarrhea is observed, but in some cases, in ammatory colitis can occur (Halet et al. 1983). Finally, EAEC pathotype increases the production of mucus allowing a pattern autoaggregative of bacteria and forming a layer in the epithelium. Adherence and is due to aggregative adherence mbriae, especially the variant I (AAF/I) encoded by aggA gene, which in turn is located in a plasmid of60 MDa (Nataro et al. 1992 (Navarro et al. 2018), such information is necessary to assert the risk factors that could affect the safety of sheep carcasses in this country, therefore, we investigated the prevalence of DEC isolates obtained from sheep slaughtered in an abattoir in Mexico, and determined the presence of virulence factors, the phylogenetic classi cation of isolates as well as their antimicrobial resistance pro le. Therefore, the main objective of this work is to know which diarrheal pathotypes of E. coli are naturally present in sheep slaughtered in a slaughterhouse in the state of Mexico and to know if they could represent a risk factor for the consuming population.

Virulence factors
The identi cation and characterization of diarrheagenic E. coli pathotypes (STEC, EPEC, ETEC, EIEC, and EAEC) were performed by PCR. Fragments of several virulence genes were ampli ed and assigned to each pathotype employing primers and thermal  Table 1). The reaction products were visualized on 2% agarose containing ethidium bromide.

Phylogenetic group determination
A quadruplex PCR was carried out to identify the phylogenetic groups (A, B1, B2, C, D, E, and F), the chuA, yjaA, arpA, and TspE4.C2 genes were ampli ed with primers and PCR conditions according to Clermont et al. (2013) (Table 1)

Antimicrobial resistance genes
To identify antimicrobial resistance genes against β-lactams, tetracyclines, and sulfonamides, the genes bla TEM , tetA, tetB, sul1, and sul2 were analyzed by PCR technique using the primers and conditions described by Kerrn Table 3). The PCR products were visualized by electrophoresis on a 2% agarose gel stained with ethidium bromide.
Statistical analysis.
The virulence factors, serotypes, antibiotics resistance, and phylogroups results were analyzed by descriptive statistics and the generated data arranged in tables.

Virulence factors
The identi cation and characterization of diarrheagenic E. coli pathotypes (STEC, EPEC, ETEC, EIEC, and EAEC) were performed by PCR. Fragments of several virulence genes were ampli ed and assigned to each pathotype employing primers and thermal cycling conditions as described previously (Scheutz et Table 1). The reaction products were visualized on 2% agarose containing ethidium bromide.

Phylogenetic group determination
A quadruplex PCR was carried out to identify the phylogenetic groups (A, B1, B2, C, D, E, and F), the chuA, yjaA, arpA, and TspE4.C2 genes were ampli ed with primers and PCR conditions according to Clermont

Antimicrobial resistance genes
To identify antimicrobial resistance genes against β-lactams, tetracyclines, and sulfonamides, the genes bla TEM , tetA, tetB, sul1, and sul2 were analyzed by PCR technique using the primers and conditions described by Kerrn  It does not apply since the investigation was carried out with animals previously slaughtered by the municipal authorities of the slaughterhouse.
Con ict of interests.
No con ict of interest on the part of all authors.

Data availability statement
The data that support the ndings of this study are openly available in UAEMex Institutional repository at The most frequent stx subtype gene described worldwide in sheep is stx1c (Brett et al., 2003;Kumar et al., 2012;Erol et al., 2016), the results of our investigation corroborate this statement, howbeit, a small number of isolates carried stx1a and stx1d. The stx1c subtype gene is related to diarrhea without complications in humans (Beutin et al., 2007).
Recent research has shown that stx subtypes have a predilection toward different receptors, the Stx B subunit recognizes Gb3 as its principal receptor and to a lesser extent Gb4. Lee and Tesh (2019) highlight the relevance of this interaction as a key mechanism in the pathogenicity of STEC. Stx1a interacts strongly with Gb3 on the human glomerular endothelium. On the other hand, the subtype stx 2e shows predilection for Gb4 and Gb5 present in the glomerular endothelium of ruminants and pigs.
In this work stx2g gene was predominant, followed by stx2b, these subtypes are not associated with HUS and HC development in humans, which could represent a low hazard to establish disease. In contrast, the presence of stx2c and stx2d genes that were not reported in this investigation that boost the development of HUS and HC, have been reported in sheep carcasses from Turkey and Switzerland. Amezquita et al. (2014) found stx2c and stx2d genes in backyard sheep in Mexico. Prager et al. (2011) demonstrated that isolates harboring stx2g gene obtained from humans, animals and environmental sources had a close phylogenetic relationship, reinforcing the idea of human infections as a potential zoonotic disease.
Identi cation of stx subtypes is a priority as lets an early prediction of the virulence potential of each STEC isolate. This observation generated enough evidence to know that stx2a and stx2d genes are crucial determinants in the severity of HUS; furthermore, the mere presence of stx2a gene is considered an independent risk factor to develop HUS in multivariate analysis. Therefore, the identi cation of stx subtypes should be performed routinely in diagnostic laboratories. The presence of most of the DEC pathotypes of public health concern can be isolated from sheep, goats, and bovines which raise the relevance of livestock as a reservoir of these pathogens (Mora et al. 2011); precarious hygiene conditions make it possible for DEC to contaminate meat products with feces during different processes in slaughterhouses in Mexico.
The high percentage (~90-100%) of isolates belonged to phylogenetic group B1 (commensal E. coli), which is similar to that reported in other countries in isolates from sheep, goats, and bovines (Ghanbarpour et  Antimicrobial resistance (AMR) was observed in STEC isolates against AMP (72%), TET (30 %), and SXT (9%); these percentages were lower in comparison to a study carried out in Turkey where a higher frequency of AMR to AMP and TET (100% and 50% respectively) was reported (Seker and Kus 2019). In Egypt, lower levels of AMR to AMP (66.7%) but higher to SXT (73.3%) were described in a goat slaughterhouse (Elsayed et al. 2018). In Mexico, a study discovered 92% and 75% of AMR to AMP and TET respectively in bovines, this contrast with our study where both antibiotics showed a lower level of AMR. Another study in this country found AMR to cephalosporins in STEC isolates from bovines, interestingly, we did not nd AMR to these antibiotics, despite this, both studies showed AMR to TET and AMP (Martinez-Vazquez et al. 2018; Navarro et al. 2018).
In the case of a-EPEC, a lower resistance rate in comparison with this study was found in adult sheep in Spain with a 1.9%, 0, and 1% for GEN, TET, and SXT respectively (Medina et al. 2011), conversely, a study from Brazil detected higher rates of resistance against CIP (22%), AMK (4%), GEN (9%) and cephalosporins (72%) in a sheep abattoir (Rigobelo et al. 2008). In the particular case of ETEC, we nd out high resistance levels for AMP (100%) and TET (50%) in our work compared to Njoroge et al. (2013) with goat isolates in Kenia.
Multi-drug resistance (MDR) was found against 3 and 4 antibiotics (TET, NIT, AMP, and SXT) in 10 STEC isolates. The presence of MDR E. coli in the gut microbiota of the analyzed sheep could further disseminate to other microorganisms due to horizontal gene transfer (Penders et al. 2013).
In the present study, it was possible to detect resistance genes such as tetA, tetB, sul1, sul2 detected in isolates resistant to tetracycline and trimethoprim-sulfamethoxazole. Research studies around the world also reported the nding of some of these genes, in Portugal (Ramos et al., 2011) informed the presence of tetA, tetB and in a smaller number sul2, in sheep samples processed in a slaughterhouse. Medina et al, (2011) working with live sheep in Spain reported the presence of these same genes, being tetA the most frequent, nally, in France, bovine isolates harbored tetA and sul2 genes (Um et al., 2018).
This investigation is one of the few to characterize DEC from slaughtered sheep in Mexico, and to the author knowledge, the rst to be carried out in the region with the highest production, processing, and consumption of sheep meat of the country (SIAP 2016). We identi ed several serotypes related to gastrointestinal illness in Mexico, along with some stx subtypes genes that have been reported worldwide as low virulent (stx1a, stx1c, stx1d, stx2b, and stx2g), nevertheless, some serotypes are implicated in diarrhea and MDR isolates could pose a threat for treatment in case of intestinal and extra-intestinal illness in people who consume sheep meat. These ndings re ect the potential concern of sheep as a primary reservoir of STEC non-O157 and the possible transmission through the food chain.