Prevalence, Virulence Genes and Antimicrobial Proles of Escherichia Coli O157:H7 Isolated from Healthy Cattle

Background: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is associated with intestinal infection in human and considered a main cause of food-borne diseases. It was isolated from animals, human and food. The aim of the study was to assess the incidence of E. coli O157:H7 in fecal samples of healthy cattle collected in slaughterhouses (n=160) and from farms (n=100). Methods: E. coli isolates were detected on MacConkey agar. A total of 236 E. coli isolates were recovered from fecal samples of healthy cattle. We used sorbitol MacConkey to detect non-sorbitol fermenting colonies that were examined for the presence of O157 antigen by latex agglutination, and positive bacteria were screened for the existence of stx1, stx2, eaeA and ehxA by PCR as well as rfbEO157 and iCH7 genes specic for serotype O157. All isolates were examined for the susceptibility against 21antibiotics discs. Results: Of the 236 E. coli isolates, 4.2% (10/236) were positive for STEC O157:H7. Shiga toxin gene (stx2) was present in 70% of isolates, stx1 and ehxA were conrmed in 60% of the isolates, whereas eae was identied in two isolates. Other virulence factors screened (mH, sfa/focDE, cdt3, traT, iutA and hly) were present among the 10 isolates. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin and sulfamethoxazole/trimethoprim. All isolates belong to the phylo-group E. Conclusion: This is the rst study of the incidence of E. coli O157:H7 in cattle in Tunisia. Our nding proves the existence of STEC O157:H7 in healthy animals producing food for human consumption which could be a source of human contamination.

Strains belonging to the subgroup of shiga toxin-producing strains (STEC) are distinguished by certain EHEC serotypes, which are considerably linked to outbreaks in humans and causes clinical sickness. STEC is a foodborne bacteria which have been associated to many epidemics in all continents especially serotype O157:H7 (Karmali 1989). STEC strains were isolated from faeces of healthy ruminants like cattle, goats and sheep which can be natural reservoirs of these pathogens (Persad, Lejeune 2015).
E. coli O157:H7 is the dominant serotype of STEC group associated with human infections. The rst identi cation of this serotype as a pathogen was in 1982 during an outbreak of hemorrhagic colitis in Oregon and Michigan, U.S.A (Riley et al. 1983). The STEC O157:H7 can cause acute infections, with a spectrum of human illnesses ranging from abdominal pain, bloody diarrhea to fatal disease, like hemolytic-uremic syndrome (HUS) and hemorrhagic colitis (HC). The main STEC O157 infections are food borne more particularly concerning cattle sources (Atna e et al. 2017).
The STEC strains possess shiga toxins (stx1 and stx2) genes which consider the major virulence factors of these strains. Stx2 is associated more closely with the sickness than stx1 (García- Aljaro et al. 2004). Other important virulence determinants are: intimin protein, encoded by eae gene and important for attaching and effacing activity within the colonization of host intestinal mucosa and cause severe human infections, and enterohemolysin is encoded by the plasmid-and phage-carried enterohemolysin (ehxA) gene (Al-Gallas et al. 2006). STEC O157:H7 isolates have been detected in north Africa from humans, animals and food products. An Algerian study identi ed a rate of 7% from bovine carcasses (Chahed et al. 2006). In Morocco, a prevalence of STEC O157:H7 was 9%, 9.1% and 11.1% from raw meat products, dairy products and marketed meat respectively (Beneduce et al. 2008,Benkerroum et al. 2004. A Tunisian study com rmed that 3.4% of E. coli isolates among human stool samples were STECand 0.3% was E. coli O157:H7 (Al- Gallas et al. 2006). In Egypt, a survey con rmed that the prevalence among beef samples, chicken samples and lamb samples was 6%, 4% and 4% respectively (Abdul- Raouf et al. 1996).
An increasing rate of STEC O157 outbreaks, is related to the human consumption of fruits and vegetables contaminated with domestic or wild animal faeces. E. coli O157:H7 is transmitted to human by consumption of contaminated foods like raw meat, undercooked meat and raw milk. Contaminated water and foods by faecal material and cross-contamination through food production and processing, will lead to STEC infection (Lupindu 2018). Therefore, the objective of our study was to assess the incidence, virulence genes and antimicrobial resistance pro les of E. coli O157:H7 in fecal samples of healthy cattle. To the best of our knowledge, this is the rst detection report of E. coli O157 in healthy cattle in the Tunisia.

Samples Collection
The sample collection in this study was conducted on two types; rstly, faecal samples from 160 cattle intended for slaughter collected between December 2016 and April 2017.

Agglutination Test Of O157
Each non-sorbitol-fermenting colony isolated on SMAC plates was examined for the existence of the O157 antigens by agglutination latex reagent (Oxoid).
A rmation ofE. coli O157by PCR All non-sorbitol fermenting E. coli isolates and O157 agglutination-positive were examined for the existence of rfbEO157 gene and iCH7 by simplex PCR (Gannon et al. 1997). The PCR condition was as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 45 sec, annealing at speci c temperature for 45 sec (Table 1), extension at 72°C for 45 sec; and a nal extension (72°C, 7 min).
A multiplex PCR for stx1, stx2, uidA, ehxA and eae was achieved for the O157:H7 strains and primers arelisted in Table 1 (Al-Ajmi et al. 2020). The thermal cycling program of multiplex PCR was as follows, the denaturation: 95°C for 5 min followed by 25 cycles of 95°C for 1 min, annealing at 56°C for 1 min and the extension at 72°C for 1 min and the nal extension at 72°C for 5 min. The gel electrophoresis was used to separated PCR products by using 2 % agarose gel containing ethidium bromide.
The stx1 and stx2 ampli cations were sequenced in order to prove that the amplicon matched to the stx1 and stx2 sequences. The gained sequences were aligned with the data sequences in NCBI (http://www.ncbi.nlm.nih.gov).

Antimicrobial Susceptibility Testing
The antimicrobial susceptibility was determined by the disk-diffusion method on Mueller-Hinton agar plates as recommended by the Antibiogram Committee of the French Society (CA-SFM; www.sfm-microbiologie.org) using antibiotic disc panels comprising µg/disk: twelve β-lactam (amoxicillin (25)

Detection Of Phylogenetic Groups
The phylogenetic groups (A, B1, B2, C, D, E, F) were detected among the isolates by the quadruplex PCR method developed by Clermont et al. (Clermont et al. 2013). The phylo-groups determination established on the existence of the chuA, yjaA genes and TspE4-C2 fragment by the quadruplex PCR to detect (A, B1, B2, D) and C, E were further identi ed by using speci c primer sets (Table 1).

Results
In our study, 236 E. coli isolates were collected from the examination of 250 faecal samples of healthy cattle in Tunisia. Out of 236 E. coli isolates, 159 were from cattle in slaughterhouses and 77 from cattle from farms. Of these E. coli strains, 100% were positive for methyl-red, lactose and indol, and 100% were negative for urease, citrate and H2S. The results revealed that 10 E. coli were nonfermenting of sorbitol on CT-SMAC and these 10 (4.2%) strains were E. coli O157:H7. Out the 10 strains; 6 isolates were isolated from healthly cattle in slaughterhouses and 4 from healthy cattle from farms.
The con rmation of E. coli O157 by latex agglutination testing reveal that all isolates were O157 positive. All of these isolates were con rmed as E. coli O157:H7 via screening of rfbO157 and icH7 genes by speci c primers.
PCR analysis of the 10 E. coli O157 isolates reveals that uidA, icH7 and O157 genes were present in all strains. Stx2 gene was present in 7 isolates (70%), stx1 and ehxA were con rmed in six isolates (60%) whereas eae was identi ed in two isolates.
We found four isolates carrying three virulence genes as follow; three strains harbored stx2, stx1 and ehxA and one strain harbored stx2, eae and ehxA ( Table 2). All E. coli O157 isolates belong to the phylo-group E.
The O157 isolates were further tested for 13 virulence factors. All isolates carried at least one virulence gene tested. Out of 10 isolates, 60% carried more than three virulence gene tested. The mH was the most frequent virulence gene and was detected in 90% (9/10) of the isolates, followed by sfa/focDE 60%. The frequency of cdt3, traT, and iutA among the isolates was 50%, 50%, and 40% respectively, wherase, hly was the lowest virulence genes of E. coli isolates which was found in one isolates ( Table 2). None of the isolates harbored cnf1, aer, papA, bfpA, papG allele III, ibeA and fyuA. A total of 236 E. coli isolates were collected from faecal samples of healthy cattle in Tunisia during a ve-month time period in 2017 and nine months in 2018, and were evaluated for the incidence of E. coli O157 and antimicrobial pro les. This is the rst report concerning the presence of E. coli O157:H7 in cattle in Tunisia.
Our nding exhibited that among 236 E. coli isolates, ten E. coli O157:H7 were detected with a rate of 4.2 %. These isolates were cultured on CT-SMAC agar as non-sorbitol fermenters and were con rmed as STEC O157 by using latex agglutination and PCR. This is in agreement with other studies investigating E. ). An Algerian study reported an occurence of E. coli O157 inmore than 7% of bovine carcasses (Chahed et al. 2006). In Morocco, the incidence of E. coli O157:H7 in dairy products and marketed meat products was 9.1% and 11.1% respectively (Benkerroum et al. 2004). In Tunisia, 327 E. coli strains were isolated from diarrheic and non-diarrheic people. By using PCR techniques it has been demonstraed that 11 isolates (3.4%) express the stx gene encoding for STEC (EHEC) and only one (0.3%) was con rmed as E. coli O157:H7 (Al-Gallas et al. 2006).
In Africa, the highest incidence in cattle was 31.2% representive in four studies. In Asian countries, the highest rates was 12.22% in Jordian cattle and the lowest (0.13%) was evaluated in Taiwan. In Europe, the highest estimated occurrence was demonstrated from Italy (10.45%) and the lowest from Norway (0.25%). Furthermore, the USA incidence estimate was 7.  On the other hand, more than 40% of the isolates were resistant to cefuroxime and streptomycin, perhaps via inappropriate or wide use of drug for prophylactic purpose and treating infections. Our study showed that 9 STEC strains harbored mH and half isolates harbored sfa/focDE, cdt3, traT, and iutA. These factors were identi ed in a previous study among E. coli from dairy farms in America (Pereira et al. 2011).
In an Iraian study of STEC, they found papA, cnf1, traT and cnf2 the highest virulence genes (Momtaz et al. 2012). The detected factors contribute to virulence which affect of host cell processes and contribute to bacterial pathogenesis. The ndings of these virulence factors in our isolates in associated with high prevalence of stx1, stx2 and ehxA suggest that STEC O157 in Tunisian calves may pose a serious public health concern.
The ndings of our study revealed that all E. coli O157 isolates belonged to phylogroup E. This was identical to the report of Tenaillon et al. (Tenaillon et al. 2010). A study in Brazil demonstrated that E. coli belonging to phylogroups E and B1 were isolated from cattle, wherease phylogroups A and F were from poultry and B2 and D were associated with isolates from water buffalo (Morcatti Coura et al. 2015).

Conclusions
The prevalence of E. coli O157:H7 in healthy cattle with some antibiotics resistance indicate a possibly risk to public health concern. The existence of STEC O157:H7 in animal feces intended to slaughter highlighted the possible contamination of meat products prepared for human consumption. The high prevalence of stx1, stx2 and ehxA with other virulence factors suggest that STEC O157 in Tunisian calves may pose a serious public health concern. Our study reveals the necessity for a regular screening of E. coli O157:H7 in animal in order to control this pathogen. It is important to take necessary measures in the slaughterhouse during the slaughter and skinning of animals to prevent cross contamination of meat by this pathogen.