Occurrence of Escherichia coli as a Causative Agent of Enteritis in Dogs with Special Reference to Their Multidrug Resistance and Virulence

| Escherichia coli (E. coli) massively causes deaths all around the world. Human–pet proximity may inadvertently harm humans. Dogs are susceptible to virulent E. coli strains causing enteric infections in humans. In the present study, a total of 90 rectal swabs were collected from dogs of different ages (suffered from diarrhea with fever, nausea, chills, loss of appetite, and bloating) at different veterinary hospitals and clinics in Cairo to determine the incidence rate of E. coli and their virulence and resistance genes. Bacteriological examination revealed an overall E. coli occurrence rate of 67.7% (61/90). The highest isolation rate of 75.5% was from puppies that were 3 months and more. Serotyping of ten E. coli isolates showed that they belonged to seven serogroups O18, O27, O55, O126, O148, O158, and O166 and other untypable three strains. All E. coli isolates were subjected to disc diffusion sensitivity tests and were resistant to tetracycline, trimethoprim/ sulphamethoxazole (100% for each), cefotaxime (95.1%), and erythromycin (93.4%), while they were sensitive to amikacin only (88.5%). The occurrence of virulence genes in the seven tested E. coli isolates was conducted using PCR. It was revealed that the stx1 gene was detected in O18, O126, O148, O158, and O166 serogroups while the stx2 gene was detected in O18 and O27 only. The eaeA gene was detected in O27, O55, O148, and O158 serogroups. Also, the antibiotic-resistant blaTEM and tetA genes were detected in all the seven tested serotypes. These combined results indicate that pet animals may harbor E. coli causing diarrhea at different ages.

gene encodes for intimin, the adherence protein whose receptor is required for virulence (Ben Said et al., 2019). The ability of STEC to induce serious diseases correlates to the emittance of one or more Shiga-like toxins) stx1, stx2, or other variants); they inhibit the host cells' synthesis of protein, leading to cell death (Ahsan et al., 2020).
Dogs that carry multidrug-resistant E. coli strain (MDREC) in their feces could contaminate the surrounding environment, as a result of transmitting these bacteria to humans as well as other animals (Warren et al., 2001). E. coli is often prone to various antibiotics; however, by time and antibiotics' excessive use, drug-resistant strains emerged. Moreover, extended-spectrum β-lactamases (ESBL) that induce enteric pathogens is a serious problem (Mathur et al., 2002).
This work aims to detect the occurrence rate of E.coli in diarrheic dogs of different ages, identify the isolates by VITEK2 compact, serological testing, and serogroup, as well as investigate the presence of the virulence (stx1, stx2, and eae) and drug resistance (bla TEM and tet A) genes.

sampLes COLLeCtiOn
A total of 90 rectal swabs were collected from dogs of different ages (one month to over one year) from different veterinary hospitals and clinics in Cairo. The animals were suffered from diarrhea with fever, bloating, chills, loss of appetite, and nausea. Each sample was collected using a sterile swab that was kept in an icebox immediately after collection and then sent to the laboratory without delay. All samples were taken with care about ethical guideline for sampling and also during working all biosafety measures were done. The experimental protocols were conducted according to guidelines of Ethics of Animal Use in Research Committee, Faculty of Veterinary Medicine, Zagazig University, Egypt.

isOLatiOn and identifiCatiOn Of E. coli
Rectal swabs were cultivated into nutrient broth (Difco) tubes and incubated aerobically at 37°C for 18 hours. A loopful from each broth culture was inoculated onto Mac-Conkey agar (Oxoid) (Cruickshanak et al., 1975) and Eosin Methylene Blue (EMB) agar (Oxoid) (Koneman et al., 1988). Agar plates were incubated aerobically for 24 hours at 37°C and then were observed to detect if any bacterial colonies were present. Suspected colonies were assessed for Gram`s reaction. Gram-negative bacilli colonies were found. Each sample was further subjected to biochemical testing such as catalase, urease test, Triple Sugar Iron agar (TSI), methyl red test, indole test, citrate test, and Voges-Proskauer (IMViC) (McFaddin, 1985) as the tradition-al method of identification. Then, results were confirmed by VITEK2 compact identification and serotyping.  (Pincus, 2006).

serOLOGiCaL identifiCatiOn
Ten E. coli isolates were submitted to slide agglutination test via polyvalent antisera per instruction of manufactures procedures (E. coli antisera Denka Seiken Co. LTD) (Collee et al., 1996).

antimiCrObiaL susCeptibiLity testinG
Fresh cultures of all isolates of E. coli were tested for antimicrobial susceptibility via the standard disk diffusion method as per guidelines of the Clinical Laboratory Standards Institute (CLSI, 2017). The following antimicrobial discs were used: amikacin (AK, 30 μg), amoxicillin/ clavulanic acid (AMC, 20/10 μg), ampicillin (Amp, 15 μg), cephalothin (KF, 30 μg), ciprofloxacin (CIP, 30 μg), doxycycline (DO, 30 μg), erythromycin (E, 5μg), neomycin (N, 30 μg), tetracycline (TE, 30 μg), and trimethoprim/ sulphamethoxazole (SXT, 25 μg) (Oxoid). deteCtiOn Of sOme ViruLenCe and antibiOtiC resistanCe Genes Of E. coli by pCr DNA was extracted from the seven E. coli serotypes to detect virulence and antibiotic resistance genes via the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) according to the manufacturer's recommendations. 200 μl of the sample suspension was incubated with 200 μl of lysis buffer and 10 μl of proteinase K at 56 O C for 10 minutes. Afterward, 200 μl of ethanol (100%) was added to the lysate. The sample was rinsed and centrifuged as per the instructions of the manufacturer. Nucleic acid was eluted with 100 μl of elution buffer found in the kit. The used primers sets were procured from Metabion (Germany). Table 1 illustrates the cycling parameters.
Primers were applied in a 25-μl reaction mixture of 12.5 μl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 μl of each 20 pmol primer concentrations, 6 μl of DNA template and 4.5 μl of water. PCR outcomes were dispersed by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature utilizing gradients of 5V/cm. For gel examination, 20 μl of the PCR products were loaded in each gel slot. Gelpilot 100 bp DNA Ladder (Qiagen, Germany, GmbH) was adopted to know the fragment sizes. A gel documentation system (Alpha Innotech, Biomet Journal of Animal Health and Production 2021 | Volume 9 | Special Issue 1 | Page 9 ra) was used to get the gel photograph. Computer software was utilized to analyze the data.

rESuLtS
Out of the 90 examined rectal swabs, 61 were positive for E. coli (67.7%) at different ages. The highest isolation rate was in puppies of less than three months old and recorded as 75.5% and the lowest frequency was 57.1% was recorded from dogs of more than one year old as shown in Table 2. Ten isolates were representing seven serogroups including O18, O27, O55, O126, O148, O158, and O166 in addition to 3 untypable groups. All isolates of E. coli subjected to disc diffusion sensitivity test showed high drug resistances levels (100%) was against tetracycline and trimethoprim/ sulphamethoxa-zole, followed by cephalothin, (95.1%) and erythromycin (93.4%), while the highest sensitivity (88.5%) was to amikacin (Table 3). The E. coli virulence genes of the tested isolates showed amplicons of 779, 614, and 248 bp for stx1, stx2, and eae genes, respectively. Of the seven-tested E. coli isolates (seven serogroups) five strains were positive to stx1 gene, two strains to stx2 gene, and four strains to eae gene (Table 4 and Figures 1 and 2). While the bla TEM and tetA antibio-Journal of Animal Health and Production 2021 | Volume 9 | Special Issue 1 | Page 10 tic-resistant genes representing 516 and 576 bp size, respectively, were detected in all the seven tested isolates (seven serogroups) ( Table 4 and Figures 3 and 4).

Figure 1:
Agarose gel electrophoresis showed PCR products of E.coli stx1and stx2 genes amplified at 614 and 779 bp respectively from seven isolates. L: representing the molecular size marker (100 pb plus ladder). Lane 1 was positive to stx2, lanes 2,3,4, and 5 were positive to stx1. Lane 6 was positive to stx1 and stx2, and lane 7 was negative to stx1 and stx2. N: represents control negative, P: control positive Figure 2: Agarose gel electrophoresis showed PCR product of E.coli eae gene amplified at 248 bp from seven isolates, N: represents negative control; P: represents positive control of eae gene (248 bp). L: represents the molecular size marker (100pb plus ladder). Lanes 1, 4, 5, and 7 were positive to eae gene. Lanes 2, 3, and 6 were negative to eae gene.

dIScuSSIon
Dogs and puppies are one of the critical carriers of E. coli, one of the chief causatives of diarrhea and other diseases in humans (Hasan et al., 2016). Out of the 90 examined Journal of Animal Health and Production 2021 | Volume 9 | Special Issue 1 | Page 11 rectal swabs, 61 were positive for E. coli (67.7%) at different ages. The highest isolation rate was in puppies of less than three months old and recorded as 75.5% and the lowest frequency was 57.1% was recorded from dogs of more than one year old.
In this study, E. coli was isolated from diarrheic dogs at different ages, with total isolation percentages of 67.7% (61/90). Puppies under three months old were the most susceptible to E. coli infection; 75.5% of this pool agrees with Broes et al. (1988) who reported that E. coli was present in diarrheic puppies from one month to one month and a half old. Dogs older than one year were less susceptible to E. coli infection ( Table 2). This conforms to various studies exhibiting that the incidence peak of enteritis always occurred within few days after the inception of the weaning period (one month) and the majority of EPEC infections take place in the first three months of lifespan as mentioned by Janke et al. (1989).
From all suspected isolates proved by traditional bacteriological examination to be E. coli, ten random isolates were reinvestigated using the VITEK2 compact system via Gram-negative cards and the results confirm the isolation of E. coli with an incidence of 100%. The VITEK2 compact system is a highly automated new tool for accurate and rapid identification of E. coli. The ten random isolates were subjected to serotyping and the results showed that these ten isolates were representing seven serogroups including O18, O27, O55, O126, O148, O158, and O166 in addition to 3 untypable groups.
On contrary, in other studies in Trinidad, Adesiyun et al. (1997) reported that the prevalence of EPEC strains was not significantly associated with age. Moreover, Marjanca et al. (2002) isolated 24 strains of hemolytic E. coli from dogs with diarrhea. The detected serogroup detected in this study were O18, O27, O55, O126, O148, O158, and O166. On the other hand, Maniam et al. (2020) reported that the most prevalent E. coli serogroup isolated from dogs from feces of healthy ones were O104:H4 and O102:H18. Also, Michele et al. (2014) found that E. coli O145: H 28 was isolated from stray dogs.
The E. coli virulence genes of the tested isolates showed amplicons of 779, 614, and 248 bp for stx1, stx2, and eae genes, respectively. Of the seven-tested E. coli isolated, seven serogroups: five strains were positive to stx1 gene, two strains to stx2 gene, and four strains to eae gene. While the bla TEM and tet A antibiotic-resistant genes representing 516 and 576 bp size, respectively, were detected in all the seven tested isolates (seven serogroups).
In this study, virulence genes have been detected in E.coli isolated of dog origin where the stx1 and stx2 genes were detected in five E. coli serogroup (O166, O126, O158, O148, and O18) and two E. coli groups (O27 and O18), respectively. stx 1 and stx 2 genes maintain the same pattern in the other reports (Staats et al., 2003;Nakazato et al., 2004;Bentancor et al., 2007).
In our results, the blaTEM gene was detected in seven tested isolates by PCR at 516 bp, which agrees with the earlier findings (Younis et al., 2015;Delmani et al., 2017). The blaTEM gene found in a plasmid showed a strong correlation between the genogroup conferred resistance determined by PCR and antibiotic susceptibility patterns. Moreover, tetA gene was found in seven tested isolates at 576 bp amplicon which agrees with (Nde and Logue, 2008) and (Torkan et al., 2015) who suggested that tetA could be the predominant tetracycline gene of resistance harbored by pathogenic E. coli present in dogs in Iran. There are other tetracycline-resistant genes thought to induce resistance via enzymatic inactivation and ribosomal protection.

concLuSIon
Virulence genes of E. coli isolated from dogs were stx1, stx2, and eae, detected in current study, which probably influence the E. coli enteritis pathogenicity. Moreover, multidrug resistance genes were blaTEM and Tet A detected in E. coli isolates. The results suggested the pet owners to adopt hygiene practice to prevent infection, such as proper food-preparing methods, avoiding drinking water from potentially contaminated sources, thoroughly cooking meat before feeding dogs, and washing hands frequently and thoroughly.