Samples were collected from 124 cattle from a bovine slaughterhouse located in the center of the state of Tamaulipas, Mexico. This slaughterhouse receives cattle from nearby municipalities. One specimen was randomly selected per farm for the fecal sample and the carcass sample later to be taken.

A total of 124 bovine fecal samples were collected by direct rectal retrieval using disposable gloves. Each glove was inverted and sealed immediately after collection.

A total of 124 carcass samples were collected by sterile sponge from each carcass 12 h after evisceration: a minimum of 100 cm² from inside the round area from the navel–plate–brisket–foreshank areas. All carcass samples were collected according to the national regulations described in NOM-SSA1-1994 (15). All samples were placed in separate sterile plastic bags to prevent cross-contamination, labeled, and transported in ice to the laboratory.

Twenty-five grams of each sample was enriched by inoculation in 225 ml of peptone water. They were then homogenized and incubated at 37°C. After 24 h, the samples were plated onto eosin methylene blue and were incubated at 37°C for 18–24 h. Six colonies of presumptive E. coli strains were randomly picked from each sample, and each was subsequently transferred separately to tryptic soy agar and incubated for 24 h at 37°C to obtain a pure culture. All suspect colonies were confirmed by biochemical identification, including the sugar fermentation test, the indole production test, and the citrate test (16).

Antimicrobial susceptibility was tested by the agar disc diffusion method, following the Clinical and Laboratory Standards Institute guidelines (17). The antimicrobials used were tetracycline (TE; 30 μg), ampicillin (AM; 10 μg), levofloxacin (LEV; 5 μg), cephalothin (CF; 30 μg), cefotaxime (30 μg), gentamicin (GE; 10 μg), cefepime (30 μg), trimethoprim/sulfamethoxazole (25 μg), netilmicin (NET, 30 μg), amikacin (30 μg), ceftriaxone (30 μg), and streptomycin (STR, 30 μg) (Sensi-disc BBL). These antimicrobials represent the major classes of antimicrobial drugs relevant in both veterinary and human medicine. Each E. coli strain colony was evaluated based on the diameter of the clear inhibition zone, and the results were interpreted following the criteria provided by the CLSI. E. coli ATCC 25922 was used as a control.

Bacterial DNA was obtained from a pure culture on tryptic soy agar (BD Becton Dickinson & Co., Cuautitlán Izcalli, Mexico) by lysis of a bacterial cell suspension at 95°C for 15 min, followed by centrifugation at 13,000 × g for 3 min. Six antibiotic resistance genes were screened by PCR: genes encoding tetracycline efflux pump (tetA and tetB), streptomycin phosphotransferases (strA), aminoglycoside adenyltransferase (aadA), and beta-lactamase resistance (bla_TEM and bla_SHV) (18–20). PCR conditions were performed in a total volume of 25 μl containing 1× buffer, 25 mM MgCl₂, 10 mM dNTPs (Bioline), 10 mM of each primer, 5 U of Taq polymerase (Uniparts), and sterile water. PCR amplification was conducted following these conditions: initial denaturation at 95°C for 7 min, followed by 30 cycles of denaturation at 95°C for 45 s, annealing at 42 at 59°C for 45 s, and extension at 72°C for 45 s and a final cycle of amplification at 72°C for 7 min. The PCR products were analyzed by gel electrophoresis in 2.5% agarose (Sigma Aldrich, Germany), followed by visualization on a transilluminator.

The detection of virulence factor genes was performed according to the method described by Canizalez et al. (21). PCR was carried out using target gene-specific primers, including shiga toxin (stx1 and stx2), intimin (eaeA), bundle-forming pilus (bfp), and enterohemolysin A (hlyA). The PCR reaction mixture contained buffer 1X, MgCl₂ 25 mM, dNTPs 10 mM, primer 10 mM, and Taq DNA polymerase 5 U. The volume of this mix was adjusted to 25 μl with sterile water. The PCR amplification conditions were as follows: initial denaturation at 95°C for 1 min, followed by 30 cycles of denaturation at 95°C for 45 s, annealing at 54–60°C for 45 s, and extension at 72°C for 45 s and a final cycle of amplification at 72°C for 7 min. Verification of PCR products was performed in horizontal electrophoresis using 2.5% agarose gel with 0.5× TBE and Sybr gold at 100 V for 45 min. A molecular marker was run concurrently (100 pb Promega). The negative control consisted of all contents of the reaction mixture excluding template DNA, which was substituted with 1 μl sterile water. The DNA bands were visualized and photographed under UV light.

Out of 248 samples tested, E. coli strains were detected in 85.9% (213/248); 87.9% (109/124) were confirmed as E. coli strains in fecal samples, and 83.8% (104/124) were from carcass samples (Table 1).

Among the 213 E. coli isolates tested, 94.8% (202/213) showed resistance for at least one antimicrobial, mainly AM (83.0%; 177/213), CF (76.0%; 162/213), and TE (69.0%; 147/213) (Table 1). The percentage of resistance to the other antimicrobial agents was in all cases below 36%. The results showed resistance to AM in 68.8% of fecal strains (75/109) and 98.0% of carcass strains (102/104). Phenotypic resistance to CF E. coli was confirmed in 74.3% of fecal isolates (81/109) and 77.8% of carcass isolates (81/104). The isolates that showed resistance to TE were 71.5% of fecal strains (78/109) and 66.3% of carcass strains (69/104). The percentage of sensitivity to LEV (90.6%; 193/213), GE (85.9%; 183/213), and NET (80.7%; 172/213) shown in this work was higher than 80%. Fifty-seven antibiogram resistance patterns were detected in this study (46 fecal and 11 carcasses; Table 2). The detection of 72.7% (155/213) strains which showed a MDR phenotype (104 fecal and 82 carcasses) is remarkable.

Of the isolates, 19.2% (41/213) were negative for all the resistant genes tested. The tet(A) and tet(B) genes were detected in 43.1% of isolates (92/213; 35 fecal and 57 carcasses). Both tet genes were detected in 12.6% (27/213) of isolates (17 fecal and 10 carcasses). The strA and aadA1 genes were detected in 17.3% (37/213; eight fecal and 29 carcasses) and 51.6% (110/213; six fecal and 104 carcasses) of isolates, respectively. β-Lactamic genes were detected in 10.3% (22/213; 18 fecal and four carcasses) from bla_TEM and in 0.4% (1/213; one fecal and zero carcasses) from bla_SHV (Table 3).

Of the isolates, 14.0% (30/213) showed one or two genes encoding a virulence factor (stx1, stx2, or hlyA). The stx genes were carried by 11.2% (24/213; 12 fecal and 12 carcasses) of the studied isolates. From these stx positives, 4.2% (9/213; three fecal and six carcasses) were stx1, and 7.0% (15/213; nine fecal and six carcasses) were stx2. Only three strains showed both genes stx1 and stx2 (1.4%; 3/213). However, hlyA was detected in only 2.8% (6/213; four fecal and two carcasses). None of the isolates contained the virulence genes eaeA and bfp.