Escherichia coli (E. coli) O157 and non-O157 among ruminants in Peninsular Malaysia: pathogenicity, antibiotic resistance, and survival under sub-lethal stress conditions.

Pathogenic Escherichia coli (E. coli) are a common cause of diarrhoea and a broad range of extra-intestinal diseases in humans. Among these pathogenic strains, Shiga toxin producing E. coli (STEC), has emerged as one of the most virulent virotypes associated with cases of food borne disease in humans. Ruminants are considered an important reservoir of these pathogens. In Malaysia, data on the prevalence and characterisation of E. coli O157 and the non-O157 serogroups in ruminants is limited. Thus, E. coli including STEC O157 and serogroups of non-O157 STEC were isolated and characterised based on their virulence, antibiotic resistance and survival under sub-lethal heat, cold and acid stress conditions. A total of 136 ruminant feces samples were collected from six different farms in Peninsular Malaysia for the isolation and characterisation of E. coli O157 and the non-O157 serogroups O26, O103, O111, O121, O45 and O145. STEC O157:H7 was isolated from six (4.4 %) samples, from which 32 O157:H7 strains were obtained. All 32 STEC O157:H7 strains from this study were motile, carried stx2c, eaeA-γ1 and ehxA, belonged to the less virulent lineage II, contained an occupied sbcB locus, and were negative for Stx production. Non-O157 STEC was isolated from 2 (1.5 %) samples, from which 2 non-O157 strains of unknown serotype were obtained. One of the STEC non-O157 strains carried stx1a, stx2a, stx2c, and ehxA and produced moderate amounts of Stx, while the other carried stx1a alone and the production of Stx was below the level of detection. A total of six Shiga toxin producing E. coli strains including two STEC O157 and one non-O157 STEC strain obtained from Malaysia were used in the isolation of stx bacteriophage and subsequent infection of non-STEC. However, no infection was demonstrated from any of the induced stx bacteriophage from the STEC strains. In addition to the STEC strains, a total of 161 non-STEC strains including ten strains of serogroup O157, ten strains of serogroup O103 and five strains of serogroup O26 were obtained from the 136 ruminant feces collected. Of these E. coli strains, a total of 153 strains representing each feces sample and each serougroup obtained were used to determine antibiotic resistance. Analysis of antibiotic resistance among these E. coli indicated that complete resistance was most common for more traditional antibiotics such as tetracycline (11/153, 7.2 %), trimethoprim (6/153, 3.9 %), ampicillin (5/153, 3.3 %), and streptomycin (3/153, 2.0 %), while they were completely susceptible to more modern antibiotics such as cefuroxime and cetazidime. Multi-antibiotic resistance was observed in only 5.9 % (9/153) of the strains. Plasmid mediated resistance was apparent for tetracycline – tet(A), trimethoprim – dhfr I, V, VII, and XIII, ampicillin and cephalothin – blaTEM, streptomycin – aadA and strA–strB, and chloramphenicol – floR and cmlA. Conjugation assays with selected strains indicated the transmissibility of antibiotic resistance determinants such as ampicillin, tetracycline, trimethoprim and streptomycin. The effect of sub-lethal heat, cold and acid adaptation stress on the survival of two multi-antibiotic resistant E. coli strains, one positive (EC27) and the other negative for stx (EC135), in subsequent acidic conditions mimicking the acidic environment in the human stomach was also investigated. Acid adaptation and cold-stress adaptation of strain EC27 increased its susceptibility to subsequent acid stress, while strain EC135 was susceptible to acid stress regardless of pre-exposure to acid and cold stress. Heat shock treatment did not have any impact on the survival of both strains under post acid stress conditions. The results of this study highlight that ruminants in Malaysia are a potential source of STEC and a significant reservoir of antibiotic resistance determinants with the propensity of antibiotic resistance dissemination. However, the presence of STEC O157 and non-O157 in a small percentage of ruminants in this study together with their virulence characteristics suggests that they may have limited impact on public health. Moreover, the lack of E. coli strains with resistance towards more modern antibiotics such as cefuroxime and ceftazidime and the reduced survival observed from the multi-antibiotic resistant E. coli strains under severe stress conditions further indicates their low pathogenic potential towards humans.