METHODS: Escherichia coli was isolated from 453 collected samples, including 210 cloacal swabs and 243 environmental samples. The antimicrobial susceptibility profile of the E. coli isolates was assessed for sixteen antibiotics using the disc diffusion method. The E. coli isolates were evaluated for phenotypic ESBL production using modified double disc synergy. After extraction of genomic DNA, ESBL resistance genes, phylogenetic group, and virulence genes were detected by PCR using appropriate primers. ESBL genes were further confirmed by sequencing. The molecular typing of E. coli strains was determined by Multilocus Sequence Typing (MLST).

RESULTS: A total of 93.8% (425/453) E. coli were isolated from the collected samples. Out of 334 E. coli isolates screened, 14.7% (49/334) were phenotypically ESBL producers. All the ESBL-EC were resistant to tetracycline, ciprofloxacin, and ampicillin. Thus, 100% of the ESBL-EC were multidrug resistant. Of the ESBL-EC 81.6% were positive for at least one ESBL encoding gene. The most prevalent ESBL gene detected was blaTEM (77.6%; 38/49) followed by blaCTX-M (32.7%; 16/49) and blaSHV (18.4%; 9/49). The majority of ESBL-EC belonged to phylogenic groups A followed by B1 accounting for 44.9% and 12.2%, respectively. The most frequently identified sequence types were ST10 (n = 3) and ST206 (n = 3). The most detected virulence genes in the E. coli isolates were astA (33.3%; 22/66) followed by iss (15.2%; 10/66).

RESULTS: Four hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 μg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia.