Multidrug resistance, biofilm formation, and virulence genes of Escherichia coli from backyard poultry farms

Background and Aim: Backyard chicken flocks have traditionally been regarded as an essential food source in developed countries; however, they may act as reservoirs and spread various zoonotic bacterial pathogens. This study was designed to investigate the prevalence, phenotypic resistance, biofilm formation (BF), and pathotypes of Escherichia coli isolates from backyard poultry farms. Materials and Methods: Cloacal swabs (n=150) and internal organs (n=150) were collected from 30 backyard chicken flocks; 20 of them were experiencing systemic infection, and the other ten were apparently healthy. Samples were bacteriologically examined for E. coli isolation. Isolates were identified biochemically by the VITEK® 2 COMPACT system (BioMérieux, France). For molecular identification, 16S rRNA was amplified and sequenced. Ten antimicrobials were selected for E. coli antimicrobial susceptibility testing. The minimum inhibitory concentration for each antimicrobial was determined. The extended-spectrum β-lactamase activity in isolates was investigated using cephalosporin/clavulanate combination disks. The ability of isolates for BF was determined by the microtiter plate method. Thirteen virulence genes linked to different E. coli pathotypes and two serotype-related genes were investigated by real-time polymerase chain reaction. Results: Eighty-six E. coli strains were isolated from 30 backyard chicken flocks. The isolates were biochemically identified to the species level. Genetically, sequences of the 16S rRNA gene showed >98% identity with E. coli in the National Center for Biological Information database. The frequency of isolation from diseased flocks was significantly higher (p<0.05) than apparently healthy flocks; 63.9% of the isolates were recovered from cloacal swabs and 36.04% were recovered from internal organs. E. coli isolates showed high resistance to ampicillin (AMP; 75.6%), gentamicin (39.5%), and tetracycline (29.1%). However, none of the isolates were resistant to imipenem. A variable drug resistance profile for E. coli isolates was reported. Twenty-one (24.4%) isolates were sensitive to all ten antimicrobials. Seven (8.1%) isolates were resistant only to AMP, and 28 (32.6%) were resistant to two antimicrobials, whereas the remaining 30 (34.9%) isolates showed multidrug resistance (MDR). Of the 86 isolates, 8 (9.3%) were confirmed as extended-spectrum β-lactamase (ESBL)-producing E. coli by the combination disk diffusion method. All ESBL isolates were MDR with an MDR index of 0.5-0.6. Fifty-seven (66.3%) isolates were capable of forming biofilms; 22 (25.6%) of them were strong biofilm producers, 24 (27.9%) moderate producers, and 11 (12.8%) weak producers. A statistically significant pairwise correlation was obtained for MDR versus BF (r=0.512) and MDR index versus BF (r=0.556). Based on virulence gene profiles, five pathotypes were identified, including enteropathogenic E. coli (39.5%), avian pathogenic E. coli (32.53%), enterohemorrhagic E. coli (EHEC; 9.3%), enterotoxigenic E. coli (ETEC; 5.8%), and enteroaggregative E. coli (EAEC; 1.2%). The lower frequency of EAEC and ETEC was statistically significant than other pathotypes. Three isolates were identified as O157 based on the detection of the rbfO157 gene. Conclusion: This study reported a high prevalence of MDR, suggesting the misuse of antimicrobials in backyard chicken farms. The emergence of ESBL and EHEC isolates in backyard chickens is a public health concern. Furthermore, the backyard flocks environment may harbor different pathogenic bacteria that may enhance the persistence of infection and the transmission to in-contact humans. Regular monitoring for the occurrence of MDR and the zoonotic pathotypes among E. coli in backyard chicken flocks is recommended, as these bacteria can transmit to humans through food products or contaminated environments.