Genomic Characterization, Antimicrobial Resistance Profiles, and tetA Nucleotide Substitutions of Escherichia coli Isolated from Healthy Dogs in Thailand

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) from companion dogs is an emerging One Health concern because dogs may serve as asymptomatic reservoirs of multidrug-resistant (MDR) and potentially pathogenic bacteria that can be transmitted to humans. This study aimed to determine antimicrobial susceptibility profiles, characterize antimicrobial resistance and virulence associated genes, and identify nucleotide substitutions in resistance determinants of E. coli isolated from healthy dogs in Thailand. Fecal samples (n = 200; 100 samples per province) were collected from healthy dogs attending a total of 50 small animal clinics located in Nakhon Si Thammarat and Chiang Mai provinces between January and March 2026. E. coli isolates were identified by culture and polymerase chain reaction (PCR) confirmation. Antimicrobial susceptibility was evaluated against 12 antimicrobial agents using the disk diffusion method according to CLSI guidelines. Thirty randomly selected isolates were further screened for resistance and virulence-associated genes by PCR, and the tetA gene was sequenced to identify nucleotide substitutions. Differences in gene distributions between provinces were assessed using Fisher’s exact test. A total of 66 E. coli isolates were recovered. High resistance rates were observed for ampicillin (100%), piperacillin (84.85%), ceftriaxone (60.61%), tetracycline (56.06%), and aztreonam (46.97%). Multidrug resistance was identified in 47% of the isolates. The 30 isolates exhibiting the highest levels of antimicrobial resistance were selected for molecular characterization. Among these, the β-lactam resistance gene blaTEM was significantly more prevalent in Nakhon Si Thammarat than in Chiang Mai (60% vs. 0%; p = 0.022). The virulence gene stx2 was also detected significantly more frequently in isolates from Nakhon Si Thammarat (93.33% vs. 26.67%; p < 0.001). Sequence analysis of tetA revealed multiple nucleotide substitutions in two isolates, suggesting ongoing genetic variation within tetracycline resistance determinants that may contribute to the evolution and persistence of antimicrobial resistance. These findings demonstrate that healthy dogs in Thailand can act as reservoirs of MDR and potentially virulent E. coli. The observed geographic variation in resistance and virulence gene distributions highlights the importance of antimicrobial stewardship and continuous molecular surveillance in companion animals within a One Health framework.