Multilevel surveillance of antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae from companion animals: Evidence from a Veterinary University Hospital before and after EU Regulation 2022/1255

Antimicrobial resistance (AMR) is a major One Health challenge requiring integrated surveillance across human and veterinary medicine. This study presents a multilevel surveillance of AMR in Escherichia coli and Klebsiella pneumoniae in an Italian Veterinary University Hospital over 54 months, evaluating temporal trends in relation to internal interventions and the implementation of the EU Regulation 2022/1255. Passive surveillance of clinical isolates from dogs and cats was integrated with active surveillance of patients' intestinal microbiota and the hospital environment, molecular detection of resistance genes, and monitoring of injectable antimicrobial use (AMU). Resistance patterns, directly attributable mortality, carriage and environmental presence of third-generation cephalosporin-resistant (3GCR) and carbapenem-resistant (CR) E. coli and K. pneumoniae and AMU were analyzed over nine consecutive semesters. K. pneumoniae represented a minority of clinical isolates (9.4% n = 80/847) but exhibited significantly higher resistance rates than E. coli. Resistance to ampicillin, enrofloxacin, and piperacillin-tazobactam, as well as simultaneous resistance to class B and class A antimicrobials, showed significant decreasing trends over time. High frequencies of 3GCR and CR carriage were detected in patients' intestinal microbiota, particularly at discharge/death, though both declined significantly during the study period. Environmental contamination remained low. Piperacillin-tazobactam use progressively declined and ceased after the regulation, while ampicillin-sulbactam use increased, suggesting a substitution effect. Interrupted time-series analyses indicated that most favorable trends were already established before regulation, which appeared to consolidate the improvements. Overall, the findings support the value of continuous multilevel AMR surveillance combined with stewardship and regulatory measures in veterinary healthcare settings.