Valutatore di Tesi di Dottorato in Veterinary and Animal Science Class XXXVII - Department of Veterinary Medicine and Animal Sciences - Università degli Studi di Milano: 'Extended-spectrum β-lactamase-, AmpC- and carbapenemase-producing bacteria in dogs and cats'

Antimicrobial resistance (AMR) is a critical global health challenge, compromising the efficacy of antibiotics and threatening both human and animal health. The rise of multidrug-resistant (MDR) bacteria compromises the efficacy of treatments for infections in humans and animals, leading to prolonged illnesses, increased mortality, and substantial economic burdens. Companion animals are increasingly recognized as reservoirs and potential sources of antimicrobial-resistant pathogens, contributing to the dissemination of resistance genes across ecosystems and posing a zoonotic risk. Understanding the prevalence, genetic mechanisms, and risk factors associated with AMR in dogs and cats is essential for the development of effective control strategies within a One Health approach. This thesis primarily focused on fecal shedding of extended-spectrum β-lactamase (ESBL)-, AmpC-, and carbapenemase-producing Escherichia coli, considered a key indicator of antimicrobial resistance in bacterial populations. Fecal samples from dogs and cats, including both pets and stray animals were investigated for the presence, genetic determinants, and associated risk factors of these bacteria. An additional investigation focused on dogs with urinary tract infections and analyzed ESBL-, AmpC-, and carbapenemase-producing E. coli as well as Klebsiella pneumoniae, a clinically significant pathogen often linked to resistance to the highest-priority critically important antimicrobials, along with other AMR mechanisms such as biofilm formation in these bacteria isolated from urinary samples. This thesis is based on four studies. The first study focused on cats, which have been studied less frequently than dogs, and investigated the presence of ESBL/AmpC-producing E. coli in both pet and stray cats. Fecal samples from 97 cats revealed that six (6.2%) carried these resistant bacteria. Genetic analysis detected blaCTX-M in all isolates, along with blaTEM (83.3%),blaSHV (16.7%), and chromosomal AmpC over expression (1%). All isolates were multi-drug resistant (MDR). Statistical analysis demonstrated significant associations between ESBL/AmpC carriage and unhealthy status or previous antibiotic therapy, underscoring the potential role of cats as reservoirs for resistant bacteria. The second study focused exclusively on stray cats and included the study of carbapenemase producing E. coli, due to their importance according to the One Health concept. Among the 94 stray cats analyzed, 18 (19.1%) tested positive for ESBL-/AmpC-/carbapenemase-producing E. coli. Notably, besides ESBL-/AmpC-producing E. coli, this study showed the presence of carbapenemase genes blaNDM and blaOXA-48 in seven (7.4%) samples. Among the isolates analyzed, 17 (94.4%) were identified as MDR. Hospitalization, prior antibiotic treatment, and unhealthy status were identified as significant risk factors. Moreover, ESBL-producing E. coli were detected in healthy stray cats which had not been hospitalized and had likely not been previously treated. Overall results highlight the complex epidemiology of AMR in stray animals suggesting the need for further investigation on the source of infection also focusing on environmental contamination. Carbapenemase-producing E. coli fecal carriage was further investigated in the third study that focused on dogs, in which carbapenemase-producing bacteria were less frequently studied. The fecal carriage of ESBL-/AmpC-/carbapenemase-producing E. coli was detected in 14/100 (14%) dogs, with 92.9% of isolates exhibiting MDR. Genetic analysis identified blaCTX-M in ESBL-producing isolates, blaCMY-2 in AmpC-producing isolates, and blaOXA-48 in carbapenemase-producing E. coli. Antibiotic treatment was confirmed as a significant risk factor for fecal shedding of these bacteria, emphasizing the need for antimicrobial stewardship in veterinary medicine. The fourth study investigated AMR bacteria in dogs with bacterial urinary tract infections (UTIs) focusing on the detection of ESBL-/AmpC-/carbapenemase-producing E. coli and K. pneumoniae, including evaluation of biofilm production of isolates, a factor that contributes to bacterial persistence and resistance. Among 133 urine samples analyzed, 53 (39.8%) were culture-positive. E. coli was the predominant pathogen (28; 21.1%) and six (4.5%) samples tested positive for K. pneumoniae. ESBL production, driven by blaCTX-M group 1, was detected in 4/34 (11.8%) E. coli/K. pneumoniae isolates and was significantly associated with K. pneumoniae. Furthermore, 11 (32.4%) isolates exhibited MDR. Biofilm formation was observed in 23 (67.6%) isolates, with moderate biofilm production significantly associated with K. pneumoniae. These findings emphasize the need for in vitro susceptibility testing and careful antibiotic use in treating canine UTIs. Overall, these studies underline the critical role of companion animals in the spread of antimicrobial-resistant bacteria. The detection of MDR and carbapenemase-producing E. coli is of concern and reinforces the need for enhanced surveillance programs and antimicrobial stewardship within a One Health framework, evaluating the risk of transmission to humans due to the close interaction of pets with their owners. Addressing AMR in companion animals is essential to safeguard both veterinary and public health, reinforcing the need for a collaborative One Health approach.