Multidrug Resistance and Production of Extended Spectrum β-lactamases and Plasmid-mediated AmpC β-lactamases in Enterobacteriaceae isolates from diseased cats in Sicily

Introduction: In human and veterinary medicine, Enterobacteriaceae are common causes of enteric and extra-intestinal opportunistic infections and their resistance to multiple antimicrobials is a major global threat. Multidrug-resistant (MDR) Enterobacteriaceae are increasingly reported in companion animals, thus raising great concerns for animal and public health (Bogaerts et al., 2015). The β-lactam resistance in Enterobacteriaceae is associated mainly with production of enzymes hydrolyzing these antibiotics, among which the extended-spectrum β-lactamases (ESBLs), Plasmid-mediated AmpC β-lactamases (pAmpC) and carbapenemases are the most important resistance mechanisms (Rubin and Pitout, 2014). Objectives: This study aimed to investigate the antimicrobial resistance of Enterobacteriaceae isolates from a Sicilian population of cats affected by diseases commonly encountered in practice, with emphasis on multidrug resistance, and to detect the occurrence of ESBLs and Plasmid-mediated AmpC β-lactamases pAmpC producers. Materials and Methods: Clinical samples were collected from n=101 cats affected by several clinical conditions (58.4% diarrhoea, 30.7% rhinitis, 3.9% otitis, 2.9% conjunctivitis, 1% abscess, 2% stomatitis, 1% cystitis). Bacterial susceptibility testing to n=8 antimicrobial classes and interpretation were performed according to EUCAST clinical breakpoints (EUCAST, 2015). ESBLs and pAmpC genes were identified by PCR and DNA sequencing. Phylogenetic groups of Escherichia coli (E. coli) harbouring resistance genes were determined according to Doumith et al. (2012). Results: A total of n=125 Enterobacteriaceae were isolated from n=90 cats. E. coli (52%) was the most frequently isolated, followed by Enterobacter spp. (16%), Proteus spp. (10%) and Citrobacter spp. (10%). The higher prevalences of resistance among isolates were against amoxicillin-clavulanic acid (49%) and third-generation cephalosporines (40%). Although lower, resistance to aztreonam (32%), ciprofloxacin (23%), amikacin (31%), chloramphenicol (24%) and sulphamethoxazole –thrimetoprim (37%) were also significant, whereas all isolates were susceptible to meropenem. Forty-five percent (n=56) of isolates were multidrug-resistant, showing n= 29 different MDR profiles, and were isolated from the 47% (n=42) of cats. PCR and DNA sequencing confirmed a total of n= 26 MDR isolates as ESBLs/pAmpC β-lactamases producers, representing the 21% of total isolates and recovered from the 20% (n =18) of cats, affected by diarrhoea, rhinitis, abscess, otitis, stomatitis and cystitis. Twenty-three isolates were confirmed as ESBLs-producers, harbouring several bla genes, namely: blaCTX-M-group1 (n=12), -group2 (n=1) and –group9 (n=1); blaSHV (n=1), blaTEM (n=8) and blaOXA-1 (n=6). Ten isolates were pAmpC blaCMY-producers, with n=7 isolates also harbouring blaTEM (n=4), blaCTX-M (n=2) and blaOXA-1 (n=1). ESBL/pAmp C-producing E. coli (n=12) belonged to phylogenetic groups B2 and D and were collected from n=6 diarrheic cats, n=1 cat with rhinitis, n=1 with cystitis and n=1 with otitis. Two MDR non β-lactamase producing E. coli belonged to phylogenetic groups B2 and D as well and were isolated from n=1 cat with rhinitis and n=1 cat with diarrhea. Six E. coli belonged to phylogenetic groups A and B1and were isolated from n=3 cats with rhinitis, n=1 cat with diarrhea, n=1 with abscess and n=1 with stomatitis. One MDR non β-lactamase producing E. coli belonged to phylogenetic group B1 was isolated from n=1 cat with diarrhea. Discussion and Conclusions: This study showed the prevalence of MDR and β-lactamases producing Enterobacteriaceae isolated in a variety of common clinical conditions in a feline population in Southern Italy, with a high degree of diversity between antimicrobial resistance profiles. To the best of knowledge, occurrence of MDR ESBLs/pAmpC producing E. coli in cats affected by rhinitis and detection of gene blaCTX-M-79 in a member of Enterobacteriaceae isolated from companion animals are described for the first time in literature. The emergence of ESBL/pAmpC-producing MDR Enterobacteriaceae poses major limitations in companion animals’ therapeutic options. Furthermore, it raises great concerns regarding the bi-directional transmission of MDR bacteria between pets and humans, and awareness should be raised among companion animal practitioners. Resort to appropriate bacteriological isolation, identification and susceptibility testing is essential to address antimicrobial treatment of commonly encountered bacterial infections. This could avoid the resort to ineffective compounds, thus reducing selective pressure excerted by antimicrobials on resistant strains, helping the control and monitoring of antimicrobial resistance in companion nimals’medicine.