Deadly Puppy Infection Caused by an MDR Escherichia coli O39 blaCTX–M–15, blaCMY–2, blaDHA–1, and aac(6)-Ib-cr – Positive in a Breeding Kennel in Central Italy

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired β-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring bla TEM–1b , bla CTX–M–15 , bla OXA–1 , aph(6)-Ib , aac(6′)Ib-cr , aac(3)-Ila , aph(6)-Id , aadA1 , qnrB1 , sul2 , catA1 , catB3 , tetA , and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, bla DHA–1 , qnrB4 , mph(A) , sul1 , and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A bla CMY–2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA , iss , astA , and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 bla CTX–M–15 , bla CMY–2 , bla DHA–1 , and aac(6′)-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir.