Deadly Puppy Infection Caused by an MDR Escherichia coli O39 bla CTX-M-15, bla CMY-2, bla DHA-1, and aac(6)-Ib-cr - Positive in a Breeding Kennel in Central Italy

Abstract

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.

Keywords: CMY-2; CTX-M-15; DHA-1; E. coli; plasmids; whole genome sequencing.

FIGURE 1

Circular map of pIV_IncI1_CMY-2 plasmid. Green arrow represent conjugal transfer system of the plasmid, red arrow represents bla_CMY–2, yellow arrows represent mobile elements, white arrows represent hypothetical proteins, purple arrow represents the replication protein rep_IncI1, and gray arrows represent plasmid partitioning proteins. Furthermore, the black-arced box represents the plasmid antimicrobial resistance island (ARI).

FIGURE 2

Linear map of pIV_IncFII_DHA. Arrows show the direction of transcription of ORFs while rectangles show truncated ORFs. Replicons, partitioning genes, mobile elements, conjugal transfer genes, antibiotic resistance, bla_DHA–1, and other remaining genes are designated by violet, gray, yellow, green, red, pink, and white, respectively. Antimicrobial resistance island (ARI) is marked by a horizontal black line.

FIGURE 3

Linear map of pIV_IncHI2_CTX_M_15. Arrows show the direction of transcription of ORFs while rectangles show truncated ORFs. Replicons, partitioning genes, mobile elements, conjugal transfer genes, antibiotic resistance, stability genes, and other remaining genes are designated by violet, gray, yellow, green, red, orange, and white, respectively. Antimicrobial resistance island (ARI) is marked by a horizontal black line.