Klebsiella are bacterial pathogens that can bring about a selection of severe bacterial infections in humans, primarily due to K. pneumoniae (KP) [1], [two] and to a lesser diploma to K. oxytoca (KO) [3], [4]. KP is also a properly-recognized causal agent of mastitis in cattle and bacteraemia in calves, cervicitis and metritis in mares, pneumonia and septicemia in foals, pneumonia, urinary tract an infection (UTI) and septicemia in canines [five], [6], [7]. Increasing antimicrobial resistance, especially to aminoglycosides, (fluoro)quinolones, third and fourth generation cephalosporins, cephamycins, and carbapenems have been described in the final 10 years [eight], [nine], [ten], and poses serious therapeutic problems when dealing with Klebsiella bacterial infections in individuals. In veterinary medicine, scarce facts is documented on the incidence of extended spectrum beta-lactamases (ESBLs), AmpC beta-lactamases and plasmid mediated quinolone resistance (PMQR) in Klebsiella isolates from companion animals [11], [twelve]. The purpose of the review was to offer molecular characterization of extended-spectrum cephalosporin (ESC) resistance and PMQR in Klebsiella isolates from clinical instances or lesions in necropsied animals of canine and feline origin in Italy. A additional aim was to ascertain phenotype and genotype of co-resistances, and to provide plasmid identification and genetic relatedness by Multilocus Sequence Typing (MLST) and Pulsed Subject Gel Electrophoresis (PFGE) among the isolates, to examine possible clustering of ESC, PMQR, and other resistance genes amongst clones.
In between 2006 and 2012, the Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana (IZSLT) investigated samples from 1555 dogs and 429 cats of medical instances and necropsy specimens with suspicious bacterial bacterial infections, submitted by veterinarians practising mostly in central Italy, and some practising in northern Italy. Presumptive optimistic Klebsiella isolates had been determined making use of the API 20E identification method (bioMer?ieux, Craponne, France). Antimicrobial susceptibility screening was done as minimal inhibitory concentrations (MIC) by micro-broth dilution in 96-well microtitre plates (Trek Diagnostic Devices, Westlake, OH, United states). The next antimicrobials were tested: ampicillin, cefotaxime, ceftazidime, ciprofloxacin, chloramphenicol, florfenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfonamides, tetracycline, and trimethoprim. The effects had been interpreted according to the European Committee on Antibiotic Susceptibility Testing (EUCAST) epidemiological minimize-offs (www.eucast.org) and to Medical Laboratory Regular Institute [16] or EUCAST medical breakpoints for individuals medicine for which epidemiological cutoffs have not been produced readily available (kanamycin, chloramphenicol, sulfamethoxazole, trimethoprim). For streptomycin, a slice-off of sixteen mg/L was used, according to EUCAST MIC distributions. Confirmatory examination for the detection of ESBLs were being executed on isolates resistant to cefotaxime or ceftazidime according to Clinical Laboratory Standard Institute (CLSI) tips [16].
The 15 KP isolates investigated by MLST ended up assigned to four different Sequence Forms (ST): ST11 (n = 1), ST340 (n = two), ST101 (n = eight), and ST15 (n = four) (Determine 1). ST11 and its singlelocus (tonB) variant (SLV) ST340 (3/15, twenty%), each belonging to CC11, had been detected in 2012. The separation of the isolates based mostly on MLST corresponded properly with PFGE benefits grouping the identical isolates (Determine 1). A total of eleven distinct PFGE designs were being observed which includes two clusters of two and 4 indistinguishable isolates, respectively (Figure 1). The cluster of the two isolates equally belonged to ST340 and was connected (80% similarity) to a one isolate exhibiting a unique PFGE pattern and belonging to ST11. The other cluster of 4 indistinguishable isolates was extremely relevant (from 99% to 80% similarity) to more four isolates in the identical PFGE team, all belonging to ST101 (Determine 1). No clustering was noticed related to time, animal origin, nor infection, but some to the existence of resistance genes (Table 1). No MLST was assigned to the 4 KO isolates. However, the 4 isolates revealed 3 distinct PFGE patterns of which one was a cluster of two equivalent isolates (Figure 2). The 3 patterns appeared not to be linked, indicating a similarity of forty five% and fifty five% to the pattern of the two clustering isolates. Curiously, the two isolates of the identical PFGE sample had been the two from puppies and isolated within just the similar calendar year, but it could be the final result of a random impact (Determine 2).
