Phenotypic and Genetic Characterization of Klebsiella pneumoniae Isolates from Wild Animals in Central Italy

SIMPLE SUMMARY: Klebsiella pneumoniae is an opportunistic bacterium known as a nosocomial pathogen, but it is able to colonize a multitude of ecological niches, both free-living and host-associated. For this reason, the presence of antimicrobial resistance genes in wildlife is an indicator that resistant bacteria of human- or livestock-origin are widespread in the environment. Wild animals can act as efficient reservoirs and epidemiological links between humans, livestock, and natural environments. Despite the renewal of interest in epidemiology, there is a critical lack of an unbiased ecological approaches to defining reservoirs and sources of infections. The aims of the present study were to investigate the occurrence of K. pneumoniae in wildlife, to characterize antimicrobial features through phenotypic and whole genome sequencing approaches, and to identify similar available sequences that could suggest a transmission event. ABSTRACT: Despite Klebsiella pneumoniae being widely recognized as a nosocomial pathogen, there is a critical lack in defining its reservoirs and sources of infections. Most studies on risk factors have focused on multidrug-resistant (MDR) isolates and clinically-oriented questions. Over a two-year period, we sampled 131 wild animals including mammal and bird species from three regions of Central Italy. All typical colonies isolated from the analytical portions were confirmed by real-time PCR and identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). All confirmed K. pneumoniae isolates were tested for antimicrobial susceptibility to 29 antimicrobials and subjected to whole genome sequencing. Typical colonies were detected in 17 samples (13%), which were identified as K. pneumoniae (n = 16) and as K. quasipneumoniae (n = 1) by MALDI-TOF MS. The antimicrobial susceptibility profile showed that all the isolates were resistant to β-lactams (ceftobiprole, cloxacillin, cefazolin) and tetracycline; resistance to ertapenem and trimethoprim was observed and nine out of 16 K. pneumoniae isolates (56.2%) were classified as MDR. Genomic characterization allowed the detection of fluoroquinolone resistance-associated efflux pumps, fosfomycin and β-lactamase resistance genes, and virulence genes in the overall dataset. The cluster analysis of two isolates detected from wild boar with available clinical genomes showed the closest similarity. This study highlights the link between humans, domestic animals, and wildlife, showing that the current knowledge on this ecological context is lacking and that the potential health risks are underestimated.