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

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.

Keywords: K. pneumoniae; antimicrobial resistance; whole genome sequencing; wildlife.

Figure 1

K. pneumoniae positive (red) and negative (green) samples isolated from wild animals in the Abruzzo, Molise, and Lazio regions.

Figure 2

Heatmap encompassing antimicrobial susceptibility testing results of K. pneumoniae strains (blue) and K. quasipneumionae strain (green). Red color indicates resistance, yellow indicates an intermediate phenotype, and white indicates susceptibility. AMP: ampicillin, POD: cefpodoxime, CD: cefpodoxime-clavulanic acid, P/T4:piperacillin-tazobactam constant 4, FOX: cefoxitin, BPR: ceftobiprole, AZT: aztreonam, ETP: ertapenem, MERO: meropenem, AMI: amikacin, GEN: gentamicin, NET: netilmicin, TOB: tobramycin, KAN: kanamycin, CIP: ciprofloxacin, NAL: nalidixic acid, TMP: trimethoprim, SXT: trimethoprim-sulfamethoxazole, CLO: cloxacillin, TET: tetracycline, A/S2: ampicillin-sulbactam, FAZ: cefazolin, FEP: cefepime, TAZ: ceftazidime, AXO: ceftriaxone, FUR: cefuroxime, TIM2: ticarcillin-clavulanic acid constant 2, TGC: tigecycline.

Figure 3

Neighbor joining (NJ) tree resulting from the cgMLST analysis performed on the 16 K. pneumoniae draft assemblies according to the Pasteur Institute’s scheme (629 loci). The tree is annotated with metadata (source and year of isolation), antimicrobial resistance gene (blue color indicates the presence), virulence score, virulence genes (red color indicates the presence), and plasmid (grey color indicates the presence). For novel sequence types we attributed the symbol “*”.

Figure 4

Cluster analysis performed with public genomes available on Pathogenwatch platform. (a) Core-genome MLST (cgMLST) analysis of the isolate 2021.TE.17 with 45 available clinical genomes belonging to the ST23. In the red circle are highlighted the ST23 public genomes carrying carbapenemase resistance genes. (b) cgMLST analysis of isolate 2021.TE.18 with four clinical genomes (ERR1217151, SRR5386627, SRR10058597, and ERR1541571). In the branches it is reported the numbers of allelic differences.

Figure 4

Cluster analysis performed with public genomes available on Pathogenwatch platform. (a) Core-genome MLST (cgMLST) analysis of the isolate 2021.TE.17 with 45 available clinical genomes belonging to the ST23. In the red circle are highlighted the ST23 public genomes carrying carbapenemase resistance genes. (b) cgMLST analysis of isolate 2021.TE.18 with four clinical genomes (ERR1217151, SRR5386627, SRR10058597, and ERR1541571). In the branches it is reported the numbers of allelic differences.