Genomic characterization of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) strains circulating in three university hospitals in Northern Italy over three years

Abstract

Objectives: Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment.

Methods: Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters.

Results: Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying bla_KPC-3 (48, 55.2%), bla_KPC-2 (38, 43.7%), and in one case (1.2%) bla_KPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p<0.001). While ST307 was associated with infection events, a trend associated with colonization events could be observed for ST20.

Conclusions: Integration of genomic, resistance score, and clinical data allowed us to define a relative diversification of KPC-Kp in Northern Italy between 2019 and 2021, characterized by few large transmission chains and rare inter-hospital transmission. Our results also provided initial evidence of correlation between KPC-Kp genomic signatures and higher MIC levels to some antimicrobial agents or colonization/infection status, once again underlining WGS's importance in bacterial surveillance.

Keywords: K. pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp); Antimicrobial resistance (AMR); Genomic epidemiology; WGS.

Fig. 1

Estimated Maximum Likelihood phylogenetic analysis of KPC-Kp (N=87) isolated from three hospitals in Northern Italy and reference genomes (N=16). The Maximum Likelihood tree was inferred from a core-SNP alignment of 25,322 bp. The phylogeny was estimated with IqTree using the best-fit model of nucleotide substitution GTR+F+G4 with 1,000 replicates and fast bootstrapping. The numbers on the leaves represent the sample IDs. Reference genomes are displayed in grey. Bootstrap values higher than 90 are displayed on branches. Information regarding samples is annotated: hospital of origin, isolation date, isolation site, sequence type (ST), capsular locus (K locus), lipooligosaccharide locus (O locus), and the presence (solid figures) or absence of antimicrobial resistance genes, virulence factors and transposons, number of plasmids and bacteriophages, colonization, or infection status

Fig. 2

Minimum Spanning Trees (MSTs) of the pairwise SNP distance of strains within the 5 predominant STs. Branches display the pairwise SNP distances, while nodes report the strains ID and are colored based on the hospital of origin, blue= P, green=H, salmon=S. Strains colored in red are strains described in a recent Italian outbreak [22]. A) ST307, constructed on a coreSNP of 773 bp; B) ST512/1519, constructed on a coreSNP of 489 bp; C) ST20, constructed on a coreSNP of 587 bp; D ST101, constructed on a coreSNP of 271 bp; E) ST258, constructed on a coreSNP of 229 bp