Genomic epidemiology of Klebsiella pneumoniae in Italy and novel insights into the origin and global evolution of its resistance to carbapenem antibiotics

Abstract

Klebsiella pneumoniae is at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated with K. pneumoniae infections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniae strains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology of K. pneumoniae in Italy. Thirty-one strains were carbapenem-resistant K. pneumoniae carbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity of K. pneumoniae worldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼ 1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group of K. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding of K. pneumoniae evolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

FIG 1

Maximum likelihood phylogeny of Klebsiella pneumoniae, based on 319 genomes. The phylogeny was reconstructed starting from an alignment of 94,812 core SNPs, using the software RAxML with a generalized time-reversible (GTR) model and 100 bootstraps, which are not shown for the sake of figure clarity. (A) Circular representation of the phylogeny, obtained using iTOL (itol.embl.de), ignoring branch length. Color circles indicate, from the innermost to the outermost, presence/absence of KPC variants, geographic location in terms of continents, ST based on multilocus sequence typing, and presence in the genome of genes from four beta-lactamase families. The red arrow indicates the origin of the clonal complex 258 clade. (B) Unrooted representation of the phylogeny showing the branch lengths, highlighting the genetic uniformity of clonal complex 258.

FIG 2

Uneven clustering of core SNPs in the clonal complex 258 clade. The phylogenetic reconstruction of the 206 representatives of the clonal complex 258 clade is shown on the left, while the core SNP frequency is shown on the right in shades of red, representing the number of core SNPs per 1,000-bp window for each genome. Detected recombinations are indicated at the top of the figure, and main clades of the clonal complex are indicated on the right side of the figure.

FIG 3

Hypothesis of recombinations occurring in the clonal complex 258 clade. Schematic representation based on the results of the analyses presented. Main nodes of interest are shown, highlighting the hypothesized pattern of three recombination events leading to the current state of clonal complex 258. Dates are inferred based on the molecular clock analysis depicted in Fig. 4.

FIG 4

Estimation of divergence times in clonal complex 258. A schematic version of the time-scaled phylogeny was obtained using BEAST software with an uncorrelated log-normal relaxed clock and GTR model with no correction for site rate heterogeneity. The analysis was run for 1,000,000,000 steps, with sampling every 10,000 steps and 25% burn-in. The Italian monophyla are highlighted in blue, while the sequence type 11 (ST11) Asian clade is highlighted in green. All of the phyla with no indication of ST are comprised mainly of isolates of ST258. The dates indicated in the figure, for selected branches and nodes, were inferred from the analysis described above; for a comparison with the dates of isolation of strains, see Discussion.