Genomic insights into the diversity, virulence and resistance of Klebsiella pneumoniae extensively drug resistant clinical isolates

Abstract

Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília's hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10 366 genes, with a core-genome size of ~4476 genes found in 95 % of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. β-Lactamase production provided the most notable mechanism associated with K. pneumoniae. Each strain presented two or three different β-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D β-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.

Keywords: CC258; Klebsiella pneumoniae; ST11; XDR; pan-genome; β-lactamase.

Fig. 1.

Core-genome SNP phylogeny and gene presence–absence matrix of 70 K. pneumoniae isolates from Brasília’s hospitals between 2010 and 2014, and 20 diverse K. pneumoniae reference genomes. Using the pan-genome analysis with Roary, we identified 4173 genes present in >95 % of strains (core genes), and an additional 14 316 genes present in the accessory genome. Core-genome SNPs were subsequently used to generate maximum-likelihood phylogenetic trees using RAxML. (a) Unrooted core-genome phylogeny with STs of the Brasília isolates highlighted in different colours. (b) The gene presence–absence matrix from the Roary pan-genome. Scale bar is a maximum likelihood estimate of the number of substitutions that have occurred on average per site between two nodes in a tree.

Fig. 2.

Distribution of ST, capsule biosynthesis (K) and LPS (O) loci. (a) Number of isolates of a particular ST sampled per year from Brasília’s hospitals, and (b) capsule and O-antigen serotype prediction mapped onto a circular core-genome SNP phylogeny, with tips labelled by sampling year (shape) and ST group (colour). Twenty reference-quality K. pneumoniae isolates (*) are included in the SNP phylogeny (b). For both (a) and (b), colour indicates ST groups, with Brasília isolates at the top and reference strains at the bottom.

Fig. 3.

Presence of the yersiniabactin- and colibactin-encoding mobile element ICEKp in a subset of Brasília K. pneumoniae isolates. The yersiniabactin STs (YbSTs) and colibactin STs (CbSTs) were identified by Kleborate. Core-genome SNP phylogeny of these isolates is shown on the left, with the tips of the tree labelled with the year of isolation (indicated by shapes) and hospital (indicated by colours). The majority of Brasília K. pneumoniae isolates do not have virulence genes.

Fig. 4.

Heat map of the antibiotic-sensitivity profile of Brasília K. pneumoniae clinical isolates as assessed by Vitek 2. Resistance (red) or sensitivity (blue) against a particular antibiotic is defined using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoint at the time of isolation. Missing antibiotic information is shown in white. Antibiotics tested included antibiotics from the following classes (indicated by Roman numerals). (I) Penicillins, which included amp_sulbac (ampicillin/sulbactam), ampicillin, aztreonam, pip_tazo (piperacillin/tazobactam) and piperacillin. (II) Carbapenems, which included ertapenem, imipenem and meropenem. (III) Second-generation cephalosporins, which included cefalotin, cefuroxime and cefotetan. (IV) Third-generation cephalosporins, which included cefotaxime, ceftazidime and ceftriaxone. (V) Fourth-generation cephalosporins, which included cefepime. (VI) Quinolones, which included levofloxacin and ciprofloxacin. (VII) Aminoglycosides, which included amikacin, gentamicin, tobramycin. The complete profile with MIC values is available in Table S7.

Fig. 5.

Antimicrobial resistance of K. pneumoniae clinical isolates from 12 Brasília hospitals. Brasília isolates sampled between 2010 and 2014 (with year of isolation shown by different shapes and hospital by different colours) mapped onto a core-genome SNP phylogeny reconstructed from 70 Brasília isolates and 20 diverse reference-quality K. pneumoniae strains. The presence of different resistance genes is coloured by class. The majority of the isolates have genes from at least seven or more antibiotic-resistance classes.