Comparative Genomic Analysis of a Clinical Isolate of Klebsiella quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 Beta-Lactamases Producer Harboring Two Drug-Resistance Plasmids from Southeast Brazil

Abstract

The aim of this study was to unravel the genetic determinants responsible for multidrug (including carbapenems) resistance and virulence in a clinical isolate of Klebsiella quasipneumoniae subsp. similipneumoniae by whole-genome sequencing and comparative analyses. Eighty-three clinical isolates initially identified as carbapenem-resistant K. pneumoniae were collected from nosocomial infections in southeast Brazil. After RAPD screening, the KPC-142 isolate, showing the most divergent DNA pattern, was selected for complete genome sequencing in an Illumina HiSeq 2500 instrument. Reads were assembled into scaffolds, gaps between scaffolds were resolved by in silico gap filling and extensive bioinformatics analyses were performed, using multiple comparative analysis tools and databases. Genome sequencing allowed to correct the classification of the KPC-142 isolate as K. quasipneumoniae subsp. similipneumoniae. To the best of our knowledge this is the first complete genome reported to date of a clinical isolate of this subspecies harboring both class A beta-lactamases KPC-2 and OKP-B-6 from South America. KPC-142 has one 5.2 Mbp chromosome (57.8% G+C) and two plasmids: 190 Kbp pKQPS142a (50.7% G+C) and 11 Kbp pKQPS142b (57.3% G+C). The 3 Kbp region in pKQPS142b containing the bla_KPC-2 was found highly similar to that of pKp13d of K. pneumoniae Kp13 isolated in Southern Brazil in 2009, suggesting the horizontal transfer of this resistance gene between different species of Klebsiella. KPC-142 additionally harbors an integrative conjugative element ICEPm1 that could be involved in the mobilization of pKQPS142b and determinants of resistance to other classes of antimicrobials, including aminoglycoside and silver. We present the completely assembled genome sequence of a clinical isolate of K. quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 beta-lactamases producer and discuss the most relevant genomic features of this important resistant pathogen in comparison to several strains belonging to K. quasipneumoniae subsp. similipneumoniae (phylogroup II-B), K. quasipneumoniae subsp. quasipneumoniae (phylogroup II-A), K. pneumoniae (phylogroup I), and K. variicola (phylogroup III). Our study contributes to the description of the characteristics of a novel K. quasipneumoniae subsp. similipneumoniae strain circulating in South America that currently represent a serious potential risk for nosocomial settings.

Keywords: KPC-2; Klebsiella quasipneumoniae subsp similipneumoniae; OKP-B-6; complete genome sequence; nosocomial infection; silver resistance.

Figure 1

Genomic comparison between the plasmid pKQPS142a of Kqps142 and plasmid pKPN-332 of K. pneumoniae KPNIH39 performed by Gview program, using BlastN (>70% identity and E-values < 10⁻⁵). Black line represents GC content. (A) region of ter loci; (B) arsenical operon, and (C) copper-resistance operon and silver resistance genes.

Figure 2

Comparison among the plasmids pKQPS142b of Kqps142, K. pneumoniae strain Kp13 (pKp13d), K. pneumoniae strain A60136 (p60136), and E. coli (IncQ pRSF1010). Homologous genes to plasmid pKPC142b carrying bla_KPC−2 were obtained by BLASTn, and represented by the same color in other species, each color defined to a specific gene. Black line represents the GC content. Features of each plasmid were obtained using the GView program, with modifications.^*The pKp13d is significantly larger than other compared elements. The genome length scales were adapted to emphasize only the region with similarity to pKQPS142b.

Figure 3

(A) Maximum likelihood tree for the matrix consisting of the concatenated allele sequences for seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) loci, constructed with PhyML, using the GTR+I+G model of nucleotide substitution. 1,000 bootstrap replicates were calculated to assess robustness. 1,000 bootstrap replicates were calculated to assess robustness, and support values >50% are shown. (B) Split decomposition analysis using the seven MLST alleles reveals a network-like structure, suggestive of recombination events. “||” represent long branches that were truncated to fit (see Figure S3 for the complete network). In both panels, KPC-142 is labeled on a yellow background and in the (panel A) the ST number of each isolate/strain is in brackets.

Figure 4

Genomic Atlas showing the common or exclusives chromosomal genes of Kqps142 (inner circle, in red), in comparison to: eight K. quasipneumoniae subsp. similipneumoniae strains (in order, inner to outer circles—HKUOPA4, HKUOPJ4, HKUOPL4, ATCC 700603, 07A044, MGH44, 193 KOXY, and 385 ECLO); nine K. quasipneumoniae subsp. quasipneumoniae (in order, inner to outer circles—UCICRE14, 01A030, FI_HV_2014, MGH96, 18A069, ARLG2711, PO1285, AKSD007, and 21_GR_13); nine K. pneumoniae (in order, inner to outer circles—DSM_30104, KP13, 1084, CAV1193, HS11286, KCTC-2242, KP617, MGH 78578, and NTUH-K2044); and nine K. variicola (in order, inner to outer circles–BZ19, GJ1, UCICRE10, MGH40, MGH20, UCI18, BIDMC61, BIDMC88, and MGH80). Comparison was obtained by Gview program, using BlastN (>70% identity and E-values < 10⁻⁵). Black line represents GC content. Regions containing interesting genes or features (named R1 to R7) are highlighted. R7 contains phage sequences and the integrative conjugative element ICEPm1. Genes of capsular polysaccharide synthesis are shown in details. Kqs, K. quasipneumoniae subsp. similipneumoniae; Kqq, K. quasipneumoniae subsp. quasipneumoniae; Kpn, K. pneumoniae; and Kv, K. variicola.

Figure 5

Virulence genes in Klebsiella strain panel, including Kqps142. Presence of the virulence genes per genome (from 0 to 9) of K. pneumoniae (Kpn), K. quasipneumoniae subsp. quasipneumoniae (Kqq), K. quasipneumoniae subsp. similipneumoniae (Kqs), and K. variicola (Kv).