A Retrospective Whole-Genome Sequencing Analysis of Carbapenem and Colistin-Resistant Klebsiella Pneumoniae Nosocomial Strains Isolated during an MDR Surveillance Program

Abstract

Multidrug-resistant Klebsiella pneumoniae (MDR Kp), in particular carbapenem-resistant Kp (CR-Kp), has become endemic in Italy, where alarming data have been reported on the spread of colistin-resistant CR-Kp (CRCR-Kp). During the period 2013-2014, 27 CRCR-Kp nosocomial strains were isolated within the Modena University Hospital Policlinico (MUHP) multidrug resistance surveillance program. We retrospectively investigated these isolates by whole-genome sequencing (WGS) analysis of the resistome, virulome, plasmid content, and core single nucleotide polymorphisms (cSNPs) in order to gain insights into their molecular epidemiology. The in silico WGS analysis of the resistome revealed the presence of genes, such as blaKPC, related to the phenotypically detected resistances to carbapenems. Concerning colistin resistance, the plasmidic genes mcr 1-9 were not detected, while known and new genetic variations in mgrB, phoQ, and pmrB were found. The virulome profile revealed the presence of type-3 fimbriae, capsular polysaccharide, and iron acquisition system genes. The detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX3, and IncFII(K) types. The cSNPs genotyping was consistent with the multi locus sequence typing (MLST) and with the distribution of mutations related to colistin resistance genes. In a nosocomial drug resistance surveillance program, WGS proved to be a useful tool for elucidating the spread dynamics of CRCR-Kp nosocomial strains and could help to limit their diffusion.

Keywords: Klebsiella pneumoniae; MDR nosocomial spread; genetic relatedness; whole-genome sequencing.

Figure 1

Resistome data. Resistome data obtained by ResFinder-2.1 software, Pasteur MLST Kp database and running BLAST are here summarized. In particular, in the left box, grey and white colors represent gene presence and absence respectively; in the right box the colistin-resistance related mutations are grouped.

Figure 2

Virulome data and plasmid content. Virulome data were obtained by Pasteur MLST Kp database; plasmid content data were obtained by PlasmidFinder-1.3. Grey and white colors represent gene presence and absence respectively. ND, Not Defined.

Figure 3

Core SNPs analysis data incorporated into the epidemiological metadata. Core SNPs tree (left) shows two major lineages corresponding to the ST512 (blue) and the ST258 (red). Clusters within ST512 lineage are indicated by capital letters (A, B) followed by a number in the minor branches (B1, B2) and by additional lowercase letters in the subgroups (B2a, B2b). The right box represents patients’ movements inside the hospital, each color represents a ward. ICU, red; Infectious Disease, cyan, Long Term Care, pink; Medicine II, yellow; Medicine ICU, blue; Nephrology, dark green; Oncology, brown; Orthopedics, light green; Otolaryngology, orange; Pneumology, grey; Transplant Unit, fuchsia. ICU, Intensive Care Unit; Δ, deletion; IS, Insertion Sequence; ins, insertion; wt, wild type. * days 15–31.