Whole genome sequencing for the molecular characterization of carbapenem-resistant Klebsiella pneumoniae strains isolated at the Italian ASST Fatebenefratelli Sacco Hospital, 2012-2014

Abstract

Background: The emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment.

Methods: Sixty-eight carbapenemase-producing K. pneumoniae strains isolated at Luigi Sacco University Hospital-ASST Fatebenefratelli Sacco (Milan, Italy) between 2012 and 2014 were characterised microbiologically and molecularly. They were tested for drug susceptibility and carbapenemase phenotypes, investigated by means of repetitive extra-genic palindromic polymerase chain reaction (REP-PCR), and fully sequenced by means of next-generation sequencing for the in silico analysis of multi-locus sequence typing (MLST), their resistome, virulome and plasmid content, and their core single nucleotide polymorphism (SNP) genotypes.

Results: All of the samples were resistant to carbapenems, other β-lactams and ciprofloxacin; many were resistant to aminoglycosides and tigecycline; and seven were resistant to colistin. Resistome analysis revealed the presence of blaKPC genes and, less frequently blaSHV, blaTEM, blaCTX-M and blaOXA, which are related to resistance to carbapenem and other β-lactams. Other genes conferring resistance to aminoglycoside, fluoroquinolone, phenicol, sulphonamide, tetracycline, trimethoprim and macrolide-lincosamide-streptogramin were also detected. Genes related to AcrAB-TolC efflux pump-dependent and pump-independent tigecycline resistance mechanisms were investigated, but it was not possible to clearly correlate the genomic features with tigecycline resistance because of the presence of a common mutation in susceptible, intermediate and resistant strains. Concerning colistin resistance, the mgrB gene was disrupted by an IS5-like element, and the mobile mcr-1 and mcr-2 genes were not detected in two cases. The virulome profile revealed type-3 fimbriae and iron uptake system genes, which are important during the colonisation stage in the mammalian host environment. The in silico detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX1, IncX3, IncFII(K), IncN, IncL/M(pMU407) and IncFIA(HI1). REP-PCR showed five major clusters, and MLST revealed six different sequence types: 512, 258, 307, 1519, 745 and 101. Core SNP genotyping, which led to four clusters, correlated with the MLST data. Isolates of the same sequencing type often had common genetic traits, but the SNP analysis allowed greater strain tracking and discrimination than either the REP-PCR or MLST analysis.

Conclusion: Our findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution.

Keywords: Bacteria epidemiology; Carbapenem-resistant Klebsiella pneumoniae; Whole-genome sequencing.

Fig. 1

Dendrogram analysis, virtual gel image of REP-PCR fingerprint patterns and antibiotic resistance profiles of 68 carbapenem-resistant K. pneumoniae strains. Strains were considered to be genetically related if their fingerprint band patterns were ≥95% similar. Strains resistant, intermediate and sensitive to antibiotics are shown in black, gray and white, respectively. P: Pattern, indicates samples with indistinguishable electrophoretic profile. G: Group, indicates isolates differing for less than three electrophoretic bands

Fig. 2

Core SNP tree, resistome, virulome and plasmid replicon analysis of 68 carbapenem-resistant K. pneumoniae strains. The maximum likelihood core SNP tree (left) was constructed by using K. pneumoniae 342 as reference genome. Major genetically distinct lineages are indicated by capital letters and branches labelled in different colours on the basis of the MLST type. The resistome, virulome and plasmid replicon analysis boxes (right) show the presence (grey) or absence (white) of the relevant genes for each of the isolates