Abstract
BackgroundThe emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment.MethodsSixty-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.ResultsAll 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.ConclusionOur findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution.
Highlights
The emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment
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
Summary
The emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment. Klebsiella pneumoniae bacteria are normal gastrointestinal flora but, in people with weakened immune systems and/or debilitating diseases, can cause severe infections whose management has been complicated by increasing antimicrobial resistance over the last 20 years. The recent worldwide spread of strains that are resistant to carbapenems is even more worrying because these antibiotics are often the last line of effective treatment of the infections caused by multidrug-resistant K. pneumoniae [4]. The currently few treatment options for carbapenemresistant K. pneumoniae (CRKP) infections include polymyxins, tigecycline, aminoglycosides and fosfomycin, but their use is limited by concerns about their efficacy and safety [8], and there are increasing reports of resistance to colistin and tigecycline [9, 10]. Since 2010, Italy has seen a remarkable increase in the occurrence of CRKP, which greatly contributed to the epidemic spread of carbapenem-resistant Enterobacteriaceae (CRE) recorded by a countrywide cross-sectional survey, the results of which were published in 2013 [12]
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