Estudo molecular de clones de alto risco de Klebsiella pneumoniae produtoras de carbapenemase do tipo KPC resistentes a colistina

Abstract

The emergence of Klebsiella pneumoniae strains resistant to carbapenems and polymyxins (polymyxin B and colistin) is a threat to antimicrobial treatment. In this study, we investigated the molecular basis of colistin resistance in 23 Klebsiella pneumoniae KPC-producing strains (KPC-KP), of colonization and infection, from an outbreak that occurred in a public teaching hospital in Brazil. Whole genome sequencing (WGS) analysis was used to determine the mechanisms of resistance to colistin in a representative strain of each clone determined from the Pulsed Field Gel Electrophoresis (PFGE) technique. In addition, genotype analyzes were performed from the Polymerase Chain Reaction (PCR) for the following resistance genes (blaKPC, blaCTX-M, blaNDM, blaIMP, blaVIM and mcr-1) and virulence (fimH, fimA, wabG, khe, ecpA, entB, iutA, mrkD, rmpA, kfu, ybtS and allS) and phenotypic experiments to evaluate the biofilm formation capacity (adhesion assay, viable cell quantification and biofilm formation) and the hypermucoviscosity (string test). This study evidenced concerns regarding the use of antibiotics, mainly broad-spectrum cephalosporins, carbapenems and polymyxins, which may be related to the emergence of colistin-resistant KPC-KP strains. In total, five patterns of clonal profiles were identified with most strains belonging to clone A (82.6%). The clonality study also showed that most recurrent infections were caused by the same strain that was previously colonizing or infecting, and was demonstrated the ability of multiple clones to coexist in the same patient. Among the sequenced strains (N = 4, clones A, B, C and D) different sequence types were observed, (STs: ST11, ST37, ST 340 and ST 1484). It was possible to observe a complex resistome with high frequencies of resistance and virulence genes , and together with phenotypic tests was documented the presence of highly virulent strains, being one also hypermucoviscous. In general, the strains were biofilm producers, regardless of colistin resistance, clonal profile and isolation site, increasing their persistence and making difficult eradication. For the first time in Brazil, a fragment of 3,055 base pairs inserted in the mgrB gene, composed of the insertion sequence ISEcp1 and the gene blaCTX-M-15 in the 40KPC strain (representative of the dominant clone A) was identified, which resulted in acquired resistance concomitantly with broad-spectrum cephalosporins and colistin. The WGS allowed the identification of a species characterized as Klebsiella quasipneumoniae subsp. similipneumoniae, highly virulent, hypermucoviscous and colistin-resistant, which may be another important reservoir of resistance genes. The aspects presented here reinforce K. pneumoniae as a successful pathogen in causing infections, resulting in its rapid expansion and persistence in hospital settings. Rapid detection of carbapenem and colistin-resistant strains, together with the implementation of effective infection control measures, are crucial important in preventing the dissemination of high-risk clones of KP-KPC resistant to colistin, as evidenced in this study.