Disseminação de Klebsiella pneumoniae multirresistente: produção de biofilme, avaliação da virulência e do fitness bacteriano em amostras clínicas produtoras de KPC

Abstract

Klebsiella pneumoniae is often involved with healthcare-associated infections (HAI) in Brazil, and has a great ability to develop or acquire antimicrobial resistance. This study investigated the clonal dissemination and prevalence of sequence types (STs) in clinical strains of K. pneumoniae carrying blaCTX-M e blaKPC genes, as well as the presence of genes encoding virulence factors and the ability of these strains to produce biofilms. In addition, the impact of carbapenem and polymyxin-resistance on bacterial fitness of carbapenemase-producing K. pneumoniae (KPC) strains was also examined. We randomly selected non-duplicated K. pneumoniae isolates from a collection recovered from inpatients at the Clinical Hospital of the Federal University of Uberlândia (HC-UFU) from June 2009 to July 2015. The study included broad-spectrum cephalosporin and/or carbapenems-resistant strains. For investigation of the blaCTX-M and blaKPC resistance genes and its association with virulence genes (fimH, fimA, wabG, iucC, rmpA, ecpA, mrkD e khe), the strains were evaluated by PCR. DNA sequencing was performed to confirm the presence of blaKPC-2 gene. The pulsotypes, STs and clonal complexes (CCs) were determined by PFGE and MLST, respectively. Initial adhesion and biofilm formation were examined by quantitative assays and the results confirmed by scanning electron microscopy. For fitness evaluation, in vitro pairwise competition experiments were carried out using three strains of K. pneumoniae: one resistant to carbapenem, harboring 5 of the 8 evaluated virulence genes, another less virulent, but resistant to carbapenem and polymyxin, and ATCC 10031 multisensitive strain. For epidemiological evaluations, sixty K. pneumoniae strains were randomly selected, of which 30 carbapenem-sensitive (KpSC) strains isolated from infections; 30 carbapenem-resistant K. pneumoniae (KpRC) strains, 20 isolated from infections and 10 from colonizations. Significant differences were found when patients infected by KpSC and KpRC were compared, especially the high previous use of β-lactams (53%), carbapenems (73.3%) and polymyxin B (43.3%). Inadequate therapy, although prevalent in 56.7% of the patients, was much higher (70%) among those with KpRC infections. In total, 75% of the strains were characterized as multiresistant. Furthermore, we observed high consumption of cefepime, ceftriaxone and carbapenems by defined daily doses analysis, with an upward trend between the beginning and the end of the period of study. Regarding the presence of resistance genes, 80% of strains carried blaCTX-M gene and 100% of carbapenem-resistant isolates the blaKPC-2 gene. Virulence genes were detected with high frequencies in both groups, unrelated to carbapenem resistance. However, among the KpRC strains, the fimH, fimA and wabG genes were predominant (83%). It was observed polyclonal dissemination of strains with predominance of MLST STs 11 and 340, belonging to the clonal complex 258. All K. pneumoniae strains evaluated could adhere to an unmodified polystyrene surface. However, the statistical analysis showed that three strains had remarkably higher adhesion rates than the others (p<0.001). Compared to control, all strains had the ability to produce biofilm, without significant differences. However, the evaluation of biomass by crystal violet showed that 60% of the isolates were weak biofilm producers, results confirmed by scanning electron microscopy. According to the fitness results, the polymyxin-resistant strain was found to have more significant growth rates (p=0.030) and, in competition experiments between the two multiresistant clinical strains, the carbapenem-resistant strain showed a significantly lower fitness cost when compared to the carbapenem and polymyxin-resistant strain. The knowledge of the virulence factors and the pathogenic potential of ESBL and carbapenemases-producing K. pneumoniae contributes to a better understanding of colonization, pathogenicity and persistence of these microorganisms in the hospital environment and can provide tools to improve the treatment of serious infections as well as subsidies to improve infection control and prevention measures.