Estudo de vias alternativas aos sistemas LiaFSR e YycFGHIJ que levam à diminuição da sensibilidade à daptomicina em Enterococcus faecium

Abstract

Enterococcus is one of the most important multi-drug resistant nosocomial pathogens. Infections caused by vancomycin resistant Enterococci (VRE) are becoming increasingly difficult to treat, often with prolonged hospital outbreaks that are real challenges. Daptomycin (DAP) is a drug of last resort for treating infections caused by VRE; mainly E. faecium belonged to clonal complex 17, called "high risk" clone. In this study, samples of E. faecium from the Hospital de Base de São Jose do Rio Preto were analyzed by molecular typing and the susceptibility profile to several antimicrobials was determined. A high-risk vancomycin-resistant clone with reduced susceptibility to daptomycin has been identified spreading in the hospital. The mechanism of DAP resistance is not fully elucidated. For the purpose of studying the mechanism of resistance to DAP in the clinical isolates of this hospital and to identify new pathways to resistance, DAP hyper-susceptible E. faecium HBSJRP18 clinical isolate was used to perform in vitro selections until resistant mutants were obtained. Subsequently, genes with mutation in these selected isolates, as well as those already described in the literature, were analyzed in the clinical samples. Genomes of these mutants were sequenced and compared to the DAP hyper-susceptible E. faecium HBSJRP18 to detect the presence of single nucleotide polymorphism (SNP) in genes that may be related to resistance to DAP. The mutated/intact genes were overexpressed in the hyper-susceptible/resistant strain, thus DAP MIC was determined to investigate their roles in resistance to that antibiotic. The genes that showed mutations in the three BHI selections were lafB-like, a glycosyltransferase possibly involved in the LTA biosynthesis, and dhaK, a dihydroxyacetone kinase possibly involved in the metabolism of glycerol. Cloning and overexpression of the genes of interest were performed, which enabled the evidence of the role of lafB-like in hyper-susceptibility to DAP. dhaK still remains unclear if it is actually involved in resistance. Mutations in these genes were also found in all clinical samples, as well as the liaFSR genes already described in the literature. The findings of this study have a particular importance considering that vancomycin-resistant E. faecium infections have grown frighteningly in the present day. The lafB-like gene may be a potential therapeutic target and should be better studied for this.