RESUMO
Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a "last-resort" antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 µg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120 â Ala and Trp73 â Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus ß-lactam combinations and continue to question the DAP breakpoint of 4 µg/ml.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Enterococcus faecium/efeitos dos fármacos , Genes Reguladores , Genoma Bacteriano , Substituição de Aminoácidos , Ampicilina/farmacologia , Proteínas de Bactérias/metabolismo , Compostos de Boro , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Enterococcus faecium/química , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Corantes Fluorescentes , Expressão Gênica , Testes de Sensibilidade Microbiana , Vancomicina/farmacologia , Resistência a Vancomicina/genéticaRESUMO
We report the case of a patient from Brazil with a bloodstream infection caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA) that was susceptible to vancomycin (designated BR-VSSA) but that acquired the vanA gene cluster during antibiotic therapy and became resistant to vancomycin (designated BR-VRSA). Both strains belong to the sequence type (ST) 8 community-associated genetic lineage that carries the staphylococcal chromosomal cassette mec (SCCmec) type IVa and the S. aureus protein A gene (spa) type t292 and are phylogenetically related to MRSA lineage USA300. A conjugative plasmid of 55,706 bp (pBRZ01) carrying the vanA cluster was identified and readily transferred to other staphylococci. The pBRZ01 plasmid harbors DNA sequences that are typical of the plasmid-associated replication genes rep24 or rep21 described in community-associated MRSA strains from Australia (pWBG745). The presence and dissemination of community-associated MRSA containing vanA could become a serious public health concern.