RESUMO

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-ß-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (107 CFU/ml) was used as a reference standard (cutoff ≥16 µg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (the most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 µg/ml, the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ The overall negative predictive value ranged from 81.4% to 95.6% in Latin American countries using published prevalence rates of the CzIE. MSSA isolates from the United States were genetically diverse, with no distinguishing genomic differences from Latin American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high inoculum, the rapid test is inexpensive, feasible, and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

Assuntos

Cefazolina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefazolina/farmacologia , Testes Diagnósticos de Rotina , Humanos , América Latina , Meticilina , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/genética

RESUMO

Little is known about the population structure of vancomycin-resistant Enterococcus faecium (VREfm) in Latin America (LATAM). Here, we provide a complete genomic characterization of 55 representative Latin American VREfm recovered from 1998-2015 in 5 countries. The LATAM VREfm population is structured into two main clinical clades without geographical clustering. Using the LATAM genomes, we reconstructed the global population of VREfm by including 285 genomes from 36 countries spanning from 1946 to 2017. In contrast to previous studies, our results show an early branching of animal related isolates and a further split of clinical isolates into two sub-clades within clade A. The overall phylogenomic structure of clade A was highly dependent on recombination (54% of the genome) and the split between clades A and B was estimated to have occurred more than 2,765 years ago. Furthermore, our molecular clock calculations suggest the branching of animal isolates and clinical clades occurred ~502 years ago whereas the split within the clinical clade occurred ~302 years ago (previous studies showed a more recent split between clinical an animal branches around ~74 years ago). By including isolates from Latin America, we present novel insights into the population structure of VREfm and revisit the evolution of these pathogens.

Assuntos

Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Infecções por Bactérias Gram-Positivas/epidemiologia , Enterococos Resistentes à Vancomicina/genética , Vancomicina/farmacologia , Antibacterianos , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Surtos de Doenças , Genômica/métodos , Genótipo , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Epidemiologia Molecular/métodos , Filogenia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos

RESUMO

The ferric uptake regulator (Fur) plays a major role in controlling the expression of iron homeostasis genes in bacterial organisms. In this work, we fully characterized the capacity of Fur to reconfigure the global transcriptional network and influence iron homeostasis in Enterococcus faecalis. The characterization of the Fur regulon from E. faecalis indicated that this protein (Fur) regulated the expression of genes involved in iron uptake systems, conferring to the system a high level of efficiency and specificity to respond under different iron exposure conditions. An RNAseq assay coupled with a systems biology approach allowed us to identify the first global transcriptional network activated by different iron treatments (excess and limited), with and without the presence of Fur. The results showed that changes in iron availability activated a complex network of transcriptional factors in E. faecalis, among them global regulators such as LysR, ArgR, GalRS, and local regulators, LexA and CopY, which were also stimulated by copper and zinc treatments. The deletion of Fur impacted the expression of genes encoding for ABC transporters, energy production and [Fe-S] proteins, which optimized detoxification and iron uptake under iron excess and limitation, respectively. Finally, considering the close relationship between iron homeostasis and pathogenesis, our data showed that the absence of Fur increased the internal concentration of iron in the bacterium and also affected its ability to produce biofilm. These results open new alternatives in the field of infection mechanisms of E. faecalis.

RESUMO

We report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate of Staphylococcus aureus recovered from a patient in Brazil. Emergence of vancomycin resistance in methicillin-susceptible S. aureus would indicate that this resistance trait might be poised to disseminate more rapidly among S. aureus and represents a major public health threat.

Assuntos

Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Bacteriemia/microbiologia , Brasil/epidemiologia , Humanos , Meticilina/farmacologia , Meticilina/uso terapêutico , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Vancomicina/uso terapêutico , Resistência a Vancomicina/imunologia

RESUMO

Daptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane-targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides.

Assuntos

Anti-Infecciosos/farmacologia , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Peptídeos/farmacologia , Animais , Antibacterianos/farmacologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Cardiolipinas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Testes de Sensibilidade Microbiana/métodos , Deleção de Sequência/genética

RESUMO

Treatment of severe infections caused by vancomycin-resistant enterococci (VRE) is challenging due to the scarcity of reliable therapeutic alternatives. In this context, daptomycin (DAP), a lipopeptide antibiotic, has emerged as an interesting alternative as it is one of the few compounds that retain in vitro bactericidal activity against VRE isolates, although it has not been approved for this purpose by regulatory agencies. In this review, we will summarise the clinical, animal and in vitro evidence evaluating the efficacy of DAP for the management of deep-seated VRE infections. In addition, we will address important clinical concerns such as the emergence of DAP resistance during therapy and reports of therapeutic failure with DAP monotherapy. Finally, we will discuss possible future strategies (such as the use of higher doses and/or combination therapies) to optimise the use of this antibiotic against VRE.

Assuntos

Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Daptomicina/uso terapêutico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Enterococos Resistentes à Vancomicina/isolamento & purificação , Animais , Bacteriemia/microbiologia , Modelos Animais de Doenças , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Resultado do Tratamento

RESUMO

High-dose daptomycin (DAP) therapy failed in a neutropenic patient with bloodstream infection caused by a DAP-susceptible Enterococcus faecium (minimum inhibitory concentration, 3 µg/mL) harboring genetic changes associated with DAP resistance, with persistent bacteremia and selection of additional resistances. Daptomycin monotherapy should be used cautiously against DAP-susceptible E. faecium strains with minimum inhibitory concentrations >2 µg/mL.

Assuntos

Antibacterianos , Bacteriemia , Daptomicina , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas , Adulto , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Neutropenia Febril Induzida por Quimioterapia , Daptomicina/administração & dosagem , Daptomicina/uso terapêutico , Enterococcus faecium/isolamento & purificação , Evolução Fatal , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Leucemia-Linfoma Linfoblástico de Células Precursoras , Falha de Tratamento , Adulto Jovem

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 → Ala and Trp73 → 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/genética

RESUMO

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.

Assuntos

Bacteriemia/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Resistência a Vancomicina/genética , Adulto , Brasil , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Masculino , Testes de Sensibilidade Microbiana , Família Multigênica , Micose Fungoide/complicações , Plasmídeos/genética , Análise de Sequência de DNA

RESUMO

Several reports have implicated the inoculum effect that some strains of type A beta-lactamase (Bla)-producing, methicillin-susceptible Staphylococcus aureus (MSSA) show against cefazolin as the cause for clinical failures in certain serious deep-seated infections. Here, using a previously reported MSSA strain displaying this phenotype (TX0117), we obtained a Bla-cured derivative (TX0117c) with a combination of novobiocin and high temperature. Both isolates were then used in a rat endocarditis model and treated with cefazolin, nafcillin, and daptomycin, given to simulate human dosing. Animals were treated for 3 days and either sacrificed at 24 h after the last antibiotic dose (standard group) or left untreated for an additional 3 days (relapse group). With TX0117 in the standard treatment group, daptomycin and nafcillin were both significantly better than cefazolin in reducing CFU/g of vegetations, achieving mean log10 reductions compared to levels in untreated rats of 7.1, 5.3, and 1.8, respectively (cefazolin versus daptomycin, P < 0.0001; cefazolin versus nafcillin, P = 0.005; daptomycin versus nafcillin, P = 0.053). In addition, cefazolin was significantly more effective in reducing vegetation titers of TX0117c than of TX0117 (mean log10 reduction of 1.4 versus 5.5, respectively; P = 0.0001). Similar results were observed with animals in the relapse group. Thus, these data show that there can be an in vivo consequence of the in vitro inoculum effect that some MSSA strains display against cefazolin and indicate a specific role for Bla production using a Bla-cured derivative strain against which cefazolin regained both in vitro and in vivo activity.