RESUMO
A phase 2 study of gepotidacin demonstrated the safety and efficacy of 3 gepotidacin doses (750 mg every 12 h [q12h], 1,000 mg q12h, and 1,000 mg every 8 h [q8h]) in hospitalized patients with suspected/confirmed Gram-positive acute bacterial skin and skin structure infections (ABSSSIs). Evaluating microbiology outcomes and responses were secondary endpoints. Pretreatment isolates recovered from infected lesions underwent susceptibility testing per Clinical and Laboratory Standards Institute guidelines. Staphylococcus aureus accounted for 78/102 (76%) of Gram-positive isolates; 54/78 (69%) were methicillin-resistant S. aureus (MRSA), and 24/78 (31%) were methicillin-susceptible S. aureus (MSSA). Posttherapy microbiological success (culture-confirmed eradication of the pretreatment pathogen or presumed eradication based on a clinical outcome of success) for S. aureus was 90% for the gepotidacin 750-mg q12h group, 89% for the 1,000-mg q12h, and 73% in the 1000-mg q8h group. For 78 S. aureus isolates obtained from pretreatment lesions, gepotidacin MIC50/MIC90 values were 0.25/0.5 µg/ml against both MRSA and MSSA. Isolates recovered from the few patients with posttreatment cultures showed no significant reduction in gepotidacin susceptibility (≥4-fold MIC increase) between pretreatment and posttreatment isolates. Two of the 78 S. aureus isolates from pretreatment lesions had elevated gepotidacin MICs and had mutations known to occur in quinolone-resistant S. aureus (GyrA S84L, ParC S80Y, and ParE D422E) or to confer elevated MICs to novel bacterial topoisomerase inhibitors (GyrA D83N, both isolates; ParC V67A, one isolate). This first report of microbiological outcomes and responses of gepotidacin in patients with ABSSSIs supports further evaluation of gepotidacin as a novel first-in-class antibacterial agent. (This study has been registered at ClinicalTrials.gov under identifier NCT02045797.).
Assuntos
Acenaftenos/farmacologia , Antibacterianos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Mutação/genética , Pele/microbiologia , Dermatopatias Infecciosas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.
Assuntos
DNA Topoisomerases Tipo II/genética , Farmacorresistência Bacteriana/genética , Evolução Molecular , Transferência Genética Horizontal , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Anti-Infecciosos/farmacologia , Sequência de Bases , DNA Girase/genética , Fluoroquinolonas/farmacologia , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Metionina tRNA Ligase/antagonistas & inibidores , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Metionina tRNA Ligase/genética , Metionina tRNA Ligase/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Streptococcus pneumoniae/genéticaRESUMO
Streptococcus pneumoniae is an important human pathogen capable of causing serious infections. NADH oxidase, a factor necessary for infection, was previously identified as part of a signature-tagged mutagenesis screen of a S. pneumoniae clinical isolate, 0100993. The mutant, with a plasmid insertion disrupting the nox gene, was attenuated for virulence in a murine respiratory tract infection model. A complete refined nox deletion mutant was generated by allelic-replacement mutagenesis and found to be attenuated for virulence 10(5)-fold in the murine respiratory tract infection model and at least 10(4)-fold in a Mongolian gerbil otitis media infection model, confirming the importance of the NADH oxidase for both types of S. pneumoniae infection. NADH oxidase converts O(2) to H(2)O. If O(2) is not fully reduced, it can form superoxide anion (O2(-)) and hydrogen peroxide (H(2)O(2)), both of which can be toxic to cells. Bacterial cell extracts from the allelic-replacement mutant were found to lack NADH oxidase activity and the mutant was unable to grow exponentially under conditions of vigorous aeration. In contrast, the mutant displayed normal growth characteristics under conditions of limited aeration. The S. pneumoniae nox gene was cloned and expressed in E. coli. The purified His-tagged NADH oxidase was shown to oxidize NADH with a K:(m) of 32 microM, but was unable to oxidize NADPH. Oxidation of NADH was independent of exogenous FAD or FMN.
Assuntos
Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Infecções Pneumocócicas/fisiopatologia , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/patogenicidade , Alelos , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Otite Média/microbiologia , Otite Média/fisiopatologia , Filogenia , Infecções Pneumocócicas/microbiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/fisiopatologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , VirulênciaRESUMO
Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Staphylococcus aureus/genética , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Bacitracina/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Modelos Animais de Doenças , Feminino , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos CBA , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Infecções Pneumocócicas/microbiologia , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/patogenicidade , VirulênciaRESUMO
The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.
Assuntos
Amidoidrolases , Antibacterianos/farmacologia , Fucose/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Regiões Promotoras Genéticas/genética , Streptococcus pneumoniae/efeitos dos fármacos , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Dados de Sequência Molecular , Óperon , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/metabolismoRESUMO
Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.