RESUMO
BACKGROUND: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is increasing worldwide, with Mycobacterium avium complex (MAC) and Mycobacterium abscessus as the predominant pathogens. Current treatments are poorly tolerated and modestly effective, highlighting the need for new treatments. SPR719, the active moiety of the benzimidazole prodrug SPR720, inhibits the ATPase subunits of DNA gyrase B, a target not exploited by current antibiotics, and therefore, no cross-resistance is expected with standard-of-care (SOC) agents. OBJECTIVES: To evaluate the in vitro activity of SPR719 against MAC and M. abscessus clinical isolates, including those resistant to SOC agents, and in vivo efficacy of SPR720 in murine non-tuberculous mycobacteria (NTM) pulmonary infection models. METHODS: NTM isolates were tested for susceptibility to SPR719. Chronic C3HeB/FeJ and severe combined immunodeficient murine models of pulmonary infection were used to assess efficacy of SPR720 against MAC and M. abscessus, respectively. RESULTS: SPR719 was active against MAC (MIC90, 2â mg/L) and M. abscessus (MIC90, 4â mg/L) clinical isolates. Efficacy of SPR720 was demonstrated against MAC pulmonary infection, both as a monotherapy and in combination with SOC agents. SPR720 monotherapy exhibited dose-dependent reduction in bacterial burden, with the largest reduction observed when combined with clarithromycin and ethambutol. Efficacy of SPR720 was also demonstrated against M. abscessus pulmonary infection where monotherapy exhibited a dose-dependent reduction in bacterial burden with further reductions detected when combined with SOC agents. CONCLUSIONS: In vitro activity of SPR720 against common NTM pathogens and efficacy in murine infections warrant the continued clinical evaluation of SPR720 as a new oral option for the treatment of NTM-PD.
Assuntos
Pneumopatias , Infecções por Mycobacterium não Tuberculosas , Pneumonia , Humanos , Animais , Camundongos , Micobactérias não Tuberculosas , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Modelos Animais de Doenças , Complexo Mycobacterium avium , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Pneumopatias/tratamento farmacológico , Pneumonia/tratamento farmacológicoRESUMO
The continued evolution of bacterial resistance to the ß-lactam class of antibiotics has necessitated countermeasures to ensure continued effectiveness in the treatment of infections caused by bacterial pathogens. One relatively successful approach has been the development of new ß-lactam analogs with advantages over prior compounds in this class. The carbapenems are an example of such ß-lactam analogs possessing improved stability against ß-lactamase enzymes and, therefore, a wider spectrum of activity. However, all carbapenems currently marketed for adult patients are intravenous agents, and there is an unmet need for an oral agent to treat patients that otherwise do not require hospitalization. Tebipenem pivoxil hydrobromide (tebipenem-PI-HBr or SPR994) is an orally available prodrug of tebipenem, a carbapenem with activity versus multidrug-resistant (MDR) Gram-negative pathogens, including quinolone-resistant and extended-spectrum-ß-lactamase-producing Enterobacterales Tebipenem-PI-HBr is currently in development for the treatment of complicated urinary tract infections (cUTI). Microbiological data are presented here that demonstrate equivalency of tebipenem with intravenous carbapenems such as meropenem and support its use in infections in which the potency and spectrum of a carbapenem are desired. The results from standard in vitro microbiology assays as well as efficacy in several in vivo mouse infection models suggest that tebipenem-PI-HBr could be a valuable oral agent available to physicians for the treatment of infections, particularly those caused by antibiotic-resistant Gram-negative pathogens.
Assuntos
Carbapenêmicos , Infecções Urinárias , Adulto , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Carbapenêmicos/farmacologia , Humanos , Meropeném , Camundongos , Infecções Urinárias/tratamento farmacológicoRESUMO
Tebipenem (SPR859) is the microbiologically active form of SPR994 (tebipenem-pivoxil), an orally available carbapenem with activity against extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae Measurement of the relative binding of SPR859 to the bacterial cell targets revealed that it is a potent inhibitor of multiple penicillin-binding proteins (PBPs) but primarily a Gram-negative PBP 2 inhibitor, similar to other compounds in this class. These data support further clinical development of SPR994.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Carbapenêmicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Humanos , Testes de Sensibilidade Microbiana/métodos , Proteínas de Ligação às Penicilinas/metabolismo , beta-Lactamases/metabolismoRESUMO
The antimicrobial activity of tebipenem and other carbapenem agents were tested in vitro against a set of recent clinical isolates responsible for urinary tract infection (UTI), as well as against a challenge set. Isolates were tested by reference broth microdilution and included Escherichia coli (101 isolates), Klebsiella pneumoniae (208 isolates), and Proteus mirabilis (103 isolates) species. Within each species tested, tebipenem showed equivalent MIC50/90 values to those of meropenem (E. coli MIC50/90, ≤0.015/0.03 mg/liter; K. pneumoniae MIC50/90, 0.03/0.06 mg/liter; and P. mirabilis MIC50/90, 0.06/0.12 mg/liter) and consistently displayed MIC90 values 8-fold lower than imipenem. Tebipenem and meropenem (MIC50, 0.03 mg/liter) showed equivalent MIC50 results against wild-type, AmpC-, and/or extended-spectrum ß-lactamase (ESBL)-producing isolates. Tebipenem also displayed MIC50/90 values 4- to 8-fold lower than imipenem against the challenge set. All carbapenem agents were less active (MIC50, ≥8 mg/liter) against isolates carrying carbapenemase genes. These data confirm the in vitro activity of the orally available agent tebipenem against prevalent UTI Enterobacteriaceae species, including those producing ESBLs and/or plasmid AmpC enzymes.
Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Enterobacteriaceae/patogenicidade , Enterobacteriaceae/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
Nontuberculous mycobacterium (NTM) infections are increasing globally. The Mycobacterium avium complex (MAC) and Mycobacterium abscessus are the most frequently encountered NTM among clinical laboratories, and treatment options are extremely limited. In this study, the in vitro potency of a novel benzimidazole, SPR719, the microbiologically active form of the orally available prodrug SPR720, was tested against several species of NTM. MICs were determined for 161 isolates of NTM of 13 taxa (seven species, three subspecies, and three groups/complexes) in cation-adjusted Mueller-Hinton Broth, as described and recommended by the Clinical and Laboratory Standards Institute (CLSI M24-A2). Comparator antimicrobials included amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, minocycline, moxifloxacin, tigecycline, and trimethoprim-sulfamethoxazole (TMP-SMX) for the rapidly growing mycobacteria (RGM), amikacin and clarithromycin for the MAC, and amikacin, ciprofloxacin, clarithromycin, doxycycline, linezolid, moxifloxacin, rifabutin, rifampin, and TMP-SMX for the other slowly growing NTM. SPR719 was found to be potent against multiple clinical strains of NTM with an MIC50 range of 0.25 to 4 µg/ml for several species of NTM. These findings support the further advancement of SPR720 for the treatment of NTM disease.
Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Micobactérias não Tuberculosas/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana/métodos , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológicoRESUMO
BACKGROUND: In small series or individual reports, SNPs within the mprF ORF and dysregulation of its expression in Staphylococcus aureus have been linked to daptomycin resistance (DAP-R) via a proposed gain-in-function mechanism. Similarly, dysregulation of dltABCD has also been associated with DAP-R. METHODS: Using 22 well-characterized, isogenic daptomycin-susceptible (DAP-S)/DAP-R clinical MRSA strain pairs, we assessed potential relationships of the DAP-R phenotype with: (i) regulation of mprF transcription; (ii) regulation of dltABCD transcription; (iii) expression of the two-component regulatory system, graRS (upstream regulator for both mprF and dltABCD transcription); (iv) SNPs within the graRS promoter or its ORF; and (v) altered mprF transcription and lysyl-phosphatidylglycerol (L-PG) synthesis. RESULTS: Enhanced expression of mprF occurred with SNPs in highly distinct and well-chronicled MprF domain 'hot spots' and rarely occurred without such mutations. Increased expression and/or dysregulation of mprF and dltABCD were not uncommon in DAP-R strains, occurring in 27% of strains for each gene. In these latter strains, neither graRS expression profiles nor polymorphic sequences within the graRS promoter or ORF could be significantly linked to altered transcription of mprF or dlt. CONCLUSIONS: Although graRS can co-regulate mprF and dltABCD expression, loci outside of this regulon appear to be involved in dysregulation of these latter two genes and the DAP-R phenotype. Finally, DAP-R strains exhibiting significantly altered mprF transcription profiles produced significantly increased levels of L-PG.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Daptomicina/farmacologia , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP(s))/daptomycin-resistant (DAP(r)) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP(r) strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP(r) strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP(r) strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP(r) strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus.
Assuntos
Aminoaciltransferases/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Meticilina/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Fenótipo , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been commonly observed in daptomycin-resistant (DAP(r)) Staphylococcus aureus strains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if such mprF SNPs are causal in DAP(r) strains or are merely a biomarker for this phenotype. In this study, we used an isogenic set of S. aureus strains: (i) Newman, (ii) its isogenic ΔmprF mutant, and (iii) several in trans plasmid complementation constructs, expressing either a wild-type or point-mutated form of the mprF ORF cloned from two isogenic DAP-susceptible (DAP(s))-DAP(r) strain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprF strain with singly point-mutated mprF genes (mprFS295L or mprFT345A) revealed that (i) individual and distinct point mutations within the mprF ORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding to S. aureus by a charge repulsion mechanism. Thus, for these two DAP(r) strains, the defined mprF SNPs appear to be causally related to this phenotype.
Assuntos
Aminoaciltransferases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Farmacorresistência Bacteriana/genética , Polimorfismo de Nucleotídeo Único , Staphylococcus aureus/efeitos dos fármacos , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Teste de Complementação Genética , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Fases de Leitura Aberta , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Eletricidade EstáticaRESUMO
Daptomycin (DAP) is a cyclic lipopeptide antibiotic used for the treatment of certain Staphylococcus aureus infections. Although rare, strains have been isolated that are DAP resistant. These strains usually have mutations in mprF, a gene encoding a membrane protein with both lysylphosphatidylglycerol (LPG) synthase and flippase activities. Because ΔmprF strains have increased DAP susceptibility, the mechanism of resistance is not likely due to a loss of mprF function. In this study, we developed an LC-MS assay to examine the effect of different mprF mutations on the ratio of phosphatidylglycerol (PG) to LPG in the membrane. Our assay demonstrated that some, but not all, mutations in the flippase and synthase domains result in small but reproducible increases in the proportion of LPG relative to PG. Techniques described herein represent a higher throughput and more sensitive method for measuring relative phospholipids levels. These results offer guidance in the understanding of how mprF confers DAP resistance; namely, mprF-mediated resistance may be through more than one mechanism, including increased overall LPG synthesis and increased LPG present on the outer leaflet of the cytoplasmic membrane.
Assuntos
Aminoaciltransferases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Mutação , Fosfolipídeos/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Cromatografia Líquida , Íons/química , Lisina/análise , Lisina/química , Testes de Sensibilidade Microbiana , Fosfatidilgliceróis/análise , Fosfatidilgliceróis/química , Fosfolipídeos/análise , Staphylococcus aureus/química , Espectrometria de Massas em TandemRESUMO
Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains.
Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/tratamento farmacológico , Aminoaciltransferases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Teste de Complementação Genética , Genótipo , Humanos , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Microscopia Eletrônica , Mutação , Fenótipo , Plasmídeos , Infecções Estafilocócicas/microbiologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Transformação BacterianaRESUMO
Mutations in mprF have been shown to result in reduced susceptibility to daptomycin and other cationic antibacterials. An mprF antisense-inducible plasmid was constructed and used to demonstrate that depletion of mprF can reestablish susceptibility to daptomycin. Inducing antisense to mprF also resulted in increased susceptibility to vancomycin and gentamicin but, paradoxically, decreased susceptibility to oxacillin. These results suggest that mprF mutations that reduce susceptibility to cationic antibacterials result in a gain-of-function phenotype.
Assuntos
Antibacterianos/farmacologia , Daptomicina/farmacologia , Farmacorresistência Bacteriana/genética , RNA Antissenso/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Northern Blotting , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Vancomicina/farmacologiaRESUMO
We investigated the hypothesis that methicillin-resistant Staphylococcus aureus (MRSA) isolates developing reduced susceptibilities to daptomycin (DAP; a calcium-dependent molecule acting as a cationic antimicrobial peptide [CAP]) may also coevolve reduced in vitro susceptibilities to host defense cationic antimicrobial peptides (HDPs). Ten isogenic pairs of clinical MRSA DAP-susceptible/DAP-resistant (DAP(s)/DAP(r)) strains were tested against two distinct HDPs differing in structure, mechanism of action, and origin (thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]) and one bacterium-derived CAP, polymyxin B (PMB). Seven of 10 DAP(r) strains had point mutations in the mprF locus (with or without yyc operon mutations), while three DAP(r) strains had neither mutation. Several phenotypic parameters previously associated with DAP(r) were also examined: cell membrane order (fluidity), surface charge, and cell wall thickness profiles. Compared to the 10 DAP(s) parental strains, their respective DAP(r) strains exhibited (i) significantly reduced susceptibility to killing by all three peptides (P < 0.05), (ii) increased cell membrane fluidity, and (iii) significantly thicker cell walls (P < 0.0001). There was no consistent pattern of surface charge profiles distinguishing DAP(s) and DAP(r) strain pairs. Reduced in vitro susceptibility to two HDPs and one bacterium-derived CAP tracked closely with DAP(r) in these 10 recent MRSA clinical isolates. These results suggest that adaptive mechanisms involved in the evolution of DAP(r) also provide MRSA with enhanced survivability against HDPs. Such adaptations appear to correlate with MRSA variations in cell membrane order and cell wall structure. DAP(r) strains with or without mutations in the mprF locus demonstrated significant cross-resistance profiles to these unrelated CAPs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Daptomicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Genótipo , Gentamicinas/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Oxacilina/farmacologia , Polimixina B/farmacologia , Vancomicina/farmacologia , alfa-Defensinas/farmacologia , beta-Tromboglobulina/farmacologiaRESUMO
The antimicrobial activities of several ß-lactam agents were tested by broth microdilution alone and in combination with a new polymyxin analog, SPR741 (at a fixed concentration of 8 mg/L), against a challenge set of clinical isolates (202 Escherichia coli and 221 Klebsiella pneumoniae isolates). Using Clinical and Laboratory Standards Institute (CLSI) or European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility criteria for each partner antibiotic, mecillinam-SPR741, temocillin-SPR741, and piperacillin-tazobactam-SPR741 combinations had susceptibility rates higher (85.6-100.0%) than the respective agents tested alone (47.5-88.7%) against extended-spectrum ß-lactamase (ESBL)-producing E. coli and K. pneumoniae. Temocillin-SPR741 (97.8% susceptible) had MIC50 (minimum inhibitory concentration) and MIC90 results of 0.5 and 2 mg/L, respectively, against K. pneumoniae carbapenemase (KPC)-producing E. coli, 8- to 16-fold lower than temocillin alone (MIC50/90, 8/16 mg/L; 65.2% susceptible). The mecillinam MIC50/MIC90 results dropped to 1/4 mg/L (from 128/>256 mg/L when tested alone) against metallo-ß-lactamase (MBL)-producing E. coli. These MICs for mecillinam-SPR741 resulted in a susceptibility rate of 96.9% versus 9.4% for mecillinam. In general, a decrease in MICs for ß-lactams (MIC90, >32 mg/L) in the presence of SPR741 was not observed against KPC-, MBL- or OXA-48-like-producing K. pneumoniae. These study results indicate that some agents had a significant increase in in vitro activity in the presence of SPR741 and could become potential strategic options for treating serious infections caused by multidrug-resistant Enterobacteriaceae.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Polimixinas/farmacologia , beta-Lactamas/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/metabolismo , Escherichia coli/metabolismo , Humanos , Klebsiella pneumoniae/metabolismo , Testes de Sensibilidade Microbiana/métodos , Penicilinas/farmacologia , Combinação Piperacilina e Tazobactam/farmacologia , beta-Lactamases/metabolismoRESUMO
Therapeutics targeting Gram-negative bacteria have the challenge of overcoming a formidable outer membrane (OM) barrier. Here, we characterize the action of SPR741, a novel polymyxin B (PMB) analogue shown to potentiate several large-scaffold antibiotics in Gram-negative pathogens. Probing the surface topology of Escherichia coli using atomic force microscopy revealed substantial OM disorder at concentrations of SPR741 that lead to antibiotic potentiation. Conversely, very little cytoplasmic membrane depolarization was observed at these same concentrations, indicating that SPR741 acts predominately on the OM. Truncating the lipopolysaccharide (LPS) core with genetic perturbations uniquely sensitized E. coli to SPR741, suggesting that LPS core residues keep SPR741 at the OM, where it can potentiate a codrug, rather than permit its entry to the cytoplasmic membrane. Further, a promoter activity assay revealed that SPR741 challenge induced the expression of RcsAB, a stress sensor for OM perturbation. Together, these results indicate that SPR741 interacts predominately with the OM, in contrast to the dual action of PMB and colistin at both the outer and cytoplasmic membranes.
Assuntos
Antibacterianos , Polimixina B , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Escherichia coli/genética , Bactérias Gram-Negativas , Polimixina B/farmacologiaRESUMO
Our previous studies of clinical daptomycin-resistant (Dap(r)) Staphylococcus aureus strains suggested that resistance is linked to the perturbations of several key cell membrane (CM) characteristics, including the CM order (fluidity), phospholipid content and asymmetry, and relative surface charge. In the present study, we examined the CM profiles of a well-known methicillin-resistant Staphylococcus aureus (MRSA) strain (MW2) after in vitro selection for DAP resistance by a 20-day serial passage in sublethal concentrations of DAP. Compared to levels for the parental strain, Dap(r) strains exhibited (i) decreased CM fluidity, (ii) the increased synthesis of total lysyl-phosphatidylglycerol (LPG), (iii) the increased flipping of LPG to the CM outer bilayer, and (iv) the increased expression of mprF, the gene responsible for the latter two phenotypes. In addition, we found that the expression of the dlt operon, which also increases positive surface charge, was enhanced in the Dap(r) mutants. These phenotypic and genotypic changes correlated with reduced DAP surface binding, mirroring observations made in clinical Dap(r) isolates. In this strain, serial exposure to DAP induced an increase in vancomycin MICs into the vancomycin-intermediate S. aureus (VISA) range (4 microg/ml) in parallel with increasing DAP MICs. Also, this Dap(r) strain exhibited significantly thicker cell walls than the parental strain, potentially correlating with the coevolution of the VISA phenotype and implicating cell wall structure and/or function in the Dap(r) phenotype. Importantly, despite the overexpression of mprF and dlt, the relative net positive surface charge was decreased in the Dap(r) mutants, suggesting that other factors contribute to the surface charge alterations and that a simple charge repulsion mechanism could not entirely explain the Dap(r) phenotype in these strains.
Assuntos
Antibacterianos/farmacologia , Membrana Celular/química , Daptomicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Fluidez de Membrana , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Fenótipo , Fosfolipídeos/análiseRESUMO
Carbapenems are potent antibacterials with broad-spectrum activity. However, poor oral absorption generally confines this important drug class to in-hospital use by intravenous (IV) administration. The continued rise in drug resistant pathogens creates a need for alternative oral therapies with broad-spectrum activity. SPR994 is a novel formulation of the orally bioavailable pivoxil prodrug of SPR859 (tebipenem) and is being developed as the first oral carbapenem for treatment of complicated urinary tract infections (cUTIs) in adults. Herein, we describe characteristics beneficial to oral administration and compare the in vitro and in vivo activity of SPR859 or SPR994 with IV carbapenems.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Carbapenêmicos/administração & dosagem , Carbapenêmicos/farmacocinética , Administração Intravenosa , Administração Oral , Adulto , Animais , Antibacterianos/química , Antibacterianos/uso terapêutico , Disponibilidade Biológica , Carbapenêmicos/química , Carbapenêmicos/uso terapêutico , Modelos Animais de Doenças , Microbioma Gastrointestinal/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hospitalização , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Resultado do Tratamento , Infecções Urinárias/tratamento farmacológicoRESUMO
The discovery of urgently needed antibiotics is hindered by challenges to information sharing. To help address this challenge, The Pew Charitable Trusts launched SPARK: the Shared Platform for Antibiotic Research and Knowledge. SPARK is an online, publicly available, interactive database designed to help scientists build on previous research and generate new insights to advance the field's understanding of Gram-negative permeability. This Viewpoint details how data are selected and integrated into the platform, how scientists can use SPARK to share their data, and the ways the scientific community can access and use these data to develop hypotheses.
Assuntos
Antibacterianos , Bases de Dados Factuais , Descoberta de Drogas , Disseminação de Informação , Colaboração Intersetorial , Pesquisa , Instituições de Caridade , Saúde Global , Bactérias Gram-Negativas/efeitos dos fármacos , HumanosRESUMO
Alkyl quinolone molecules 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) are important quorum sensing signals, which play a mediatory role in the pathogenesis of acute and chronic Pseudomonas aeruginosa infection. A targeted approach inhibiting the bacterial 'multiple virulence factor regulon' (MvfR) protein complex, offers the possibility to block the synthesis of MvfR-dependant signal molecules. Here, a high throughput bioanalytical method was developed using LC-MS/MS detection for the selective determination of HHQ and PQS in mouse tissue homogenate, over a sensitive range of 1-5000 and 10-5000pg/mL, respectively. Chromatographic peak distortion of the iron chelator PQS was overcome with the applied use of a bidentate chelator mobile phase additive 2-Picolinic acid at 0.2mM concentration, giving an improved separation and response for the analyte, whilst maintaining overall MS system robustness. Following thigh infection with P. aeruginosa strain 2-PA14 in mice, the concentration and time course of HHQ and PQS (4-hydroxy-2-alkyl-quinolone (HAQ) biomarkers) residing in the biophase were evaluated, and exhibited a low level combined with a substantial inter-individual variability. Quantifiable levels could be obtained from approximately 15h post infection, to the study termination at 21-22h. A dose dependant reduction in HAQ tissue concentrations at selected time points were obtained following MvfR inhibitor administration versus drug vehicle (p<0.01, Kruskal-Wallis-one way ANOVA) and meta -analyses of several studies enabled an inhibitory concentration (IC50) of 80nM free drug to be determined. However, due to the experimental limitations a defined time profile for in-vivo HAQ production could not be characterised. Microsomal stability measurements demonstrated a rapid metabolic clearance of both alkyl quinolone biomarkers in the bacterial host, with a hepatic extraction ratio greater than 0.96 (the measurable assay limit). High clearance underpinned the low concentrations present in the well-perfused thigh tissue. Along with method development and validation details, this paper considers the kinetics of in-vivo HAQ bio-synthesis during Pseudomonas infection; and risks of biomarker over-estimation from samples which contain an exogenous population of bacteria.
Assuntos
4-Quinolonas/análise , Infecções por Pseudomonas , Pseudomonas aeruginosa/química , Percepção de Quorum , Espectrometria de Massas em Tandem/métodos , 4-Quinolonas/metabolismo , Animais , Cromatografia Líquida/métodos , Humanos , Camundongos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Músculo Esquelético/microbiologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/fisiologiaRESUMO
Daptomycin resistance (DAP(R)) in Staphylococcus aureus is associated with mutations in genes that are also implicated in staphylococcal pathogenesis. Using a laboratory-derived series of DAP exposed strains, we showed a relationship between increasing DAP MIC and reduced virulence in a Galleria mellonella infection model. Point mutations in walK and rpoC led to cumulative reductions in virulence and simultaneous increases in DAP MIC. A point mutation to mprF did not impact on S.aureus virulence; however deletion of mprF led to virulence attenuation and hyper-susceptibility to DAP. To validate our findings in G. mellonella, we confirmed the attenuated virulence of select isolates from the laboratory-derived series using a murine septicaemia model. As a corollary, we showed significant virulence reductions for clinically-derived DAP(R) isolates compared to their isogenic, DAP-susceptible progenitors (DAP(S)). Intriguingly, each clinical DAP(R) isolate was persistent in vivo. Taken together, it appears the genetic correlates underlying daptomycin resistance in S. aureus also alter pathogenicity.
Assuntos
Antibacterianos/farmacologia , Daptomicina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Genótipo , Larva/microbiologia , Testes de Sensibilidade Microbiana , Mariposas/microbiologia , Fenótipo , Mutação Puntual , Staphylococcus aureus/genética , VirulênciaRESUMO
Daptomycin is an extensively used anti-staphylococcal agent due to the rise in methicillin-resistant Staphylococcus aureus, but the mechanism(s) of resistance is poorly understood. Comparative genome sequencing, transcriptomics, ultrastructure, and cell envelope studies were carried out on two relatively higher level (4 and 8 µg/ml(-1)) laboratory-derived daptomycin-resistant strains (strains CB1541 and CB1540 respectively) compared to their parent strain (CB1118; MW2). Several mutations were found in the strains. Both strains had the same mutations in the two-component system genes walK and agrA. In strain CB1540 mutations were also detected in the ribose phosphate pyrophosphokinase (prs) and polyribonucleotide nucleotidyltransferase genes (pnpA), a hypothetical protein gene, and in an intergenic region. In strain CB1541 there were mutations in clpP, an ATP-dependent protease, and two different hypothetical protein genes. The strain CB1540 transcriptome was characterized by upregulation of cap (capsule) operon genes, genes involved in the accumulation of the compatible solute glycine betaine, ure genes of the urease operon, and mscL encoding a mechanosensitive chanel. Downregulated genes included smpB, femAB and femH involved in the formation of the pentaglycine interpeptide bridge, genes involved in protein synthesis and fermentation, and spa encoding protein A. Genes altered in their expression common to both transcriptomes included some involved in glycine betaine accumulation, mscL, ure genes, femH, spa and smpB. However, the CB1541 transcriptome was further characterized by upregulation of various heat shock chaperone and protease genes, consistent with a mutation in clpP, and lytM and sceD. Both strains showed slow growth, and strongly decreased autolytic activity that appeared to be mainly due to decreased autolysin production. In contrast to previous common findings, we did not find any mutations in phospholipid biosynthesis genes, and it appears there are multiple pathways to and factors in daptomycin resistance.