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1.
Nat Commun ; 12(1): 3577, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117249

RESUMO

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.


Assuntos
Antibacterianos/farmacologia , Diterpenos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Lincosamidas/farmacologia , Compostos Policíclicos/farmacologia , Estreptograminas/farmacologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Farmacorresistência Bacteriana/genética , Bactérias Gram-Positivas/genética , Modelos Moleculares , Peptidil Transferases/metabolismo , Conformação Proteica , RNA Mensageiro , Ribossomos/metabolismo
2.
J Antimicrob Chemother ; 76(6): 1467-1471, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33755133

RESUMO

BACKGROUND: To address the growing antibiotic resistance problem, new antibacterial drugs must exert activity against pathogens resistant to agents already in use. With a view to providing a rapid means for deselecting antibacterial drug candidates that fail to meet this requirement, we report here the generation and application of a platform for detecting cross-resistance between established and novel antibacterial agents. METHODS: This first iteration of the cross-resistance platform (CRP) consists of 28 strains of defined resistance genotype, established in a uniform genetic background (the SH1000 strain of the clinically significant pathogen Staphylococcus aureus). Most CRP members were engineered through introduction of constitutively expressed resistance determinants on a low copy-number plasmid, with a smaller number selected as spontaneous resistant mutants. RESULTS: Members of the CRP collectively exhibit resistance to many of the major classes of antibacterial agent in use. We employed the CRP to test two antibiotics that have been proposed in the literature as potential drug candidates: γ-actinorhodin and batumin. No cross-resistance was detected for γ-actinorhodin, whilst a CRP member resistant to triclosan exhibited a 32-fold reduction in susceptibility to batumin. Thus, a resistance phenotype that already exists in clinical strains mediates profound resistance to batumin, implying that this compound is not a promising antibacterial drug candidate. CONCLUSIONS: By detecting cross-resistance between established and novel antibacterial agents, the CRP offers the ability to deselect compounds whose activity is substantially impaired by existing resistance mechanisms. The CRP therefore represents a useful addition to the antibacterial drug discovery toolbox.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Descoberta de Drogas , Humanos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/genética
3.
Structure ; 28(10): 1087-1100.e3, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32857965

RESUMO

Acinetobacter baumannii is a Gram-negative bacterium primarily associated with hospital-acquired, often multidrug-resistant (MDR) infections. The ribosome-targeting antibiotics amikacin and tigecycline are among the limited arsenal of drugs available for treatment of such infections. We present high-resolution structures of the 70S ribosome from A. baumannii in complex with these antibiotics, as determined by cryoelectron microscopy. Comparison with the ribosomes of other bacteria reveals several unique structural features at functionally important sites, including around the exit of the polypeptide tunnel and the periphery of the subunit interface. The structures also reveal the mode and site of interaction of these drugs with the ribosome. This work paves the way for the design of new inhibitors of translation to address infections caused by MDR A. baumannii.


Assuntos
Acinetobacter baumannii/citologia , Amicacina/química , Antibacterianos/química , Ribossomos/química , Tigeciclina/química , Acinetobacter baumannii/química , Sítios de Ligação , Microscopia Crioeletrônica , Modelos Moleculares , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Ribossomos/metabolismo
4.
Chem Commun (Camb) ; 56(58): 8047-8050, 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32538401

RESUMO

The feasibility of using activity-directed synthesis to drive antibacterial discovery was investigated. An array of 220 Pd-catalysed microscale reactions was executed, and the crude product mixtures were evaluated for activity against Staphylococcus aureus. Scale-up of the hit reactions, purification and evaluation, enabled expansion of a class of antibacterial quinazolinones. The novel antibacterials had MICs from 0.016 µg mL-1 (i.e. 38 nM) to 2-4 µg mL-1 against S. aureus ATCC29213.


Assuntos
Anilidas/farmacologia , Antibacterianos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Anilidas/síntese química , Anilidas/química , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
5.
Nat Rev Microbiol ; 18(11): 637-648, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32587401

RESUMO

Antibiotic resistance is mediated through several distinct mechanisms, most of which are relatively well understood and the clinical importance of which has long been recognized. Until very recently, neither of these statements was readily applicable to the class of resistance mechanism known as target protection, a phenomenon whereby a resistance protein physically associates with an antibiotic target to rescue it from antibiotic-mediated inhibition. In this Review, we summarize recent progress in understanding the nature and importance of target protection. In particular, we describe the molecular basis of the known target protection systems, emphasizing that target protection does not involve a single, uniform mechanism but is instead brought about in several mechanistically distinct ways.


Assuntos
Antibacterianos , Proteínas de Bactérias , Farmacorresistência Bacteriana/fisiologia , Modelos Moleculares , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/química , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo
6.
J Infect Dis ; 221(6): 1000-1016, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31628459

RESUMO

Prolonging the clinical effectiveness of ß-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between ß-lactams and DCS. DCS resensitized MRSA to ß-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to ß-lactam antibiotics.


Assuntos
Alanina/metabolismo , Antibacterianos/farmacologia , Ciclosserina/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , beta-Lactamas/farmacologia , Animais , Antimetabólitos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Transporte Biológico , Feminino , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Camundongos , Mutação , Polissacarídeos/química , Polissacarídeos/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
7.
mBio ; 10(5)2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31662453

RESUMO

Sporadic literature reports describe isolates of pathogenic bacteria that harbor an antibiotic resistance determinant but remain susceptible to the corresponding antibiotic as a consequence of a genetic defect. Such strains represent a source from which antibiotic resistance may reemerge to cause treatment failure in patients. Here, we report a systematic investigation into the prevalence and nature of this phenomenon, which we term silencing of antibiotic resistance by mutation (SARM). Instances of SARM were detected among 1,470 Staphylococcus aureus isolates through side-by-side comparison of antibiotic resistance genotype (as determined by whole-genome sequencing) versus phenotype (as assessed through susceptibility testing). Of the isolates analyzed, 152 (10.3%) harbored a silenced resistance gene, including 46 (3.1%) that exhibited SARM to currently deployed antistaphylococcal drugs. SARM resulted from diverse mutational events but most commonly through frameshift mutation of resistance determinants as a result of point deletion in poly(A) tracts. The majority (∼90%) of SARM strains reverted to antibiotic resistance at frequencies of ≥10-9; thus, while appearing antibiotic sensitive in the clinical microbiology laboratory, most S. aureus isolates exhibiting SARM will revert to antibiotic resistance at frequencies achievable in patients. In view of its prevalence in a major pathogen, SARM represents a significant potential threat to the therapeutic efficacy of antibiotics.IMPORTANCE Antibiotic resistance hinders the treatment of bacterial infection. To guide effective therapy, clinical microbiology laboratories routinely perform susceptibility testing to determine the antibiotic sensitivity of an infecting pathogen. This approach relies on the assumption that it can reliably distinguish bacteria capable of expressing antibiotic resistance in patients, an idea challenged by the present study. We report that the important human pathogen Staphylococcus aureus frequently carries antibiotic resistance genes that have become inactivated ("silenced") by mutation, leading strains to appear antibiotic sensitive. However, resistance can rapidly reemerge in most such cases, at frequencies readily achievable in infected patients. Silent antibiotic resistance is therefore prevalent, transient, and evades routine detection, rendering it a significant potential threat to antibacterial chemotherapy.


Assuntos
Farmacorresistência Bacteriana/genética , Inativação Gênica , Mutação , Staphylococcus aureus/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Sequenciamento Completo do Genoma
8.
J Antimicrob Chemother ; 74(4): 907-911, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30590494

RESUMO

OBJECTIVES: Bacterial biofilms represent a major impediment to healing in chronic wounds and are largely refractory to the antibacterial agents currently used in wound management. From a repurposing screen of compounds considered safe for topical application in humans, we report the identification of the personal care product preservatives bronopol and bronidox as broad-spectrum antibiofilm agents and potential candidates for reducing biofilm burden in chronic wounds. METHODS: Antibiofilm activity was assessed by viable counting against single-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa in the Calgary Biofilm Device, and against mixed-species biofilms of the two organisms growing on nitrocellulose discs. RESULTS: Bronopol and bronidox exhibited broad-spectrum antibiofilm activity that encompassed the two major wound pathogens, S. aureus and P. aeruginosa. When impregnated into gauze dressings at their existing maximum authorized concentrations for safe use and placed onto an established mixed-species biofilm, bronopol and bronidox completely eradicated P. aeruginosa and achieved an ∼5 log10 reduction in the S. aureus population. The antibiofilm action of bronopol and bronidox was attributed to their ability to kill slow- or non-growing bacteria found in biofilms, and both compounds exhibited synergistic antibiofilm effects in combination with established wound-treatment agents. CONCLUSIONS: Bronopol and bronidox kill bacteria regardless of growth state, a property that endows them with broad-spectrum antibiofilm activity. As this effect is observed at concentrations authorized for use on human skin, these compounds represent promising candidates for the treatment of chronic wounds.


Assuntos
Anti-Infecciosos Locais/farmacologia , Biofilmes/efeitos dos fármacos , Dioxanos/farmacologia , Conservantes Farmacêuticos/farmacologia , Propilenoglicóis/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Reposicionamento de Medicamentos , Humanos , Viabilidade Microbiana/efeitos dos fármacos
9.
mSphere ; 3(6)2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30541781

RESUMO

Resistance to the lantibiotic nisin (NIS) arises readily in Staphylococcus aureus as a consequence of mutations in the nsaS gene, which encodes the sensor kinase of the NsaRS two-component regulatory system. Here we present a series of studies to establish how these mutational changes result in reduced NIS susceptibility. Comparative transcriptomic analysis revealed upregulation of the NsaRS regulon in a NIS-resistant mutant of S. aureus versus its otherwise-isogenic progenitor, indicating that NIS resistance mutations prompt gain-of-function in NsaS. Two putative ABC transporters (BraDE and VraDE) encoded within the NsaRS regulon that have been reported to provide a degree of intrinsic protection against NIS were shown to be responsible for acquired NIS resistance; as is the case for intrinsic NIS resistance, NIS detoxification was ultimately mediated by VraDE, with BraDE participating in the signaling cascade underlying VraDE expression. Our study revealed new features of this signal transduction pathway, including that BraDE (but not VraDE) physically interacts with NsaRS. Furthermore, while BraDE has been shown to sense stimuli and signal to NsaS in a process that is contingent upon ATP hydrolysis, we established that this protein complex is also essential for onward transduction of the signal from NsaS through energy-independent means. NIS resistance in S. aureus therefore joins the small number of documented examples in which acquired antimicrobial resistance results from the unmasking of an intrinsic detoxification mechanism through gain-of-function mutation in a regulatory circuit.IMPORTANCE NIS and related bacteriocins are of interest as candidates for the treatment of human infections caused by Gram-positive pathogens such as Staphylococcus aureus An important liability of NIS in this regard is the ease with which S. aureus acquires resistance. Here we establish that this organism naturally possesses the cellular machinery to detoxify NIS but that the ABC transporter responsible (VraDE) is not ordinarily produced to a degree sufficient to yield substantial resistance. Acquired NIS resistance mutations prompt activation of the regulatory circuit controlling expression of vraDE, thereby unmasking an intrinsic resistance determinant. Our results provide new insights into the complex mechanism by which expression of vraDE is regulated and suggest that a potential route to overcoming the resistance liability of NIS could involve chemical modification of the molecule to prevent its recognition by the VraDE transporter.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Inativação Metabólica , Redes e Vias Metabólicas , Nisina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mutação , Nisina/metabolismo , Transdução de Sinais , Staphylococcus aureus/genética
10.
Genome Biol ; 19(1): 94, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-30021593

RESUMO

BACKGROUND: Fitness costs imposed on bacteria by antibiotic resistance mechanisms are believed to hamper their dissemination. The scale of these costs is highly variable. Some, including resistance of Staphylococcus aureus to the clinically important antibiotic mupirocin, have been reported as being cost-free, which suggests that there are few barriers preventing their global spread. However, this is not supported by surveillance data in healthy communities, which indicate that this resistance mechanism is relatively unsuccessful. RESULTS: Epistasis analysis on two collections of MRSA provides an explanation for this discord, where the mupirocin resistance-conferring mutation of the ileS gene appears to affect the levels of toxins produced by S. aureus when combined with specific polymorphisms at other loci. Proteomic analysis demonstrates that the activity of the secretory apparatus of the PSM family of toxins is affected by mupirocin resistance. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production becomes necessary. This hidden fitness cost provides a likely explanation for why this mupirocin-resistance mechanism is not more prevalent, given the widespread use of this antibiotic. CONCLUSIONS: With dwindling pools of antibiotics available for use, information on the fitness consequences of the acquisition of resistance may need to be considered when designing antibiotic prescribing policies. However, this study suggests there are levels of depth that we do not understand, and that holistic, surveillance and functional genomics approaches are required to gain this crucial information.


Assuntos
Antibacterianos/farmacologia , Epistasia Genética , Aptidão Genética/efeitos dos fármacos , Genoma Bacteriano , Isoleucina-tRNA Ligase/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Mupirocina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Farmacorresistência Bacteriana , Evolução Molecular , Loci Gênicos , Isoleucina-tRNA Ligase/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Proteômica/métodos
11.
Front Microbiol ; 9: 129, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29472905

RESUMO

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies.

12.
ACS Med Chem Lett ; 9(2): 84-88, 2018 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-29456792

RESUMO

N-Leucinyl benzenesulfonamides have been discovered as a novel class of potent inhibitors of E. coli leucyl-tRNA synthetase. The binding of inhibitors to the enzyme was measured by using isothermal titration calorimetry. This provided information on enthalpy and entropy contributions to binding, which, together with docking studies, were used for structure-activity relationship analysis. Enzymatic assays revealed that N-leucinyl benzenesulfonamides display remarkable selectivity for E. coli leucyl-tRNA synthetase compared to S. aureus and human orthologues. The simplest analogue of the series, N-leucinyl benzenesulfonamide (R = H), showed the highest affinity against E. coli leucyl-tRNA synthetase and also exhibited antibacterial activity against Gram-negative pathogens (the best MIC = 8 µg/mL, E. coli ATCC 25922), which renders it as a promising template for antibacterial drug discovery.

13.
ACS Infect Dis ; 4(3): 239-246, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29376318

RESUMO

Members of the ATP-binding cassette (ABC)-F protein subfamily collectively mediate resistance to a broader range of clinically important antibiotic classes than any other group of resistance proteins and are widespread in pathogenic bacteria. Following over 25 years' of controversy regarding the mechanism by which these proteins work, it has recently been established that they provide antibiotic resistance through the previously recognized but underappreciated phenomenon of target protection; they bind to the ribosome to effect the release of ribosome-targeted antibiotics, thereby rescuing the translation apparatus from antibiotic-mediated inhibition. Here we review the ABC-F resistance proteins with an emphasis on their mechanism of action, first exploring the history of the debate about how these proteins work and outlining our current state of knowledge and then considering key questions to be addressed in understanding the molecular detail of their function.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/metabolismo , Bactérias/enzimologia , Farmacorresistência Bacteriana , Biossíntese de Proteínas , Ribossomos/metabolismo , Bactérias/efeitos dos fármacos
14.
Egypt J Chem ; 61(Conference Iss): 9-25, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31937997

RESUMO

AS THE RESISTANCE of Staphylococcus aureus to antibiotics represents a major threat to global health, anti-infectives with novel mechanisms must be developed. Novel compounds were generated as potential phenylalanine tRNA synthetase (PheRS) inhibitors based on the published homology model of S. aureus PheRS to aid the design process using Molecular Operating Environment (MOE) software. PheRS was selected as it is structurally unique enzyme among the aminoacyl-tRNA synthetases (aaRS), it is considerably different from human cytosolic and human mitochondrial aaRS and it is essential and conserved across bacterial species. The designed compounds were synthesized according to different clear schemes. The compounds were confirmed by 1H NMR, 13C NMR, HRMS and/or microanalysis, and they were microbiologically evaluated.

15.
Sci Rep ; 7(1): 17419, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29234001

RESUMO

Of the thousands of natural product antibiotics discovered to date, only a handful have been developed for the treatment of bacterial infection. The clinically unexploited majority likely include compounds with untapped potential as antibacterial drugs, and in view of the ever-growing unmet medical need for such agents, warrant systematic re-evaluation. Here we revisit the actinorhodins, a class that was first reported 70 years ago, but which remains poorly characterized. We show that γ-actinorhodin possesses many of the requisite properties of an antibacterial drug, displaying potent and selective bactericidal activity against key Gram-positive pathogens (including Staphylococcus aureus and enterococci), a mode of action distinct from that of other agents in clinical use, an extremely low potential for the development of resistance, and a degree of in vivo efficacy in an invertebrate model of infection. Our findings underscore the utility of revisiting unexploited antibiotics as a source of novel antibacterial drug candidates.


Assuntos
Antibacterianos/farmacologia , Animais , Antraquinonas/farmacologia , Candida albicans/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Descoberta de Drogas , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Humanos , Lactonas/farmacologia , Lepidópteros , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Streptomyces coelicolor/efeitos dos fármacos
16.
J Antimicrob Chemother ; 72(11): 3043-3046, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28981647

RESUMO

Objectives: To assess the prevalence of cryptic silver (Ag+) resistance amongst clinical isolates of Gram-negative bacteria, and to examine how overt Ag+ resistance becomes activated in such strains. Methods: Established methods were used to determine the susceptibility of 444 recent clinical isolates to Ag+, and to evaluate the potential for overt Ag+ resistance to emerge in susceptible isolates by spontaneous mutation. The genetic basis for Ag+ resistance was investigated using PCR amplification and DNA sequencing. Results: None of the isolates tested displayed overt Ag+ resistance. However, upon silver challenge, high-level Ag+ resistance (silver nitrate MIC >128 mg/L) was selected at high frequency (10-7 to 10-8) in 76% of isolates of Enterobacter spp., ∼58% of isolates of Klebsiella spp. and ∼0.7% of isolates of Escherichia coli. All strains in which Ag+ resistance could be selected harboured the sil operon, with resistance apparently resulting from activation of this system as a consequence of single missense mutations in silS. By contrast, Ag+ resistance was not selected in isolates lacking sil, which included all tested representatives of Pseudomonas aeruginosa, Acinetobacter spp., Citrobacter spp. and Proteus spp. Conclusions: Whilst overt Ag+ resistance in Gram-negative pathogens is uncommon, cryptic Ag+ resistance pertaining to the sil operon is prevalent and readily activated in particular genera (Enterobacter and Klebsiella).


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Nitrato de Prata/farmacologia , Prata/farmacologia , Enterobacter/efeitos dos fármacos , Escherichia coli/genética , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Klebsiella/efeitos dos fármacos , Mutação de Sentido Incorreto/efeitos dos fármacos , Óperon , Prevalência
19.
Antimicrob Agents Chemother ; 60(10): 6359-61, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27431224

RESUMO

Bacterial aminoacyl-tRNA synthetases (aaRSs) represent promising antibacterial drug targets. Unfortunately, the aaRS inhibitors that have to date reached clinical trials are subject to rapid resistance development through mutation, a phenomenon that limits their potential clinical utility. Here, we confirm the intuitively correct idea that simultaneous targeting of two different aaRS enzymes prevents the emergence of spontaneous bacterial resistance at high frequency, a finding that supports the development of multitargeted anti-aaRS therapies.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Compostos de Boro/farmacologia , Diaminas/farmacologia , Testes de Sensibilidade Microbiana , Terapia de Alvo Molecular , Mupirocina/farmacologia , Taxa de Mutação , Staphylococcus aureus/genética , Tiofenos/farmacologia
20.
PLoS One ; 11(5): e0155139, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27167126

RESUMO

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies.


Assuntos
Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Carbazóis/química , Carbazóis/farmacologia , Divisão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Ácidos Graxos/biossíntese , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Genes Bacterianos , Cinética , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lipossomos/química , Substâncias Macromoleculares/metabolismo , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mutação/genética , Fosfolipídeos/metabolismo , Pseudomonas aeruginosa/genética , Análise de Sequência de DNA , Staphylococcus aureus/genética , Fatores de Tempo
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