RESUMO
Staphylococcus aureus has the ability to invade cortical bone osteocyte lacuno-canalicular networks (OLCNs) and cause osteomyelitis. It was recently established that the cell wall transpeptidase, penicillin-binding protein 4 (PBP4), is crucial for this function, with pbp4 deletion strains unable to invade OLCNs and cause bone pathogenesis in a murine model of S. aureus osteomyelitis. Moreover, PBP4 has recently been found to modulate S. aureus resistance to ß-lactam antibiotics. As such, small molecule inhibitors of S. aureus PBP4 may represent dual functional antimicrobial agents that limit osteomyelitis and/or reverse antibiotic resistance. A high throughput screen recently revealed that the phenyl-urea 1 targets PBP4. Herein, we describe a structure-activity relationship (SAR) study on 1. Leveraging in silico docking and modeling, a set of analogs was synthesized and assessed for PBP4 inhibitory activities. Results revealed a preliminary SAR and identified lead compounds with enhanced binding to PBP4, more potent antibiotic resistance reversal, and diminished PBP4 cell wall transpeptidase activity in comparison to 1.
Assuntos
Antibacterianos , Simulação de Acoplamento Molecular , Proteínas de Ligação às Penicilinas , Staphylococcus aureus , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Antibacterianos/farmacologia , Antibacterianos/química , Relação Estrutura-Atividade , Staphylococcus aureus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ureia/química , Ureia/farmacologia , Ureia/análogos & derivados , Animais , Camundongos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidoresRESUMO
PURPOSE: Antimicrobial resistance is a global health threat, compounded by the reduction in the discovery of new antibiotics. A repurposed drugs-based approach could provide a viable alternative for the treatment of multidrug-resistant (MDR) bacterial infections. In this study, we sought to evaluate the in vitro efficacy of a novel drug combination, polymyxin B/trimethoprim (PT) + rifampin on MDR isolates from patients with bacterial keratitis in India. METHODS: Forty-three isolates, which included 20 Staphylococcus aureus , 19 Pseudomonas aeruginosa , 3 Pseudomonas stutzeri , and 1 Acinetobacter baumannii , were evaluated for their antibiotic resistance by minimum inhibitory concentration (MIC). Fractional Inhibitory Concentration Index (FICI) testing was performed to measure the antimicrobial impact of PT + rifampin in combination. RESULTS: Among S. aureus isolates, 100% were resistant to at least 1 antibiotic class, 12 (60%) were MDR, and 14 (70%) were classified as methicillin-resistant. Among the gram-negative isolates, >90% were classified as MDR. Fractional Inhibitory Concentration (FIC) testing revealed that PT + rifampin was effective in completely inhibiting growth of all isolates while also displaying additive or synergistic activity in approximately 70% of the strains. Mean FICI values were 0.753 ± 0.311 and 0.791 ± 0.369 for S. aureus and gram-negative isolates, respectively, and a >2-fold reduction in MIC was measured for both PT and rifampin when tested in combination versus alone. CONCLUSIONS: Our data demonstrate the ability of PT + rifampin to eliminate all isolates tested, even those conferring MDR, highlighting the promise of this drug combination for the treatment of bacterial keratitis.
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Antibacterianos , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla , Infecções Oculares Bacterianas , Testes de Sensibilidade Microbiana , Polimixina B , Rifampina , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/tratamento farmacológico , Rifampina/farmacologia , Rifampina/uso terapêutico , Polimixina B/farmacologia , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico , Quimioterapia CombinadaRESUMO
Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.
Assuntos
Brucelose , Pequeno RNA não Traduzido , Animais , Camundongos , Brucella abortus/metabolismo , Regulação da Expressão Gênica , Macrófagos , Camundongos Endogâmicos BALB C , Proteômica , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismoRESUMO
Acinetobacter baumannii is a nosocomial pathogen of serious healthcare concern that is becoming increasingly difficult to treat due to antibiotic treatment failure. Recent studies have revealed that clinically defined antibiotic-susceptible strains upregulate the expression of a repertoire of putative drug efflux pumps during their growth under biologically relevant conditions, e.g., in human serum, resulting in efflux-associated resistance to physiologically achievable antibiotic levels within a patient. This phenomenon, termed Adaptive Efflux Mediated Resistance (AEMR), has been hypothesized to account for one mechanism by which antibiotic-susceptible A. baumannii fails to respond to antibiotic treatment. In the current study, we sought to identify genetic determinants that contribute to A. baumannii serum-associated AEMR by screening a transposon mutant library for members that display a loss of the AEMR phenotype. Results revealed that mutation of a putative pirin-like protein, YhaK, results in a loss of AEMR, a phenotype that could be complemented by a wild-type copy of the yhaK gene and was verified in a second strain background. Ethidium bromide efflux assays confirmed that the loss of AEMR phenotype due to pirin-like protein mutation correlated with reduced overarching efflux capacity. Further, flow cytometry and confocal microscopy measures of a fluorophore 7-(dimethylamino)-coumarin-4-acetic acid (DMACA)-tagged levofloxacin isomer, ofloxacin, further verified that YhaK mutation reduces AEMR-mediated antibiotic efflux. RNA-sequencing studies revealed that YhaK may be required for the expression of multiple efflux-associated systems, including MATE and ABC families of efflux pumps. Collectively, the data indicate that the A. baumannii YhaK pirin-like protein plays a role in modulating the organism's adaptive efflux-mediated resistance phenotype.
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Methicillin-resistant Staphylococcus aureus (MRSA) is a global healthcare concern. Such resistance has historically been attributed to the acquisition of mecA (or mecC), which encodes an alternative penicillin binding protein, PBP2a, with low ß-lactam affinity. However, recent studies have indicated that penicillin binding protein 4 (PBP4) is also a critical determinant of S. aureus methicillin resistance, particularly among community-acquired MRSA strains. Thus, PBP4 has been considered an intriguing therapeutic target as corresponding inhibitors may restore MRSA ß-lactam susceptibility. In addition to its role in antibiotic resistance, PBP4 has also recently been shown to be required for S. aureus cortical bone osteocyte lacuno-canalicular network (OLCN) invasion and colonization, providing the organism with a niche for re-occurring bone infection. From these perspectives, the development of PBP4 inhibitors may have tremendous impact as agents that both reverse methicillin resistance and inhibit the organism's ability to cause chronic osteomyelitis. Accordingly, using a whole-cell high-throughput screen of a 30,000-member small molecule chemical library and secondary assays we identified putative S. aureus PBP4 inhibitors. Quantitative reverse transcriptase mediated PCR and PBP4 binding assays revealed that hits could be further distinguished as compounds that reduce PBP4 expression versus compounds that are likely to affect the protein's function. We also showed that 6.25 µM (2.5 µg/mL) of the lead candidate, 9314848, reverses the organism's PBP4-dependent MRSA phenotype and inhibits its ability to traverse Microfluidic-Silicon Membrane-Canalicular Arrays (µSiM-CA) that model the OLCN orifice. Collectively, these molecules may represent promising potential as PBP4-inhibitors that can be further developed as adjuvants for the treatment of MRSA infections and/or osteomyelitis prophylactics.
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Purpose: Pseudomonas aeruginosa is a leading cause of corneal infections. Recently, we discovered an antimicrobial drug combination, polymyxin B/trimethoprim (PT) + rifampin, that displayed impressive efficacy toward P. aeruginosa in both in vitro and in vivo studies. As such, this combination was further evaluated as a potential keratitis therapeutic through testing the combination's efficacy against a diverse set of P. aeruginosa clinical isolates. Methods: Minimum inhibitory concentrations (MICs) of moxifloxacin, levofloxacin, erythromycin, tobramycin, PT, polymyxin B (alone), trimethoprim (alone), and rifampin were determined for 154 ocular clinical P. aeruginosa isolates, 90% of which were derived from corneal scrapings. Additionally, the efficacy of PT + rifampin was evaluated utilizing fractional inhibitory concentration (FIC) testing. Results: While 100% of isolates were resistant to erythromycin (average MIC 224 ± 110 µg·mL-1) and trimethoprim (alone) (206 ± 67.3 µg·mL-1), antibiotic resistance was generally found to be low: moxifloxacin (2% of isolates resistant; average MIC 1.08 ± 1.61 µg·mL-1), levofloxacin (3.9%; 1.02 ± 2.96 µg·mL-1), tobramycin (1%; 0.319 ± 1.31 µg·mL-1), polymyxin B (0%; 0.539 ± 0.206 µg·mL-1), PT (0%; 0.416 ± 0.135 µg·mL-1), and rifampin (0%; 23.4 ± 6.86 µg·mL-1). Additionally, FIC testing revealed that PT + rifampin eradicated 100% of isolates demonstrating additive or synergistic activity in 95% of isolates (average FIC index 0.701 ± 0.132). Conclusions: The drug combination of PT + rifampin was effective against a large panel of clinically relevant P. aeruginosa strains and, as such, may represent a promising therapeutic for P. aeruginosa keratitis. Translational Relevance: This work furthers the preclinical development of a novel antibiotic combination for the treatment of corneal infections (bacterial keratitis).
Assuntos
Ceratite , Infecções por Pseudomonas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bacitracina/farmacologia , Bacitracina/uso terapêutico , Combinação de Medicamentos , Eritromicina/farmacologia , Eritromicina/uso terapêutico , Framicetina/farmacologia , Framicetina/uso terapêutico , Humanos , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Levofloxacino/farmacologia , Levofloxacino/uso terapêutico , Moxifloxacina/farmacologia , Moxifloxacina/uso terapêutico , Polimixina B/farmacologia , Polimixina B/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Rifampina/farmacologia , Rifampina/uso terapêutico , Tobramicina/farmacologia , Tobramicina/uso terapêutico , Trimetoprima/farmacologia , Trimetoprima/uso terapêuticoRESUMO
Staphylococcus aureus is a major cause of ocular infections, often resulting in devastating vision loss. Despite the significant morbidity associated with these infections, little is yet known regarding the specific strain types that may have a predilection for ocular tissues nor the set of virulence factors that drive its pathogenicity in this specific biological niche. Whole genome sequencing (WGS) can provide valuable insight in this regard by providing a prospective, comprehensive assessment of the strain types and virulence factors driving disease among specific subsets of clinical isolates. As such, a set of 163-member S. aureus ocular clinical strains were sequenced and assessed for both common strain types (multilocus sequence type (MLST), spa, agr) associated with ocular infections as well as the presence/absence of 235 known virulence factors in a high throughput manner. This ocular strain set was then directly compared to a fully sequenced 116-member non-ocular S. aureus strain set curated from NCBI in order to identify key differences between ocular and non-ocular S. aureus isolates. The most common sequence types found among ocular S. aureus isolates were ST5, ST8 and ST30, generally reflecting circulating non-ocular pathogenic S. aureus strains. However, importantly, ocular isolates were found to be significantly enriched for a set of enterotoxins, suggesting a potential role for this class of virulence factors in promoting ocular disease. Further genomic analysis revealed that these enterotoxins are located on mobile pathogenicity islands, thus horizontal gene transfer may promote the acquisition of enterotoxins, potentially amplifying S. aureus virulence in ocular tissues.
Assuntos
Olho/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Genes Bacterianos/genética , Genômica/métodos , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma/métodos , Adulto JovemRESUMO
Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N-aryl-2-nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (e.g., HP 29, against methicillin-resistant Staphylococcus aureus: MIC = 0.075 µM; MBEC = 2.35 µM), and transcriptional analysis revealed that HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34, MIC = 0.80 µM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.
Assuntos
Compostos de Anilina/química , Antibacterianos/síntese química , Fenazinas/química , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Halogenação , Humanos , Ferro/química , Deficiências de Ferro , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Fenazinas/farmacologia , Fenazinas/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Relação Estrutura-Atividade , Cicatrização/efeitos dos fármacosRESUMO
Antibiotic resistance is spreading worldwide and it has become one of the most important issues in modern medicine. In this context, the bacterial RNA degradation and processing machinery are essential processes for bacterial viability that may be exploited for antimicrobial therapy. In Staphylococcus aureus, RnpA has been hypothesized to be one of the main players in these mechanisms. S. aureus RnpA is able to modulate mRNA degradation and complex with a ribozyme (rnpB), facilitating ptRNA maturation. Corresponding small molecule screening campaigns have recently identified a few classes of RnpA inhibitors, and their structure activity relationship (SAR) has only been partially explored. Accordingly, in the present work, using computational modeling of S. aureus RnpA we identified putative crucial interactions of known RnpA inhibitors, and we used this information to design, synthesize, and biologically assess new potential RnpA inhibitors. The present results may be beneficial for the overall knowledge about RnpA inhibitors belonging to both RNPA2000-like thiosemicarbazides and JC-like piperidine carboxamides molecular classes. We evaluated the importance of the different key moieties, such as the dichlorophenyl and the piperidine of JC2, and the semithiocarbazide, the furan, and the i-propylphenyl ring of RNPA2000. Our efforts could provide a foundation for further computational-guided investigations.
RESUMO
Staphylococcus aureus is well-recognized to cause debilitating bacterial infections that are difficult to treat due to the emergence of antibiotic resistance. As such, there is a need to develop new antimicrobials for the therapeutic intervention of S. aureus disease. To that end, S. aureus RnpA is an essential enzyme that is hypothesized to participate in two required cellular processes, precursor tRNA (ptRNA) maturation and mRNA degradation. Corresponding high throughput screening campaigns have identified the phenylcarbamoyl cyclic thiopenes as a chemical class of RnpA inhibitors that display promising antibacterial effects by reducing RnpA ptRNA and mRNA degradation activities and low human cell toxicity. Herein, we perform a structure activity relationship study of the chemical scaffold. Results revealed that the cycloalkane ring size and trifluoroacetamide moiety are required for antibacterial activity, whereas modifications of the para and/or meta positions of the pharmacophore's phenyl group allowed tuning of the scaffold's antimicrobial performance and RnpA inhibitory activity. The top performing compounds with respect to antimicrobial activity also did not exhibit cytotoxicity to human cell lines at concentrations up to 100 µM, greater than 100-fold the minimum inhibitory concentration (MIC). Focused studies of one analog, RNP0012, which exhibited the most potent antimicrobial and inhibition of cellular RnpA activities revealed that the compound reduced bacterial burden in a murine model of S. aureus disease. Taken together, the results presented are expected to provide an early framework for optimization of next-generation of RnpA inhibitor analogues that may represent progenitors of a new class of antimicrobials.
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Small molecule target identification is a critical step in modern antibacterial drug discovery, particularly against multi-drug resistant pathogens. Albocycline (ALB) is a macrolactone natural product with potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) whose mechanism of action has been elusive to date. Herein, we report biochemical and genomic studies that reveal ALB does not target bacterial peptidoglycan biosynthesis or the ribosome; rather, it appears to modulate NADPH ratios and upregulate redox sensing in the cell consistent with previous studies at Upjohn. Owing to the complexity inherent in biological pathways, further genomic assays are needed to identify the true molecular target(s) of albocycline.
Assuntos
Antibacterianos/farmacologia , NADP/genética , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Relação Dose-Resposta a Droga , Lactonas/química , Lactonas/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , NADP/metabolismo , Relação Estrutura-Atividade , Resistência a Vancomicina/efeitos dos fármacosRESUMO
Albocycline (ALB) is a unique macrolactone natural product with potent, narrow-spectrum activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate (VISA), and vancomycin-resistant S. aureus (VRSA) strains (MIC = 0.5-1.0 µg/mL). Described herein is the synthesis and evaluation of a novel series analogs derived from albocycline by functionalization at three specific sites: the C2-C3 enone, the tertiary carbinol at C4, and the allylic C16 methyl group. Exploration of the structure-activity relationships (SAR) by means of minimum inhibitory concentration assays (MICs) revealed that C4 ester analog 6 was twice as potent as ALB, which represents a class of lead compound that can be further studied to address multi-drug resistant pathogens.
Assuntos
Antibacterianos/farmacologia , Produtos Biológicos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Lactonas/síntese química , Lactonas/química , Lactonas/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Acinetobacter baumannii is a nosocomial pathogen of urgent concern for public health due to rising rates of multidrug and pandrug resistance. In the context of environmental cues such as growth in human serum, A. baumannii is known to display adaptive efflux, in which a multitude of efflux-associated genes are upregulated, resulting in efflux-mediated drug tolerance in strains that are otherwise susceptible to antibiotic therapy. Previously, we identified a sulfonamide-containing scaffold molecule (ABEPI1) that reversed serum-associated antibiotic tolerance in A. baumannii. Herein, we present structure-activity relationship studies on 29 newly synthesized analogues. These molecules were characterized for their ability to potentiate multiple antibiotics in serum, reduce serum-associated ethidium bromide efflux and depolarize bacterial cell membranes. In addition, they were assessed for toxicity to mammalian cells. Collectively, these molecules may represent promising potential adjuvants for use in combination with new and existing antibiotics to treat A. baumannii bacterial infections.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Sulfonamidas/farmacologia , Antibacterianos/sangue , Antibacterianos/química , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Tolerância a Medicamentos , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/sangue , Sulfonamidas/químicaRESUMO
BACKGROUND: When grown in human serum, laboratory isolates of Pseudomonas aeruginosa exhibit tolerance to antibiotics at inhibitory concentrations. This phenomenon, known as serum-associated antibiotic tolerance (SAT), could lead to clinical treatment failure of pseudomonal infections. Our purpose in this study was to determine the prevalence and clinical impact of SAT in Pseudomonas isolates in hospitalized children. METHODS: The SAT phenotype was assessed in patients aged <18 years admitted with respiratory or blood cultures positive for P. aeruginosa. The SAT phenotype was a priori defined as a ≥2-log increase in colony-forming units when grown in human serum compared with Luria-Bertani medium in the presence of minocycline or tobramycin. RESULTS: SAT was detected in 29 (64%) patients. Fourteen patients each (34%) had cystic fibrosis (CF) and tracheostomies. Patient demographics and comorbidities did not differ by SAT status. Among CF patients, SAT was associated with longer duration of intravenous antibiotics (10 days vs 5 days; Pâ <â .01). CONCLUSIONS: This study establishes that SAT exists in P. aeruginosa from human serum and may be a novel factor that contributes to differences in clinical outcomes. Future research should investigate the mechanisms that contribute to SAT in order to identify novel targets for adjunctive antimicrobial therapies.
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Fibrose Cística , Infecções por Pseudomonas , Antibacterianos/uso terapêutico , Criança , Fibrose Cística/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa , TobramicinaRESUMO
Bacterial keratitis causes significant blindness, yet antimicrobial resistance has rendered current treatments ineffective. Polymyxin B-trimethoprim (PT) plus rifampin has potent in vitro activity against Staphylococcus aureus and Pseudomonas aeruginosa, two important causes of keratitis. Here we further characterize this combination against P. aeruginosa in a murine keratitis model. PT plus rifampin performed comparably to or better than moxifloxacin, the gold standard, suggesting that the combination may be a promising therapy for bacterial keratitis.
Assuntos
Antibacterianos/farmacologia , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Córnea/efeitos dos fármacos , Córnea/microbiologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Quimioterapia Combinada/métodos , Infecções Oculares Bacterianas/microbiologia , Feminino , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana/métodos , Polimixina B/farmacologia , Rifampina/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Trimetoprima/farmacologiaRESUMO
BACKGROUND AND OBJECTIVES: Hospital-acquired infections (HAIs) and multidrug resistant bacteria pose a significant threat to the U.S. healthcare system. With a dearth of new antibiotic approvals, novel antimicrobial strategies are required to help solve this problem. Violet-blue visible light (400-470 nm) has been shown to elicit strong antimicrobial effects toward many pathogens, including representatives of the ESKAPE bacterial pathogens, which have a high propensity to cause HAIs. However, phototherapeutic solutions to prevention or treating infections are currently limited by efficient and nonobtrusive light-delivery mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Here, we investigate the in vitro antimicrobial properties of flexible Corning® light-diffusing fiber (LDF) toward members of the ESKAPE pathogens in a variety of growth states and in the context of biological materials. Bacteria were grown on agar surfaces, in liquid culture and on abiotic surfaces. We also explored the effects of 405 nm light within the presence of lung surfactant, human serum, and on eukaryotic cells. Pathogens tested include Enterococcus spp, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Staphylococcus epidermidis, Streptococcus pyogenes, Candida albicans, and Escherichia coli. RESULTS: Overall, the LDF delivery of 405 nm violet-blue light exerted a significant degree of microbicidal activity against a wide range of pathogens under diverse experimental conditions. CONCLUSIONS: The results exemplify the fiber's promise as a non-traditional approach for the prevention and/or therapeutic intervention of HAIs. Lasers Surg. Med. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.
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Candida albicans/efeitos da radiação , Infecção Hospitalar/prevenção & controle , Desinfecção/métodos , Bactérias Gram-Negativas/efeitos da radiação , Bactérias Gram-Positivas/efeitos da radiação , Lasers Semicondutores/uso terapêutico , Fibras Ópticas , Candidíase/prevenção & controle , Desinfecção/instrumentação , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Positivas/prevenção & controle , Humanos , Terapia com Luz de Baixa Intensidade , Testes de Sensibilidade MicrobianaRESUMO
Staphylococcus aureus RnpA is thought to be a unique dual functional antimicrobial target that is required for two essential cellular processes, precursor tRNA processing and messenger RNA degradation. Herein, we used a previously described whole cell-based mupirocin synergy assay to screen members of a 53,000 compound small molecule diversity library and simultaneously enrich for agents with cellular RnpA inhibitory activity. A medicinal chemistry-based campaign was launched to generate a preliminary structure activity relationship and guide early optimization of two novel chemical classes of RnpA inhibitors identified, phenylcarbamoyl cyclic thiophene and piperidinecarboxamide. Representatives of each chemical class displayed potent anti-staphylococcal activity, limited the protein's in vitro ptRNA processing and mRNA degradation activities, and exhibited favorable therapeutic indexes. The most potent piperidinecarboxamide RnpA inhibitor, JC2, displayed inhibition of cellular RnpA mRNA turnover, RnpA-depletion strain hypersusceptibility, and exhibited antimicrobial efficacy in a wax worm model of S. aureus infection. Taken together, these results establish that the whole cell screening assay used is amenable to identifying small molecule RnpA inhibitors within large chemical libraries and that the chemical classes identified here may represent progenitors of new classes of antimicrobials that target RnpA.
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Osteomyelitis is a chronic bone infection that is often treated with adjuvant antibiotic-impregnated poly(methyl methacrylate) (PMMA) cement spacers in multi-staged revisions. However, failure rates remain substantial due to recurrence of infection, which is attributed to the poor performance of the PMMA cement as a drug release device. Hence, the objective of this study was to design and evaluate a bioresorbable calcium phosphate scaffold (CaPS) for sustained antimicrobial drug release and investigate its efficacy in a murine model of femoral implant-associated osteomyelitis. Incorporating rifampin and sitafloxacin, which are effective against bacterial phenotypes responsible for bacterial persistence, into 3D-printed CaPS coated with poly(lactic co-glycolic) acid, achieved controlled release for up to two weeks. Implantation into the murine infection model resulted in decreased bacterial colonization rates at 3- and 10-weeks post-revision for the 3D printed CaPS in comparison to gentamicin-laden PMMA. Furthermore, a significant increase in bone formation was observed for 3D printed CaPS incorporated with rifampin at 3 and 10 weeks. The results of this study demonstrate that osteoconductive 3D printed CaPS incorporated with antimicrobials demonstrate more efficacious bacterial colonization outcomes and bone growth in a single-stage revision in comparison to gentamicin-laden PMMA requiring a two-stage revision.
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Staphylococcus aureus and Pseudomonas aeruginosa are two of the most common causes of bacterial keratitis and corresponding corneal blindness. Accordingly, such infections are predominantly treated with broad-spectrum fluoroquinolones, such as moxifloxacin. Yet, the rising fluoroquinolone resistance has necessitated the development of alternative therapeutic options. Herein, we describe the development of a polymyxin B-trimethoprim (PT) ophthalmic formulation containing the antibiotic rifampin, which exhibits synergistic antimicrobial activity toward a panel of contemporary ocular clinical S. aureus and P. aeruginosa isolates, low spontaneous resistance frequency, and in vitro bactericidal kinetics and antibiofilm activities equaling or exceeding the antimicrobial properties of moxifloxacin. The PT plus rifampin combination also demonstrated increased efficacy in comparison to those of either commercial PT or moxifloxacin in a murine keratitis model of infection, resulting in bacterial clearance of 70% in the animals treated. These results suggest that the combination of PT and rifampin may represent a novel antimicrobial agent in the treatment of bacterial keratitis.
Assuntos
Antibacterianos/farmacologia , Infecções Oculares Bacterianas/tratamento farmacológico , Ceratite/tratamento farmacológico , Polimixina B/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Rifampina/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Trimetoprima/farmacologia , Animais , Córnea/efeitos dos fármacos , Córnea/microbiologia , Córnea/patologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/patologia , Feminino , Humanos , Ceratite/microbiologia , Ceratite/patologia , Camundongos , Camundongos Endogâmicos BALB C , Moxifloxacina/farmacologia , Soluções Oftálmicas/farmacologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Resultado do TratamentoRESUMO
Drug repurposing offers an expedited and economical route to develop new clinical therapeutics in comparison to traditional drug development. Growth-based high-throughput screening is concomitant with drug repurposing and enables rapid identification of new therapeutic uses for investigated drugs; however, this traditional method is not compatible with microorganisms with abnormal growth patterns such as Staphylococcus aureus small-colony variants (SCV). SCV subpopulations are auxotrophic for key compounds in biosynthetic pathways, which result in low growth rate. SCV formation is also associated with reduced antibiotic susceptibility, and the SCV's ability to revert to the normal cell growth state is thought to contribute to recurrence of S. aureus infections. Thus, there is a critical need to identify antimicrobial agents that are potent against SCV in order to effectively treat chronic infections. Accordingly, here we describe adapting an adenylate kinase (AK)-based cell death reporter assay to identify members of a Food and Drug Administration (FDA)-approved drug library that display bactericidal activity against S. aureus SCV. Four library members, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, exhibited potent SCV bactericidal activity against a stable S. aureus SCV. Further investigation showed that sitafloxacin was potent against methicillin-susceptible and -resistant S. aureus, as well as S. aureus within an established biofilm. Taken together, these results demonstrate the ability to use the AK assay to screen small-molecule libraries for SCV bactericidal agents and highlight the therapeutic potential of sitafloxacin to be repurposed to treat chronic S. aureus infections associated with SCV and/or biofilm growth states.IMPORTANCE Conventional antibiotics fail to successfully treat chronic osteomyelitis, endocarditis, and device-related and airway infections. These recurring infections are associated with the emergence of SCV, which are recalcitrant to conventional antibiotics. Studies have investigated antibiotic therapies to treat SCV-related infections but have had little success, emphasizing the need to identify novel antimicrobial drugs. However, drug discovery is a costly and time-consuming process. An alternative strategy is drug repurposing, which could identify FDA-approved and well-characterized drugs that could have off-label utility in treating SCV. In this study, we adapted a high-throughput AK-based assay to identify 4 FDA-approved drugs, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, which display antimicrobial activity against S. aureus SCV, suggesting an avenue for drug repurposing in order to effectively treat SCV-related infections. Additionally, this screening paradigm can easily be adapted for other drug/chemical libraries to identify compounds bactericidal against SCV.