RESUMO
Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals.Hypothesis. Apramycin may have in vitro activity against Mtb.Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value.Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay.Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC50 of 0.5 µg ml-1 and an MIC90 of 1 µg ml-1. We determined the tentative ECOFF value as 1 µg ml-1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12â% (4/33), 20.69â% (6/29) and 66.67â% (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides.Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.
Assuntos
Antituberculosos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Nebramicina , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Humanos , Antituberculosos/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Farmacorresistência Bacteriana MúltiplaRESUMO
BACKGROUND: The aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, the potential of apramycin in the treatment of drug-resistant bloodstream infections (BSIs) has not yet been assessed. METHODS: The resistance gene annotations of 40â888 blood-culture isolates were analysed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for a total of nine Escherichia coli and Klebsiella pneumoniae isolates. RESULTS: Genotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, colistin resistance was shown in 46.4% and apramycin in 2.1%. Apramycin activity against methylated ribosomes was > 100-fold higher than that for other aminoglycosides. Frequencies of resistance were < 10-9 at 8 × minimum inhibitory concentration (MIC). Tentative epidemiological cut-offs (TECOFFs) were determined as 8 µg/mL for E. coli and 4 µg/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log colony-forming unit (CFU) reduction in the blood and peritoneum. Two doses of 80 mg/kg resulted in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans and led to complete eradication of carbapenem- and aminoglycoside-resistant bacteraemia. CONCLUSION: Encouraging coverage and potent in vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants the conduct of clinical studies to validate apramycin as a drug candidate for the prophylaxis and treatment of BSI.
Assuntos
Aminoglicosídeos , Antibacterianos , Carbapenêmicos , Modelos Animais de Doenças , Escherichia coli , Infecções por Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Nebramicina , Animais , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Nebramicina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Camundongos , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Aminoglicosídeos/farmacologia , Aminoglicosídeos/uso terapêutico , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Peritonite/tratamento farmacológico , Peritonite/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Humanos , Feminino , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Farmacorresistência BacterianaRESUMO
Antibiotic resistance genes (ARGs) are a kind of emerging environmental contamination, and are commonly found in antibiotic application situations, attracting wide attention. Fish skin mucosal surface (SMS), as the contact interface between fish and water, is the first line of defense against external pollutant invasion. Antibiotics are widely used in aquaculture, and SMS may be exposed to antibiotics. However, what happens to SMS when antibiotics are applied, and whether ARGs are enriched in SMS are not clear. In this study, Zebrafish (Danio rerio) were exposed to antibiotic and antibiotic resistant bacteria in the laboratory to simulate the aquaculture situation, and the effects of SMS on the spread of ARGs were explored. The results showed that SMS maintained the stability of the bacterial abundance and diversity under apramycin (APR) and bacterial exposure effectively. Until 11 days after stopping APR exposure, the abundance of ARGs in SMS (mean value was 3.32 × 10-3 copies/16S rRNA copies) still did not recover to the initial stage before exposure, which means that enriched ARGs in SMS were persistently remained. Moreover, non-specific immunity played an important role in resisting infection of external contamination. Besides, among antioxidant proteins, superoxide dismutase showed the highest activity. Consequently, it showed that SMS became a barrier of antibiotic resistance genes under APR exposure, and ARGs in SMS were difficult to remove once colonized. This study provided a reference for understanding the transmission, enrichment process, and ecological impact of antibiotics and ARGs in aquatic environments.
Assuntos
Antibacterianos , Nebramicina , Pele , Peixe-Zebra , Animais , Peixe-Zebra/genética , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Pele/efeitos dos fármacos , Pele/microbiologia , Resistência Microbiana a Medicamentos/genética , Mucosa/efeitos dos fármacos , Mucosa/microbiologia , Poluentes Químicos da Água/toxicidadeRESUMO
OBJECTIVES: To evaluate the in vitro activity of the combination of apramycin with colistin, meropenem, minocycline or sulbactam, against some well-characterized XDR Acinetobacter baumannii clinical isolates from Greece, to understand how apramycin can be best incorporated into clinical practice and optimize effectiveness. METHODS: In vitro interactions of apramycin (0.5×, 1× and 2× the MIC value) with colistin (2â mg/L), meropenem (30â mg/L), minocycline (3.5â mg/L) or sulbactam (24â mg/L) were tested using time-kill methodology. Twenty-one clinical A. baumannii isolates were chosen, exhibiting apramycin MICs of 4-16â mg/L, which were at or below the apramycin preliminary epidemiological cut-off value of 16â mg/L. These isolates were selected for a range of colistin (4-32â mg/L), meropenem (16-256â mg/L), minocycline (8-32â mg/L) and sulbactam (8-32â mg/L) MICs across the resistant range. Synergy was defined as a ≥2â log10â cfu/mL reduction compared with the most active agent. RESULTS: The combination of apramycin with colistin, meropenem, minocycline or sulbactam was synergistic, at least at one of the concentrations of apramycin (0.5×, 1× or 2× MIC), against 83.3%, 90.5%, 90.9% or 92.3% of the tested isolates, respectively. Apramycin alone was bactericidal at 24â h against 9.5% and 33.3% of the tested isolates at concentrations equal to 1× and 2× MIC, while the combination of apramycin at 2× MIC with colistin, meropenem or sulbactam was bactericidal against all isolates tested (100%). The apramycin 2× MIC/minocycline combination had bactericidal activity against 90.9% of the tested isolates. CONCLUSIONS: Apramycin combinations may have potential as a treatment option for XDR/pandrug-resistant (PDR) A. baumannii infections and warrant validation in the clinical setting, when this new aminoglycoside is available for clinical use.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Testes de Sensibilidade Microbiana , Nebramicina , Nebramicina/análogos & derivados , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Grécia , Antibacterianos/farmacologia , Humanos , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Nebramicina/farmacologia , Sulbactam/farmacologia , Sinergismo Farmacológico , Meropeném/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Viabilidade Microbiana/efeitos dos fármacos , Minociclina/farmacologiaRESUMO
OBJECTIVES: Apramycin is an aminoglycoside (AG) with a unique structure that is little affected by plasmid-mediated mechanisms of AG resistance, including most AG-modifying enzymes and 16S rRNA methyltransferases (16S-RMTases). We evaluate the activity of apramycin against a collection of 16S-RMTase-producing isolates, including Enterobacterales, non-fermenting bacteria, and carbapenemase producers. METHODS: In total, 164 non-duplicate 16S-RMTase-producing isolates, including 84 Enterobacterales, 53 Acinetobacter baumannii and 27 Pseudomonas aeruginosa isolates, were included in the study. Whole-genome sequencing (WGS) was performed on all isolates with Illumina technology. The minimum inhibitory concentration (MIC) of apramycin was determined by broth microdilution with customized Sensititre plates (Thermo Fisher Scientific, Dardilly, France). RESULTS: We found that 95% (156/164) of the 16S-RMTase-producing isolates were susceptible to apramycin, with a MIC50 of 4 mg/L and a MIC90 of 16 mg/L, respectively. Resistance rates were higher in P. aeruginosa (11%) than in A. baumannii (4%) or Enterobacterales (4%) (P < 0.0001 for each comparison). Eight isolates were resistant to apramycin, including one isolate with an MIC >64 mg/L due to the acquisition of the aac(3)-IV gene. The genetic environment of the aac(3)-IV gene was similar to that in the pAH01-4 plasmid of an Escherichia coli isolate from chicken in China. CONCLUSION: Resistance to apramycin remains rare in 16S-RMTase-producing isolates. Apramycin may, therefore, be an interesting alternative treatment for infections caused by 16S-RMTase and carbapenemase producers.
Assuntos
Antibacterianos , Nebramicina , RNA Ribossômico 16S/genética , Antibacterianos/farmacologia , Aminoglicosídeos/farmacologia , Nebramicina/farmacologia , Escherichia coliRESUMO
The purpose of this study was to establish the clinical breakpoint (CBP) of apramycin (APR) against Salmonella in swine and evaluate its effect on intestinal microbiota. The CBP was established based on three cutoff values of wild-type cutoff value (COWT), pharmacokinetic-pharmadynamic (PK/PD) cutoff value (COPD) and clinical cutoff value (COCL). The effect of the optimized dose regimen based on ex vivo PK/PD study. The evolution of the ileum flora was determined by the 16rRNA gene sequencing and bioinformatics. This study firstly established the COWT, COPD in ileum, and COCL of APR against swine Salmonella, the value of these cutoffs were 32 µg/mL, 32 µg/mL and 8 µg/mL, respectively. According to the guiding principle of the Clinical Laboratory Standards Institute (CLSI), the final CBP in ileum was 32 µg/mL. Our results revealed the main evolution route in the composition of ileum microbiota of diarrheic piglets treated by APR. The change of the abundances of Bacteroidetes and Euryarchaeota was the most obvious during the evolution process. Methanobrevibacter, Prevotella, S24-7 and Ruminococcaceae were obtained as the highest abundance genus. The abundance of Methanobrevibacter increased significantly when APR treatment carried and decreased in cure and withdrawal period groups. The abundance of Prevotella in the tested groups was significantly lower than that in the healthy group. A decreased of abundance in S24-7 was observed after Salmonella infection and increased slightly after cure. Ruminococcaceae increased significantly after Salmonella infection and decreased significantly after APR treatment. In addition, the genera of Methanobrevibacter and Prevotella were defined as the key node. Valine, leucine and isoleucine biosynthesis, D-Glutamine and D-glutamate metabolism, D-Alanine metabolism, Peptidoglycan and amino acids biosynthesis were the top five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the ileum microbiota of piglets during the Salmonella infection and APR treatment process. Our study extended the understanding of dynamic shift of gut microbes during diarrheic piglets treated by APR.
Assuntos
Microbioma Gastrointestinal , Nebramicina , Animais , Íleo , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Prevotella , Salmonella , SuínosRESUMO
Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's inherent resistance to clinically available antimicrobials. The low bactericidal potency of currently available treatment regimens is of concern and testifies to the poor therapeutic outcomes for pulmonary M. abscessus infections. Mechanistically, we demonstrate here that the acetyltransferase Eis2 is responsible for the lack of bactericidal activity of amikacin, the standard aminoglycoside used in combination treatment. In contrast, the aminoglycoside apramycin, with a distinct structure, is not modified by any of the pathogen's innate aminoglycoside resistance mechanisms and is not affected by the multidrug resistance regulator WhiB7. As a consequence, apramycin uniquely shows potent bactericidal activity against M. abscessus. This favorable feature of apramycin is reflected in a mouse model of pulmonary M. abscessus infection, which demonstrates superior activity, compared with amikacin. These findings encourage the development of apramycin for the treatment of M. abscessus infections and suggest that M. abscessus eradication in pulmonary disease may be within therapeutic reach.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Nebramicina , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Camundongos , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Nebramicina/uso terapêuticoRESUMO
BACKGROUND: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). METHODS: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. FINDINGS: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. INTERPRETATION: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.
Assuntos
Antibacterianos/uso terapêutico , Concentração de Íons de Hidrogênio , Nebramicina/análogos & derivados , Pielonefrite/tratamento farmacológico , Infecções Urinárias/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Nebramicina/farmacologia , Nebramicina/uso terapêutico , Pielonefrite/etiologia , Ratos , Resultado do Tratamento , Infecções Urinárias/etiologiaRESUMO
Introduction. The emergence of multidrug-resistant Salmonella Typhimurium strains has increased the need for safe, alternative therapies from natural sources with antibacterial properties.Hypothesis/Gap Statement. There are no published data regarding the use of chitosan propolis nanocomposite (CPNP) either alone or in combination with antibiotics as antimicrobials against S. Typhimurium, especially in Egypt.Aim. This study evaluated the antibacterial activities of five antimicrobials [apramycin, propolis, chitosan nanoparticles (CNPs), chitosan propolis nanocomposite (CPNP) and CPNP +apramycin] against ten virulent and multidrug-resistant (MDR) S. Typhimurium field strains recovered from diarrheic rabbits through in vitro and in vivo study.Methodology. The expression levels of three virulence genes of S. Typhimurium strains were determined by quantitative reverse-transcription PCR (RT-qPCR) after exposure to sub-inhibitory concentrations of apramycin, propolis, CNPs, CPNP alone, and CPNP +apramycin. Additionally, 90 New Zealand rabbits were divided into control and experimentally S. Typhimurium-infected groups. The infected rabbits were orally administered saline solution (infected-untreated); 10 mg apramycin/kg (infected-apramycin-treated); 50 mg propolis/kg (infected-propolis-treated); 15 mg CPNP/kg (infected-CPNP-treated) and 15 mg CPNP +10 mg apramycin/kg (infected-CPNP +apramycin-treated) for 5 days.Results. The RT-qPCR analysis revealed different degrees of downregulation of all screened genes. Furthermore, the treatment of infected rabbits with CPNP or CPNP +apramycin significantly improved performance parameters, and total bacterial and Salmonella species counts, while also modulating both oxidative stress and altered liver and kidney parameters.Conclusion. This work demonstrates the use of CPNP alone or in combination with apramycin in the treatment of S. Typhimurium in rabbits.
Assuntos
Antibacterianos/uso terapêutico , Quitosana/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Nanocompostos/uso terapêutico , Própole/química , Infecções por Salmonella/tratamento farmacológico , Salmonella typhimurium/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antioxidantes/metabolismo , Carga Bacteriana/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quitosana/farmacologia , Quitosana/uso terapêutico , Chlorocebus aethiops , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Nanocompostos/química , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Nebramicina/uso terapêutico , Própole/farmacologia , Própole/uso terapêutico , Coelhos , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Células Vero , Virulência/genéticaRESUMO
Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and florfenicol. The susceptibility to apramycin and florfenicol was evaluated by disk diffusion of five S. aureus collections, comprising 352 isolates of animal (n = 265) and human (n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ≥15 mm (ECOFF = 15 mm) for apramycin and ≥21 mm for florfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas five NWT isolates were identified for florfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and florfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.
Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/normas , Nebramicina/análogos & derivados , Staphylococcus aureus/efeitos dos fármacos , Tianfenicol/análogos & derivados , Farmacorresistência Bacteriana , Nebramicina/farmacologia , Tianfenicol/farmacologia , Medicina VeterináriaRESUMO
Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row. The downstream errors in a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a distinct class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/genética , Proteoma/genética , Ribossomos/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Espectrometria de Massas , Mutação de Sentido Incorreto , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Fator Tu de Elongação de Peptídeos/genética , Peptídeos/genética , Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteômica , Proteínas Recombinantes , Ribossomos/efeitos dos fármacos , Estreptomicina/farmacologia , Estresse Fisiológico/genéticaRESUMO
Florfenicol, apramycin, and danofloxacin are antibiotics approved only for veterinary use and that have good therapeutic effects on chicken respiratory infections caused by Escherichia coli. We established epidemiological cutoff values (ECV) for these antibiotics using 363 E. coli isolates from tracheal samples of chickens in 5 veterinary clinics in Guangdong Province, China. The minimum inhibitory concentrations (MIC) were determined using the agar dilution method as per Clinical and Laboratory Standards Institution guidelines. The ECV were then calculated using the statistical method and verified by normalized resistance interpretation and ECOFFinder software programs. The ECV of florfenicol, apramycin, and danofloxacin against E. coli were 16, 16, and 0.125 µg/mL, respectively. Susceptibility tests indicated that these isolates were resistant to florfenicol (66.7%), apramycin (22.3%), and danofloxacin (92.3%). Strains carrying floR were distributed in the range of MIC ≥32 µg/mL for florfenicol. Apramycin resistance was found in 77 strains (77/363, 21.1%), and isolates that carried aac(3)-IV were all in the range of MIC ≥512 µg/mL. Danofloxacin resistance was found in the range of MIC ≤0.125 µg/mL, but there were no mutations in the quinolone resistance-determining regions and plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrC, qnrD, aac-(6')-Ib-cr, qep, and oqxB. The presence of the qnrS gene was verified in a few of the strains with an MIC of 0.06 µg/mL. The establishment of ECV was significant for monitoring of resistance development and therapy guidance.
Assuntos
Antibacterianos/farmacologia , Galinhas , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Doenças das Aves Domésticas/tratamento farmacológico , Infecções Respiratórias/veterinária , Animais , Antibacterianos/uso terapêutico , China/epidemiologia , Farmacorresistência Bacteriana , Escherichia coli/genética , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Fluoroquinolonas/farmacologia , Testes de Sensibilidade Microbiana/veterinária , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/microbiologia , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Tianfenicol/análogos & derivados , Tianfenicol/farmacologiaRESUMO
The value of model-based translation in drug discovery and development is now effectively being recognized in many disease areas and among various stakeholders. Such quantitative approaches are expected to facilitate the selection on which compound to prioritize for successful development, predict the human efficacious dose based on preclinical data with adequate precision, guide design, and de-risk later development stages. The importance of time-dependencies, which are typically species-dependent due to different turnover rates of biological processes, is, however, often neglected. For bacterial infections, the choice of dosing regimen is typically relying on preclinical pharmacokinetic (PK) and pharmacodynamic (PD) data, because the bacterial load and disease severity, and consequently the PK/PD relationship, cannot be quantified well on clinical data, given the low-information end points used. It is time to recognize the limitations of using time-collapsed approaches for translation (i.e., methods where targets are based on summary measures of exposure and response). Models describing the full time-course captures important quantitative information of drug distribution, bacterial growth, antibiotic killing, and resistance development, and can account for species-differences in the PK profiles driving the killing. Furthermore, with a model-based approach for translation, we can take a holistic approach in development of a joint model for in vitro, in vivo, and clinical data, as well as incorporating information on the contribution of the immune system. Such advancements are anticipated to facilitate rational decision making during various stages of drug development and in the optimization of treatment regimens for different groups of patients.
Assuntos
Anti-Infecciosos/farmacologia , Desenvolvimento de Medicamentos/organização & administração , Modelos Biológicos , United States Food and Drug Administration/organização & administração , Aminoglicosídeos/farmacologia , Anti-Infecciosos/farmacocinética , Antivirais/farmacologia , Carbapenêmicos/farmacologia , Relação Dose-Resposta a Droga , Aprovação de Drogas/organização & administração , Descoberta de Drogas/organização & administração , Avaliação Pré-Clínica de Medicamentos/métodos , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/fisiologia , Quimioterapia Combinada , Humanos , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Projetos de Pesquisa , Estados Unidos , United States Food and Drug Administration/normasRESUMO
Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1::aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Halomonas/efeitos dos fármacos , Cloreto de Sódio/metabolismo , Aminoglicosídeos/análise , Antibacterianos/análise , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Gentamicinas/farmacologia , Halomonas/genética , Halomonas/metabolismo , Canamicina/farmacologia , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Cloreto de Sódio/análiseRESUMO
Apramycin represents a subclass of aminoglycoside antibiotics that has been shown to evade almost all mechanisms of clinically relevant aminoglycoside resistance. Model-informed drug development may facilitate its transition from preclinical to clinical phase. This study explored the potential of pharmacokinetic/pharmacodynamic (PK/PD) modeling to maximize the use of in vitro time-kill and in vivo preclinical data for prediction of a human efficacious dose (HED) for apramycin. PK model parameters of apramycin from four different species (mouse, rat, guinea pig, and dog) were allometrically scaled to humans. A semimechanistic PK/PD model was developed from the rich in vitro data on four Escherichia coli strains and subsequently the sparse in vivo efficacy data on the same strains were integrated. An efficacious human dose was predicted from the PK/PD model and compared with the classical PK/PD index methodology and the aminoglycoside dose similarity. One-compartment models described the PK data and human values for clearance and volume of distribution were predicted to 7.07 L/hour and 26.8 L, respectively. The required fAUC/MIC (area under the unbound drug concentration-time curve over MIC ratio) targets for stasis and 1-log kill in the thigh model were 34.5 and 76.2, respectively. The developed PK/PD model predicted the efficacy data well with strain-specific differences in susceptibility, maximum bacterial load, and resistance development. All three dose prediction approaches supported an apramycin daily dose of 30 mg/kg for a typical adult patient. The results indicate that the mechanistic PK/PD modeling approach can be suitable for HED prediction and serves to efficiently integrate all available efficacy data with potential to improve predictive capacity.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Nebramicina/análogos & derivados , Animais , Antibacterianos/farmacocinética , Área Sob a Curva , Técnicas Bacteriológicas , Cães , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Cobaias , Camundongos , Modelos Biológicos , Nebramicina/administração & dosagem , Nebramicina/farmacocinética , Nebramicina/farmacologia , RatosRESUMO
We describe the convergent synthesis of a 5-O-ß-D-ribofuranosyl-based apramycin derivative (apralog) that displays significantly improved antibacterial activity over the parent apramycin against wild-type ESKAPE pathogens. In addition, the new apralog retains excellent antibacterial activity in the presence of the only aminoglycoside modifying enzyme (AAC(3)-IV) acting on the parent, without incurring susceptibility to the APH(3') mechanism that disables other 5-O-ß-D-ribofuranosyl 2-deoxystreptamine type aminoglycosides by phosphorylation at the ribose 5-position. Consistent with this antibacterial activity, the new apralog has excellent 30â nM activity (IC50 ) for the inhibition of protein synthesis by the bacterial ribosome in a cell-free translation assay, while retaining the excellent across-the-board selectivity of the parent for inhibition of bacterial over eukaryotic ribosomes. Overall, these characteristics translate into excellent inâ vivo efficacy against E. coli in a mouse thigh infection model and reduced ototoxicity vis à vis the parent in mouse cochlear explants.
Assuntos
Antibacterianos/farmacologia , Cóclea/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Nebramicina/análogos & derivados , Animais , Antibacterianos/síntese química , Antibacterianos/química , Configuração de Carboidratos , Cóclea/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Nebramicina/síntese química , Nebramicina/química , Nebramicina/farmacologiaRESUMO
OBJECTIVES: Novel therapeutics are urgently required for the treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) causing critical infections with high mortality. Here we assessed the therapeutic potential of the clinical-stage drug candidate EBL-1003 (crystalline free base of apramycin) in the treatment of CRAB lung infections. METHODS: The genotypic and phenotypic susceptibility of CRAB clinical isolates to aminoglycosides and colistin was assessed by database mining and broth microdilution. The therapeutic potential was assessed by target attainment simulations on the basis of time-kill kinetics, a murine lung infection model, comparative pharmacokinetic analysis in plasma, epithelial lining fluid (ELF) and lung tissue, and pharmacokinetic/pharmacodynamic (PKPD) modelling. RESULTS: Resistance gene annotations of 5451 CRAB genomes deposited in the National Database of Antibiotic Resistant Organisms (NDARO) suggested >99.9% of genotypic susceptibility to apramycin. Low susceptibility to standard-of-care aminoglycosides and high susceptibility to EBL-1003 were confirmed by antimicrobial susceptibility testing of 100 A. baumannii isolates. Time-kill experiments and a mouse lung infection model with the extremely drug-resistant CRAB strain AR Bank #0282 resulted in rapid 4-log CFU reduction both in vitro and in vivo. A single dose of 125 mg/kg EBL-1003 in CRAB-infected mice resulted in an AUC of 339 h × µg/mL in plasma and 299 h × µg/mL in ELF, suggesting a lung penetration of 88%. PKPD simulations suggested a previously predicted dose of 30 mg/kg in patients (creatinine clearance (CLCr) = 80 mL/min) to result in >99% probability of -2 log target attainment for MICs up to 16 µg/mL. CONCLUSIONS: This study provides proof of concept for the efficacy of EBL-1003 in the treatment of CRAB lung infections. Broad in vitro coverage, rapid killing, potent in vivo efficacy, and a high probability of target attainment render EBL-1003 a strong therapeutic candidate for a priority pathogen for which treatment options are very limited.
Assuntos
Infecções por Acinetobacter , Antibacterianos , Nebramicina/análogos & derivados , Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Animais , Antibacterianos/farmacologia , Colistina/farmacologia , Pulmão , Camundongos , Testes de Sensibilidade Microbiana , Nebramicina/farmacocinética , Nebramicina/farmacologiaRESUMO
BACKGROUND: Apramycin is used exclusively for the treatment of Escherichia coli (E.coli) infections in swine around the world since the early 1980s. Recently, many research papers have demonstrated that apramycin has significant in vitro activity against multidrug-resistant E.coli isolated in hospitals. Therefore, ensuring the proper use of apramycin in veterinary clinics is of great significance of public health. The objectives of this study were to develop a wild-type cutoff for apramycin against E.coli using a statistical method recommended by Clinical and Laboratory Standards Institute (CLSI) and to investigate the prevalence of resistance genes that confer resistance to apramycin in E. coli. RESULTS: Apramycin susceptibility testing of 1230 E.coli clinical isolates from swine were determinded by broth microdilution testing according to the CLSI document M07-A9. A total number of 310 E.coli strains from different minimum inhibitory concentration (MIC) subsets (0.5-256 µg/mL) were selected for the detection of resistance genes (aac(3)-IV; npmA; apmA) in E. coli by PCR. The percentage of E. coli isolates at each MIC (0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 µg/mL) was 0.08, 0.08, 0.16, 2.93, 31.14, 38.86, 12.85, 2.03, 1.46, and 10.41%. The MIC50 and MIC90 were 16 and 64 µg/mL. All the 310 E.coli isolates were negative for npmA and apmA gene, and only the aac(3)-IV gene was detected in this study. CONCLUSIONS: The wild-type cutoff for apramycin against E.coli was defined as 32 µg/mL. The prevelance of aac(3)-IV gene mainly concentrated in these MIC subsets 'MIC ≥ 64 µg/ mL', which indicates that the wild-type cutoff established in our study is reliable. The wild-type cutoff offers interpretion criteria of apramycin susceptibility testing of E.coli.
Assuntos
Farmacorresistência Bacteriana/genética , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Nebramicina/análogos & derivados , Animais , Antibacterianos/farmacologia , Escherichia coli/genética , Infecções por Escherichia coli/tratamento farmacológico , Testes de Sensibilidade Microbiana/veterinária , Nebramicina/farmacologia , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologiaRESUMO
Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure-activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections.
Assuntos
Acetiltransferases/genética , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Acetiltransferases/química , Acetiltransferases/metabolismo , Aminoglicosídeos/química , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Domínio Catalítico , Bases de Dados Genéticas , Genoma Bacteriano/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Epidemiologia Molecular , Mutagênese Sítio-Dirigida , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Padrão de Cuidado , Relação Estrutura-AtividadeRESUMO
Actinomycetales, such as the genus Streptomyces, are well-known cell factories employed to produce a wide variety of secondary metabolites for industrial use. However, not only is the genetic engineering of Streptomyces more complicated and tedious than other model laboratory species, such as Escherichia coli, there is also a considerable lack of genetic tools, hindering its adoption as a common chassis for synthetic biology. In this work, 23 novel shuttle vectors are presented that follow the canonical SEVA (Standard European Vector Architecture) common architecture with the goal of increasing the genetic toolbox repertoire for Streptomyces and other actinomycetes. The ORI module of these plasmids is composed of the combination of two origins of replication, one for Gram-negative bacteria and the other for Streptomyces, a Gram-positive bacteria. Origins of replication have been included in the collection for integrative, low-copy number, and medium-to-high-copy number vectors for Streptomyces. Also, a new selection marker has been developed that confers resistance to apramycin. The functionality of these plasmids was tested via the heterologous expression of GFP and the heterologous production of the plant flavonoid apigenin in Streptomyces albus J1074, with successful results in both cases, therefore expanding the current repertoire of genetic manipulation tools in Streptomyces species.