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As an obligate aerobe, Mycobacterium tuberculosis relies on its branched electron transport chain (ETC) for energy production through oxidative phosphorylation. Regimens targeting ETC exhibit promising potential to enhance bactericidal activity against M. tuberculosis and hold the prospect of shortening treatment duration. Our previous research demonstrated that the bacteriostatic drug candidate TB47 (T) inhibited the growth of M. tuberculosis by targeting the cytochrome bc1 complex and exhibited synergistic activity with clofazimine (C). Here, we found synergistic activities between C and sudapyridine (S), a structural analog of bedaquiline (B). S has shown similar anti-tuberculosis efficacy and may share a mechanism of action with B, which inhibits ATP synthesis and the energy metabolism of bacteria. We evaluated the efficacy of SCT in combination with linezolid (L) or pyrazinamide (Z) using a well-established murine model of tuberculosis. Compared to the BPa(pretomanid)L regimen, SCT and SCTL demonstrated similar bactericidal and sterilizing activities. There was no significant difference in activity between SCT and SCTL. In contrast, SCZ and SCTZ showed much higher activities, with none of the 15 mice experiencing relapse after 2 months of treatment with either SCZ or SCTZ. However, T did not contribute to the activity of the SCZ. Our findings emphasize the efficacy and the potential clinical significance of combination therapy with ETC inhibitors. Additionally, cross-resistance exists not only between S and B but also between S/B and C. This is supported by our findings, as spontaneous S-resistant mutants exhibited mutations in Rv0678, which are associated with cross-resistance to B and C.
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Procambarus clarkii (crayfish) accumulates a high concentration of Arsenic (As) from the aquatic environment and causes considerable human health risks. In this study, Limosilactobacillus fermentum GR-3 strain was isolated from "Jiangshui" and applied for As(III) adsorption and antioxidant abilities. Strain GR-3 removed 50.67% of 50 mg/L As(III) and exhibited the high antioxidant potential of DPPH (1,1-Diphenyl-2-picrylhydrazyl) (87.63%) and hydroxyl radical (74.51%) scavenging rate in vitro. P. clarkii was feed with strain GR-3, the results showed that As(III) concentration reduced, and residual level in hepatopancreas was decreased by 36%, compared to As(III)-exposed group (control). Gut microbial sequencing showed that strain GR-3 restores gut microbiota dysbiosis caused by As(III) exposure. Further application in the field scale was performed and revealed a decrease in As(III) accumulation and increasing 50% aquaculture production of the total output. In summary, feed-additive probiotic is recommended as a novel strategy to minimize aquaculture foods toxicity and safe human health.
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Arsênio , Limosilactobacillus fermentum , Animais , Arsênio/metabolismo , Arsênio/toxicidade , Astacoidea , Hepatopâncreas/metabolismo , Humanos , Estresse OxidativoRESUMO
TB47, a new drug candidate targeting QcrB in the electron transport chain, has shown a unique synergistic activity with clofazimine and forms a highly sterilizing combination. Here, we investigated the sterilizing effects of several all-oral regimens containing TB47 plus clofazimine and linezolid as a block and the roles of fluoroquinolones and pyrazinamide in them. All these regimens cured tuberculosis within 4 to 6 months in a well-established mouse model, and adding pyrazinamide showed a significant difference in bactericidal effects.
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Mycobacterium tuberculosis , Tuberculose , Animais , Antituberculosos/uso terapêutico , Clofazimina/uso terapêutico , Modelos Animais de Doenças , Linezolida , Camundongos , Camundongos Endogâmicos BALB C , Tuberculose/tratamento farmacológicoRESUMO
The increasing clinical significance of Mycobacterium abscessus is owed to its innate high-level, broad-spectrum resistance to antibiotics and therefore rapidly evolves as an important human pathogen. This warrants the identification of novel targets for aiding the discovery of new drugs or drug combinations to treat M. abscessus infections. This study is inspired by the drug-hypersensitive profile of a mutant M. abscessus (U14) with transposon insertion in MAB_1915. We validated the role of MAB_1915 in intrinsic drug resistance in M. abscessus by constructing a selectable marker-free in-frame deletion in MAB_1915 and complementing the mutant with the same or extended version of the gene and then followed by drug susceptibility testing. Judging by the putative function of MAB_1915, cell envelope permeability was studied by ethidium bromide accumulation assay and susceptibility testing against dyes and detergents. In this study, we established genetic evidence of the role of MAB_1915 in intrinsic resistance to rifampicin, rifabutin, linezolid, clarithromycin, vancomycin, and bedaquiline. Disruption of MAB_1915 has also been observed to cause a significant increase in cell envelope permeability in M. abscessus. Restoration of resistance is observed to depend on at least 27 base pairs upstream of the coding DNA sequence of MAB_1915. MAB_1915 could therefore be associated with cell envelope permeability, and hence its role in intrinsic resistance to multiple drugs in M. abscessus, which presents it as a novel target for future development of effective antimicrobials to overcome intrinsic drug resistance in M. abscessus. IMPORTANCE: This study reports the role of a putative fadD (MAB_1915) in innate resistance to multiple drugs by M. abscessus, hence identifying MAB_1915 as a valuable target and providing a baseline for further mechanistic studies and development of effective antimicrobials to check the high level of intrinsic resistance in this pathogen.
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Discovery of novel antitubercular drugs is an effective strategy against drug-resistant tuberculosis (TB). Our previous study has identified LPX-16j as a novel antitubercular compound. Herein, we perform a comprehensive structure-activity relationship (SAR) based on LPX-16j, indicating that the central pyrimidine ring moiety was crucial for the antitubercular activities of its derivatives, and replacing the naphthyl group with hydrophobic substitutes was well tolerated. The representative derivative 5a exhibited potent activity against H37Ra, H37Rv, and clinical drug-resistant TB with minimum inhibitory concentration (MIC) values of 0.5-1.0 µg/mL. Meanwhile, 5a showed an acceptable safety in vivo and displayed a favorable oral bioavailability with a value of 40.7%. The differential scanning fluorescence, isothermal titration calorimetry, and molecular docking assays indicated that PknB could be one of the targets of compound 5a. Overall, this study identified 5a as a novel promising lead compound with the potential to develop candidates for the treatment of drug-resistant TB.
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Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Simulação de Acoplamento Molecular , Antituberculosos/farmacologia , Relação Estrutura-Atividade , Pirimidinas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The World Health Organization (WHO) reports that tuberculosis (TB) is one of the top 10 leading causes of global mortality. The increasing incidence of multidrug-resistant TB highlights the urgent need for an intensified quest to discover innovative anti-TB medications In this study, we investigated four new derivatives from the quinoxaline-2-carboxylic acid 1,4-dioxide class. New 3-methylquinoxaline 1,4-dioxides with a variation in substituents at positions 2 and 6(7) were synthesized via nucleophilic aromatic substitution with amines and assessed against a Mycobacteria spp. Compound 4 showed high antimycobacterial activity (1.25 µg/mL against M. tuberculosis) and low toxicity in vivo in mice. Selection and whole-genomic sequencing of spontaneous drug-resistant M. smegmatis mutants revealed a high number of single-nucleotide polymorphisms, confirming the predicted mode of action of the quinoxaline-2-carboxylic acid 1,4-dioxide 4 as a DNA-damaging agent. Subsequent reverse genetics methods confirmed that mutations in the genes MSMEG_4646, MSMEG_5122, and MSMEG_1380 mediate resistance to these compounds. Overall, the derivatives of quinoxaline-2-carboxylic acid 1,4-dioxide present a promising scaffold for the development of innovative antimycobacterial drugs.
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Clofazimine (CFZ) and bedaquiline (BDQ) are currently used for the treatment of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains. In recent years, adding CFZ and BDQ to tuberculosis (TB) drug regimens against MDR Mtb strains has significantly improved treatment results, but these improvements are threatened by the emergence of MDR and extensively drug-resistant (XDR) Mtb strains. Recently, CFZ and BDQ have attracted much attention for their strong clinical efficacy, although very little is known about the mechanisms of action, drug susceptibility test (DST), resistance mechanisms, cross-resistance, and pharmacokinetics of these two drugs. In this current review, we provide recent updates on the mechanisms of action, DST, associated mutations with individual resistance and cross-resistance, clinical efficacy, and pharmacokinetics of CFZ and BDQ against Mtb strains. Presently, known mechanisms of resistance for CFZ and/or BDQ include mutations within the Rv0678, pepQ, Rv1979c, and atpE genes. The cross-resistance between CFZ and BDQ may reduce available MDR-/XDR-TB treatment options. The use of CFZ and BDQ for treatment in the setting of limited DST could allow further spread of drug resistance. The DST and resistance knowledge are urgently needed where CFZ and BDQ resistance do emerge. Therefore, an in-depth understanding of clinical efficacy, DST, cross-resistance, and pharmacokinetics for CFZ and BDQ against Mtb can provide new ideas for improving treatment outcomes, reducing mortality, preventing drug resistance, and TB transmission. Along with this, it will also help to develop rapid molecular diagnostic tools as well as novel therapeutic drugs for TB.
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The emergence of drug resistance in pathogens leads to a loss of effectiveness of antimicrobials and complicates the treatment of bacterial infections. Quinoxaline 1,4-dioxides represent a prospective scaffold for search of new compounds with improved chemotherapeutic characteristics. Novel 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides with alteration of substituents at position 2 and 6 were synthesized via nucleophilic substitution with piperazine moiety and evaluated against a broad panel of bacteria and fungi by measuring their minimal inhibitory concentrations. Their mode of action was assessed by whole-genomic sequencing of spontaneous drug-resistant Mycobacterium smegmatis mutants, followed by comparative genomic analysis, and on an original pDualrep2 system. Most of the 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides showed high antibacterial properties against Gram-positive strains, including mycobacteria, and the introduction of a halogen atom in the position 6 of the quinoxaline ring further increased their activity, with 13c being the most active compound. The mode of action studies confirmed the DNA-damaging nature of the obtained quinoxaline 1,4-dioxides, while drug-resistance may be provided by mutations in redox homeostasis genes, encoding enzymes potentially involved in the activation of the compounds. This study extends views about the antimicrobial and antifungal activities of the quinoxaline 1,4-dioxides and can potentially lead to the discovery of new antibacterial drugs.
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Introduction: Infections caused by non-tuberculosis mycobacteria are significantly worsening across the globe. M. fortuitum complex is a rapidly growing pathogenic species that is of clinical relevance to both humans and animals. This pathogen has the potential to create adverse effects on human healthcare. Methods: The MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The morphological studies, comparative genomic analysis, and drug resistance profiles along with variants detection were performed in this study. In addition, comparative analysis of virulence genes led us to understand the pathogenicity of this organism. Results: Bacterial growth kinetics and morphology confirmed that MF GZ001 is a rapidly growing species with a rough morphotype. The MF GZ001 contains 6413573 bp genome size with 66.18 % high G+C content. MF GZ001 possesses a larger genome than other related mycobacteria and included 6156 protein-coding genes. Molecular phylogenetic tree, collinearity, and comparative genomic analysis suggested that MF GZ001 is a novel member of the M. fortuitum complex. We carried out the drug resistance profile analysis and found single nucleotide polymorphism (SNP) mutations in key drug resistance genes such as rpoB, katG, AAC(2')-Ib, gyrA, gyrB, embB, pncA, blaF, thyA, embC, embR, and iniA. In addition, the MF GZ001strain contains mutations in iniA, iniC, pncA, and ribD which conferred resistance to isoniazid, ethambutol, pyrazinamide, and para-aminosalicylic acid respectively, which are not frequently observed in rapidly growing mycobacteria. A wide variety of predicted putative potential virulence genes were found in MF GZ001, most of which are shared with well-recognized mycobacterial species with high pathogenic profiles such as M. tuberculosis and M. abscessus. Discussion: Our identified novel features of a pathogenic member of the M. fortuitum complex will provide the foundation for further investigation of mycobacterial pathogenicity and effective treatment.
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Farmacorresistência Bacteriana , Mycobacteriaceae , Animais , Humanos , Pessoa de Meia-Idade , Testes de Sensibilidade Microbiana , Filogenia , Farmacorresistência Bacteriana/genética , Mycobacteriaceae/efeitos dos fármacos , Mycobacteriaceae/genéticaRESUMO
Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.
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Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Tuberculose , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Etambutol/uso terapêutico , Humanos , Imipenem/farmacologia , Imipenem/uso terapêutico , Linezolida/uso terapêutico , Camundongos , Camundongos Knockout , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/genética , Pentosiltransferases , Permeabilidade , Rifabutina/farmacologia , Rifabutina/uso terapêuticoRESUMO
Buruli ulcer (BU), caused by Mycobacterium ulcerans, is currently treated with rifampin-streptomycin or rifampin-clarithromycin daily for 8 weeks recommended by World Health Organization (WHO). These options are lengthy with severe side effects. A new anti-tuberculosis drug, TB47, targeting QcrB in cytochrome bc1:aa3 complex is being developed in China. TB47-containing regimens were evaluated in a well-established murine model using an autoluminescent M. ulcerans strain. High-level TB47-resistant spontaneous M. ulcerans mutants were selected and their qcrB genes were sequenced. The in vivo activities of TB47 against both low-level and high-level TB47-resistant mutants were tested in BU murine model. Here, we show that TB47-containing oral 3-drug regimens can completely cure BU in ≤2 weeks for daily use or in ≤3 weeks given twice per week (6 doses in total). All high-level TB47-resistant mutants could only be selected using the low-level mutants which were still sensitive to TB47 in mice. This is the first report of double mutations in QcrB in mycobacteria. In summary, TB47-containing regimens have promise to cure BU highly effectively and prevent the emergence of drug resistance. Novel QcrB mutations found here may guide the potential clinical molecular diagnosis of resistance and the discovery of new drugs against the high-level resistant mutants.
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Klebsiella pneumoniae is an opportunistic pathogen that is responsible for community acquired infections and nosocomial infections. Antibiotic-resistant K. pneumoniae and/or hypervirulent K. pneumoniae are emerging as a serious threat to public health. For the sake of alleviating and conquering current dilemma, discovery of effective new drugs against K. pneumoniae is a tough challenge. However, traditional anti-K. pneumoniae drug discovery methods cost considerable amount of time, animals, labor and so on. So an efficient technique for in vitro and in vivo drug screening with the least time duration, animals and labor cost is highly needed for the discovery of new effective compounds. Hence, in this study we constructed a selectable marker-free autoluminescent K. pneumoniae (SfAlKp) harboring luxCDABE by combining Tn7 transposon and Xer-dif system. SfAlKp can be used for discovery of new drugs via detecting luminescence intensity as a surrogate marker. The energy-consuming autoluminescent reaction catalyzed by the LuxAB enzymes which use the substrates produced by LuxCDE using the metabolites of the bacteria. Tn7 can insert exogenous genes into the bacterial genome and the DNA fragment in between dif sequences can be recognized and removed by endogenous XerCD recombinases of K. pneumoniae. The drug susceptibility and growth rate of SfAlKp are identical to its parent strain, meanwhile the luminescence intensity and stability are also significant characteristics of SfAlKp. Compared to conventional techniques, the autoluminescence-based measurement is more applicable to high throughput screening for compounds both in vitro as well as in vivo in animal model.
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Técnicas Biossensoriais , Infecções por Klebsiella , Preparações Farmacêuticas , Animais , Antibacterianos/farmacologia , Biomarcadores , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/genética , CamundongosRESUMO
Pseudomonas aeruginosa is an increasingly prevalent pathogen that has become a serious health concern due to an increasing incidence of multidrug-resistant (MDR) hospital-acquired infections. The emergence of MDR-P. aeruginosa coupled with shrinking antibiotic pipelines has increased the demand for new antimicrobials and therapeutics. An effective tool for drug screening both in vitro and in vivo can facilitate the discovery of drugs and regimens for treating P. aeruginosa infection. Here, for the first time, we combined the mini-Tn7 system and Xer/dif recombinase system to construct a stable and selectable marker-free autoluminescent P. aeruginosa (SfAlPa) by one step. Afterwards, in vitro and in vivo activities of several antibiotics including amikacin, biapenem, levofloxacin and polymyxin B were assessed using SfAlPa. This study demonstrated that the use of SfAlPa could significantly facilitate rapid real-time evaluating the activities of compounds. Compared to prevailing methods, this method reduces the time, effort, animals and costs consumed in the discovery of new drugs against P. aeruginosa. Additionally, the methodology described in this study could be easily modified for construction of selectable marker-free reporter strain in other Gram-negative bacteria.