RESUMEN
Mycobacterium infection gives rise to granulomas predominantly composed of inflammatory M1-like macrophages, with bacteria-permissive M2 macrophages also detected in deep granulomas. Our histological analysis of Mycobacterium bovis bacillus Calmette-Guerin-elicited granulomas in guinea pigs revealed that S100A9-expressing neutrophils bordered a unique M2 niche within the inner circle of concentrically multilayered granulomas. We evaluated the effect of S100A9 on macrophage M2 polarization based on guinea pig studies. S100A9-deficient mouse neutrophils abrogated M2 polarization, which was critically dependent on COX-2 signaling in neutrophils. Mechanistic evidence suggested that nuclear S100A9 interacts with C/EBPß, which cooperatively activates the Cox-2 promoter and amplifies prostaglandin E2 production, followed by M2 polarization in proximal macrophages. Because the M2 populations in guinea pig granulomas were abolished via treatment with celecoxib, a selective COX-2 inhibitor, we propose the S100A9/Cox-2 axis as a major pathway driving M2 niche formation in granulomas.
RESUMEN
Infections of CTX-M extended-spectrum ß-lactamase-producing Enterobacterales are a severe threat in clinical settings. CTX-M genes on plasmids have been transferred to many Enterobacterales species, and these species have spread, leading to the global problem of antimicrobial resistance. Here, we developed a lateral flow immunoassay (LFIA) based on an anti-CTX-M rabbit monoclonal antibody. This antibody detected CTX-M variants from the CTX-M-9, CTX-M-2, and CTX-M-1 groups expressed in clinical isolates. The LFIA showed 100% sensitivity and specificity with clinical isolates on agar plates, and its limit of detection was 0.8 ng/mL recombinant CTX-M-14. The rabbit monoclonal antibody did not cross-react with bacteria producing other class A ß-lactamases, including SHV. In conclusion, we developed a highly sensitive and specific LFIA capable of detecting CTX-M enzyme production in Enterobacterales. We anticipate that our LFIA will become a point-of-care test enabling rapid detection of CTX-M in hospital and community settings as well as a rapid environmental test.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Enterobacteriaceae/aislamiento & purificación , beta-Lactamasas/análisis , Animales , Enterobacteriaceae/metabolismo , Inmunoensayo , Pruebas en el Punto de Atención , Conejos , Sensibilidad y Especificidad , beta-Lactamasas/biosíntesisRESUMEN
Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of M. leprae have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of M. leprae, and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. Methodology/Principal Finding: To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of M. leprae. An in silico study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against M. leprae DNA gyrase than others. Furthermore, analysis using quinolone-resistant M. leprae DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an in silico study. Conclusions/Significance: The inhibitory effect of WQ compounds that have a new R1 group against M. leprae DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant M. leprae and other bacterial pathogens.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium leprae/efectos de los fármacos , Mycobacterium leprae/genética , Quinolonas/farmacología , Azetidinas/farmacología , Fluoroquinolonas/farmacología , Genes Bacterianos , Pruebas de Sensibilidad MicrobianaRESUMEN
Aims: Quinolone-resistant nontyphoidal Salmonella having serine replaced by isoleucine at the 83rd amino acid in GyrA (GyrA-Ser83Ile) has recently been found in Asian countries. In this study, we aimed to examine the direct effect of substitution Ser83Ile on DNA gyrase activity and/or resistance to quinolones. Materials and Methods: Using 50% of the maximal inhibitory concentrations (IC50s) of quinolones, recombinant wild type (WT) and seven mutant DNA gyrases having amino acid substitutions, including Ser83Ile, were screened for enzymatic activity that causes supercoils in relaxed plasmid DNA and resistance to quinolones. Results: Little differences in supercoiling activity were observed between WT and mutant DNA gyrases. By contrast, the IC50s of ciprofloxacin and norfloxacin against GyrA-Ser83Ile/GyrB-WT were 11.6 and 73.3 µg/mL, respectively, which were the highest used against the DNA gyrases examined in this study. Conclusion: Ser83Ile in GyrA was shown to confer high-level quinolone resistance to DNA gyrases of nontyphoidal Salmonella, with no loss of supercoiling activity. Salmonella strain carrying GyrA with Ser83Ile may emerge under a high-concentration pressure of quinolones and easily spread even with no selection bias by quinolones. Hence, avoiding the overuse of quinolones is needed to prevent the spread of Salmonella with Ser83Ile in GyrA.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/genética , Farmacorresistencia Bacteriana/genética , Quinolonas/farmacología , Salmonella typhimurium/genética , Sustitución de Aminoácidos , Genes Bacterianos/genética , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro antimycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guérin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Azetidinas/farmacología , Proteínas Bacterianas/metabolismo , Girasa de ADN/metabolismo , Farmacorresistencia Bacteriana , Fluoroquinolonas/farmacología , Mycobacterium bovis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Proteínas Bacterianas/genética , Girasa de ADN/genética , Farmacorresistencia Bacteriana/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Etambutol/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium bovis/enzimología , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genéticaRESUMEN
BACKGROUND: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. METHODOLOGY/PRINCIPAL FINDINGS: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. CONCLUSIONS/SIGNIFICANCE: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens.
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Antibacterianos/farmacología , Azetidinas/farmacología , Girasa de ADN/efectos de los fármacos , Fluoroquinolonas/farmacología , Mycobacterium leprae/efectos de los fármacos , Antibacterianos/uso terapéutico , Azetidinas/uso terapéutico , Farmacorresistencia Bacteriana/efectos de los fármacos , Fluoroquinolonas/uso terapéutico , Humanos , Lepra/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Ofloxacino/farmacologíaRESUMEN
Quinolones show excellent antibacterial activity against Salmonella isolates. Recently, however, quinolone resistance has been increasing in bacteria. This study aimed to examine in vitro, and compare the activity of DC-159a against Salmonella enterica serovar Typhimurium with that of ciprofloxacin and nalidixic acid. Inhibitory effects of quinolones were assessed by the drug concentration needed to inhibit the supercoiling activity of recombinant DNA gyrases by 50% (IC50). Dilution methods were used to determine the minimum inhibitory concentration (MIC) of quinolones against two different strains, Salmonella Typhimurium and Salmonella Enteritidis. The IC50s of DC-159a against mutant DNA gyrases were much lower than those of nalidixic acid and ciprofloxacin. In particular, the IC50 of DC-159a against DNA gyrase with double mutation was less than 1/50 that of ciprofloxacin and nalidixic acid. MICs of DC-159a were higher than those of ciprofloxacin but lower than those of nalidixic acid. However, the estimated MICs of DC-159a against Salmonella strains with mutant DNA gyrase were lower than those of ciprofloxacin and nalidixic acid. Therefore, DC-159a can be suggested as an antibiotic candidate for treating salmonellosis caused by quinolone-resistant S. Typhimurium.
Asunto(s)
Aminopiridinas/farmacología , Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Salmonella enterica/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Ciprofloxacina/farmacología , Girasa de ADN/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación/genética , Ácido Nalidíxico/farmacología , Quinolonas/farmacología , Infecciones por Salmonella/tratamiento farmacológico , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genéticaRESUMEN
We have recently reported a self-threading polythiophene as a new family of insulated molecular wires. Herein, we focused on the structure-property relationships of the unique three-dimensional architecture of the monomer. We have synthesized nine self-threading bithiophene monomers that have cyclic side-chains of different size and flexibility: i.e., 21-, 22-, 23-, 24-, 26-, and 30-membered rings composed of paraffinic, olefinic, or alkynic chains. To investigate their structure-property relationships, (1) Hâ NMR spectroscopy, UV absorption, and fluorescence spectroscopy measurements were conducted. We found that cyclic side-chains define the movable range of the dihedral angle of the bithiophene backbone, thereby affecting its photophysical properties. Therefore, the ability to design a structure with atomic precision as described herein would lead to the fine-tuning of the electronic properties of insulated molecular wires.