novobiocin and Gram-Negative-Bacterial-Infections

Multidrug efflux is a major contributor to antibiotic resistance in Gram-negative bacterial pathogens. Inhibition of multidrug efflux pumps is a promising approach for reviving the efficacy of existing antibiotics. Previously, inhibitors targeting both the efflux transporter AcrB and the membrane fusion protein AcrA in the Escherichia coli AcrAB-TolC efflux pump were identified. Here we use existing physicochemical property guidelines to generate a filtered library of compounds for computational docking. We then experimentally test the top candidate coumpounds using in vitro binding assays and in vivo potentiation assays in bacterial strains with controllable permeability barriers. We thus identify a new class of inhibitors of E. coli AcrAB-TolC. Six molecules with a shared scaffold were found to potentiate the antimicrobial activity of erythromycin and novobiocin in hyperporinated E. coli cells. Importantly, these six molecules were also active in wild-type strains of both Acinetobacter baumannii and Klebsiella pneumoniae, potentiating the activity of erythromycin and novobiocin up to 8-fold.

Topics: Acinetobacter baumannii; Anti-Bacterial Agents; Anti-Infective Agents; Carrier Proteins; Computational Biology; Drug Resistance, Bacterial; Drug Synergism; Erythromycin; Escherichia coli Proteins; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Humans; Klebsiella pneumoniae; Lipoproteins; Membrane Transport Proteins; Molecular Docking Simulation; Multidrug Resistance-Associated Proteins; Novobiocin

A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23 kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Anti-Bacterial Agents; Bacterial Proteins; Bacterial Vaccines; Blotting, Western; Catfishes; DNA Gyrase; DNA-Directed RNA Polymerases; Drug Resistance, Bacterial; Fish Diseases; Fructose-Bisphosphate Aldolase; Gram-Negative Bacterial Infections; Mutation; Novobiocin

Three attenuated Aeromonas hydrophila vaccines were developed from the virulent 2009 West Alabama isolates through selection for resistance to both novobiocin and rifampicin. When channel catfish (Ictalurus punctatus) were IP injected with 4×105 colony-forming unit (CFU) of the mutants, no fish died. However, when the same age and size matched channel catfish were IP injected with similar amount of their virulent parents, 80-100% fish died. Similarly, when Nile tilapia (Oreochromis niloticus) were IP injected with 2×108 CFU of the mutants, no fish died. However, when Nile tilapia were IP injected with similar amount of the mutants, all fish died. Vaccination of channel catfish with the mutants at dose of 4×105 CFU/fish offered 86-100% protection against their virulent parents at 14 days post vaccination (dpv). Vaccination of Nile tilapia with the mutants at dose of 2×108 CFU/fish offered 100% protection against their virulent parents at 14, 28, and 56 dpv. Agglutination assay results suggested that protection elicited by the mutants was partially due to antibody-mediated immunity. Taken together, our results suggest that the three attenuated vaccines might be used to protect channel catfish and Nile tilapia against the highly virulent 2009 West Alabama isolates of A. hydrophila.

Topics: Aeromonas hydrophila; Alabama; Animals; Anti-Bacterial Agents; Antibodies, Bacterial; Bacterial Vaccines; Cichlids; Drug Resistance, Bacterial; Fish Diseases; Gram-Negative Bacterial Infections; Ictaluridae; Mutation; Novobiocin; Rifampin; Survival Analysis; Vaccines, Attenuated; Virulence

Topics: Aeromonas; Agar; Anti-Bacterial Agents; Carbanilides; Cefsulodin; Culture Media; Electron Transport Complex IV; Feces; Gram-Negative Bacterial Infections; Humans; Microbial Sensitivity Tests; Novobiocin

Polymyxin B nonapeptide, derived by cleavage of the fatty acyl diaminobutyric acid from polymyxin B, is considerably less toxic, lacks bactericidal activity, and retains its ability to render gram-negative bacteria susceptible to several antibiotics by permeabilizing their outer membranes. The peptide rendered all 53 polymyxin-susceptible strains tested more susceptible to novobiocin, lowering the MIC of novobiocin eightfold or more. The combination of polymyxin B nonapeptide with novobiocin or with erythromycin administered intraperitoneally in multiple doses synergistically protected mice infected with gram-negative bacteria. This combination may be clinically useful because of the apparent rarity of the acquisition of resistance.

Topics: Animals; Anti-Bacterial Agents; Cell Membrane Permeability; Drug Resistance, Microbial; Drug Synergism; Drug Therapy, Combination; Erythromycin; Female; Gram-Negative Bacterial Infections; Humans; Mice; Mice, Inbred ICR; Novobiocin; Polymyxin B