Assessment of polymyxin B-doxycycline in combination against Pseudomonas aeruginosa in vitro and in a mouse model of acute pneumonia.

The increasing prevalence of antibiotic resistance in Pseudomonas aeruginosa has created an urgent need for suitable therapy. This study explored the pairing of doxycycline with other antipseudomonal antibiotics, and found that polymyxin B in combination with doxycycline had a synergistic effect against clinical strains of P. aeruginosa. This synergistic combination was studied by checkerboard assays and time-kill curve analysis. Further, in-vitro biofilm disruption, pyoverdine inhibition assays were performed. The efficacy of polymyxin B-doxycycline in combination, administered by inhalation, was evaluated using a mouse model of acute pneumonia. The combination was found to have a synergistic effect in both in-vitro and in-vivo studies. The combination decreased biofilms of P. aeruginosa and reduced the level of pyoverdine, an important siderophore of P. aeruginosa. In addition, the combination decreased the P. aeruginosa population by 3 log10 (P<0.01) in the mouse model of acute pneumonia, and showed an improvement in lung function by inhalation. To the best of the authors' knowledge, this is the first in-vivo study to evaluate the efficacy of polymyxin B in combination with doxycycline against P. aeruginosa, showing a possible promising option for acute pneumonia due to multi-drug-resistant P. aeruginosa.

The small molecule IITR08027 restores the antibacterial activity of fluoroquinolones against multidrug-resistant Acinetobacter baumannii by efflux inhibition.

Efflux pumps are one of the major determinants of multiple drug resistance. In this study, AbeM, a multidrug efflux pump in Acinetobacter baumannii, was cloned into a multicopy plasmid (pUC18) and was transformed into an efflux-deficient mutant of Escherichia coli (KAM32). The elevated resistance profile of the recombinant E. coli for ciprofloxacin was utilised to screen a small-molecule library of 8000 molecules to identify IITR08027, a small molecule that is not inhibitory on its own but that could potentiate the activity of ciprofloxacin. When used in combination against A. baumannii, the molecule improves the killing efficiency of ciprofloxacin, extends its post-antibiotic effect and causes a decrease in frequency of resistant mutant selection with the antibiotic. Quinacrine-based fluorescence quenching and membrane depolarisation assays revealed that IITR08027 functions as a proton gradient inhibitor. This extends the activity of IITR08027 against other H+-driven efflux pumps such as AbeS. MTT assay against HeLa and HEK293 cells suggested that the molecule is non-toxic at its minimum effective concentration. We propose that, in combination with fluoroquinolones (ciprofloxacin and norfloxacin), IITR08027 may be effective against multidrug-resistant A. baumannii expressing AbeM or other efflux pumps that use the proton gradient as an efflux mechanism.

Effect of intermittent aeration on microbial diversity in an intermittently aerated IFAS reactor treating municipal wastewater: A field study.

In the present study, the effect of three intermittent aeration (IA) cycles on treatment performance and microbial diversity was investigated in an integrated fixed film activated sludge (IFAS) reactor treating municipal wastewater. The results showed that IA strategies were able to achieve efficient removal of organics and nitrogen ranging between 90 and 95% and 70 and 80%, respectively, however the phosphorus removal was found to be inversely proportional to the duration of aeration off time in each IA cycle. The microscopic analysis revealed that the suspended and attached biomass had compact morphology and open floc structure, respectively. For each gram of volatile suspended solids, 165 and 148 mg of extracellular polymeric substances (EPS) were extracted from attached and suspended biomass, respectively, constituting carbohydrates (∼24%), proteins (∼31%), humic acids (∼28%), DNA (∼2%) and unknown substances (∼12%). The microbial diversities of suspended biomass in IFAS reactor were investigated using culture-dependent approach, which confirmed the presence of Clostridium spp., Pseudomonas spp., Bacillus spp., Escherichia coli spp., Nitrosococcus spp., Streptococcus spp., Acinetobacter spp., Betaproteobacteria outliers, Klebsiella pneumoniae, Klebsiella aerogenes, Serratia marcescens, Micrococcus, Proteus vulgaris spp., Actinomycetes spp., and Actinobacteria including Micromonospora spp. and Streptomyces spp. Molecular tools for diversity analyses were used for ammonia and nitrite oxidizer identification, such as Nitrospira and Nitrosococcus species. Denitrifiers include the species of Pseudomonas, Betaproteobacteria and Flavobacterium. Acinetobacter, Betaproteobacteria and Gammaproteobacteria were responsible for the phosphorus removal in the present system. Overall, the system performed efficiently showing Proteobacteria (59%), Acinetobacter (12%) and Bacteroidetes (11%) as the dominant bacterial groups. However, the dominance of the bacterial diversity varied with the IA cycle time numerating the maximum percentage of bacterial species during IA1 phase i.e. 2.5 h aeration/0.5 h non-aeration.