Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa.

Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

Molecular diversity assessed by MALDI mass spectrometry of two scorpion species venom from two different locations in Morocco.

Scorpion venom is a cocktail of molecules whose composition is remarkably plastic, controlled by several factors. The Moroccan scorpion fauna is characterized by its richness and high rate of endemism and the venom molecular variability of many species is not yet well characterized. The aim of the present study was to highlight the molecular variability of the venom composition of Androctonus amoreuxi and Buthacus stockmanni (endemic species), both belonging to the Buthidae family, collected from two Moroccan regions, Zagora and Tan-tan. Characterization of the molecular mass fingerprints (MFPs) of each specimen was performed by Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) using a sandwich (Sand) and a dried-droplet (DD) sample preparation and dilutions. Considering these two methods, a total of 828 ion signals were detected, and Sand method produced more adducts (56%) than DD (44%). We observed interspecific variations in the venom composition between these two species showing they share 235 ion signals, while 226 and 367 are specific for these two species, respectively. Moreover, B. stockmanni specimens showed a clear difference in their MFPs between the two geographical areas studied, suggesting intraspecific variations. Moreover, specimens from each population also show an intraspecific variability. In addition, for the same individual, a variation in the venom composition was also recorded depending on the milking frequency. Our results confirmed the presence of characteristic components in each extracted venom sample. In conclusion, MFPs assessed by MALDI-MS represent a fast, non-supervised, sensitive, reliable and cost-efficient approach for taxonomic identification and molecular variability characterization. This study undoubtedly represents a step forward for understanding the scorpion venom plasticity, intra/inter variations, and their temporal and geographical variability.

Personalized aerosolised bacteriophage treatment of a chronic lung infection due to multidrug-resistant Pseudomonas aeruginosa.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient's isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.

Combined Bacteriophage and Antibiotic Treatment Prevents Pseudomonas aeruginosa Infection of Wild Type and cftr- Epithelial Cells.

With the increase of infections due to multidrug resistant bacterial pathogens and the shortage of antimicrobial molecules with novel targets, interest in bacteriophages as a therapeutic option has regained much attraction. Before the launch of future clinical trials, in vitro studies are required to better evaluate the efficacies and potential pitfalls of such therapies. Here we studied in an ex vivo human airway epithelial cell line model the efficacy of phage and ciprofloxacin alone and in combination to treat infection by Pseudomonas aeruginosa. The Calu-3 cell line and the isogenic CFTR knock down cell line (cftr-) infected apically with P. aeruginosa strain PAO1 showed a progressive reduction in transepithelial resistance during 24 h. Administration at 6 h p.i. of single phage, phage cocktails or ciprofloxacin alone prevented epithelial layer destruction at 24 h p.i. Bacterial regrowth, due to phage resistant mutants harboring mutations in LPS synthesis genes, occurred thereafter both in vitro and ex vivo. However, co-administration of two phages combined with ciprofloxacin efficiently prevented PAO1 regrowth and maintained epithelial cell integrity at 72 p.i. The phage/ciprofloxacin treatment did not induce an inflammatory response in the tested cell lines as determined by nanoString® gene expression analysis. We conclude that combination of phage and ciprofloxacin efficiently protects wild type and cftr- epithelial cells from infection by P. aeruginosa and emergence of phage resistant mutants without inducing an inflammatory response. Hence, phage-antibiotic combination should be a safe and promising anti-Pseudomonas therapy for future clinical trials potentially including cystic fibrosis patients.