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An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections.

ABSTRACT: Bacterial persisters are a quasidormant subpopulation of cells that are tolerant to antibiotic treatment. The combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an antipersister strategy that is aimed at reducing recurrent Pseudomonas aeruginosa infections by enhancing the killing of P. aeruginosa persisters. Stationary-phase cultures of P. aeruginosa were used to generate persister cells. A range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions, including a range of pH values and in the presence of azithromycin or cystic fibrosis (CF) patient sputum. In addition, 96-well dish biofilm and colony biofilm assays were performed, and the cytotoxicity of the tobramycin-fumarate combination was determined utilizing a lactate dehydrogenase (LDH) assay. Enhanced killing of up to 6 orders of magnitude of P. aeruginosa persisters over a range of CF isolates, including mucoid and nonmucoid strains, was observed for the tobramycin-fumarate combination compared to killing with tobramycin alone. Furthermore, significant fumarate-mediated potentiation was seen in the presence of azithromycin or CF patient sputum. Fumarate also reduced the cytotoxicity of tobramycin-treated P. aeruginosa to human epithelial airway cells. Finally, in mucoid and nonmucoid CF isolates, complete eradication of P. aeruginosa biofilm was observed in the colony biofilm assay due to fumarate potentiation. These data suggest that a combination of tobramycin with fumarate as an antibacterial potentiator may be an attractive therapeutic for eliminating recurrent P. aeruginosa infections in CF patients through the eradication of bacterial persisters.

SUBMITTER: Koeva M

PROVIDER: S-EPMC5700368 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections.

Koeva Martina M Gutu Alina D AD Hebert Wesley W Wager Jeffrey D JD Yonker Lael M LM O'Toole George A GA Ausubel Frederick M FM Moskowitz Samuel M SM Joseph-McCarthy Diane D

Antimicrobial agents and chemotherapy 20171122 12

Bacterial persisters are a quasidormant subpopulation of cells that are tolerant to antibiotic treatment. The combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an antipersister strategy that is aimed at reducing recurrent Pseudomonas aeruginosa infections by enhancing the killing of P. aeruginosa persisters. Stationary-phase cultures of P. aeruginosa were used to generate persister cells. A range of tobramycin concentrations ...[more]

PMID: 28923873

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Project description:Traditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance in Pseudomonas aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t = 2), 4 (t = 4), and 6 days (t = 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one antibiotic in monotherapy.

Project description:RationalePseudomonas aeruginosa, the predominant cause of chronic airway infections of patients with cystic fibrosis, exhibits extensive phenotypic diversity among isolates within and between sputum samples, but little is known about the underlying genetic diversity.ObjectivesTo characterize the population genetic structure of transmissible P. aeruginosa Liverpool Epidemic Strain in chronic infections of nine patients with cystic fibrosis, and infer evolutionary processes associated with adaptation to the cystic fibrosis lung.MethodsWe performed whole-genome sequencing of P. aeruginosa isolates and pooled populations and used comparative analyses of genome sequences including phylogenetic reconstructions and resolution of population structure from genome-wide allele frequencies.Measurements and main resultsGenome sequences were obtained for 360 isolates from nine patients. Phylogenetic reconstruction of the ancestry of 40 individually sequenced isolates from one patient sputum sample revealed the coexistence of two genetically diverged, recombining lineages exchanging potentially adaptive mutations. Analysis of population samples for eight additional patients indicated coexisting lineages in six cases. Reconstruction of the ancestry of individually sequenced isolates from all patients indicated smaller genetic distances between than within patients in most cases.ConclusionsOur population-level analysis demonstrates that coexistence of distinct lineages of P. aeruginosa Liverpool Epidemic Strain within individuals is common. In several cases, coexisting lineages may have been present in the infecting inoculum or assembled through multiple transmissions. Divergent lineages can share mutations via homologous recombination, potentially aiding adaptation to the airway during chronic infection. The genetic diversity of this transmissible strain within infections, revealed by high-resolution genomics, has implications for patient segregation and therapeutic strategies.

Project description:The widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used in vitro evolution and analyzed the inpatient microevolution of Pseudomonas aeruginosa, an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance in vitro, and we present evidence that similar processes shape antibiotic exposure in human patients. Our data suggest that during chronic infections, P. aeruginosa first acquires moderate drug tolerance before following distinct evolutionary trajectories that lead to high-level multidrug tolerance or to antibiotic resistance. Our studies propose that the development of antibiotic tolerance predisposes bacteria for the acquisition of resistance at early stages of infection and that both mechanisms independently promote bacterial survival during antibiotic treatment at later stages of chronic infections.IMPORTANCE Over the past decades, pan-resistant strains of major bacterial pathogens have emerged and have rendered clinically available antibiotics ineffective, putting at risk many of the major achievements of modern medicine, including surgery, cancer therapy, and organ transplantation. A thorough understanding of processes leading to the development of antibiotic resistance in human patients is thus urgently needed. We show that drug tolerance, the ability of bacteria to survive prolonged exposure to bactericidal antibiotics, rapidly evolves in the opportunistic human pathogen Pseudomonas aeruginosa upon recurrent exposures to antibiotics. Our studies show that tolerance protects P. aeruginosa against different classes of antibiotics and that it generally precedes and promotes resistance development. The rapid evolution of tolerance during treatment regimens may thus act as a strong driving force to accelerate antibiotic resistance development. To successfully counter resistance, diagnostic measures and novel treatment strategies will need to incorporate the important role of antibiotic tolerance.

Project description:Pseudomonas aeruginosa is one of the first causes of acute nosocomial and chronic infections in patients with underlying respiratory pathologies such as cystic fibrosis (CF). It has been proposed that P. aeruginosa accumulates mutations driving to peptidoglycan modifications throughout the development of the CF-associated infection, as a strategy to lower the immune detection hence ameliorating the chronic persistence. As well, some studies dealing with peptidoglycan modifications driving to a better survival within the host have been published in other gram-negatives. According to these facts, the gram-negative peptidoglycan could be considered as a pathogen-associated molecular pattern with very important implications regarding the host's detection-response, worthy to dissect in detail. For this reason, in this work we characterized for the first time the peptidoglycans of three large collections [early CF, late CF and acute infection (bloodstream) P. aeruginosa strains] from qualitative (HPLC), quantitative and inflammatory capacity-related perspectives. The final goal was to identify composition trends potentially supporting the cited strategy of evasion/resistance to the immune system and providing information regarding the differential intrinsic adaptation depending on the type of infection. Although we found several punctual strain-specific particularities, our results indicated a high degree of inter-collection uniformity in the peptidoglycan-related features and the absence of trends amongst the strains studied here. These results suggest that the peptidoglycan of P. aeruginosa is a notably conserved structure in natural isolates regardless of transitory changes that some external conditions could force. Finally, the inverse correlation between the relative amount of stem pentapeptides within the murein sacculus and the resistance to immune lytic attacks against the peptidoglycan was also suggested by our results. Altogether, this work is a major step ahead to understand the biology of peptidoglycan from P. aeruginosa natural strains, hopefully useful in future for therapeutic alternatives design.

Dataset Information

An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections.

Publications

An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections.

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