Novel treatment strategies for chronic respiratory infections based on the analysis of antimicrobial resistance dynamics in pseudomonas aeruginosa biofilms

Abstract

Pseudomonas aeruginosa is the major cause of chronic respiratory infections (CRI) and the main driver of morbidity and mortality in cystic fibrosis (CF) patients. The establishment of P. aeruginosa CRI requires a complex adaptive process that includes the selection of an important number of mutations required for long-term persistence and the transition from the planktonic to biofilm mode of growth, a hallmark of chronic infections. The complex spatial structure of the biofilm and the antibiotic selective pressure lead to evolve of heterogeneous communities conformed by different subpopulations that coexist and interact establishing diverse social behaviors. Moreover, the prolonged antibiotic treatments required in CRI unavoidably entail an increase of resistance. Traditional therapeutic strategies to control P. aeruginosa infection in CF patients based on the use of a single nebulized antibiotic seem to be ineffective over time. First, this work determined whether antibiotic resistant mutants display selfish or altruistic behaviors in mixed P. aeruginosa biofilms exposed to antibiotics. ECFP-tagged strain PAO1 and its EYFP-tagged derivatives hyperproducing the β-lactamase AmpC or the efflux pump MexAB-OprM were used to develop single or mixed biofilms. Mature biofilms were challenged with different concentrations of β-lactams to monitor biofilm structural dynamics, using confocal laser scanning microscopy (CLSM), and population dynamics, through enumeration of viable cells. While exposure of single wild-type PAO1 biofilms to β-lactams lead to a major reduction in bacterial load, it had little effect on biofilms formed by the resistant mutants. However, the most revelling finding was that bacterial load of wild-type PAO1 was significantly increased when growing in mixed biofilms compared to single biofilms. In agreement with CFU enumeration data, CLSM images revealed the amplification of the resistant mutants and their protection of susceptible populations. These findings show that mutants expressing diverse resistance mechanisms, including β-lactamases, but also, as evidenced for the first time, efflux pumps, protect the whole biofilm community, preserving susceptible populations from the effect of antibiotics. Finally, this work also evaluated the therapeutic efficacy and dynamics of antibiotic resistance in P. aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Biofilms of laboratory and clinical strains were developed using the flow cell system. Mature biofilms were challenged with different concentrations of ATM and TOB and their alternations at different time point. The number of viable cells and resistant mutants were determined, and biofilm structural dynamics were monitored by CLSM. TOB monotherapy produced an intense decrease of CFUs that was not always correlated with a reduction of biomass and/or bactericidal effect on biofilms, particularly for the CF strains. 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 individual regimens, potentiating the bactericidal effect and/or the reduction of biomass. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to current maintenance treatments with just one antibiotic in monotherapy. On the whole, this work provides new insights into the complexity of resistance development in biofilms, highlighting the significance of resistant mutants within heterogenous populations, and also represent a step forward for the development of new strategies for combating P. aeruginosa CRI.