Longitudinal Evolution of the Pseudomonas-Derived Cephalosporinase (PDC) Structure and Activity in a Cystic Fibrosis Patient Treated with ß-Lactams.

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C ß-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A "gain of function" of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC ß-lactamase is the main mechanism driving resistance in this notorious pathogen to ß-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the ß-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

Dealing with biofilms of Pseudomonas aeruginosa and Staphylococcus aureus: In vitro evaluation of a novel aerosol formulation of silver sulfadiazine.

The risk of infection of skin and soft tissue chronic wounds by gram-negative and gram-positive pathogens growing in biofilms is a major health-care concern. In this study we test a formulation of silver sulfadiazine, vitamin A and lidocaine (AF-SSD) for aerosol administration against biofilms of Pseudomonas aeruginosa and biofilms of methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains of Staphylococcus aureus. The aerosol allows the administration of AF-SSD without the direct contact with the wound and avoids contamination of the product after reiterative usage. We evaluated in vitro the anti-biofilm activity of AF-SSD by carrying out different technical approaches such as resazurin assays to measure metabolic activity/viability, crystal violet staining assays to determine biofilm biomass, counting of CFUs and live/dead staining for confocal microscopy analysis. AF-SSD clearly affected biofilm viability, biomass and structure, in the three bacterial strains tested. AF-SSD displayed a strong anti-biofilm effect, showing total bactericidal activity on biofilms of P. aeruginosa at a 400-fold dilution of the product, and after a 100-fold and 10-fold dilution for MRSA and MSSA, respectively. Considering the benefits of aerosol administration, our results support this kind of formulation as a potential improvement over conventional treatments with silver sulfadiazine.