Study of the clinical evolution of β-lactamase PDC in a hypermutable lineage of Pseudomonas aeruginosa

Congreso; XVII Congreso Argentino de Microbiología General; 2022

SAMIGE

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, and 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. We have previously explored the in-patient adaptive longterm evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of cystic fibrosis patient treated with different antibiotics during more than 25 years of chronic infection. Chronological tracking of mutations from different subpopulations demonstrated parallel evolution events in the PDC β-lactamase (Pseudomonas-derived cephalosporinase). Multiple mutations within blaPDC shaped diverse coexisting alleles, depending on the antibiotic selective pressures. Importantly, thecombination of the cumulative mutations in blaPDC resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance leading to a ?gain of function? of collateral resistance towards ceftolozane (TOL), a fifth generation cephalosporin that was notprescribed to this patient.Despite the diversity of coexisting alleles along the years, there were three most prevalent variants (PDC-461, 462 and 463), which share three conserved mutations (A89V, Q120K and V211A) combined in triple and quadruple mutant alleles, but no simple nor double mutants could be found. These enzymes showed greater resistance and catalytic efficiencies against both cephalosporines, withrespect to parental enzyme PDC-3. In fact, the mutant PDC-461 containing the core of three conserved mutations showed the highest resistance and activity. Docking and molecular dynamics calculations showed an expansion in the cavity of the active site of PDC-461, which is due to Q120K mutation that opens the active site pocket.Here, to elucidate the role of the conserved mutations and to reconstruct the evolutionary pathways, we engineered single and double mutants that combines the three core mutations and study their evolution of the resistance against CAZ. Importantly, the Q120K single mutant conferred the same MIC that the evolved enzyme PDC-461. However, it was much less stable than the rest of the mutantsin its native periplasm environment, indicating that the PDC variants evolve towards accumulation of compensatory mutations to restore the protein stability.Our current investigations aim at exploring the tradeoff between PDC activity and stability in order to gain a deeper understanding of theevolution of P. aeruginosa resistance driven by decades of antibiotic treatment in the natural CF environmental setting.