Long-term evolution of beta-lactamase AmpC isolated from Pseudomonas aeruginosa cystic fibrosis chronic airway infections

G. HEDEMANN; C. A. COLQUE; S. MOLIN; R. A. HICKMAN; H. K. JOHANSEN; A. G. ALBARRACÍN ORIO; L. M. SOMMER; A. M. SMANIA

San Luis

Congreso; XIII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2018

Antibiotic resistance has emerged as a global health concern with serious economic, social and political implications. Among high-risk pathogens, Pseudomonas aeruginosa is one of the most feared cause of nosocomial infections and is responsible for about 10-20% of hospital-acquired infections. As an opportunistic pathogen, P. aeruginosa causes acute and chronic infections, and represents the main cause of morbidity and mortality in immunocompromised patients suffering from cystic fibrosis (CF). P. aeruginosa from chronic CF infections provide unique opportunities to get insights into long-term bacterial evolution, and an extraordinary natural scenario to explore evolution of antibiotic resistance. By whole-genome sequencing, we previously evaluated the genetic changes undergone by mutator populations of P. aeruginosa during long-term chronic infections. Remarkably, P. aeruginosa isolates from one patient (CFD), who was intensively treated with β-lactam antibiotics, showed accumulation of mutations within the ampC gene with evidence of at least 4 different allelic variants coexisting in the same P. aeruginosa population, suggesting that this gene underwent a high evolutionary pressure in the CF lung. Moreover, ampC showed convergent evolution across the different sub-lineages, suggesting a role of this mutagenic process in the pathogenic fitness of P. aeruginosa. In this work, we aimed to characterize the evolution of the spontaneous mutations acquired in the ampC gene during the long-term adaptation of P. aeruginosa to the CF airways. To explore the genetic diversity within ampC as well as the dynamics of their allelic variants in the population, we used a sequential collection of isolates obtained from single sputum samples from the CFD patient, spanning 26 years of chronic infection history. Likewise, in order to understand the plasticity of the enzymes for β-lactam hydrolysis and to identify potential therapeutic implications of mutations, we explored the impact of ampC mutations from the different allelic variants on β-lactam MICs. Finally, we performed Amplicon Sequencing to study the genetic diversity of ampC and common tendencies across different CF patients. Our results show that evolution is still occurring and driven by antibiotic treatment, and that the ampC sequence is highly diverse across populations. Furthermore, some dominant ampC allelic variants are associated to high resistance towards cephalosporins and monobactams. Remarkably, we show that some positions in the ampC sequence are frequently hit by mutations across different CF patients suggesting a key role of these mutations in antibiotic resistance.