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

Congreso; XIII Congreso Argentino de Microbiología General SAMIGE; 2020

Antibiotic resistance have emerged as a global health concern with serious economic, social and political implications. Due to the increase prevalence of nosocomial infections, millions of people will die every year, which severely compromises the selection of appropriate treatments (O?Neill J 2016). 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 in different parts of the world (Samore MH et al 1999). P. aeruginosa can cause acute and chronic infections, and it 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 insight into long-term bacterial evolution in natural environments, 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 (Feliziani S 2014). Remarkably, P. aeruginosa isolates from one of the patients (CFD), who was intensively treated with β-lactam antibiotics, show accumulation of mutations within ampC gene with evidence of at least 4 different variants coexisting in the same P. aeruginosa population, indicating that this gene appeared to be under high evolutionary pressure in the CF lung. Moreover, ampC gene underwent convergent evolution across the sub-lineages suggesting that it is involved in the optimization process of the pathogenic fitness (Feliziani S 2014). In this work, we aim to characterize the evolution of 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 the variants in the population, we used a sequential collection of isolates obtained from single sputum samples from a CF patient spanning 27 years of chronic infection history. Likewise, we explore the impact of AmpC variants on β-lactam MICs to understand the plasticity of the enzymes for β-lactam hydrolysis and to identify potential therapeutic implications of mutations. In addition, we performed Amplicon Sequencing to study population diversity of AmpC variants and common tendencies across different CF patients. Our results show that evolution is still occurring during antibiotic treatment, and that AmpC populations are highly diverse. Furthermore, there is selection of specific variants that coexist with a high frequency in the population which were associated with 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 potential roll of these mutations. Understanding the driving forces of in patient evolution and utilizing antimicrobial treatment strategies that take advantage of a growing understanding of the evolutionary paths could help clinicians to optimise treatment strategies and ensure that patients have better clinical outcomes.