Clonal epidemiology and antimicrobial resistance in pseudomonas aeruginosa chronic respiratory infectionsinterpatient transmission and resistome evolution of an international cystic fibrosis clone

Abstract

Chronic respiratory infection (CRI) by Pseudomonas aeruginosa is the main cause of morbidity and mortality in cystic fibrosis (CF). During the progression from early infection to chronic non-eradicable colonization P. aeruginosa undergoes a complex evolutionary adaptation and diversification process which eventually leads to a mixed and persistent infecting population in which multidrug resistant variants frequently rise compromising the selection of appropriate antibiotic therapies. In this work the interplay between three key microbiological aspects of these infections was investigated: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of resistance to antibiotics. Clonal epidemiology, antibiotic susceptibility profiles, contribution of P. aeruginosa classical resistance mechanisms and the role of mutator variants were investigated in two large collections of CF P. aeruginosa isolates from the Balearic Islands and Spain. As well, whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, within-host evolution, WGS mutator genotypes (mutome) and resistome of widespread P. aeruginosa clonal complex 274 (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. Finally, due to the relevance of aminoglycosides in the management of CF-CRI, the dynamics of P. aeruginosa resistance development to aminoglycosides was also studied in vitro by WGS approaches. Despite discrepancies between molecular genotyping methods, a high degree of genetic diversity was documented among CF isolates from the Balearic Islands and Spain with scarce representation of CF epidemic strains. However, for the first time in Spain, we documented a superinfection with the multidrug resistant Liverpool Epidemic Strain (LES) in a chronically colonized patient. As well, P. aeruginosa CC274, previously detected in several CF individuals from Austria, Australia and France, was detected in 5 unrelated chronically colonized patients from the Balearic Islands and, therefore, this clone-type should be added to the growing list of CF epidemic clones. Subsequent analysis of the whole genomes sequences of P. aeruginosa isolates from the CC274 P. aeruginosa collection provides evidence of interpatient dissemination of mutator sublineages and denotes their potential for unexpected short-term sequence type (ST) evolution and antibiotic resistance spread, illustrating the complexity of P. aeruginosa population biology in CF. As well, epidemiological studies demonstrated the coexistence of two divergent lineages but without evident geographical barrier. Antibiotic resistance significantly accumulated overtime accompanied by hypersusceptibility to certain antibiotics such as aztreonam, which can be explained in terms of collateral susceptibility. Correlation between phenotypes and WGS genotypes of clonal isolates from the CC274 collection allowed us to define the mutational resistome of CF P. aeruginosa which extends beyond the classical mutational resistance mechanisms. Among the new chromosomic resistance determinants encountered, mutations within the penicillin-binding-protein 3 (PBP3), shaping up beta-lactam resistance, are noteworthy as well as mutations within the fusA1 gene, coding for elongation factor G, which along with MexXY overexpresion contribute to high-level aminoglycoside resistance. Strikingly, we encountered that MexXY overexpression is dispensable for in vitro resistance development to aminoglycosides which suggests an evolutionary advantage of its overexpression in the CF respiratory tract. Altogether this work demonstrates that clonal epidemiology and antibiotic resistance evolution in the CF setting results from the complex interplay among mutation-driven resistance mechanisms, within host diversification and interpatient transmission of epidemic strains.