The common soil and aquatic bacterium Pseudomonas aeruginosa, is a model example of opportunistic pathogens causing many types of difficult-to-cure infections in various compromised patients. P. aeruginosa is the most relevant pathogen producing pulmonary chronic infections in patients with Cystic Fibrosis (CF). During CF chronic infection P. aeruginosa diversifies, yielding phenotypes with particular traits that are not displayed in isolates obtained from others infections. Between them, there are mucoids, avirulent variants, hypermutators and isolates with altered antibiotic susceptibility. Importantly, this phenotypic diversification favors P. aeruginosa long-term persistence making impossible its eradication by any known therapy. Since the acquisition of these phenotypes involves loss-of-function mutations in targets genes, it has been suggested that this phenotypic diversification is catalyzed by hypermutators with high mutation rates, mainly due to DNA mismatch repair (MMR) deficiency. Indeed, the link between antimicrobial resistance and hypermutators was mostly suggested. Furthermore, working with a P. aeruginosa MMR-deficient strain in vitro, we have previously established a relation between hypermutability and mucoid conversion as well as emergence of avirulent variants via quorum sensing inactivation.
In order to gain further insights into the role of hypermutability in P. aeruginosa phenotypic diversification, we analyzed the association between hypermutator phenotype and mucoid (mucA mutants), avirulent (lasR mutants) and multi drug resistant (mexZ mutants) variants in a collection of P. aeruginosa isolates obtained from CF patients. We examined 40 isolates from 27 child and adults patients who are chronically infected with P. aeruginosa and are assisted in local Hospitals. According to SpeI macrorestriction fragment fingerprints we observed that most patients were infected by individual P. aeruginosa clonesas keeping with previous reports. By screening for resistant rifampicin cells, we observed that 20 isolates (50%) were hypermutators and 14 patients (55.5%) had hypermutators. Afterward, the score of mutations in the coding region of the mucA, lasR and mexZ genes were performed by PCR amplification and direct nucleotide sequence, analyzing their effects by using computational methods. Only one isolate had no mutation (2,5%) in all analyzed genes, displaying at the same time a non mutator phenotype. Although 75% of the isolates showed mutations in mexZ, only 40% of them were hypermutators. Repect to mucA, we observed that 65% of isolates showed mutations in that gene, 46% of which showed a hypermutator phnotype. Interestingly, a lower percentage of isolates (35%) harbor mutations in lasR, however, 80% of them were found to be hypermutators. We are currently investigating the molecular bases of the hypermutability in the 20 hypermutator isolates by complementation assays and sequencing of the main components of MMR. Our results will shed light on the mechanisms by which P. aeruginosa evolves its adaptive repertoire leading to CF chronic infection.