CONICET | Buscador de Institutos y Recursos Humanos

Pseudomonas aeruginosa, is an opportunistic pathogen causingmany types of difficult-to-cure infections in immunological 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 into phenotypes with particular traits that are not displayed in isolates obtained from other infections. Among them, there are mucoids, avirulent variants, hypermutators and isolates with altered antibiotic susceptibility. Importantly, this phenotypic diversification favors P. Aeruginosa long-term persistence making it impossible to eradicate by any known therapy. Since the acquisition of these phenotypes mostly involves loss-of-function mutations in targets genes, it has been suggested that this phenotypic diversification is catalyzed by hypermutator strains, which are mainly deficient in the DNA mismatch repair system (MMR). Indeed, the link between antimicrobial resistance and hypermutators was strongly suggested. Moreover, we have previously established a linkage between hypermutability and mucoid conversion as well as emergence of avirulent variants via quorum sensing inactivation, working with a P. aeruginosa MMR-deficient strain in vitro. In order to gain further insights in the role of the hypermutability in P. aeruginosa phenotypic diversification in vivo, 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 patients chronically infected with P. aeruginosa who are assisted in local Hospitals. According to SpeI macrorestriction fragment profiles we observed that most patients were infected by different P. aeruginosa clones as keeping with previous reports. We observed that 20 isolates (50%) were hypermutators and 14 patients (55.5%) had hypermutators. Afterward, loss-offunction mutations in the coding region of mucA, lasR and mexZ were scored by PCR amplification and direct nucleotide sequence analysis. Only one isolate (2,5%) showed unaltered sequences in the three genes, displaying at the same time a non mutator phenotype. Also, 75% of the isolates showed mutations in mexZ of which 40% were also hypermutators. In addition, we observed that 65% of isolates showed mutations in mucA, of which 46% showed a hypermutator phenotype. Interestingly, a lower percentage of isolates (35%) harboured 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 MMR components