O sistema de reparo do DNA em Pseudomonas aeruginosa: caracterização molecular e ocorrência natural de mutantes

Abstract

Pulmonary infection is a major cause of morbidity and mortality in patients with Cystic Fibrosis (CF). The prognosis of these patients is influenced by the number of annual exacerbations of the infectious processes and the microbial load in their respiratory secretions. Pseudomonas aeruginosa is the main CF pathogen, both in importance and predominance. Recently, highly transmissible P. aeruginosa strains were reported among CF patients from different CF Health Care centers. The persistence of P. aeruginosa in the lung of CF patients is associated, at leat in part, with the presence of strains with hipermutability (HPM), a phenomenon caused mainly by defects in genes involved in the DNA repair system. In this report, 43 (24%) out of 179 P. aeruginosa isolates recovered from 17 (85%) out of 20 CF patients included in the study were characterized as HPM, according to the criteria established by Maciá and collegues. The HPM subpopulations exhibited higher rates of antimicrobial resistance. By using the RAPD technique, bacteria included in our study were shown to exhibit high genetic variability and could be grouped in 49 different clones. However, a few clones were shared by different patients. No similarity was detected among these clones and epidemic strains from others countries. Of the 43 isolates classified as HPM by Maciá´s criteria, only 3 exhibited mutation frequencies allowing their inclusion in the HPM group. By sequencing the DNA from these bacteria we found mutations in the genes mutS, mutD and urvD that resulted in changes in the aminoacids sequences. We did not observe mutations in the genes mutM, mutt and mutY that could have altered the aminoacid sequence from synthesized protein. Since there are only a few report in the literature on the influence of HPM on bacterial phenotypic alterations other than antimicrobial resistance, we also compared 81 bacterial isolates from the wild populations and HPM subpopulations in their virulence properties No difference among the microorganisms were detected considering their nutricional variations, mobility and ability of biofilm formation. In contrast, HPM subpopulations exhibited decreased LasA activity and increased production of pyocianyn. These results reinforced our hypothesis that bacterial mutants may have some advantage in the pulmonary environment of CF patients. Due to the difference in the results of the two sorts of test used to identify HPM, we propose the test of diffusion in modified agar as a trial test to be included in laboratories of clinical microbiology.