Genetic Variability of P. aeruginosa Multirestistant Strains Isolated from Patiens with Acute Infections

Increasing and spreading of antibiotic resistance are the result of natural selection as a manifestation of the Darwin principle of fitness. One of the major P. aeruginosa characteristic is its remarkable ability to acquire antibiotics resistance through several ways, from spontaneous mutation to the acquisition of resistance genes by lateral gene transfer mechanisms, to the generalized increase of the spontaneous mutation rate (until three orders of magnitude) during chronic infections. This hypermutability is correlated with the presence of mutations in mismatches repair genes (MMR) facilitating the selection of mutator strains. In order to determine whether the hypermutability state, prevalently observed only in strain from cystic fibrosis patients, occurs also in strains isolated from patients with acute infections, we analyzed the mutS gene sequence from P. aeruginosa multiresistant bacterial samples derived from patients of Rome Hospitals. These mutS genes were amplified, sequenced and compared with the wild type sequence. This analysis showed the occurrence of missense mutations affecting prevalently three of the five protein domains. In particular, the effects of the T781A mutation, found in various samples, were carefully analyzed to correlate its presence with antibiotic multiresistance of the bacterial strain. Due to its localization in the HTH motif of domain V, necessary for protein homodimerization after the recognition of the DNA mismatch, for the recruitment of the MMR proteins, the first step was to determine the dimerization ability of the mutated protein by the prokaryotic two hybrid assay. Then its ability to in vivo localize on DNA mismatches was analyzed by fluorescence microscopy inducing the expression of the GFP-MutS fusion protein. Lastly, the mutation frequency induced by this mutated MutS protein was determined. Our results suggest that P. aeruginosa multiresistant bacterial strains found in patients with acute infections could derive from the spread of ipermutated strains originated in chronic patients. Keywords: antibiotic multiresistance, hypermutability state, MMR system, genetic variability, P. aeruginosa