Caracterización del mutante nfxB de Pseudomonas aeruginosapapel en la resistencia antibiótica de los biofilms e interacción con los mecanismos de resistencia intrínsecos.

Abstract

Azithromycin has shown promising results in the treatment of Pseudomonas aeruginosa chronic respiratory infections during the last years. Although it does not show bactericidal activity against planktonic cells, this macrolide is known to inhibit biofilm formation due to its ability to block or interfere with the bacterial quorum sensing. During chronic infections, mutants showing high level of adaptation to the lung environment and antibiotics are often selected, such as mutants in the gene nfxB, which encodes the negative regulator of the efflux pump MexCD-OprJ. This phenotype is frequently isolated in the respiratory tract of Cystic Fibrosis patients, but also in COPD and bronchiectasis, and it is characterized by showing antimicrobial resistance to those antimicrobials that can act as a substrate (such as ciprofloxacin, cefepime, and azithromycin), and for presenting a marked hypersusceptibility to nonsubstrate beta-lactams and aminoglycosides. Therefore, one of the aims of this work was to assess if these nfxB mutants are selected during prolonged exposure to azithromycin in both planktonic and biofilm growth. Furthermore, we evaluated the effect of alginate overproduction and hypermutation on the bactericidal activity and the dynamics of development of resistance to azithromycin and the possible emergence of cross-resistance. In this work we have also characterized the nfxB mutant as well as the relationship between MexCD-OprJ overexpression (driven by nfxB inactivation), the activity of the chromosomal betalactamase AmpC and the major efflux pumps MexAB-OprM and MexXY-OprM. To carry out these objectives, strains PAO1, its mucA mutant (PAOMA), and their respective mutS-deficient hypermutable derivatives (PAOMS and PAOMSA) were constructed using the cre-lox system. The biofilms formed by these strains were treated with different azithromycin concentrations for several days and the dynamics of antibiotic resistance development was evaluated. Azithromycin resistant mutants were characterized, their susceptibility profiles were determined, their relative mexD expression was quantified by real-time PCR and finally its regulatory gene nfxB was sequenced, revealing several inactivating mutations. In summary, our results demonstrate that nfxB mutants are readily selected during azithromycin treatment of biofilms in wildtype strains, alginate hyperproducers and particularly in hypermutable strains. Then, the knockout nfxB mutant was constructed and its biological features characterized, the susceptibility to the complement of human serum, its rifampicin resistance mutation rate and finally its whole genome gene expression was analyzed, showing that apparently there are no other overexpressed genes involved in antimicrobial resistance except for those belonging to MexCD-OprJ operon. Although no differences in the penicillin binding protein (PBP) profile between the nfxB mutant and the reference strain were found, analysis of the outer membrane protein profile revealed a reduced expression of OprM, which is responsible for the hypersensitivity to aminoglycosides and beta-lactams, except imipenem. Likewise, a decrease in the OprM expression did not explain the reversion of the high beta-lactam resistance phenotype in the nfxB-dacB double mutant. However, beta-lactamase activity determination in different cell fractions (crude extract, periplasm, and supernatant) of nfxB and nfxB-dacB mutants revealed a dramatically decreased periplasmic beta-lactamase activity in contrast with an increased activity in supernatant. Then we assessed whether this difference was beneficial in a betalactam treated biofilm, showing that in this scenario the nfxB mutant yield higher survival rates than their respective parent strains in both induced and constitutively overexpressed AmpC conditions driven by inactivation of dacB. Therefore, our data show that hypersusceptibility to beta-lactams is due to a deficient AmpC accumulation in the periplasmic space, resulting in a leakage out of the cell. However a deeper analysis has allowed us to demonstrate how that leakage was protective in biofilm growth, where AmpC could be confined in the extracellular matrix. Subsequent nfxB inactivation in clinical isolates showed that this finding is not specific to PAO1. Finally, preliminary data suggest that the AmpC leakage observed in the nfxB mutant also occurs with other betalactamases, particularly in B and D classes.