Response to photo-oxidative stress of Pseudomonas aeruginosa PAO1 mutants impaired in different functions.

Clinicians often have to deal with infections that are difficult to control because they are caused by superbugs resistant to many antibiotics. Alternatives to antibiotic treatment include antimicrobial photodynamic therapy (aPDT). The photodynamic process causes bacterial death, inducing oxidative stress through the photoactivation of photosensitizer molecules in the presence of oxygen. No PDT-resistant bacteria have been selected to date, thus the response to photo-oxidative stress in non-phototrophic bacteria needs further investigation. The opportunistic pathogen Pseudomonas aeruginosa, in particular, has been shown to be more tolerant to PDT than other micro-organisms. In order to find any genetic determinants involved in PDT-tolerance, a panel of transposon mutants of P. aeruginosa PAO1 involved in the quorum sensing signalling system and membrane cytoplasmic transport were photoinactivated as part of this study. Two pseudomonas quinolone signalling (PQS) knock-out mutants, pqsH- and pqsC-, were as PDT-sensitive as the PAO1 wild-type strains. Two PQS hyperproducer variants, pqsA- and rsaL-, were shown to be more tolerant to photo-oxidative stress than the wild-type strain. In the pqsA- mutant, the hyperpigmentation due to the presence of phenazines could protect cells against PDT stress, while in rsaL- no pigmentation was detectable. Furthermore, a mutant impaired in an ATP-binding cassette transport involved in maintaining the asymmetry of the outer membrane was significantly more tolerant to photo-oxidative stress than the wild-type strain. These observations support the involvement of quorum sensing and the importance of the bacterial cell envelope when dealing with photo-oxidative stress induced by photodynamic treatment.

Photosensitized oxidation and inactivation of pyocyanin, a virulence factor of Pseudomonas aeruginosa.

Pyocyanin (PyO-) (1-hydroxy-5-methylphenazine) is a cytotoxic compound secreted by Pseudomonas aeruginosa, an omnipresent bacterium and a human pathogen. We report that visible light illumination in the presence of rose bengal, or riboflavin, in aerated solutions (pH 7.0-7.2) induces irreversible loss of the pigment's characteristic absorption band at 690 nm, indicating its oxidation. This photobleaching was paralleled by generation of a multiline Electron Paramagnetic Resonance (EPR) spectrum attributed to a PyO(-)-derived radical. The reaction was dependent on the presence of air, sensitizers and light, was inhibited by sodium azide and was unaffected by ethanol. This suggests that PyO- was oxidized largely via singlet oxygen and that hydroxyl radicals were not involved. The photochemically modified pigment was less efficient in oxidizing NAD(P)H and generated less superoxide (by approximately 50%) than the intact PyO-, indicating its partial inactivation. 1-Methoxy-5-methylphenazine, a PyO- analog in which the -O- moiety was replaced by the methoxy group (-OMe), was resistant to oxidation, suggesting that oxidation of PyO- involves its phenolate moiety. These results also suggest that photosensitization could be a potentially useful method for inactivation of PyO- and, possibly, detoxification of superficial wounds (skin, eye) infected with P. aeruginosa.

Light-mediated changes in pigmentation of Pseudomonas aeruginosa cultures.

Cultures of Pseudomonas aeruginosa PAO grown under uninterrupted broad-spectrum light showed different pigmentation from dark-grown cultures. Whereas dark-grown bacteria produced pigments which resulted in blue-purple coloured agar, light-grown organisms produced red coloured plates. Extraction and quantification of pigments showed that both dark- and light-grown cultures produced similar concentrations of pyorubrin (red) and pyoverdin (yellow). In contrast, the concentration of pyocyanin (blue) was substantially reduced under certain lighting conditions. This decrease was dependent on both the light intensity and wavelength and occurred with light in the ultraviolet and violet region of the spectrum. After its release from bacteria, pyocyanin was rapidly and nonreversibly photoinactivated with first-order kinetics to produce colourless photoproduct(s).