The resistome and antibiotic susceptibility of Pseudomonas aeruginosa biofilms after antimicrobial photodynamic therapy.

Abstract

Pseudomonas aeruginosa, a gram negative bacteria, accounts for 7% of all hospital-acquired infections. Despite advances in medicine and antibiotic therapy, P. aeruginosa infection still results in high mortality rates of up to 62% in certain patient groups. This bacteria is also known to form biofilms, that could be 10 to 1000 times more resistant to antibiotics compared to their free-floating counterparts. Photodynamic Inactivation (PDI) has been proved to be an effective antimicrobial technique for microbial control. This method involves the incubation of the pathogen with a photosensitizer (PS), then, a light in a appropriated wavelength is applied, leading to the production of reactive oxygen species that are toxic to the microbial cells. PDI has demonstrated effective inactivation results against bacteria, fungi, and virus, using a range of PS. Besides that, an advantage of aPDT resides in its improbable capacity to cause resistance, since the aPDT action is target unspecific. Recently, a study demonstrated an innovative feature added to aPDT was its ability to modify the degree of bacterial susceptibility to antibiotics according to dosages, thus reducing resistance and persistence of microorganisms. However, the exact mechanism how the susceptibility is altered needs to be elucidated. One hypothesis is that aPDT downregulated the expression of resistant genes of the bacteria. Taking these aspects in consideration, the present work will evaluate the changes in the resistome of a resistant P. aeruginosa strain after different aPDT protocols. For this, biofilms will receive two diffent aPDT protocols (mediated by curcumin- Cur, or methylene blue - MB): one aPDT protocol will consist of 5 successive low efficacy sessions, reaching bacteria reductions around 0.5 - 1 log, at the end of the treatments. The other aPDT protocol will consist of one aPDT session with high efficacy, reaching reductions equivalent to 2 - 3 log. Then, the following tests will be performed: colony counting (plating), genomic DNA, analysis of the mutational resistome and MIC (susceptibility testing).