Antimicrobial and antibiofilm activity of drug combinations and violet-blue light against clinically relevant bacterial pathogens causing airway infections

Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Staphylococcus aureus lung colonization is critical in cystic fibrosis (CF) and other chronic lung diseases, contributing to disease progression. Biofilm growth and a propensity to evolve multidrug resistance phenotypes drastically limit the available therapeutic options. In this perspective, there has been growing interest in evaluating both combination therapies, especially for drugs administrable by nebulization allowing to achieve high lung concentrations while reducing systemic toxicity, and non-antibiotic therapies based on the using of visible light capable of generating reactive oxygen species leading to bacterial killing. In this doctoral thesis, the potential synergism of N-acetylcysteine (NAC) (a mucolytic agent with antioxidant and anti-inflammatory properties) in combination with colistin (among the last-resort agents for the treatment of infections caused by multidrug resistant Gram-negative bacteria) against S. maltophilia grown in planktonic and biofilm phase, and P. aeruginosa biofilms was investigated. The transcriptomic response of a P. aeruginosa CF strain to NAC exposure was also studied. A wide collection of S. maltophilia and P. aeruginosa clinical isolates (comprising strains isolated from CF patients and colistin-resistant strains) was included in the study. On the other hand, the potential in vitro activity of violet-blue light (415 nm wavelength) against planktonic and biofilm cultures of P. aeruginosa and S. aureus strains (including strains isolated from CF patients) was investigated. The potentiation of the antimicrobial activity of light at 415 nm in the presence of potassium iodide (KI) against planktonic cultures was also evaluated. The latter investigations were part of the follow-up activities of the European project Light4Lungs aimed at develops a novel antimicrobial therapy for the treatment of chronic lung infections using inhalable light sources. Checkerboard assays carried out with S. maltophilia strains showed a synergism of NAC-colistin combinations against the strains exhibiting colistin Minimum Inhibitory Concentration (MIC) >2 mg/L (n=13), suggesting that NAC could antagonize the mechanisms involved in colistin resistance. Nonetheless, time-kill assays revealed that NAC might potentiate colistin activity also in case of lower colistin MICs. A dose-dependent potentiation of colistin activity by NAC was clearly observed against S. maltophilia biofilms, also at sub-MIC concentrations. Biofilm susceptibility testing performed against P. aeruginosa showed a limited and strain-dependent antibiofilm activity of NAC alone (8,000 mg/L). However, a relevant antibiofilm synergism of NAC-colistin combinations was observed with the majority of the P. aeruginosa strains tested. Synergism was also confirmed with the artificial sputum medium model. RNA sequencing of NAC-exposed planktonic cultures revealed that NAC (8,000 mg/L) mainly induced (i) a Zn2+ starvation response (known to induce attenuation of P. aeruginosa virulence), (ii) downregulation of genes of the denitrification apparatus, and (iii) downregulation of flagellar biosynthesis pathway. NAC-mediated inhibition of P. aeruginosa denitrification pathway and flagellum-mediated motility were confirmed experimentally. A potential antimicrobial activity of the light at 415 nm against all the tested strains (n=4) was observed. A dose-dependent effect was detected against P. aeruginosa strains grown in planktonic phase, while only a scant or no effect was observed against S. aureus cultures. Nevertheless, the addition of KI to the planktonic cultures potentiated the photokilling activity of 415 nm LED light leading to eradication of starting inocula in three out of four cases, confirming the involvement of KI in the bacterial cell death. An antibiofilm activity of the light at 415 nm was observed against both P. aeruginosa strains and S. aureus strains, with the major effects evident against clinical isolates compared to the reference strains, underlining the differences of response to oxidative stress between diverse physiological states of growth. NAC-colistin combinations, at concentrations likely achievable by topical administration, might represent a valid option for the treatment of infections caused by biofilm-associated pathogens, such as S. maltophilia and P. aeruginosa, while potentially reducing the risk of in vivo selection of colistin resistance. NAC might also have a role in reducing P. aeruginosa virulence, which could be relevant in the very early stages of lung colonization. The antibiofilm activity of violet-blue light would deserve further investigation to consolidate the obtained data for the potential clinical application of this approach, especially in biofilm-associated chronic infections. The potentiation of photokilling activity of antimicrobial light mediated by KI should be further examined.