Investigation into novel nitric oxide based anti-biofilm strategies to target Pseudomonas aeruginosa infection in Cystic Fibrosis

Pseudomonas aeruginosa infection in the cystic fibrosis (CF) lung poses an immense therapeutic burden due the formation and tolerance of biofilms. Conventional antibiotics are not effective in tackling P. aeruginosa biofilm growth leading to many patients developing chronic infection which negatively impacts on quality of life and prognosis. The recent discovery of nitric oxide (NO) as a P. aeruginosa biofilm dispersal molecule had enabled the prospect of novel NO-based anti-biofilm treatments to be developed. The main focus of this study was to further investigate the relationship between NO and P. aeruginosa, with emphasis specifically on biofilms formed by clinical CF isolates, and to conduct investigations into potential new NO-based antibiofilm strategies. By way of using a series of microbiological techniques including multiple phenotyping assays, biofilm culturing, molecular based methods, and mechanistic investigations, this study showed the response of biofilms formed by CF isolates of P. aeruginosa to NO is varied. In fact, opposing results were shown whereby isolates that demonstrated good in vitro biofilm growth were dispersed, and isolates that demonstrated poorer in vitro biofilm growth showed evidence of biofilm promotion with NO donor sodium nitroprusside (SNP). Interestingly, no obvious genetic adaptation to the NO signal was demonstrated following repeated pre-exposure to NO, and analysis of the c-di-GMP levels showed that levels were still reduced in an isolate that had biofilm formation promoted with NO, suggesting a more complex underlying cause and multiple factors to be in play. Two novel approaches to tackling P. aeruginosa biofilms were investigated in this study, one exploring the use of a group of NO-releasing prodrugs termed cephalasporin-3’-diazeniumdiolates (C3Ds), and a second looking at the use of a novel, portable NO-generating prototype device. With the latter, although we were able to demonstrate the prototype device was effective in producing gaseous NO, biofilm dispersal was not demonstrated when utilised in an in vitro system, and further studies are needed to determine if this device could be utilised as part of CF management. The C3D prodrugs were designed as a targeted pharmaceutical alternative to gaseous NO and to limit systemic exposure. Biochemical assays demonstrated the selectivity and targeted NO release from multiple C3D analogues in the presence of bacterial specific enzyme b-lactamase. Initial lead compound DEA-CP (Diethylamine NONOate Cephalosporin Prodrug) was shown to be effective in dispersing biofilms formed by clinical isolates of P. aeruginosa and was particularly effective in combination with colistin. Although effective as a dispersal and anti-biofilm agent, DEA-CP did not affect cell viability. Multiple newer-generation C3Ds were also investigated with the aim of developing an ‘all-in-one’ compound with both anti-biofilm and anti-bacterial activity. Results from antimicrobial susceptibility testing and detailed biofilm experiments using 3-dimensional microscopy, had identified one compound from the group of analogues to be particularly promising. Compound AMINOPIP2-ceftazidime was shown to be more effective than its parent antibiotic ceftazidime, both in terms of its anti-biofilm action, and anti-bacterial activity when tested against a panel of P. aeruginosa CF isolates, and therefore represents a potential new anti-biofilm therapy to be used in CF.