Abstract
The opportunistic and ubiquitous pathogen Pseudomonas aeruginosa is commonly found in the environment and hospital settings. P. aeruginosa LESB58 is a hypervirulent and multi-drug resistant strain, frequently causing severe respiratory tract infections in patients with cystic fibrosis and is especially problematic in children. Virulence factors of P. aeruginosa include production of the siderophore pyoverdine, which sequesters iron from the host, and the aggregation of bacterial cells in biofilms, adhering to infection sites and medical equipment. A nutrient required in bacterial virulence mechanisms, antibiotic resistance and essential for cellular processes is potassium. Maintenance of intracellular potassium levels occurs by transporters such as Kdp, which is only active under low environmental potassium concentrations. Last-resort treatments against complex infections caused by the multi-drug resistant P. aeruginosa, include the polymyxin and cephalosporin classes of antibiotics. Polymyxins target and disrupt the bacterial cell membrane while cephalosporins inhibit cell- wall synthesising enzymes. The aminoarabinosylation system synthesizes an aminoarabinose sugar which neutralises the net negative charge of lipid A within the outer membrane of P. aeruginosa and provides resistance to positively charged polymyxins. It has been suggested that blocking this system may increase the lifespan of polymyxin antibiotics to treat P. aeruginosa infections. In my research project I generated P. aeruginosa LESB58 mutants lacking the operons arnBCADTEF-ugd (9,380-bp) and kdpFABCDE (8,345-bp) encoding for the aminoarabinosylation and Kdp potassium transport systems, respectively. Antibiotic susceptibility assays measured the minimum concentration required to inhibit bacterial growth. Lack of the aminoarabinosylation system increased resistance to the cephalosporin antibiotics ceftazidime (by 32-fold, from 8 μg/mL to 250 μg/mL), cefepime and cefotaxime (both 8-fold, 16 μg/mL to 125 μg/mL and 63 μg/mL to >500 μg/mL, respectively) but did not significantly affect susceptibility to polymyxins. The aminoarabinosylation mutant also demonstrated increased production of virulence factors pyoverdine (~1.9-fold) and the formation of biofilms (by 32%). The Kdp potassium transporter was necessary for growth in potassium-free medium, but not survival in this medium, as growth was rescued by supplementation of potassium. My results provide evidence against the aminoarabinosylation and Kdp potassium transport systems being potential future therapeutic targets in P. aeruginosa LESB58, due to the induction of cross-resistance and increased virulence, and persistence of the pathogen under nutrient limiting conditions, respectively.