Abstract
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing severe infections. P. aeruginosa infections are commonly treated with β-lactam antibiotics but with increasing development of β-lactam resistance, treatment is becoming increasingly problematic. P. aeruginosa can become resistant to β-lactams through a large variety of mechanisms. Notably, β-lactam resistance can occur through the acquisition of β-lactamases that hydrolyse β-lactams, via horizontal gene transfer. The ftsI gene encodes penicillin-binding protein 3 (PBP3) which is an important target of β-lactam antibiotics due to being the only essential PBP of P. aeruginosa. Mutations in ftsI are believed to contribute towards β-lactam resistance because of the high proportion of β-lactam resistant P. aeruginosa isolates harbouring PBP3 sequence variations. Currently, there is very limited research on which sequence variations of PBP3 contribute towards β-lactam resistance and which β-lactams are affected. The first aim of this research was to determine whether sequence variations of PBP3 contribute towards β-lactam resistance. Nineteen PBP3 sequence variations were expressed from a vector in the antibiotic-susceptible P. aeruginosa reference strain PAO1. Five PBP3 sequence variations were also engineered into P. aeruginosa PAO1 replacing the wild-type ftsI gene in the genome. Multiple PBP3 sequence variations were shown to significantly contribute towards β-lactam resistance in P. aeruginosa through minimum inhibitory concentration (MIC) testing of a range of β-lactams. The phenotypic changes resulting from having these PBP3 sequence variations were investigated. Having PBP3 sequence variations caused significantly elongated cell morphology along with a reduction in growth. The contribution of β-lactamases to resistance has not been well characterised in P. aeruginosa. The second aim was to characterize a range of β-lactamases that have not been well characterised in P. aeruginosa. Ten β-lactamases were cloned into P. aeruginosa PAO1 and four β-lactamase genes were deleted from the genome of clinical isolates. Each β lactamase reduced susceptibility to a variety of β-lactam antibiotics with different β lactamases affecting a different selection of β-lactam antibiotics. Importantly these results also showed which β-lactams particular β-lactamases did not cause resistance to. The deletion of β-lactamases from clinical isolates was able to increase the susceptibility to β-lactams to treatable levels. In conclusion, this research improves our current understanding of the genetic basis of β lactam resistance in P. aeruginosa.