Abstract
Background: The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that can cause a wide range of acute and chronic infections. It is a frequent cause of hospital acquired infections and chronically infects the lungs of most adults with the genetic disease cystic fibrosis (CF). P. aeruginosa has a large genome that allows for many intrinsic antibiotic resistance mechanisms, such as, a diverse array efflux pumps, low membrane permeability, and production of enzymes that inactivate or modify antibiotics. As well as resisting antibiotic treatment, during chronic infections P. aeruginosa rapidly adapts to other challenges, including oxidative stress, the immune system, host withholding of micronutrients such as iron and zinc, and competition from other microbiota.
Results: The overall aim of this research was to use in silico, in vitro, and in vivo approaches to understand how antibiotic resistance evolves in P. aeruginosa. Firstly, using a lab based experimental evolution approach, the genes that contribute most to resistance to two independent classes of antibiotic were identified. Resistance during experimental evolution increased in a stepwise manner, associated with the sequential acquisition of mutations, each evolved line obtained 4-8 resistance increasing mutations. Experimental evolution experiments also identified large genomic and phenotypic changes that can occur during resistance, including large deletions of up to 8% of the genome. Combining this experimental evolution with computational analyses showed genes that mutate to confer resistance within the lab-based screen are also found commonly mutated in clinical isolates of P. aeruginosa. Secondly, a comprehensive phenotypic, genotypic, and transcriptomic analysis of a chronic P. aeruginosa isolated from a chronically infected patient was performed. This revealed the extent of the transition that occurs within the lung of a patient with cystic fibrosis, including antibiotic resistance, changes in growth, increased biofilm production, and decreased virulence factor production.
Conclusions: This study significantly expands the understanding of the genetic basis of antibiotic resistance in P. aeruginosa through both lab- and within patient-evolution. Additionally, by studying the evolution of P. aeruginosa within a patient with CF we have been able to show the genetic and regulatory changes that allow P. aeruginosa to continue infections.