The causes and consequences of antibiotic resistance evolution in microbial pathogens

The evolution of antimicrobial resistance in bacterial pathogens is a growing global health problem that is gradually making the successful treatment of infectious diseases more difficult. Antimicrobial peptides have been proposed as promising candidates for future drug development as they retain activity against bacteria resistant to conventional antibiotics and because resistance evolution is expected to be unlikely since the peptides have complex modes of action due to their interaction with the bacterial membrane. The work presented in this thesis has involved studies to increase our understanding of the regulation of cationic antimicrobial peptide (CAMP) tolerance, the genetic basis for the evolution of resistance to the CAMP colistin and how this knowledge can provide insights into the features underlying the evolution of complex resistance mechanisms. The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) was used as a model organism in these studies for the following reasons: 1) colistin is used extensively in the treatment of P. aeruginosa infections in the airways of patients suffering from cystic fibrosis (CF); 2) P. aeruginosa establishes life-long infections in CF patients; and 3) most CF-associated P. aeruginosa infections are clonal, which means that evolution of antibiotic resistance in P. aeruginosa infections in individual patients probably occurs de novo. The first study presented in this thesis investigated the regulation of CAMP tolerance in P. aeruginosa and identified a novel gene (PA5003), which was required for P. aeruginosa to sense the presence of CAMPs in the environment. In addition, the study showed that recognition of CAMPs is required for the formation of CAMP tolerant subpopulations in P. aeruginosa biofilms. The two other studies in this thesis investigated the evolution of high-level colistin resistance in P. aeruginosa. The studies showed that the evolution of colistin resistance is a complex, multistep process that requires mutation in multiple independent loci. The evolutionary paths to resistance were severely constrained due to extensive epistatic interactions between the mutations, which indicate that the evolution of complex resistance mechanisms such as high-level colistin resistance may be predictable on a genomic scale. Furthermore, mutations in the regulatory systems encoded by phoPQ and pmrAB were essential for increased resistance and had the largest phenotypic impact in all mutation combinations and thus potentiated the evolution towards increased resistance, which highlight the importance of changes in regulation for the evolution complex mechanisms of adaptation. The highlevel resistance mutations also demonstrated antagonistic pleiotropy as they conferred a decreased growth rate in the absence of colistin and also rendered the colistin resistant strains susceptible towards all tested classes of β-lactams. The results suggest that colistin/β-lactam combination therapy could be used to reduce the risk of resistance evolution during antimicrobial chemotherapy. In addition, the observation that mutations in lpxC result in increased susceptibility of P. aeruginosa to β-lactams suggests that β-lactam/LpxC inhibitor combination therapy could be a novel treatment strategy in the combat against β-lactam resistant P. aeruginosa. In relation to this, it will be important to clarify whether lpxC mutations also confer increased susceptibility to β-lactams in other Gram-negative bacteria such as Escherichia coli and Acinetobacter baumannii.