The effects of large deletions on beta-lactam resistance in Pseudomonas aeruginosa

Abstract

Background: P. aeruginosa is an opportunistic pathogen that presents a significant threat to immunocompromised individuals, particularly to those with cystic fibrosis (CF). Chronic lung infection is a major cause of premature death in individuals with CF, and treatment is often difficult due to P. aeruginosa’s inherent and acquired antibiotic resistance mechanisms. This study focusses on seven experimentally evolved meropenem-resistant lines of P. aeruginosa, each consisting of between seven and twelve strains. The lines were generated by exposing the reference strain PAO1 to increasing concentrations of the β-lactam meropenem. Whole genome sequencing revealed large deletions in six of the lines (220 – 470 kbp) that shared an overlapping region of 210 genes. The seventh line had a 1986 bp deletion within the overlapping region, spanning the gene galU. These lines also had several additional β-lactam resistance mutations. Comparably sized deletions have been also observed in clinical isolates of P. aeruginosa. Objectives: The overall aim of this study was to characterise the contribution of large deletions to β-lactam resistance in P. aeruginosa. The first objective was to quantify the effects of the deletions on β-lactam resistance in the experimentally evolved lines. The second objective was to engineer mutant strains based on the reference strain PAO1 with large deletions and no other mutations, and to compare the β-lactam resistance of these strains to the experimentally evolved strains with the same deletions. Methods: PCR was used to identify the pre-deletion and post-deletion strains in each experimentally evolved line. β-lactam resistance of all strains was determined using the agar dilution method. An allelic exchange method, with additional selectable markers, was used to generate marked deletion mutants. Results: In each of the experimentally evolved lines, the strains with deletions were between 2- and 8-fold more resistant to meropenem, Timentin and ceftazidime than the corresponding strains with no deletion. Three mutant strains were engineered from P. aeruginosa PAO1 with deletions and no other mutations. The engineered deletion mutants with the 1986 bp deletion were 4-fold more resistant to Timentin and ceftazidime compared with PAO1, and as ceftazidime resistant as the experimentally evolved strain with the 1986bp deletion. However, they were no more meropenem resistant than PAO1. The engineered deletion mutant with a 225 kbp deletion was 4-fold more resistant to meropenem, Timentin and ceftazidime compared with PAO1, however it was less resistant than the experimentally evolved strain with the same deletion. Conclusions: The higher resistance of the experimentally evolved post-deletion strains shows that large deletions can confer high levels of β-lactam resistance in combination with other β-lactam resistance mutations. The engineered deletion mutants were in most cases more β-lactam resistant than PAO1 but as not resistant as the experimentally evolved strains with the same deletions and point mutations. The findings from the deletion mutants further reinforce the importance of each strains genetic background and its interaction with large deletions. The construction of more deletion mutants will further show the impact of large deletions on β-lactam resistance.