Abstract
Increasing antibiotic resistance is one of the major current and future threats to public health on a global level. By 2050, it is expected that 10 million people will die every year due to infections with antibiotic resistant bacteria that cannot be treated. Therefore increased understanding of resistance mechanisms is crucial to develop strategies and treatment plans for infections with antibiotic resistant bacteria. This study focused on Pseudomonas aeruginosa, one of the world’s most common hospital acquired pathogens with high intrinsic resistance to a wide range of antibiotics. Colistin is used as a ‘last line of defence’ antibiotic, which is why resistance presents a serious burden for public health.
The prevalence of colistin resistance in P. aeruginosa isolates of clinical, animal and environmental origin [n = 214] was determined based on minimal inhibitory concentration testing (MIC). Genome analysis of the resistant isolates [n = 17] revealed no mobile genetic elements carrying mcr colistin resistance genes. Further, a pan genome association approach did not identify any novel colistin resistance genes. The absence of colistin resistance genes indicated that resistance in the analyzed P. aeruginosa isolates was rather derived by mutation than mobile genetic elements. To identify resistance associated mutations, an experimental evolution study of P. aeruginosa PAO1 towards colistin [n=19] was performed, followed by genome analysis and identification of single nucleotide polymorphisms (SNPs). Mutations in genes previously associated with resistance, like pmrAB, speDE2, phoQ and PA5005, were identified in the evolved mutants. Additionally, mutations in the novel potentially resistance associated genes carB, eno and PA4292 were discovered. To quantify the effects of mutations in the novel resistance associated genes, the targeted mutations were engineered into the PAO1 genome. A loss of function mutation in the carB gene resulted in no change of colistin tolerance whereas loss of function mutation in the PA4292 gene resulted in increased colistin tolerance. Epistatic effects between known and novel mutations were identified in engineered double and triple mutants. No significant cross resistances, where mutations leading to colistin resistance simultaneously resulted in increased susceptibility towards other antibiotics, were observed.
The discovery of novel genes that have so far not been associated with colistin resistance and the gene-gene interactions provided improved understanding of colistin resistance in P. aeruginosa. An advanced understanding of colistin resistance will improve the discovery and development of treatment options for patients with infections of colistin resistant P. aeruginosa and thereby relieve the burden for public health.