The high persister phenotype ofPseudomonas aeruginosais associated with increased fitness and persistence in cystic fibrosis airways

AbstractDespite intensive antibiotic treatment of cystic fibrosis (CF) patients,Pseudomonas aeruginosaoften persists in patient airways for decades, and can do so without the development of antibiotic resistance. Using a high-throughput screening assay of survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of high-persister variants (Hip) in a large patient cohort. In a screen of 467 longitudinal clinical isolates ofP. aeruginosafrom 40 CF patients, we classified 25.7% as Hip. Hip were identified in 26 patients, but only a few bacterial lineages were dominated by Hip. Instead, the emergence of Hip increased over time, suggesting that CF airways treated with ciprofloxacin select for Hip with an increased fitness in this environment. We generally observed diverse genetic changes in the Hip isolate population (as many co-occurring routes to increased fitness exist), but interestingly elevated mutation counts in the RpoN gene of 18 Hip isolates suggest that this sigma factor plays a role in shaping levels of antibiotic tolerance. To probe the impact of the Hip phenotype in a CF-similar environment, we tested the fitness properties of otherwise genotypically and phenotypically similar low-persister (Lop) and Hip isolates in co-culture using a specialized flow-cell biofilm system mimicking pharmacokinetic/-dynamic antibiotic dosing. Hip survived ciprofloxacin treatment far better than Lop isolates. The results of this investigation provide novel insights into persister dynamics and fitness contributions to survival in the CF lung, and show that the Hip phenotype of antibiotic susceptible bacteria plays an important role in long-term infections.SignificanceAntibiotic resistance is emphasized as a rapidly increasing health threat, but antibiotic tolerance via the occurrence of persister cells in antibiotic-treated bacterial populations is clinically and publicly neglected. In 40 CF patients representing a well-established human infection model – long-term lung infections byPseudomonas aeruginosa– we show the emergence and accumulation of persister variants in a clinical population heavily reliant on antibiotic therapy. We observe that the high-persister (Hip) phenotype is independent of resistance and likely the consequence of numerous genetic alterations, complicating surveillance and inhibition in the clinic. Furthermore, we find Hip are selected for over time, survive better than ‘normal’ bacteria, and can outcompete them in CF-similar conditions, ultimately affecting 65% of patients in an early disease cohort.