Bacterial persisters in long-term infection: Emergence and fitness in a complex host environment

Despite intensive antibiotic treatment, Pseudomonas aeruginosa often persists in the airways of cystic fibrosis (CF) patients for decades, and can do so without antibiotic resistance development. Using high-throughput screening assays of bacterial survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of persisters in a large patient cohort using 460 longitudinal isolates of P. aeruginosa from 39 CF patients. Isolates were classed as high persister variants (Hip) if they regrew following antibiotic treatment in at least 75% of the experimental replicates. Strain genomic data, isolate phenotyping, and patient treatment records were integrated in a lineage-based analysis of persister formation and clinical impact. In total, 19% of the isolates were classified as Hip and Hip emergence increased over lineage colonization time within 22 Hip+ patients. Most Hip+ lineages produced multiple Hip isolates, but few Hip+ lineages were dominated by Hip. While we observed no strong signal of adaptive genetic convergence within Hip isolates, they generally emerged in parallel or following the development of ciprofloxacin resistance and slowed growth. Transient lineages were majority Hip-, while strains that persisted over a clinically diagnosed 'eradication' period were majority Hip+. Patients received indistinguishable treatment regimens before Hip emergence, but Hip+ patients overall were treated significantly more than Hip- patients, signaling repeated treatment failure. When subjected to in vivo-similar antibiotic dosing, a Hip isolate survived better than a non-Hip in a structured biofilm environment. In sum, the Hip phenotype appears to substantially contribute to long-term establishment of a lineage in the CF lung environment. Our results argue against the existence of a single dominant molecular mechanism underlying bacterial antibiotic persistence. We instead show that many routes, both phenotypic and genetic, are available for persister formation and consequent increases in strain fitness and treatment failure in CF airways. Author summary The persister phenotype, the ability of bacterial cells to survive antibiotic treatment without development of antibiotic resistance, is hypothesized to be an important contributor to treatment failure in recurrent bacterial infections. Using isolates of the bacterial pathogen Pseudomonas aeruginosa collected over a decade from the airways of 39 young cystic fibrosis patients, we investigated the emergence, continuity, and contribution to fitness of the persister phenotype in a clinical scenario with high levels of antibiotic treatment. We observe high-persister variants in 56% of the patients, but no signal of adaptive genetic convergence supporting their appearance, and no difference in patient treatment regimens before variant emergence. However, bacterial lineages (distinct bacterial strains infecting a patient over time) producing high-persister variants also produce isolates with antibiotic resistance and/or slowed growth rate. These lineages are also significantly less likely to be transient and more likely to persist in patient lungs over long periods of time without detection in the clinic. In sum, we conclude that the persister phenotype can emerge by many adaptive routes and offers important fitness contributions in the complex in vivo environment of cystic fibrosis airways.