Background Pseudomonas aeruginosa with increased mutation rates due to defective DNA repair are reported in 24–73% of chronically infected patients with bronchiectasis, with or without cystic fibrosis (CF). Mutators may enhance P aeruginosa persistence in the lung by accumulating adaptive mutations including antibiotic resistance, and have been associated with worse lung function. In chronic infection the sputum can contain a heterogeneous population of P aeruginosa with different phenotypes, including variations in colony morphology (morphotype) and antibiotic susceptibility. Most studies on mutator prevalence however only tested one or two isolates per sputum. Investigation of the clinical relevance of mutator strains relies on accurately assessing the prevalence of hypermutators. We therefore performed a detailed investigation of mutators in sputum before and after antibiotic treatment for acute exacerbation.
Methods Six patients with CF, chronically infected with P aeruginosa, were tested before and after 2 weeks antibiotic treatment for exacerbation. Culture, antibiotic susceptibility testing and pulsed-field gel electrophoresis (PFGE) typing were performed using published methods.1 Mutation rates were measured for up to four colonies of each morphotype. As the original phenotypic method for detecting mutators is very laborious,2 we developed a method to screen pooled cultures using spiral plater quantitation.
Results 168 P aeruginosa isolates were tested (average 14 per sputum). Three patients had no mutators before or after antibiotics and three cultured mutators before and after antibiotics. One to four PFGE pulsotypes and two to six morphotypes were found in each sputum. Both mutators and non-mutators were present in a single sample with no association with morphotype or genotype. Mutators were more likely to be antibiotic resistant, but there was no obvious selection of mutators after 14 days antibiotics.
Conclusions With extensive testing, mutators were found in three of six patients with chronic P aeruginosa infection. The phenotypic and genotypic diversity observed means that multiple colonies of each morphotype should be tested to reliably detect mutators. Our method variation allows more extensive testing and may assist future clinical studies.
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