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To characterise Pseudomonas aeruginosa populations during chronic lung infections of non-cystic fibrosis bronchiectasis patients, we used whole-genome sequencing to 1) assess the diversity of  P. aeruginosa and the prevalence of multilineage infections; 2) seek evidence for cross-infection or common source acquisition; and 3) characterise P. aeruginosa adaptations.189 isolates, obtained from the sputa of 91 patients attending 16 adult bronchiectasis centres in the UK, were whole-genome sequenced.Bronchiectasis isolates were representative of the wider P. aeruginosa population. Of 24 patients from whom multiple isolates were examined, there were seven examples of multilineage infections, probably arising from multiple infection events. The number of nucleotide variants between genomes of isolates from different patients was in some cases similar to the variations observed between isolates from individual patients, implying the possible occurrence of cross-infection or common source acquisition.Our data indicate that during infections of bronchiectasis patients, P. aeruginosa populations adapt by accumulating loss-of-function mutations, leading to changes in phenotypes including different modes of iron acquisition and variations in biofilm-associated polysaccharides. The within-population diversification suggests that larger scale longitudinal surveillance studies will be required to capture cross-infection or common source acquisition events at an early stage., Competing Interests: Conflict of interest: None declared., (Copyright ©ERS 2017.)

The airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogen Pseudomonas aeruginosa. Several lines of evidence suggest that the infecting P. aeruginosa lineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain of P. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained a mutS mutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs of P. aeruginosa CF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates.

Pseudomonas aeruginosa is an important pathogen in cystic fibrosis patients, and a model organism for the study of nosocomially acquired infections, biofilms and intrinsic multidrug resistance. In this study we characterize ϕPA3, a new generalized transducing bacteriophage for P. aeruginosa. ϕPA3 transduced chromosomal mutations between PAO1 strains, and infected multiple P. aeruginosa clinical isolates as well as the P. aeruginosa model laboratory strains PAK and PA14. Electron microscopy imaging was used to classify ϕPA3 in the order Caudovirales and the family Myoviridae. The genome of ϕPA3 was sequenced and found to contain 309,208 bp, the second-largest bacteriophage currently deposited in GenBank. The genome contains 378 ORFs and five tRNAs. Many ORF products in the ϕPA3 genome are similar to proteins encoded by P. aeruginosa phage ϕKZ and Pseudomonas chlororaphis phage 201ϕ2-1, and so ϕPA3 was classified genetically as a member of the ϕKZ-like group of phages. This is the first report of a member of this group of phages acting as a generalized transducer. Given its wide host range, high transduction efficiency and large genome size, the 'jumbo' phage ϕPA3 could be a powerful tool in functional genomic analysis of diverse P. aeruginosa strains of fundamental and clinical importance.

Objectives: To investigate variability in colony morphology and antibiotic susceptibility in populations of Pseudomonas aeruginosa from sputa of patients with bronchiectasis without cystic fibrosis (CF) compared with P. aeruginosa isolated from patients with CF, and from other infections as controls., Methods: P. aeruginosa was cultured from 31 patients with non-CF bronchiectasis, 24 with CF, 7 ventilated patients and 9 skin swabs. Four colonies of each morphotype of P. aeruginosa were tested for susceptibility to 12 antibiotics by disc diffusion. The variability in susceptibility between the isolates in each patient's population of P. aeruginosa was investigated., Results: The classic morphotype of P. aeruginosa was cultured from control samples with an average variation in zone size of 2 mm (range 0-4 mm) for the four colonies tested. Non-CF bronchiectasis sputa contained 1-3 colonial morphotypes of P. aeruginosa; the average difference between the largest and smallest zone sizes found in all examples of the morphotypes present in each sample varied from 3 mm (1-9 mm) for colistin to 8 mm (0-24 mm) for piperacillin/tazobactam. CF sputa contained 2-6 morphotypes of P. aeruginosa with a wider variation of susceptibility. There was variation between bacteria of the same morphotype from non-CF bronchiectasis and CF sputa., Conclusions: Phenotypic variation in colonial form and antibiotic susceptibility is not unique to chronic infection in CF but is also found in non-CF bronchiectasis. This questions the use of current susceptibility testing methods for the complex populations of bacteria found in chronic lung infection.

Pseudomonas aeruginosa is the pathogen most commonly associated with morbidity and mortality in cystic fibrosis (CF) patients. The host-pathogen interactions responsible for progressive CF lung diseases are complex. However, there is growing interest in the role of hypermutable P. aeruginosa (that is, those strains with an increased mutation frequency due to mutations in mismatch repair and error prevention genes), in terms of both bacterial adaptation and antimicrobial resistance. The prevalence of hypermutable P. aeruginosa in chronic CF infection has been established, and at 37 % is surprisingly high. To the authors' knowledge, there are no reports of prevalence during the early stages of infection, in environmental pseudomonas, which are believed to be the primary source of infection, and in epidemic strains, which have emerged as a major challenge. The aim of this study was to establish the prevalence of hypermutable P. aeruginosa in these pseudomonas populations. The hypothesis was that hypermutability would be rare in early and in environmental P. aeruginosa but in contrast would explain the relatively recent emergence of epidemic strains. It was found that 10/100 (10 %) of early isolates were strong or weak mutators, suggesting that the CF lung is not the only factor influencing the existence of mutators in this group of patients. Two weak mutators (6 %) were found in 32 environmental isolates. Only two of 15 (13 %) epidemic P. aeruginosa strains were hypermutable, and although closer analysis revealed this issue to be complex, on the whole the data suggested that the atypical characteristics of these highly transmissible strains cannot solely be explained by this phenomenon. The higher than predicted prevalence of mutators in early infection, and in environmental isolates, reinforces the importance of early and aggressive treatment for P. aeruginosa infection in CF.

Objectives: To investigate the variability in antimicrobial susceptibility of Pseudomonas aeruginosa from sputa of patients with cystic fibrosis, to compare testing individual colonies of the same morphotype either separately or combined and to study the reproducibility of testing antimicrobial susceptibility within and between laboratories., Methods: One hundred and one sputa were cultured. Four colonies of each P. aeruginosa morphotype were suspended. Susceptibility to 12 agents by disc diffusion was tested individually or by pooling the four suspensions. A sputum sample containing four morphotypes of one genotype of P. aeruginosa was used to study reproducibility. Susceptibility was tested in duplicate by eight biomedical scientists in one laboratory and by routine procedures in seven different laboratories., Results: There was a mean of four morphotypes of P. aeruginosa per sputum and three antibiograms per morphotype. In some cases, all four colonies of a single morphotype had different antibiograms. The susceptibility profiles of single isolates of P. aeruginosa correlated poorly with pooled cultures, with the pooled tests missing resistance. Results from one sample tested in duplicate by eight biomedical scientists in one laboratory and in seven other laboratories did not correlate well. The wide range of zone sizes in disc diffusion tests illustrated the variation in susceptibility of 48 colonies from one sputum sample., Conclusions: The role of conventional antimicrobial susceptibility testing is questionable once P. aeruginosa chronically infects the cystic fibrosis lung. A range of susceptibility patterns is seen, even within a morphotype. Routine test results are not reproducible and underestimate resistance.