Transmissible strains of Pseudomonas aeruginosa have been described for cystic fibrosis (CF) and may be associated with a worse prognosis. Using a comprehensive strain biobank spanning 3 decades, we sought to determine the prevalence and stability of chronic P. aeruginosa infection in an adult population. P. aeruginosa isolates from sputum samples collected at initial enrollment in our adult clinic and at the most recent clinic visit were examined by a combination of pulsed-field gel electrophoresis and multilocus sequence typing and compared against a collection of established transmissible and local non-CF bronchiectasis (nCFB) isolates. A total of 372 isolates from 107 patients, spanning 674 patient-years, including 66 patients with matched isolates from initial and final encounters, were screened. A novel clone with increased antibacterial resistance, termed the prairie epidemic strain (PES), was found in 29% (31/107 patients) of chronically infected patients referred from multiple prairie-based CF centers. This isolate was not found in those diagnosed with CF as adults or in a control population with nCFB. While 90% (60/66 patients) of patients had stable infection over a mean of 10.8 years, five patients experienced strain displacement of unique isolates, with PES occurring within 2 years of transitioning to adult care. PES has been present in our cohort since at least 1987, is unique to CF, generally establishes chronic infection during childhood, and has been found in patients at the time of transition of patients from multiple prairie-based CF clinics, suggesting broad endemicity. Studies are under way to evaluate the clinical implications of PES infection.

Epidemic strains of Pseudomonas aeruginosa have been found worldwide among the cystic fibrosis (CF) patient population. Using pulse-field gel electrophoresis, the Prairie Epidemic Strain (PES) has recently been found in one-third of patients attending the Calgary Adult CF Clinic in Canada. Using multi-locus sequence typing, PES isolates from unrelated patients were found to consistently have ST192. Though most patients acquired PES prior to enrolling in the clinic, some patients were observed to experience strain replacement upon transitioning to the clinic whereby local non-epidemic P. aeruginosa isolates were displaced by PES. Here we genotypically and phenotypically compared PES to other P. aeruginosa epidemic strains (OES) found around the world as well as local non-epidemic CF P. aeruginosa isolates in order to characterize PES. Since some epidemic strains are associated with worse clinical outcomes, we assessed the pathogenic potential of PES to determine if these isolates are virulent, shared properties with OES, and if its phenotypic properties may offer a competitive advantage in displacing local non-epidemic isolates during strain replacement. As such, we conducted a comparative analysis using fourteen phenotypic traits, including virulence factor production, biofilm formation, planktonic growth, mucoidy, and antibiotic susceptibility to characterize PES, OES, and local non-epidemic isolates. We observed that PES and OES could be differentiated from local non-epidemic isolates based on biofilm growth with PES isolates being more mucoid. Pairwise comparisons indicated that PES produced significantly higher levels of proteases and formed better biofilms than OES but were more susceptible to antibiotic treatment. Amongst five patients experiencing strain replacement, we found that super-infecting PES produced lower levels of proteases and elastases but were more resistant to antibiotics compared to the displaced non-epidemic isolates. This comparative analysis is the first to be completed on a large scale between groups of epidemic and non-epidemic CF P. aeruginosa isolates.

The natural history and epidemiology of Pseudomonas aeruginosa infections in non-cystic fibrosis (non-CF) bronchiectasis is not well understood.As such it was our intention to determine the evolution of airway infection and the transmission potential of P. aeruginosa in patients with non-CF bronchiectasis.A longitudinal cohort study was conducted from 1986–2011 using a biobank of prospectively collected isolates from patients with non-CF bronchiectasis. Patients included were ≥18 years old and had ≥2 positive P. aeruginosa cultures over a minimum 6-month period. All isolates obtained at first and most recent clinical encounters, as well as during exacerbations, that were morphologically distinct on MacConkey agar were genotyped by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 203 isolates from 39 patients were analysed. These were compared to a large collection of globally epidemic and local CF strains, as well as non-CF isolates.We identified four patterns of infection in non-CF bronchiectasis including: 1) persistence of a single strain (n=26; 67%); 2) strain displacement (n=8; 20%); 3) temporary disruption (n=3; 8%); and 4) chaotic airway infection (n=2; 5%). Patterns of infection were not significant predictors of rates of lung function decline or progression to end-stage disease and acquisition of new strains did not associate with the occurrence of exacerbations. Rarely, non-CF bronchiectasis strains with similar pulsotypes were observed in CF and non-CF controls, but no CF epidemic strains were observed. While rare shared strains were observed in non-CF bronchiectasis, whole-genome sequencing refuted patient–patient transmission.We observed a higher incidence of strain-displacement in our patient cohort compared to those observed in CF studies, although this did not impact on outcomes.

Pseudomonas aeruginosa is a major pathogen in chronic lung diseases such as cystic fibrosis (CF) and non-cystic fibrosis bronchiectasis (nCFB). Much of our understanding regarding infections in nCFB patients is extrapolated from findings in CF with little direct investigation on the adaptation of P. aeruginosa in nCFB patients. As such, we investigated whether the adaptation of P. aeruginosa was indeed similar between nCFB and CF. From our prospectively collected biobank, we identified 40 nCFB patients who had repeated P. aeruginosa isolates separated by !6 months and compared these to a control population of 28 CF patients. A total of 84 nCFB isolates [40 early (defined as the earliest isolate in the biobank) and 41 late (defined as the last available isolate in the biobank)] were compared to 83 CF isolates (39 early and 44 late). We assessed the isolates for protease, lipase and elastase production; mucoid phenotype; swarm and swim motility; biofilm production; and the presence of the lasR mutant phenotype. Overall, we observed phenotypic heterogeneity in both nCFB and CF isolates and found that P. aeruginosa adapted to the nCFB lung environment similarly to the way observed in CF isolates in terms of protease and elastase expression, motility and biofilm formation. However, significant differences between nCFB and CF isolates were observed in lipase expression, which may allude to distinct characteristics found in the lung environment of nCFB patients. We also sought to determine virulence potential over time in nCFB P. aeruginosa isolates and found that virulence decreased over time, similar to CF. INTRODUCTIONBronchiectasis is a pathologic diagnosis defined by permanent dilation and widening of the respiratory airways and is common in chronic suppurative lung diseases like cystic fibrosis (CF) and non-cystic fibrosis bronchiectasis (nCFB) (Weycker et al., 2005). Patients with bronchiectasis may present with common symptoms including sputum production, recurrent respiratory infections and airway obstruction manifesting in thickened bronchial walls, establishment of chronic infections and increased levels of inflammatory markers (Mhanna et al., 2001;Seitz et al., 2010;Bergin et al., 2013;Gupta et al., 2015).Bronchiectasis arises as a consequence of complications induced by mutations in the CF transmembrane conductance regulator CFTR. However, multiple other non-CFTR mechanisms exist that culminate in bronchiectasis, termed nCFB. Typically considered an 'orphan disease', the incidence of nCFB has risen by 8.7 % annually between (Barker & Bardana, 1988Seitz et al., 2012) and is estimated to cost over $630 million annually in the US healthcare system. Causes of bronchiectasis include immune dysregulation (including autoimmune disorders), obstruction of the airways and complications from infections or injuries (Al-Shirawi et al., 2006;McShane et al., 2012).In patients with CF and nCFB, accumulation of thick mucus in the lungs and impaired mucociliary clearance allow respiratory infections (Martens et al., 2011). ...

The virulence profiles of Pseudomonas aeruginosa quorum-sensing (QS) mutants were assessed in Drosophila melanogaster feeding and nicking infection models. Functional RhlIR and LasIR QS systems were required for killing in the fly feeding infection model but were not essential in the fly nicking infection model. Mixed infections between PAO1 and strains harbouring mutations in lasR, rhlI and lasI rhlI resulted in increased lethality in the fly feeding model compared with either isolate alone. These results suggested that the parental strain could cooperate with QS mutants in the Drosophila feeding infection model. Finally, the mixed infection between PAO1 and an rhlR mutant resulted in spiteful behaviour and reduced pathogenicity of the mixed culture. INTRODUCTIONQuorum sensing (QS) regulates the cell density-dependent expression of target genes that modulate many bacterial phenotypes, and allows bacteria to behave as multicellular organisms, reaping benefits unattainable by individual bacteria. The most common QS systems in Gram-negative bacteria are composed of transcriptional regulators encoded by luxR homologues and autoinducer biosynthetic enzymes encoded by luxI homologues (de Kievit & Iglewski, 2000;Salmond et al., 1995). Pseudomonas aeruginosa contains two such QS systems, the lasIR and rhlIR regulatory systems (Pearson et al., 1995). These systems are interdependent, interact with the Pseudomonas quinolone signal (PQS), and control a wide range of genes including those for virulence factors, sigma factors and other regulators (Albus et al., 1997;Brint & Ohman, 1995;Erickson et al., 2004;McKnight et al., 2000;Ochsner et al., 1995;Toder et al., 1991;Whiteley et al.,1999). The LasIR system consists of LasI, which synthesizes N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL), and the transcriptional regulator LasR (Pearson et al., 1994). The RhlIR system consists of the transcriptional regulator RhlR and the cognate homoserine lactone synthase RhlI, which synthesizes N-butanoyl-L-homoserine lactone (C 4 -HSL) (Winson et al., 1995).QS is known to be required for P. aeruginosa virulence in many organisms that serve as infection models, suggesting that this is an integral component of bacterial pathogenesis (Lesprit et al., 2003;Pearson et al., 2000;Rumbaugh et al., 1999b; Stoltz et al., 2008;Wu et al., 2001;Zhu et al., 2004). Detection of QS system gene targets and their cognate signalling molecules suggests that these systems are active during human infection, e.g. the chronic lung infections associated with cystic fibrosis (CF) (Erickson et al., 2002). However, the role that QS plays in chronic human infections is unclear, as mutants in QS genes frequently occur in clinical isolates (Schaber et al., 2004;Smith et al., 2006). In particular, lasR mutants frequently arise in CF infections (Smith et al., 2006), and in some model infections can be maintained alongside the parental strains (D'Argenio et al., 2007; Kohler et al., 2010;Smith et al., 2006). It has been speculated that QS systems can have a...

The monitoring of epidemic Pseudomonas aeruginosa is important for cystic fibrosis (CF) infection control. The prairie epidemic strain (PES) is common in western Canadian CF clinics. Using whole-genome sequencing, we identified a novel genomic island and developed a PCR assay for PES. Against a collection of 186 P. aeruginosa isolates, the assay had 98% sensitivity and 100% specificity. Multiple epidemic strains of Pseudomonas aeruginosa have been described as infecting patients with cystic fibrosis (CF) (1-4). Many of these strains are associated with a poor prognosis, manifesting as increased rates of lung function decline, exacerbation frequency, and treatment burden; decreased quality of life; and hastened progression to end-stage lung diseases (5, 6). We recently described a novel epidemic P. aeruginosa strain, the prairie epidemic strain (PES) (multilocus sequence type 192) (7). PES was found in one-quarter of patients attending the Calgary Adult CF Clinic (CACFC) and from patients who were transferred from other Prairie-based clinics, suggesting broad endemicity in western Canada. Furthermore, its prevalence has been unchanged through 3 decades of patients who transitioned to the CACFC. PES is more likely to be antibiotic resistant than are unique strains of P. aeruginosa, and infection with PES has been associated with increased rates of lung function decline, pulmonary exacerbation frequency, and progression to death and/or transplantation (7,8).The importance of identifying epidemic P. aeruginosa strains in CF has long been recognized. Typing strategies that are commonly currently employed include pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and random amplified polymorphic DNA PCR (RAPD-PCR) (7, 9, 10). These modalities have, to varied degrees, the ability to distinguish bacterial strains from one another, identify known epidemic strains, and determine if novel strains are potentially shared. However, they are costly and laborious, precluding routine use in clinical laboratories. Accordingly, we sought to develop a simple PCR assay to distinguish PES from non-PES P. aeruginosa organisms for clinical and research purposes in areas where this strain is prevalent (western Canada). Similar assays have been developed for other epidemic P. aeruginosa strains, including Liverpool epidemic strain (LES), Midlands-1, Manchester, and Australia epidemic strain-1 (AUS-1), and they are used prospectively in infection control and research (1-4).We performed whole-genome sequencing on 8 nonsequential isolates of PFGE and MLST-confirmed PES from our prospectively maintained biobank. The isolates were sequenced on the Illumina HiSeq 2500 (150 bp, paired-end) and assembled using SPAdes 2.5.0 (11). These strains were sequenced as a part of a large sequencing study, and a more detailed description will be given in a forthcoming manuscript (M. G. Surette, unpublished data). We identified a novel 40-kb genomic island (GI) using IslandViewer (12) and GView (13) that was not present in other pub...

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