Joanne L. Fothergill

Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a childrens cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

Pseudomonas aeruginosa Population Diversity and Turnover in Cystic Fibrosis Chronic Infections

RATIONALE Pseudomonas aeruginosa isolates from chronic cystic fibrosis lung infections display multiple phenotypes indicating extensive population diversity. OBJECTIVES We aimed to examine how such diversity is distributed within and between patients, and to study the dynamics of single-strain phenotypic diversity in multiple patients through time. METHODS Sets of 40 P. aeruginosa isolates per sputum samples were analyzed for a series of phenotypic and genotypic characteristics. Population differentiation between patients, between samples within patients, and between isolates within samples was analyzed. MEASUREMENTS AND MAIN RESULTS We characterized 15 traits for a total of 1,720 isolates of an important and widely disseminated epidemic strain of P. aeruginosa from 10 chronically infected patients with cystic fibrosis multiply sampled during 2009. Overall, 43 sputum samples were analyzed and 398 haplotypes of the Liverpool Epidemic Strain were identified. The majority of phenotypic diversity occurred within patients. Such diversity is highly dynamic, displaying rapid turnover of haplotypes through time. P. aeruginosa populations within each individual sputum sample harbored extensive diversity. Although we observed major changes in the haplotype composition within patients between samples taken at intervals of several months, the compositions varied much less during exacerbation periods, despite the use of intravenous antibiotics. Our data also highlight a correlation between periods of pulmonary exacerbation and the overproduction of pyocyanin, a quorum sensing-controlled virulence factor. CONCLUSIONS These results significantly advance our understanding of the within-host population biology of P. aeruginosa during infection of patients with cystic fibrosis, and provide in vivo evidence for a link between pyocyanin production and patient morbidity.

The role of quorum sensing in chronic cystic fibrosis Pseudomonas aeruginosa infections

Studies on cultured cells and in infection models have shown that cell density-dependent quorum-sensing (QS) controls many of the known virulence factors of Pseudomonas aeruginosa. However, it is less clear what role QS plays in chronic human lung infections associated with cystic fibrosis (CF). The involvement of QS in biofilm development, crucial to the establishment of long-term infections, suggests a role in the early stages of infection. However, the accumulation of QS mutants during chronic CF infections has been taken to indicate that any role diminishes thereafter. Here, we discuss the evidence for a continuing role for QS in P. aeruginosa CF infections, including QS activity in CF sputa and CF-relevant effects of QS-regulated products, such as pyocyanin. Bacterial population behaviour in CF is complex, and the exact roles of QS remains unclear. Therapeutic strategies directed against QS suggest that a greater understanding of bacterial populations during infection would be a valuable research goal from a clinical perspective.

Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain

BackgroundSome isolates of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa exhibit an unusual virulence-related phenotype, characterized by over-production of quorum sensing-regulated exoproducts such as pyocyanin and LasA protease. Our aim was to determine the prevalence of this unusual phenotype amongst isolates of the epidemic strain, and to study other intraclonal phenotypic and genotypic variations.ResultsThe unusual phenotype was detected in at least one epidemic strain isolate from the majority of cystic fibrosis patients tested, and can be retained for up to seven years during chronic infection. Multiple sequential isolates of the epidemic strain taken from six patients over a period of up to nine years exhibited a wide range of phenotypes, including different antimicrobial susceptibilities. Our data suggest that each sputum sample contains a mixture of phenotypes and genotypes within the epidemic strain population, including within colony morphotypes. Many isolates exhibit premature (during early rather than late exponential growth) and over-production of pyocyanin, which has a number of toxic effects directly relevant to cystic fibrosis.ConclusionThe widespread occurrence of this unusual phenotype suggests that it may play an important role in the success of the epidemic strain.

Transmissible strains of Pseudomonas aeruginosa in cystic fibrosis lung infections

Pseudomonas aeruginosa chronic lung infections are the major cause of morbidity and mortality associated with cystic fibrosis. For many years, the consensus was that cystic fibrosis patients acquire P. aeruginosa from the environment, and hence harbour their own individual clones. However, in the past 15 yrs the emergence of transmissible strains, in some cases associated with greater morbidity and increased antimicrobial resistance, has changed the way that many clinics treat their patients. Here we provide a summary of reported transmissible strains in the UK, other parts of Europe, Australia and North America. In particular, we discuss the prevalence, epidemiology, unusual genotypic and phenotypic features, and virulence of the most intensively studied transmissible strain, the Liverpool epidemic strain. We also discuss the clinical impact of transmissible strains, in particular the diagnostic and infection control approaches adopted to counter their spread. Genomic analysis carried out so far has provided little evidence that transmissibility is due to shared genetic characteristics between different strains. Previous experiences with transmissible strains should help us to learn lessons for the future. In particular, there is a clear need for strain surveillance if emerging problem strains are to be detected before they are widely transmitted.

Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations

Chronic respiratory infection by Pseudomonas aeruginosa contributes significantly to the morbidity and mortality associated with cystic fibrosis (CF). Using a series of phenotypic and genotypic tests on collections of 40 isolates per sputum sample, we analysed fluctuations within sputum populations of the P. aeruginosa Liverpool epidemic strain (LES) during pulmonary exacerbations. For each of three patients, three sequential sputum samples were analysed: (1) on presentation with exacerbation at the Regional Adult Cystic Fibrosis Unit, Liverpool; (2) a few days into intravenous antibiotic treatment; (3) when the patient had recovered. Fluctuations were observed in morphotype distribution, the production of virulence-associated quorum-sensing-dependent exoproducts (the phenazine compound pyocyanin and the elastase LasA), antibiotic susceptibility profiles and levels of auxotrophy. PCR assays were used to screen isolates for the presence of novel regions of the LES genome (islands and prophages) and to detect free phages. In one patient there was an increase in the prevalence of the LESGI-5 genomic island during the sampling period from 10 to 97.5 % carriage. LES phages 2-4 were detected in either the majority or all sputum samples tested, indicating widespread phage activity during the sampling period. The results of this study are indicative that significant fluctuations occur within P. aeruginosa populations during short periods of pulmonary exacerbation and intravenous antibiotic therapy.

Characterization of N-Linked Protein Glycosylation in Helicobacter pullorum

The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the -2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.

Comparative genomics of isolates of a Pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients.

Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40–50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain’s adaptability to varying conditions in the CF lung.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Impact of Pseudomonas aeruginosa Genomic Instability on the Application of Typing Methods for Chronic Cystic Fibrosis Infections

ABSTRACT The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients in the United Kingdom and has emerged recently in North America. In this study, we report the analysis of 24 “anomalous” CF isolates of P. aeruginosa that produced inconsistent results with regard to either pulsed-field gel electrophoresis (PFGE) or PCR tests for the LES. We used a new typing method, the ArrayTube genotyping system, to determine that of the 24 anomalous isolates tested, 13 were confirmed as the LES. LES isolates could not be clearly distinguished from non-LES isolates by two other commonly used genetic fingerprinting tests, randomly amplified polymorphic DNA (RAPD) analysis and BOX-PCR, and varied considerably in their carriage of LES genomic islands and prophages. The genomic instability of the LES suggests that identification of this emerging transmissible strain could be a challenging task, and it questions whether discrimination is always a desirable feature of bacterial typing methods in the context of chronic CF infections.