Craig Winstanley

Pseudomonas genomes: diverse and adaptable

Members of the genus Pseudomonas inhabit a wide variety of environments, which is reflected in their versatile metabolic capacity and broad potential for adaptation to fluctuating environmental conditions. Here, we examine and compare the genomes of a range of Pseudomonas spp. encompassing plant, insect and human pathogens, and environmental saprophytes. In addition to a large number of allelic differences of common genes that confer regulatory and metabolic flexibility, genome analysis suggests that many other factors contribute to the diversity and adaptability of Pseudomonas spp. Horizontal gene transfer has impacted the capability of pathogenic Pseudomonas spp. in terms of disease severity (Pseudomonas aeruginosa) and specificity (Pseudomonas syringae). Genome rearrangements likely contribute to adaptation, and a considerable complement of unique genes undoubtedly contributes to strain- and species-specific activities by as yet unknown mechanisms. Because of the lack of conserved phenotypic differences, the classification of the genus has long been contentious. DNA hybridization and genome-based analyses show close relationships among members of P. aeruginosa, but that isolates within the Pseudomonas fluorescens and P. syringae species are less closely related and may constitute different species. Collectively, genome sequences of Pseudomonas spp. have provided insights into pathogenesis and the genetic basis for diversity and adaptation.

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.

A Cystic Fibrosis Epidemic Strain of Pseudomonas aeruginosa Displays Enhanced Virulence and Antimicrobial Resistance

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is a transmissible aggressive pathogen of cystic fibrosis (CF) patients. We compared transcriptome profiles of two LES isolates with each other and with a laboratory and genetic reference strain (PAO1) after growth to late exponential phase and following exposure to oxidative stress. Both LES isolates exhibited enhanced antimicrobial resistances linked to specific mutations in efflux pump genes. Although transcription of AmpC beta-lactamase was up-regulated in both, one LES isolate contained a specific mutation rendering the ampC gene untranslatable. The virulence-related quorum-sensing (QS) regulon of LES431, an isolate that caused pneumonia in the non-CF parent of a CF patient, was considerably up-regulated in comparison to either isolate LES400, associated with a chronic CF infection, or strain PAO1. Premature activation of QS genes was detected in isolates from both non-CF parents and the CF patient in a previously reported infection episode. LES isolates lacking the up-regulated QS phenotype contained different frameshift mutations in lasR. When fed to Drosophila melanogaster, isolate LES431 killed the fruit flies more readily than either isolate LES400 or strain PAO1, indicating that virulence varies intraclonally. The LES may represent a clone with enhanced virulence and antimicrobial resistance characteristics that can vary or are lost due to mutations during long-term colonization but have contributed to the successful spread of the lineage throughout the CF population of the United Kingdom.

Increased morbidity associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients

Background: Chronic pulmonary infection with transmissible Pseudomonas aeruginosa strains in individuals with cystic fibrosis (CF) has been reported, raising issues of cross infection and patient segregation. The first such strain to be described (the Liverpool epidemic strain, LES) is now widespread in many UK CF centres. However, whether such infection carries a worse prognosis is unknown. To address this, the clinical course of a group of CF patients chronically infected by LES was compared with that in patients harbouring unique strains. Methods: Using P aeruginosa strain genotyping, two cohorts of CF patients attending the Liverpool CF service were identified who were LES positive or negative in 1998 and remained so until 2002. From these, two groups of 12 patients were matched in 1998 for age, spirometric parameters, and nutritional state and their clinical course was followed for 5 years. Patients chronically infected with Burkholderia cepacia were excluded. Results: Patients chronically infected with LES had a greater annual loss of lung function than those not chronically infected by LES (mean difference between groups −4.4% (95% CI −8.1 to −0.9; p<0.02)), and by 2002 their percentage predicted forced expiratory volume in 1 second (FEV1) was worse (mean 65.0% v 82.6%, p<0.03). Their nutritional state also deteriorated over the study period (mean difference between groups in body mass index −0.7 (95% CI −1.2 to −0.2; p<0.01)), such that by 2002 they were malnourished compared with LES negative patients (mean BMI 19.4 v 22.7, p<0.02). Conclusions: Chronic infection with the Liverpool epidemic P aeruginosa strain in CF patients confers a worse prognosis than infection with unique strains alone, confirming the need for patient segregation. Since this strain is common in many CF units, strain identification in all CF centres is essential. This can only be carried out using genomic typing methods.

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.

Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections

Pseudomonas aeruginosa populations undergo a characteristic evolutionary adaptation during chronic infection of the cystic fibrosis (CF) lung, including reduced production of virulence factors, transition to a biofilm-associated lifestyle, and evolution of high-level antibiotic resistance. Populations of P. aeruginosa in chronic CF lung infections typically exhibit high phenotypic diversity, including for clinically important traits such as antibiotic resistance and toxin production, and this diversity is dynamic over time, making accurate diagnosis and treatment challenging. Population genomics studies reveal extensive genetic diversity within patients, including for transmissible strains the coexistence of highly divergent lineages acquired by patient-to-patient transmission. The inherent spatial structure and spatial heterogeneity of selection in the CF lung appears to play a key role in driving P. aeruginosa diversification.

Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B. thailandensis and B. mallei

Burkholderia pseudomallei, the causative agent of melioidosis, carries a cluster of genes closely related in organisation to the type III secretion (TTS) system gene clusters of the plant pathogens Ralstonia solanacearum and Xanthomonas spp. The TTS gene cluster (TTS1) is present only in B. pseudomallei and not in avirulent B. thailandensis. Adjacent to the gene cluster encoding putative secreton structural proteins lie a number of open reading frames (ORFs) encoding putative proteins with little or no homology to known proteins, with the exception of one predicted protein with homology to Pseudomonas syringae HrpK. In both R. solanacearum and Xanthomonas spp., genes in this location encode secreted effector proteins. RT-PCR analysis indicated that TTS genes, including two of these ORFs, are expressed in broth at 37 degrees C. Analysis of genome sequence data identified a second cluster of TTS genes (TTS2) present in both B. pseudomallei and B. mallei (99% identity). However, B. mallei appears to lack the TTS1 gene cluster. PCR assays indicated that TTS2 was also present in B. thailandensis. TTS1 and TTS2 are similar in gene organisation, but nucleotide sequences are sufficiently divergent to suggest that the two TTS systems may have different roles.

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.