Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa.
ABSTRACT: A multiresistant strain of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom. Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1. Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined. Several sequences were found only in the Liverpool epidemic strain. Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production. The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island. Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened. None of the sequences identified was present in the Manchester strain. However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain. All isolates implicated in epidemic spread and 76% of sporadic isolates contained the exoS gene. A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.
Project description:Populations of the Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa undergo extensive diversification in the cystic fibrosis (CF) lung during long-term chronic infections.We analyzed sets of 40 isolates from the sputa of five CF patients, each chronically infected with a different non-LES strain of P. aeruginosa. For each sample (two per patient), diversity was assessed by characterizing nine phenotypic traits.All P. aeruginosa populations were highly diverse, with the majority of phenotypic variation being due to within-sample diversity.Maintenance of diverse populations in the CF lung is a common feature of P. aeruginosa infections.
Project description: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.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen implicated in a myriad of infections and a leading pathogen responsible for mortality in patients with cystic fibrosis (CF). Horizontal transfers of genes among the microorganisms living within CF patients have led to highly virulent and multi-drug resistant strains such as the Liverpool epidemic strain of P. aeruginosa, namely the LESB58 strain that has the propensity to acquire virulence and antibiotic resistance genes. Often these genes are acquired in large clusters, referred to as "genomic islands (GIs)." To decipher GIs and understand their contributions to the evolution of virulence and antibiotic resistance in P. aeruginosa LESB58, we utilized a recursive segmentation and clustering procedure, presented here as a genome-mining tool, "GEMINI." GEMINI was validated on experimentally verified islands in the LESB58 strain before examining its potential to decipher novel islands. Of the 6062 genes in P. aeruginosa LESB58, 596 genes were identified to be resident on 20 GIs of which 12 have not been previously reported. Comparative genomics provided evidence in support of our novel predictions. Furthermore, GEMINI unraveled the mosaic structure of islands that are composed of segments of likely different evolutionary origins, and demonstrated its ability to identify potential strain biomarkers. These newly found islands likely have contributed to the hyper-virulence and multidrug resistance of the Liverpool epidemic strain of P. aeruginosa.
Project description:Pseudomonas aeruginosa is a predominant pathogen in chronic lung infections in individuals with cystic fibrosis (CF). Epidemic strains of P. aeruginosa, such as the Liverpool Epidemic Strain (LES), are capable of transferring between CF patients and have been associated with increased hospital visits and antibiotic treatments. Comparative genomics and phenotypic assays have shown that antibiotic resistance profiles differ among LES isolates and that genotype–phenotype associations are difficult to establish for resistance phenotypes in clinical isolates of P. aeruginosa based on these comparisons alone. We compared two LES isolates, LESlike1 and LESB58, and the common laboratory strain P. aeruginosa PAO1 using label-free quantitative proteomics to more accurately predict functional differences between strains. The proteomes of the LES isolates were found to be more similar to each other than to PAO1. However, we also observed a number of differences in the abundance of proteins involved in quorum sensing, virulence, and antibiotic resistance, including in the comparison of LESlike1 and LESB58. Specifically, the proteomic data revealed a higher abundance of proteins involved in polymyxin and aminoglycoside resistance in LESlike1. Minimum inhibitory concentration assays confirmed that LESlike1 has higher resistance to antibiotics from these classes. These findings provide an example of the ability of proteomic data to complement genotypic and phenotypic studies to understand resistance in clinical isolates.
Project description:RATIONALE:Pseudomonas aeruginosa, the predominant cause of chronic airway infections of patients with cystic fibrosis, exhibits extensive phenotypic diversity among isolates within and between sputum samples, but little is known about the underlying genetic diversity. OBJECTIVES:To characterize the population genetic structure of transmissible P. aeruginosa Liverpool Epidemic Strain in chronic infections of nine patients with cystic fibrosis, and infer evolutionary processes associated with adaptation to the cystic fibrosis lung. METHODS:We performed whole-genome sequencing of P. aeruginosa isolates and pooled populations and used comparative analyses of genome sequences including phylogenetic reconstructions and resolution of population structure from genome-wide allele frequencies. MEASUREMENTS AND MAIN RESULTS:Genome sequences were obtained for 360 isolates from nine patients. Phylogenetic reconstruction of the ancestry of 40 individually sequenced isolates from one patient sputum sample revealed the coexistence of two genetically diverged, recombining lineages exchanging potentially adaptive mutations. Analysis of population samples for eight additional patients indicated coexisting lineages in six cases. Reconstruction of the ancestry of individually sequenced isolates from all patients indicated smaller genetic distances between than within patients in most cases. CONCLUSIONS:Our population-level analysis demonstrates that coexistence of distinct lineages of P. aeruginosa Liverpool Epidemic Strain within individuals is common. In several cases, coexisting lineages may have been present in the infecting inoculum or assembled through multiple transmissions. Divergent lineages can share mutations via homologous recombination, potentially aiding adaptation to the airway during chronic infection. The genetic diversity of this transmissible strain within infections, revealed by high-resolution genomics, has implications for patient segregation and therapeutic strategies.
Project description: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.
Project description:The PANarray design (GPL13324) contains the genes of eight P. aeruginosa genomes in non-redundant format, thus allowing identification of expression of non-PAO1 and other P. aeruginosa genes. Overall design: For the series GSE28152, isogenic isolates were sequentially collected from two cystic fibrosis (CF) patients several years apart. The isolates had not been eradicated in the meantime and represent persister strains. One was an Australian Epidemic Strain-1 isolate and the other a non-epidemic strain. Strains were cultured in an artificial sputum medium (ASMDM) closely resembling CF sputum.
Project description:Pseudomonas aeruginosa is an opportunistic bacterial pathogen well known to cause chronic lung infections in individuals with cystic fibrosis (CF). Some strains adapted to this particular niche show distinct phenotypes, such as biofilm hyperproduction. It is necessary to study CF clinical P. aeruginosa isolates, such as Liverpool Epidemic Strains (LES), to acquire a better understanding of the key genes essential for in vivo maintenance and the major virulence mechanisms involved in CF lung infections. Previously, a library of 9216 mutants of the LESB58 strain were generated by signature-tagged mutagenesis (STM) and screened in the rat model of chronic lung infection, allowing the identification of 163 STM mutants showing defects in in vivo maintenance.In the present study, these 163 mutants were successively screened in two additional surrogate host models (the amoeba and the fruit fly). The STM PALES_11731 mutant was the unique non-virulent in the three hosts. A competitive index study in rat lungs confirmed that the mutant was 20-fold less virulent than the wild-type strain. This study demonstrated the pertinence to use a multi-host approach to study the genetic determinants of P. aeruginosa strains infecting CF patients.
Project description:The PANarray design (GPL13324) contains the genes of eight P. aeruginosa genomes in non-redundant format, thus allowing identification of expression of non-PAO1 and other P. aeruginosa genes. For the series GSE28152, isogenic isolates were sequentially collected from two cystic fibrosis (CF) patients several years apart. The isolates had not been eradicated in the meantime and represent persister strains. One was an Australian Epidemic Strain-1 isolate and the other a non-epidemic strain. Strains were cultured in an artificial sputum medium (ASMDM) closely resembling CF sputum.
Project description: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 children's 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.