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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.

SUBMITTER: Fothergill JL

PROVIDER: S-EPMC2884492 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Impact of Pseudomonas aeruginosa genomic instability on the application of typing methods for chronic cystic fibrosis infections.

Fothergill Joanne L JL White Judith J Foweraker Juliet E JE Walshaw Martin J MJ Ledson Martin J MJ Mahenthiralingam Eshwar E Winstanley Craig C

Journal of clinical microbiology 20100421 6

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 ...[more]

PMID: 20410349

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Project description:RationalePseudomonas 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.ObjectivesTo 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.MethodsWe 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 resultsGenome 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.ConclusionsOur 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:Epidemic strains of Pseudomonas aeruginosa are highly virulent opportunistic pathogens with increased transmissibility and enhanced antimicrobial resistance. Understanding the cellular mechanisms behind this heightened virulence and resistance is critical. Peptidoglycan (PG) is an integral component of P. aeruginosa cells that is essential to its survival and a target for antimicrobials. Here, we examined the global PG composition of two P. aeruginosa epidemic strains, LESB58 and LESlike1, and compared them to the common laboratory strains PAO1 and PA14. We also examined changes in PG composition when the strains were cultured under nutrient conditions that resembled cystic fibrosis lung infections. We identified 448 unique muropeptides and provide the first evidence for stem peptides modified with O-methylation, meso-diaminopimelic acid (mDAP) deamination, and novel substitutions of mDAP residues within P. aeruginosa PG. Our results also present the first evidence for both d,l- and l,d-endopeptidase activity on the PG sacculus of a Gram-negative organism. The PG composition of the epidemic strains varied significantly when grown under conditions resembling cystic fibrosis (CF) lung infections, showing increases in O-methylated stem peptides and decreases in l,d-endopeptidase activity as well as an increased abundance of de-N-acetylated sugars and l,d-transpeptidase activity, which are related to bacterial virulence and antibiotic resistance, respectively. We also identified strain-specific changes where LESlike1 increased the addition of unique amino acids to the terminus of the stem peptide and LESB58 increased amidase activity. Overall, this study demonstrates that P. aeruginosa PG composition is primarily influenced by nutrient conditions that mimic the CF lung; however, inherent strain-to-strain differences also exist. IMPORTANCE Using peptidoglycomics to examine the global composition of the peptidoglycan (PG) allows insights into the enzymatic activity that functions on this important biopolymer. Changes within the PG structure have implications for numerous physiological processes, including virulence and antimicrobial resistance. The identification of highly unique PG modifications illustrates the complexity of this biopolymer in Pseudomonas aeruginosa. Analyzing the PG composition of clinical P. aeruginosa epidemic strains provides insights into the increased virulence and antimicrobial resistance of these difficult-to-eradicate infections.

Dataset Information

Impact of Pseudomonas aeruginosa genomic instability on the application of typing methods for chronic cystic fibrosis infections.

Publications

Impact of Pseudomonas aeruginosa genomic instability on the application of typing methods for chronic cystic fibrosis infections.

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