Project description:Endemic strains of Legionella pneumophila sequence type 1 (ST1), in particular the ST1/Paris pulsotype, are dispersed worldwide and represent about 10% of culture-proven clinical cases of Legionnaires' disease in France. The high rate of isolation of this strain from both clinical and environmental samples makes identification of the source of infection difficult during epidemiological investigations. The full-length genome sequence of this strain was recently determined, and it revealed the presence of a CRISPR/cas complex. The aim of this study was to develop and evaluate a spoligotyping tool based on the diversity of this CRISPR locus that would allow the accurate subtyping of the L. pneumophila serogroup 1 ST1/Paris pulsotype. The CRISPR loci of 28 L. pneumophila ST1/Paris pulsotype isolates were sequenced, and 42 different spacers regions were characterized. A membrane-based spoligotyping method was developed and used to determine the subtypes of 406 L. pneumophila isolates, including 233 with the ST1/Paris pulsotype profile that were collected in France from 2000 to 2011. A total of 46 different spoligotypes were detected, and 41 of these were specifically identified in the ST1/Paris pulsotype isolates. In 27 of 33 epidemiological investigations, the environmental source of contamination was confirmed by comparing spoligotypes of clinical isolates with those of environmental isolates. With an index of discrimination of 79.72% (95% confidence interval, 75.82 to 83.63), spoligotyping of the L. pneumophila ST1/Paris pulsotype has the potential to be a useful complementary genotyping tool for discriminating isolates with undistinguishable pulsed-field gel electrophoresis (PFGE) and ST genotypes, which could help to identify environmental sources of infection.
Project description:Genome sequencing of Legionella pneumophila subsp. pneumophila isolates from 2005 and 2008 Legionellosis outbreak in Fredrikstad, Norway
Project description:The causative agent of legionellosis, Legionella pneumophila, colonizes all natural and human-made water networks, thus constituting the source of contaminated aerosols responsible for airborne human infections. Efficient control of infections, especially during epidemics, necessitates the fastest and most resolutive identification possible of the bacterial source for subsequent disinfection of reservoirs. We thus compared recognized typing approaches for Legionella with a method based on characterization of insertion sequence (IS) content. A total of 86 clinical or environmental isolates of L. pneumophila, including 84 Paris isolates, sampled from 25 clinical investigations in France between 2001 and 2007, were obtained from the Legionella National Reference Center. All strains were typed by monoclonal antibody subgrouping, sequence-based typing, pulsed-field gel electrophoresis, and restriction fragment length polymorphism based on the presence or absence of IS elements. We identified six different types of IS elements in L. pneumophila Paris and used them as genomic markers in hybridization experiments. One IS type, ISLpn11, revealed a high discriminatory power. Simpson's index of discrimination, calculated from the distribution of IS elements, was higher than that obtained with the other typing methods used for L. pneumophila Paris. Moreover, specific ISLpn11 copies were found only in strains isolated from particular cities. In more than half of the cases, each clinical isolate had an ISLpn11 profile that was recovered in at least one environmental isolate from the same geographical location, suggesting that our method could identify the infection source. Phylogenetic analysis suggests a clonal expansion for the L. pneumophila Paris strain.
Project description:Comparaison of the transcriptional profile of mutant of the lpp1663 gene and the parent strain Legionella pneumophila strain Paris. lpp1663 codes for a protein with a ProQ domain (PFAM PF04352). A mutant of this gene was obtained by deleting the lpp1663 ORF and replacing it with a gene confering resistance to kanamycin. Transcriptional profiling was done on cultures grown to exponential phase (OD=0.8). Three independent biological replicates were analysed.
Project description:Comparaison of the transcriptional profile of mutant of the lpp0148 gene and the parent strain Legionella pneumophila strain Paris. lpp0148 codes for a protein with a ProQ domain (PFAM PF04352). A mutant of this gene was obtained by inserting a premature stop codon in the lpp0148 ORF. The lpp0148 mutant is constitutively competent for natural transformation as described here http://www.ncbi.nlm.nih.gov/pubmed/26526572. Transcriptional profiling was done on cultures grown to exponential phase (OD=0.8). Three independent biological replicates were analysed.