PCR-based ‘serotyping’ of Free

Abstract

Currently, several PCR assays based on 16S rRNA and virulence-associated genes are available for detection of . So far, no genotyping method has been published that can discriminate between serogroups and monoclonal subgroups of the most common serogroup 1. Our first approach was to analyse LPS-associated genes of seven serogroup 1 strains, and we developed two PCR-based methods specific for serogroup 1. Specific DNA fragments could be amplified from all the serogroup 1 strains (=43) including the strains from the American Type Culture Collection. In contrast, none of the strains from serogroups 2–15 (=41) contained these specific gene regions. In a second approach, primers specific for the gene, encoding an -acetyltransferase, which is responsible for the presence of the LPS epitope recognized by mAb 3/1, were designed and tested for their ability to differentiate between mAb 3/1-positive and -negative strains. All mAb 3/1-positive strains (=30) contained the gene, but in turn 4 of 13 tested mAb 3/1-negative strains were also positive in the PCR. Thus, the discrimination between mAb 3/1-positive and mAb 3/1-negative subgroups could not be achieved for all strains. In a third approach, two intergenic regions expected to be specific for monoclonal subgroup Knoxville and closely related subgroups Benidorm/Bellingham were identified and used for selective genotyping. These intergenic regions could not only be amplified in every tested strain belonging to the subgroups Knoxville, Benidorm and Bellingham, but also in some strains of other unrelated subgroups. The two PCR approaches with primers specific for serogroup 1 genes definitely represent a valuable tool in outbreak investigations and for risk assessment. They also might be used for culture-independent diagnosis of legionellosis caused by serogroup 1.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.008508-0
2009-05-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/58/5/588.html?itemId=/content/journal/jmm/10.1099/jmm.0.008508-0&mimeType=html&fmt=ahah

References

  1. Bernander S., Jacobson K., Helbig J. H., Lück P. C., Lundholm M. 2003; A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroup 1 is characterized by stable genetic fingerprinting but variable monoclonal antibody patterns. J Clin Microbiol 41:2503–2508 [CrossRef]
    [Google Scholar]
  2. Brito D. A., Ramirez M., de Lencastre H. 2003; Serotyping Streptococcus pneumoniae by multiplex PCR. J Clin Microbiol 41:2378–2384 [CrossRef]
    [Google Scholar]
  3. Cazalet C., Rusniok C., Brüggemann H., Zidane N., Magnier A., Ma L., Tichit M., Jarraud S., Bouchier C. other authors 2004; Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet 36:1165–1173 [CrossRef]
    [Google Scholar]
  4. Cazalet C., Jarraud S., Ghavi-Helm Y., Kunst F., Glaser P., Etienne J., Buchrieser C. 2008; Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species. Genome Res 18:431–441 [CrossRef]
    [Google Scholar]
  5. Chien M., Morozova I., Shi S., Sheng H., Chen J., Gomez S. M., Asamani G., Hill K., Nuara J. other authors 2004; The genomic sequence of the accidental pathogen Legionella pneumophila . Science 305:1966–1968 [CrossRef]
    [Google Scholar]
  6. Fields B. S., Benson R. F., Besser R. E. 2002; Legionella and Legionnaires' disease: 25 years of investigation. Clin Microbiol Rev 15:506–526 [CrossRef]
    [Google Scholar]
  7. Fry N. K., Alexiou-Daniel S., Bangsborg J. M., Bernander S., Castellani Pastoris M., Etienne J., Forsblom B., Gaia V., Helbig J. H. other authors 1999; A multicenter evaluation of genotyping methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results from a pan-European study. Clin Microbiol Infect 5:462–477 [CrossRef]
    [Google Scholar]
  8. Fry N. K., Afshar B., Wewalka G., Harrison T. G. 2006; Epidemiological typing of Legionella pneumophila in the absence of isolates. In Legionella: State of the Art 30 Years After its Recognition pp 152–155 Edited by Cianciotto N. P., Abu Kwaik Y., Edelstein P. H., Fields B. S., Geary D. F., Harrison T. G., Joseph C. A., Ratcliff R. M., Stout J. E., Swanson M. S. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  9. Gaia V., Fry N. K., Afshar B., Lück P. C., Meugnier H., Etienne J., Peduzzi R., Harrison T. G. 2005; Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila . J Clin Microbiol 43:2047–2052 [CrossRef]
    [Google Scholar]
  10. Glöckner G., Albert-Weissenberger C., Weinmann E., Jacobi S., Schunder E., Steinert M., Hacker J., Heuner K. 2008; Identification and characterization of a new conjugation/type IVA secretion system ( trb / tra ) of Legionella pneumophila Corby localized on two mobile genomic islands. Int J Med Microbiol 298:411–428 [CrossRef]
    [Google Scholar]
  11. Helbig J. H., Kurtz J. B., Pastoris M. C., Pelaz C., Lück P. C. 1997; Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups. J Clin Microbiol 35:2841–2845
    [Google Scholar]
  12. Helbig J. H., Bernander S., Castellani Pastoris M., Etienne J., Gaia V., Lauwers S., Lindsay D., Lück P. C., Marques T. other authors 2002; Pan-European study on culture-proven Legionnaires' disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups. Eur J Clin Microbiol Infect Dis 21:710–716 [CrossRef]
    [Google Scholar]
  13. Joly J. R., McKinney R. M., Tobin J. O., Bibb W. F., Watkins I. D., Ramsay D. 1986; Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies. J Clin Microbiol 23:768–771
    [Google Scholar]
  14. Kaplan J. B., Perry M. B., MacLean L. L., Furgang D., Wilson M. E., Fine D. H. 2001; Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f. Infect Immun 69:5375–5384 [CrossRef]
    [Google Scholar]
  15. Kozak N. A., Brown E., Benson R. F., Shelton B. G., Fields B. S. 2007; Presence of a lag-1 gene as a virulence marker for Legionella pneumophila serogroup 1 strains. In Abstracts of the 107th General Meeting of the American Society for Microbiology 2007. Toronto, Canada abstract D 144 Washington, DC: American Society for Microbiology;
    [Google Scholar]
  16. Kuchana R., Uldum S. A. 2007; PCR method for detection of monoclonal subgroups of Legionella pneumophila serogroup 1. In 22nd Annual Meeting of the European Working Group on Legionella Infections, 2007. Stockholm, Sweden oral presentation
    [Google Scholar]
  17. Lück P. C., Freier T., Steudel C., Knirel Y. A., Lüneberg E., Zähringer U., Helbig J. H. 2001; A point mutation in the active site of Legionella pneumophila O-acetyltransferase results in modified lipopolysaccharide but does not influence virulence. Int J Med Microbiol 291:345–352 [CrossRef]
    [Google Scholar]
  18. Lück P. C., Ecker C., Reischl U., Linde H. J., Stempka R. 2007; Culture-independent identification of the source of an infection by direct amplification and sequencing of Legionella pneumophila DNA from a clinical specimen. J Clin Microbiol 45:3143–3144 [CrossRef]
    [Google Scholar]
  19. Lüneberg E., Zetzmann N., Alber D., Knirel Y. A., Kooistra O., Zahringer U., Frosch M. 2000; Cloning and functional characterization of a 30 kb gene locus required for lipopolysaccharide biosynthesis in Legionella pneumophila . Int J Med Microbiol 290:37–49 [CrossRef]
    [Google Scholar]
  20. Ratzow S., Gaia V., Helbig J. H., Fry N. K., Lück P. C. 2007; Addition of neuA , the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains. J Clin Microbiol 45:1965–1968 [CrossRef]
    [Google Scholar]
  21. Satola S. W., Collins J. T., Napier R., Farley M. M. 2007; Capsule gene analysis of invasive Haemophilus influenzae : accuracy of serotyping and prevalence of IS1016 among nontypeable isolates. J Clin Microbiol 45:3230–3238 [CrossRef]
    [Google Scholar]
  22. Wagner C., Krönert C., Lück P. C., Jacobs E., Cianciotto N. P., Helbig J. H. 2007; Random mutagenesis of Legionella pneumophila reveals genes associated with lipopolysaccharide synthesis and recognition by typing monoclonal antibodies. J Appl Microbiol 103:1975–1982 [CrossRef]
    [Google Scholar]
  23. Yu V. L., Plouffe J. F., Pastoris M. C., Stout J. E., Schousboe M., Widmer A., Summersgill J., File T., Heath C. M. other authors 2002; Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis 186:127–128 [CrossRef]
    [Google Scholar]
  24. Yu J., Carvalho M. da G. S., Beall B., Nahm M. H. 2008; A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR. J Med Microbiol 57:171–178 [CrossRef]
    [Google Scholar]
  25. Zou C. H., Knirel Y. A., Helbig J. H., Zähringer U., Mintz C. S. 1999; Molecular cloning and characterization of a locus responsible for O acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide. J Bacteriol 181:4137–4141
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.008508-0
Loading
/content/journal/jmm/10.1099/jmm.0.008508-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed