title: High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.
abstract: Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.
authors.affiliation: Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany.
authors.forename: Leila
authors.initials: L
authors.lastname: Kahlisch
authors.forename: Karsten
authors.initials: K
authors.lastname: Henne
authors.forename: Josefin
authors.initials: J
authors.lastname: Draheim
authors.forename: Ingrid
authors.initials: I
authors.lastname: Brettar
authors.forename: Manfred G
authors.initials: MG
authors.lastname: Höfle
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary; D004784:Environmental Monitoring / Q000379:methods*; D005838:Genotype; D005858:Germany; D016952:Legionella pneumophila / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences / Q000235:genetics; D014871:Water Microbiology; D014881:Water Supply / Q000032:analysis*
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S