High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.

2.50
Hdl Handle:
http://hdl.handle.net/10033/123882
Title:
High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.
Authors:
Kahlisch, Leila; Henne, Karsten; Draheim, Josefin; Brettar, Ingrid; Höfle, Manfred G
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.
Affiliation:
Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany.
Citation:
High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA. 2010, 76 (18):6186-95 Appl. Environ. Microbiol.
Journal:
Applied and environmental microbiology
Issue Date:
Sep-2010
URI:
http://hdl.handle.net/10033/123882
DOI:
10.1128/AEM.00416-10
PubMed ID:
20656879
Type:
Article
Language:
en
ISSN:
1098-5336
Appears in Collections:
publications of the research group vaccinology and applied microbiology (VAC)

Full metadata record

DC FieldValue Language
dc.contributor.authorKahlisch, Leilaen
dc.contributor.authorHenne, Karstenen
dc.contributor.authorDraheim, Josefinen
dc.contributor.authorBrettar, Ingriden
dc.contributor.authorHöfle, Manfred Gen
dc.date.accessioned2011-03-08T15:08:42Z-
dc.date.available2011-03-08T15:08:42Z-
dc.date.issued2010-09-
dc.identifier.citationHigh-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA. 2010, 76 (18):6186-95 Appl. Environ. Microbiol.en
dc.identifier.issn1098-5336-
dc.identifier.pmid20656879-
dc.identifier.doi10.1128/AEM.00416-10-
dc.identifier.urihttp://hdl.handle.net/10033/123882-
dc.description.abstractCentral 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.en
dc.language.isoenen
dc.subject.meshBase Sequenceen
dc.subject.meshDNA Primersen
dc.subject.meshElectrophoresis, Capillaryen
dc.subject.meshEnvironmental Monitoringen
dc.subject.meshGenotypeen
dc.subject.meshGermanyen
dc.subject.meshLegionella pneumophilaen
dc.subject.meshMolecular Sequence Dataen
dc.subject.meshPolymerase Chain Reactionen
dc.subject.meshRNA, Ribosomal, 16Sen
dc.subject.meshReverse Transcriptase Polymerase Chain Reactionen
dc.subject.meshSequence Analysis, DNAen
dc.subject.meshTandem Repeat Sequencesen
dc.subject.meshWater Microbiologyen
dc.subject.meshWater Supplyen
dc.titleHigh-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.en
dc.typeArticleen
dc.contributor.departmentDepartment of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany.en
dc.identifier.journalApplied and environmental microbiologyen

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