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

Applied and Environmental Microbiology
Leila KahlischManfred G Höfle

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 ...Continue Reading

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Mar 15, 2011·Epidemiology and Infection·S F DufresneM Laverdière
Sep 10, 2015·Clinical Chemistry and Laboratory Medicine : CCLM·Dong-Sheng Lian, Shu-Jin Zhao
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Apr 13, 2015·Water Research·Sarah Rodríguez-MartínezMalka Halpern
Apr 18, 2019·Scientific Reports·Yehonatan SharabyMalka Halpern
Oct 25, 2020·Applied and Environmental Microbiology·Sho ShimadaKazuhiro Tateda

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