Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Legionella pneumophila and Development of an Optimized Multiple-Locus VNTR Analysis Typing Scheme

Abstract

The utility of a genotypic typing assay for Legionella pneumophila was investigated. A multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) scheme using PCR and agarose gel electrophoresis is proposed based on eight minisatellite markers. Panels of well-characterized strains were examined in a multicenter analysis to validate the assay and to compare its performance to that of other genotyping assays. Excellent typeability, reproducibility, stability, and epidemiological concordance were observed. The MLVA type or profile is composed of a string of allele numbers, corresponding to the number of repeats at each VNTR locus, separated by commas, in a predetermined order. A database containing information from 99 L. pneumophila serogroup 1 strains and four strains of other serogroups and their MLVA profiles, which can be queried online, is available from http://bacterial-genotyping.igmors.u-psud.fr/.

Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease

“…Multiple-locus VNTR assays are based on the separation and sizing of amplified short to long tandem repeated sequences or microsatellites (up to 9 bp) and minisatellites (more than 9 bp) spread throughout the bacterial genome (28). This technique has an index of discrimination (28) similar to that of SBT. SBT is a powerful method based on the sequencing of seven gene loci (13,31) that is already being recognized as the new EWGLI (European Working Group for Legionella Infections) "gold standard" tool for L. pneumophila typing and for which an important database of sequence types (STs) is available at http://www.ewgli.org.…”

“…However, in the past few years, epidemiological techniques have been developed to be used directly with clinical samples, independently of strain isolation, by PCR-based typing methods such as multiple-locus variable-number tandemrepeat (VNTR) and sequence-based typing (SBT) (13,14,25,27,33). Multiple-locus VNTR assays are based on the separation and sizing of amplified short to long tandem repeated sequences or microsatellites (up to 9 bp) and minisatellites (more than 9 bp) spread throughout the bacterial genome (28). This technique has an index of discrimination (28) similar to that of SBT.…”

Spatial distribution of Legionella pneumophila MLVA-genotypes in a drinking water system

“…Legionella infection (Lück et al, 2007) and provide clues on its infection routes (Olsen et 73 al., 2010;Reuter et al, 2013). One of these methods is the Multiple-Locus Variable-number 74 tandem-repeat Assay (MLVA) (Pourcel et al, 2007, Kahlisch et al 2010. This method 75 relies on the variability found in some tandemly repeated DNA sequences which represent 76 sources of genetic polymorphism (mini-satellites).…”

“…One ml was thermally treated (50 ºC for 30 min) 162 and 0.5 ml samples were plated on two GVPC plates. The LOD for Legionella was 10 M A N U S C R I P T A C C E P T E D conducted on 62 of the environmental Legionella isolates as described by Kahlisch et al 192 2010 using the primers according to Pourcel et al (2007) In order to study the prevalence of Legionella in a drinking water system in Israel, we 230 selected seven sampling points (figure 1. A-G) that followed the water course in a premise 231 plumbing system at Kiryat Tivon, Israel.…”

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

“…The genus Legionella comprises 71 distinct serogroups from more than 50 known species [2][3][4] and new species are frequently described [5][6][7]. Legionella pneumophila, was first recognized in 1977 following an epidemic of acute pneumonia in Philadelphia [8], is the etiological agent of the majority of cases of legionellosis, and the best part of cases have been attributed to L. pneumophila serogroup 1 [3,9].…”