Characterization of a Tandem Repeat Polymorphism inLegionella pneumophilaand Its Use for Genotyping

Abstract

We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome of Legionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of other Legionella species or other bacteria used as controls. A polymorphism was observed for seven markers among the L. pneumophila strains tested. To check whether these markers could be used to compare strains of L. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies of L. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of the L. pneumophila colonies and gave a first hint as to the presence of several strains in a single sample.

Distribution and characterization of staphylococcal interspersed repeat units (SIRUs) and potential use for strain differentiation

“…The availability of multiple sequenced genomes meant that our study did not suffer from the limitations of some other studies, in that we were able to design primers in a homologous region (Pourcel et al, 2003).…”

“…These loci have a characteristic structure consisting of a variable number of near-identical repeated DNA sequences arranged consecutively. A general term for these repeats is variable-number tandem repeats (VNTRs), and true VNTRs have now been reported in Bacillus anthracis, Legionella pneumophila, Pseudomonas aeruginosa, Salmonella enterica and Escherichia coli O157 (Keim et al, 2000;Liu et al, 2003;Noller et al, 2003;Onteniente et al, 2003;Pourcel et al, 2003). With the exception of M. tuberculosis, these studies have been confined to comparisons of molecular strain differentiation techniques.…”

Characterization of a tandem repeat polymorphism in Rickettsia strains

“…The availability of whole genome sequences and appropriate algorithms for searching for repetitive sequences has led to the application of the VNTR typing approach, namely the development of multilocus VNTR analysis (MLVA) typing systems for several pathogens, such as Bacillus anthracis (Keim et al, 2000), Yersinia pestis (Klevytska et al, 2001), Francisella tularensis (Farlow et al, 2001), Borrelia burgdorferi (Farlow et al, 2002), Legionella pneumophila (Pourcel et al, 2003) and Mycobacterium tuberculosis (Spurgiesz et al, 2003). The MLVA approach has been proven to present high discriminatory power, reproducibility and portability, making it a strong candidate for the increased development of reference databases that allow online strain identification services (Supply et al, 2001;Onteniente et al, 2003).…”

Identification of Variable-Number Tandem-Repeat Loci in Leptospira interrogans Sensu Stricto

“…Analysis of variable-number tandem repeats (VNTR), also called multiple-locus VNTR analysis, has proven to be a highly powerful and discriminant method to study the population structure of bacteria (17) and to characterize isolates even from monomorphic bacterial populations (6,11,13). The genome of L. interrogans serovar Lai has recently been sequenced (20), and this allowed us to define pairs of primers flanking some VNTR-like loci.…”