Consensus Sequence-Based Scheme for Epidemiological Typing of Clinical and Environmental Isolates ofLegionella pneumophila

Abstract

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of >0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.

Variable genetic element typing: a quick method for epidemiological subtyping of Legionella pneumophila

“…The primers developed and used in this study were aligned to the four available complete genomes of L. pneumophila [10;11;12] In addition, all five variants of the Corby strains that differ in the cultivability in amoebal hosts, point mutations in the rpoB and the lag-1 genes showed the same VET results (data not shown) thus arguing that the markers detected in the VET are stable as were the SBT results [7].…”

“…For magA and traD sequences from the pathogenicity island [13] were used. DNAs from L. pneumophila strains was prepared as for sequenced based typing [7]. The VET-PCR were performed with 10pmol of each primer, 10µl of DNA-puffer, 5U of GoldTaq-Polymerase, 2µl DNA of each strain ,and 200pmol of each dNTP at a final volumes of 50µl.…”

“…At the beginning of these experiments the specificity of the PCR products was confirmed by DNA sequencing [7]. In each run the mip gene PCR of the SBT scheme were used as a positive control [7]. All VET PCR assays were run at least twice.…”

“…These data were compared to the European Working Group on Legionella Infections (EWGLI) SBT system. (EUL 135 to 139) that differ in their ability to multiply in amoebae and macrophages showed different reactivity patterns with MAbs due to a mutation in a lipopolysaccharide synthesis gene or that were resistant to rifampicin [7]. 40 strains were by definition unrelated to any other isolates in the study, i.e.…”

“…For this numerous phenotypic and genotypic typing methods have been applied to the epidemiological typing of L. pneumophila in the recent years [4]. This include monoclonal antibody (MAb) subgrouping as a rapid screening method [3]and genotyping methods such as amplified fragment length polymorphism (AFLP) [4], pulsed-field gel electrophoresis (PFGE) [4;5] and sequence based typing [6][7][8]. In contrast to band based typing methods that are difficult to standardize the developed sequence based typing (SBT) has the potential of excellent typeability, interlaboratory reproducibility and epidemiologic concordance [9].…”