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.
Clinical isolates of Legionella pneumophila, obtained from 167 patients, who acquired their illness in the community in England and Wales between January 2000 and March 2008, were compared with 276 environmental isolates of L. pneumophila obtained over the same period as part of the routine sampling of 'managed' water systems. The 443 isolates were typed by monoclonal antibody (mAb) subgrouping and the internationally standardised, seven-gene loci, sequence-based typing (SBT) scheme of the European Working Group for Legionella Infections (EWGLI). Of the clinical isolates, 97.6% were L. pneumophila serogroup (sgp) 1, compared with only 55.8% of environmental isolates (P = 0.0002); 91.6% were subgrouped as mAb3/1+ve, compared with only 8.3% of environmental isolates (P < 0.0001). The isolates were very diverse, with SBT identifying 111 sequence types (STs) (index of diversity [IOD] 0.954). Among the clinical isolates, 42 ST were seen, with one (ST47) accounting for 25.7% and three (ST47, ST37 and ST62) accounting for 46.1% of all isolates. Eighty-two STs were identified among the environmental isolates, with two (ST1 and ST79) accounting for 34.1% of these. Comparison of the STs seen among clinical and environmental isolates showed that there was very little overlap between the two populations (P < 0.0001), with common clinical strains found in the environment very infrequently: 0.4, 0.7 and 0% (ST47, ST37 and ST62, respectively), and common environmental strains rarely causing disease: 4.8 and 1.2% (ST1 and ST79, respectively). Combining phenotypic and genotypic data identified 144 phenons (IOD 0.970); 52 among clinical isolates and 101 among environmental isolates. The most abundant clinical strain, mAb 'Allentown' ST47, accounted for 22.8% of cases, but was only found once in the environment. Conversely, mAb 'Oxford/OLDA' ST1 was the most common environmental strain (17.0%), but only caused two infections. A review of the published data shows that mAb 'Allentown' ST47 is also an important cause of infection in France and possibly in the Netherlands. However, it was not found in a large study of German clinical isolates. This study confirms previous work showing that just a few strains of L. pneumophila cause the majority of community-acquired Legionella infection in England and Wales, and that these clinically significant strains are only rarely found in managed water systems. These data suggest that knowing which particular strain is present in an environment might be at least as important as knowing the quantity in which legionellae are present.
SummaryWhole-genome sequencing can be used to support or refute suspected links between hospital water systems and Legionnaires’ disease cases. However, caveats regarding the interpretation of genomic data from Legionella pneumophila are described that should be considered in future investigations.
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