PCR-based 'serotyping' of Legionella pneumophila

Abstract

Currently, several PCR assays based on 16S rRNA and virulence-associated genes are available for detection of Legionella pneumophila. So far, no genotyping method has been published that can discriminate between serogroups and monoclonal subgroups of the most common L. pneumophila serogroup 1. Our first approach was to analyse LPS-associated genes of seven L. pneumophila serogroup 1 strains, and we developed two PCR-based methods specific for serogroup 1. Specific DNA fragments could be amplified from all the serogroup 1 strains (n=43) including the strains from the American Type Culture Collection. In contrast, none of the strains from serogroups 2-15 (n=41) contained these specific gene regions. In a second approach, primers specific for the lag-1 gene, encoding an O-acetyltransferase, which is responsible for the presence of the LPS epitope recognized by mAb 3/1, were designed and tested for their ability to differentiate between mAb 3/1-positive and -negative strains. All mAb 3/1-positive strains (n=30) contained the lag-1 gene, but in turn 4 of 13 tested mAb 3/1-negative strains were also positive in the PCR. Thus, the discrimination between mAb 3/1-positive and mAb 3/1-negative subgroups could not be achieved for all strains. In a third approach, two intergenic regions expected to be specific for monoclonal subgroup Knoxville and closely related subgroups Benidorm/Bellingham were identified and used for selective genotyping. These intergenic regions could not only be amplified in every tested strain belonging to the subgroups Knoxville, Benidorm and Bellingham, but also in some strains of other unrelated subgroups. The two PCR approaches with primers specific for serogroup 1 genes definitely represent a valuable tool in outbreak investigations and for risk assessment. They also might be used for culture-independent diagnosis of legionellosis caused by L. pneumophila serogroup 1.