Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples

Abstract

Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.

FIG. 1.

Comparison of the LPS genes lpp0827 to lpp0843 of L. pneumophila Sg1 strain Paris to those of L. pneumophila Sg6 strain ATCC 33215. The comparison of the two genomic regions is based on TBlastX. Blue bars indicate homologous regions in the two gene clusters based on a level of protein similarity greater than 25%. Light blue, genes predicted in this region; green, pseudogenes.

FIG. 2.

Comparison of the LPS gene region of L. pneumophila Sg10 strain ATCC 43283 with that of L. pneumophila Sg13 strain ATCC 43736. The comparison of the two genomic regions is based on TBlastX. Blue bars indicate homologous regions in the two gene clusters based on a level of protein similarity greater than 25%. Light blue, genes predicted in this region.