Molecular Detection of Legionella: Moving on From mip

Abstract

The detection of Legionella pneumophila in environmental and clinical samples is frequently performed by PCR amplification of the mip and/or 16S rRNA genes. Combined with DNA sequencing, these two genetic loci can be used to distinguish different species of Legionella and identify L. pneumophila. However, the recent Legionella genome sequences have opened up hundreds of possibilities for the development of new molecular targets for detection and diagnosis. Ongoing comparative genomics has the potential to fine tune the identification of Legionella species and serogroups by combining specific and general genetic targets. For example, the coincident detection of LPS biosynthesis genes and virulence genes may allow the differentiation of both pathogen and serogroup without the need for nucleotide sequencing. We tested this idea using data derived from a previous genomic subtractive hybridization we performed between L. pneumophila serogroup 1 and L. micdadei. Although not yet formally tested, these targets serve as an example of how comparative genomics has the potential to improve the scope and accuracy of Legionella molecular detection if embraced by laboratories undertaking Legionella surveillance.

Specific Real-Time PCR for Simultaneous Detection and Identification of Legionella pneumophila Serogroup 1 in Water and Clinical Samples

“…Thürmer and colleagues also proposed specific sequences that could distinguish between monoclonal subgroup Knoxville and the closely related subgroups Benidorm and Bellingham, which, again, are located in the LPS gene cluster (38). Very recently, Yong and colleagues reported on the identification of a serogroup-specific gene, lpg0744 (lpp0833), again part of the LPS gene cluster, by subtractive hybridization (42). Combined with the identification of an L. pneumophila species-specific gene encoding an apyrase, lpg1905 (lpp1880), these newly designed tests could significantly enhance our ability to detect and identify species and subgroups of Legionella rapidly and accurately (42).…”

“…Very recently, Yong and colleagues reported on the identification of a serogroup-specific gene, lpg0744 (lpp0833), again part of the LPS gene cluster, by subtractive hybridization (42). Combined with the identification of an L. pneumophila species-specific gene encoding an apyrase, lpg1905 (lpp1880), these newly designed tests could significantly enhance our ability to detect and identify species and subgroups of Legionella rapidly and accurately (42). Furthermore, in the future, additional specific primers can be defined for the sequences of the LPS gene cluster regions of other L. pneumophila non-Sg1 serogroups, and in particular also for L. longbeachae, which also carries very specific LPS-encoding genes (7).…”

Prevalence ofmipvirulence gene and PCR-base sequence typing ofLegionella pneumophilafrom cooling water systems of two cities in Iran

“…Molecular methods described thus far targeted a number of sequences such as the 16S rRNA, the 23S-5S spacer regions and the mip gene of L. pneumophila. [14][15][16] However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods. 16,17 There is paucity of information regarding contamination rate of L. pneumophila in water system of different sources in Iran.…”

Molecular diversity and high virulence of Legionella pneumophilastrains isolated from biofilms developed within a warm spring of a thermal spa

“…Both genes were amplified by PCR from bacterial lysates of the 30 environmental isolates. Then, the discrimination of the specium pneumophila was performed by amplifying the gene lpg0774 [18]. Finally, Lp1 typing of seven environmental Legionellae was obtained by independent gene amplifications of lpg1905 and wzm (a gene belonging to the cluster coding for the lipopolysaccharide biosynthesis) [11,18]: LAXA21, LAXB6, LAXB8, LAXB12, LAXB22, LAB24 and LAXB25 (Table 1; Figure 1).…”