LEGIONELLA - World Health Organization

Although not all strains can be reliably identified to the species level, narrowing strains to groups
is useful, and this is usually achieved using serology. DNA–DNA hybridization best identifies
a strain of Legionella or a new species. The procedure requires DNA from the test strain to be
hybridized with DNA from all known species of Legionella, and is therefore only undertaken
by specialized laboratories. Sequence analysis of specific genes has been used for taxonomic
analysis of legionellae. Analysis of 16S rRNA genes led to the designation of Legionella within
the gamma-2 subdivision of the class Proteobacteria, and has been used to show the phylogenetic
relatedness of new species of this genus (Fry et al., 1991). A sequence-based classification
scheme that targets the mip gene has been developed for legionellae (Ratcliff et al., 1998).
This scheme can unambiguously discriminate between the 39 species of Legionella tested so
far, and it is likely that all taxonomic analysis will soon become sequence-based.
Within the genus Legionella, species can therefore be distinguished by biochemical analysis,
fatty acid profiles, protein banding patterns, serology, DNA–DNA hybridization and analysis
of 16S rRNA genes (Hookey et al., 1996; Benson & Fields, 1998; Riffard et al., 1998; Fields,
Benson & Besser, 2002).
11.4.2 Identifying Legionella colonies
Steps for identifying and confirming Legionella colonies are the same, irrespective of whether the
isolates are from clinical or environmental samples. Young, presumptive colonies of L. pneumophila
show a characteristic speckled green, blue or pink–purple iridescence. More mature colonies
(after three or four days) have entire margins, and are convex, 3–4 mm in diameter and like frosted
glass in appearance. Older colonies lose most of their iridescence. Subsequent confirmation
should be carried out using a cysteine-free agar to show dependency on L-cysteine (Barker,
Farrell & Hutchinson, 1986).
The rapid identification and separate confirmation of L. pneumophila serogroup 1, other serogroups
and some other pathogenic species is important for epidemiological investigations. Presumptive
colonies of pathogenic Legionella species from clinical or environmental samples can be confirmed
using a range of antibody reactions, such as indirect immunofluorescence, direct immunofluorescence,
immunodiffusion, crossed immunoelectrophoresis and slide agglutination.
Preliminary identification of Legionella spp. with an antibody-reaction test can be done by
routine microbiological laboratories. Commercially available latex agglutination kits may be
used for confirmation. Suspect colonies are simply emulsified as directed, and mixed with each
latex reagent separately on a disposable reaction card. Each reagent is sensitized with antibodies
specific to Legionella. In the presence of homologous antigens, the latex particles agglutinate
to give a clearly visible positive reaction for some minutes (Hart et al., 2000). Isolates that
react with specific antisera against known legionellae are confirmed legionellae. The different
serogroups of L. pneumophila may cross-react (Wilkinson et al., 1990), and when isolates fail
to react with specific antisera to all known legionellae, they must be evaluated and eventually
LEGIONELLA AND THE PREVENTION OF LEGIONELLOSIS