Problems associated with identification of Legionella species from the environment and isolation of six possible new species

Abstract

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at-70°C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionellajordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.

Detection of culturable and nonculturableLegionellaspecies from hot water systems of public buildings in Japan

“…birminghamensis was isolated from a lung biopsy specimen from a cardiac transplant recipient with no symptoms of pneumonia (Wilkinson et al. 1987) and was later found in environmental water in Australia (Wilkinson et al. 1990).…”

Interspecies sequence differences in the Mip protein from the genus Legionella: implications for function and evolutionary relatedness

“…data. For explanation of phenotypical and biochemical markers and group definitions, see the following; 1, Wilkinson, (1988);2, Brenner (1987); 3, Lambert and Moss (1989), Wilkinson et al (1990);4, Jantzen et al (1993);5, O'Connell et al (1996). This may be because of the way in which the phenotype is determined (arbitrary classification) or it may be that the character is genuinely homoplastic, i.e. subject to convergence.…”

“…The arbitrariness of some classifications for Legionella can be seen on examination of the criteria on which they are based. L. israelensis has been grouped alternately with ubiquinone group A species (Wilkinson et al, 1990) or with group D species (Lambert and Moss, 1989). The discrepancy is determined solely on a small difference in the amount of ubiquinone 13 (Q13) detected.…”

“…The present study reports the first comparison of 16S RNA-based phylogeny of Legionella with data from another gene, mip. Furthermore, the utility of generally used phenotypical and biochemical characteristics, such as autofluorescence (Wilkinson, 1988), cell wall ubiquinone and fatty acid constituents (Lambert and Moss, 1989;Wilkinson et al, 1990;Jantzen et al, 1993), are compared for their ability to discern evolutionary relationships within the genus.…”

Specific Detection of Legionella pneumophila : Construction of a New 16S rRNA-Targeted Oligonucleotide Probe

“…in natural environments has mainly been done by using antibodies, PCR, and related techniques (19,21,24). Unfortunately, the use of antibodies is often hampered by unspecific binding to other bacteria and phenotypic antigen variation (29). Furthermore, biofilms can act as a penetration barrier, making it almost impossible to detect bacteria in deeper layers of the biofilm (27).…”