Phylogenetic Characterization of Viable but-not-yet Cultured Legionella Groups Grown in Amoebic Cocultures: A Case Study using Various Cooling Tower Water Samples.

　Legionella spp. exist naturally in association with amoeba in water environments and are known to be the etiological agent of a severe form of pneumonia. To detect diverse Legionella populations in cooling tower water systems, amoebic coculturing was performed for 15 water samples obtained from five different kinds of facilities in six geographically different locations. The growth of Legionella in coculture with Acanthamoeba sp. cells was monitored by quantitative PCR targeting Legionella-specific 16S rRNA genes. Seven out of the 15 samples were positive for Legionella growth and subjected to clone library analysis. A total of 333 clones were classified into 14 operational taxonomic units composed of seven known species and seven previously undescribed groups. Four of the seven Legionella-growth-positive samples harbored detectable levels of free-living amoeba and were predominated by either L. drozanskii or L. lytica, by both L. bozemanii and L. longbeachae, or by a not-yet-described group named OTU 4. The Legionella-growth- positive samples contained higher ATP levels (>980 pM) than the growth-negative samples (<160 pM) , suggesting that ATP content would be a good indicator of the presence of viable but nonculturable Legionella populations able to grow with amoeba.