Legionella thermalis sp. nov., isolated from hot spring water in Tokyo, Japan.

Strain L-47(T) of a novel bacterial species belonging to the genus Legionella was isolated from a sample of hot spring water from Tokyo, Japan. The 16S rRNA gene sequences (1477 bp) of this strain (accession number AB899895) had less than 95.0% identity with other Legionella species. The dominant fatty acids of strain L-47(T) were a15:0 (29.6%) and the major ubiquinone was Q-12 (71.1%). It had a guanine-plus-cytosine content of 41.5 mol%. The taxonomic description of Legionella thermalis sp. nov. is proposed to be type strain L-47(T) (JCM 30970(T) = KCTC 42799(T)).

[Legionella contamination risk factors in non-circulating hot spring water].

We examined water from 182 non-circulating hot spring bathing facilities in Japan for possible Legionella occurrence from June 2005 to December 2006, finding Legionella-positive cultures in 119 (29.5%) of 403 samples. Legionellae occurrence was most prevalent in bathtub water (39.4%), followed by storage tank water (23.8%), water from faucets at the bathtub edge (22.3%), and source-spring water (8.3%), indicating no statistically significant difference, in the number of legionellae, having an overall mean of 66 CFU/100mL. The maximum number of legionellae in water increased as water was sampled downstream:180 CFU/100 mL from source spring, 670 from storage tanks, 4,000 from inlet faucets, and 6,800 from bathtubs. The majority--85.7%--of isolated species were identified as L. pneumophila : L. pneumophila serogroup (SG) 1 in 22%, SG 5 in 21%, and SG 6 in 22% of positive samples. Multivariate logistic regression models used to determine the characteristics of facilities and sanitary management associated with Legionella contamination indicated that legionellae was prevalent in bathtub water under conditions where it was isolated from inlet faucet/pouring gate water (odds ratio [OR] = 6.98, 95% confidence interval [CI] = 2.14 to 22.8). Risk of occurrence was also high when the bathtub volume exceeded 5 m3 (OR = 2.74, 95% CI = 1.28 to 5.89). Legionellae occurrence was significantly reduced when the bathing water pH was lower than 6.0 (OR = 0.12, 95% CI = 0.02 to 0.63). Similarly, occurrence was rare in inlet faucet water or the upper part of the plumbing system for which pH was lower than 6.0 (OR = 0.06, 95% CI = 0.01 to 0.48), and when the water temperature was maintained at 55 degrees C or more (OR = 0.10, 95% CI = 0.01 to 0.77). We also examined the occurrence of amoeba, Mycobacterium spp., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus in water samples.

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.

Evaluation of Duopath Legionella kit for the rapid identification of Legionella strains isolated from water samples.

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a rapid and simple immunochromatographic assay kit for the identification of Legionella species. We evaluated the precision of the kit in identifying 100 strains of Legionella and 35 strains of non-Legionella bacteria isolated from cooling tower and bath water samples. Consequently, of all the Legionella strains tested, 99 strains were judged to be Legionella, and only one strain (Legionella busanensis) was judged to be non-Legionella. All of the 35 non-Legionella strains were judged to be non-Legionella. We therefore conclude that Duopath Legionella is a useful method for the rapid identification of Legionella.

Enhanced antifungal effect of the selective medium for the detection of Legionella species by a combination of cycloheximide, amphotericin B and thiabendazole.

The accurate detection of Legionella from environmental water samples using conventional plate culture methods is often made difficult by the overgrowth of non-target microorganisms on the selective agar plates. Acid pretreatment is a very effective pretreatment to decrease the overgrowth of heterotrophic bacteria. However, acid pretreatment would not be expected to eliminate molds. We evaluated the effects of four kinds of antifungal agents, individually and in combination, on the growth of Legionella strains and molds. Consequently, it was demonstrated that the combination of cycloheximide, amphotericin B and thiabendazole was very effective in eliminating molds on agar plates, and resulted in the improved detection of Legionella. Examination of 214 cooling tower water samples using the enhanced antifungal selective medium (CAT alpha) instead of GVPC alpha selective medium demonstrated a decrease in contamination by molds from 13.6% to 1.9% without affecting the growth of Legionella.

Molecular characterization of viable Legionella spp. in cooling tower water samples by combined use of ethidium monoazide and PCR.

Viable Legionella spp. in environmental water samples were characterized phylogenetically by a clone library analysis combining the use of ethidium monoazide and quantitative PCR. To examine the diversity of Legionella spp., six cooling tower water samples and three bath water samples were collected and analyzed. A total of 617 clones were analyzed for their 16S rRNA gene sequences and classified into 99 operational taxonomic units (OTUs). The majority of OTUs were not clustered with currently described Legionella spp., suggesting the wide diversity of not-yet-cultured Legionella groups harbored in cooling tower water environments.

Detection of Legionella species in environmental water by the quantitative PCR method in combination with ethidium monoazide treatment.

We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples.

[An outbreak of legionellosis in a new facility of hot spring bath in Hiuga City].

Following cerebrating ceremony in 20 June 2002, for the completion of Hiuga Sun-Park Hot Spring Bath "Ofunade-no-Yu" facilities, Miyazaki Prefecture, Kyushu Island, 200 neighbors were invited each day to experience bathing on 20 and 21 June. The Bath "Ofunade-no-Yu" officially opened on 1 July 2002. On 18 July, Hiuga Health Center was informed that 3 suspected Legionella pneumonia patients in a hospital and all of them have bathing history of "Ofunade-no-Yu". Health Center officers notified Hiuga City, the main proprietor of the Bath business, that on-site inspection on sanitary managements will be done next day and requested the City to keep the bath facilities as they are. On 19 July, Health Center officers collected bath water from seven places and recommended voluntary-closing of "Ofunade-no-Yu" business. Because of various reasons, Hiuga City did not accept the recommendation and continued business up to 23 July. Because Legionella pneumophila serogroup 1 strains from 4 patients' sputa and several bath water specimens were determined genetically similar by Pulsed Field Gel Electrophoresis of Sfi I-cut DNA. "Ofunede-no-Yu" was regarded as the source of infection of this outbreak. On 24 July, "Ofunade-no-Yu" accepted the Command to prohibit the business. Among 19,773 persons who took the bath during the period from 20 June to 23 July, 295 became ill, and 7 died. Among them, 34 were definitely diagnosed as Legionella pneumonia due to L. pneumophila SG 1, by either one or two tests of positive sputum culture, Legionella-specific urinary antigen, and significant rise of serum antibody titer against L. pneumophila SG 1. In addition to the 8 items shown by Miyazaki-Prefecture Investigation Committee as the cause of infection. Hiuga City Investigation Committee pointed out following 3 items: 1) Insufficient knowledge and understanding of stuffs on Legionella and legionellosis; 2) Residual water in tubing system after trial runs might lead multiplication of legionellae in it; and 3) Inadequate disinfection and washing for whole circulation system prior the experience bathing. The Hiuga City Committee directed 24 measures to improve the sanitary condition of the facility including following 5 items. 1) Fix the manual for maintenance and management of the bath. 2) Keep sufficient overflow of bath water. 3) Put disinfection of filters into practice. 4) Precise measurement and control of the residual chlorine concentration in bath water. 5) Replacement of filtrating material from crushed porous ceramic into natural sand.