Microbial Contamination of Rubber Ducks Floating in Bathtubs of Bathing Facilities, and an Evaluation of Their Washing Methods.

Microbiological contamination inside rubber ducks floating in the bathtub at a "duck bath" of a bathing facility was analyzed by examining bacterial and amoebic counts. The results of microbial tests, such as standard plate count, heterotrophic plate count and Legionella-LAMP (Loopmediated isothermal amplification) , showed that microbial contamination increased in the rubber ducks over time. When the ducks were washed with sodium hypochlorite, those microbial contaminations were not detected; but when the ducks were washed with an electrolyzed water, the standard plate counts and the heterotrophic plate counts were detected in the amount of 103 per duck in the wipe samples. Without proper washing of rubber ducks, bacteria and free-living amoeba can grow and colonize biofilms, and can thereby cause infection in humans. Also, microbial contamination inside ducks may reduce chlorination of the entire bathtub and cause bacterial infection such as Legionellosis from the bathtub water.

Investigations on Contamination of Environmental Water Samples by Legionella using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing.

We analyzed the contamination of environmental water samples with Legionella spp. using a conventional culture method, real-time quantitative PCR (qPCR), and real-time qPCR combined with an amoebic co-culture method. Samples (n = 110) were collected from 19 cooling towers, 31 amenity water facilities, and 60 river water sources of tap water in Japan. Legionella was detected in only three samples (3/110, 2.7%) using the culture method. The rate of Legionella detection using amoebic co-culture followed by qPCR was 74.5%, while that using qPCR without amoebic co-culture was 75.5%. A higher than 10-fold bacterial count was observed in 19 samples (19/110, 17.3%) using real-time qPCR subsequent to amoebic co-culture, compared with identical samples analyzed without co-culture. Of these 19 samples, 13 were identified as Legionella spp., including L. pneumophila and L. anisa, and the non-culturable species were identified as L. lytica and L. rowbothamii. This study showed that the detection of Legionella spp., even in those samples where they were not detected by the culture method, was possible using real-time qPCR and an amoebic co-culture method. In addition, this analytical test combination is a useful tool to detect viable and virulent Legionella spp..

Rapid on-site monitoring of Legionella pneumophila in cooling tower water using a portable microfluidic system.

Legionnaires' disease, predominantly caused by the bacterium Legionella pneumophila, has increased in prevalence worldwide. The most common mode of transmission of Legionella is inhalation of contaminated aerosols, such as those generated by cooling towers. Simple, rapid and accurate methods to enumerate L. pneumophila are required to prevent the spread of this organism. Here, we applied a microfluidic device for on-chip fluorescent staining and semi-automated counting of L. pneumophila in cooling tower water. We also constructed a portable system for rapid on-site monitoring and used it to enumerate target bacterial cells rapidly flowing in the microchannel. A fluorescently-labelled polyclonal antibody was used for the selective detection of L. pneumophila serogroup 1 in the samples. The counts of L. pneumophila in cooling tower water obtained using the system and fluorescence microscopy were similar. The detection limit of the system was 104 cells/ml, but lower numbers of L. pneumophila cells (101 to 103 cells/ml) could be detected following concentration of 0.5-3 L of the water sample by filtration. Our technique is rapid to perform (1.5 h), semi-automated (on-chip staining and counting), and portable for on-site measurement, and it may therefore be effective in the initial screening of Legionella contamination in freshwater.

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)).

Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

Roseomonas tokyonensis sp. nov. isolated from a biofilm sample obtained from a cooling tower in Tokyo, Japan.

Strain K-20(T), a Gram-negative, nonmotile, nonspore-forming and strictly aerobic coccobacillus, which produces a pale pink pigment (R2A agar medium, 30℃, seven days) was isolated from a sample of biofilm obtained from a cooling tower in Tokyo, Japan. A phylogenetic analysis of the 16S rRNA partial gene sequences (1,439 bp) showed that the strain (accession number: AB297501) was related to Roseomonas frigidaquae CW67(T) and Roseomonas stagni HS-69(T) with 97.4% and 96.9% sequence similarity, respectively. Strain K-20(T) formed a distinct cluster with Roseomonas frigidaquae CW67(T) in the phylogenetic tree at a high bootstrap value (93%); however, distance was recognized between the strains. In addition, the DNA-DNA hybridization level between strain K-20(T) and Roseomonas frigidaquae JCM 15073(T) was 33%. The taxonomic data indicate that K-20(T) (=JCM 14634(T) =KCTC 32152(T)) should be classified in the genus Roseomonas as the type strain of a novel species, Roseomonas tokyonensis sp. nov.

Isolation of Legionella species from Noyu (unattended natural hot springs in mountains and fields) samples in Japan.

In order to understand the habitation conditions of the bacteria of the genus Legionella in Noyu (unattended natural hot springs in mountains and fields) in Japan, isolation of Legionella spp. was attempted in the Noyu samples from 11 prefectures nationwide between May and September 2012, and the following results were obtained. Overall, Legionella spp. was isolated from 16 of 43 samples (37.2%). The species was isolated from the Hokkaido region to the Chugoku region but not from the Shikoku region to the Kyushu region. The number of bacteria detected was usually small, less than 5.0 × 10(1) CFU/100 ml, as found in 11 samples (68.8%), while counts of 10(2) or more to 10(3) or less CFU/100 ml were found in two samples (12.5%). Legionella pneumophila was the most commonly found strain, with 19 strains (90.5%) found, and was the dominant species. Regarding the serogrouping, four strains (21.1%) fell under group 1, the most common grouping, followed by three strains (15.8%) in group 3, two strains (10.5%) in group 5, etc. Moreover, the detected bacterial strains other than L. pneumophila included two strains (9.5%) of L. londiniensis. The temperature of the Noyu from which Legionella spp. was isolated was between 33.1°C and 41.5°C with a pH ranging from 5.2 to 8.1. The present report is the first report to clarify the habitation conditions of strains of Legionella spp. isolated from Noyu in Japan.

A coprological survey of intestinal helminthes in stray dogs captured in osaka prefecture, Japan.

This study aimed to investigate intestinal helminth infection in stray dogs in Osaka Prefecture by surveying coprological samples from dogs captured from 2006-2011. Of 212 fecal samples collected, overall prevalence of infection was 39.2%. The most common species was Toxocara canis (25.0%), followed by Trichuris vulpis (8.0%), Spirometra erinaceieuropaei (3.3%), Taeniidae (2.4%), Ancylostoma caninum (1.9%) and Toxascaris leonine (0.5%). In the molecular analysis, all of the taeniid eggs were negative for Echinococcus multilocularis and were identified as other taeniid species (e.g., Taenia pisiformis). Our results suggest that stray dogs remain important infection reservoirs of zoonotic parasites in Osaka Prefecture. Therefore, control of stray dogs is crucial for reducing the risk of public health problems due to parasitic infections.

[Isolation of Legionella species from hot springs used for foot-soaking].

OBJECTIVES: We aimed to investigate the presence of Legionella species in hot-spring baths for feet, which have been rapidly increasing in number in Japan in recent years. METHODS: The investigations were conducted between March 2009 and November 2011, and hot springs throughout the country were sampled. Legionella isolates were confirmed on the basis of the method described in the "Manual for the countermeasure to legionellosis, 3rd Edition." In this method, the samples were concentrated and smeared on GVPCalpha agar medium after acid treatment and cultured for 7 days at 36 degrees C. Gram-negative rods that required L-cysteine were determined to be Legionella species. After the first identification using Duopath Legionella (Merck Ltd. Japan), isolates were identified on the basis of agglutination reaction of an immune serum or genetic examination. RESULTS: Legionella was isolated from 56 of the 196 samples (28.6%) and was confirmed to widely inhabit hot-spring baths from Hokkaido to Kyushu. The isolation rates were the highest (40.9%) in facilities installed around railway stations, including those on platforms. The average microbial density of Legionella species per 100 ml of hot spring water was 1.0 x 10(1) CFU, with a maximum value of 1.0 x 10(4) CFU, although the microbial density in most of the samples (34 samples; 60.7%) was less than 10(2) CFU. Legionella pneumophila was the dominant strain, and 16 strains (23.9%) of serogroup 1 were isolated. In addition, 7 strains (10.4%) of Legionella londiniensis and 4 strains (6.0%) of Legionella rubrilucens were isolated. CONCLUSION: Legionella species inhabit approximately 30% of all hot springs for foot-soaking in the country. Although the number of viable organisms is small, the dominant presence of Legionella pneumophila, a major pathogen responsible for legionnaire's disease, raises the possibility of legionnaire's disease in users of these hot springs. Therefore, each institute should understand the present distribution of Legionella species in these hot springs and undertake appropriate sanitary measures.

Identification of Legionella rubrilucens isolated from a hot spring for foot-soaking in Niigata, Japan.

In May 2011, strain HYNE-20 (=JCM 17837) was isolated from a sample of hot spring water from a foot spa in Niigata, Japan, by a plating method using glycine vancomycin polymyxin B cycloheximide α-ketoglutarate (GVPCα) medium at 36°C for 7 d. The 16S rDNA sequences (1,469bp) of this strain (accession number: AB638719) had high (99.7%) similarity to Legionella rubrilucens, and we identified that this strain was indeed Legionella rubrilucens. When this strain was cultured on buffered charcoal yeast extract α-ketoglutarate (BCYEα) agar at 36°C for 7 d, it exhibited red autofluorescence under UV light (365 nm) . The dominant cellular fatty acids of the strain HYNE-20 were 16:1ω7c (29.9%) , and the guanine-plus-cytosine (G+C) content of DNA was 49.0 mol%. This is the first report that Legionella rubrilucens was isolated from a hot spring for foot soaking.

The first case of Legionella nagasakiensis isolation from hot spring water.

In August, 2010, strain HYMO-6 was isolated from a sample of hot spring water in Aomori, Japan. The 16S rDNA sequences (1,496bp) of this strain (accession number: AB597175) had a similarity of less than 96.6% to other Legionella species, prompting us to hypothesize that this strain might be a novel species belonging to the genus Legionella. However, in March of 2011, it was became clear that the HYMO-6 strain (=JCM 17450 =KCTC 23560 =DSM 24727) was Legionella nagasakiensis CDC-1796-JAP-E(T) (=ATCC BAA-1557(T) =JCM 15315(T)). When this strain was cultured on BCYEα agar at 36°C for 7 d, no long cells were observed. The dominant fatty acids of strain HYMO-6 were 16:1ω7c (32.4%), and the DNA G+C content was 42.0 mol%.

Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan.

Here, we carried out a survey to determine the prevalence of free-living amoebae (FLA) in tap-water sources from rivers and water treatment plants located in Osaka Prefecture, Japan. A total of 374 raw water samples were collected from 113 sampling points. The samples were filtrated and transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli and incubated for 2 to 7 days at 30 degrees C or 42 degrees C. The plates were examined by microscopy to morphologically identify FLA families, and polymerase chain reaction and sequence analysis were then performed to define the species of the detected Naegleria and Acanthamoeba isolates. A total of 257 of 374 samples (68.7%) were positive for FLA by microscopy, and among these there were 800 FLA isolates, including Acanthamoeba and Naegleria species. Sequence analysis identified five Acanthamoeba spp. isolates of the known pathogenic T4 genotype and 43 Naegleria australiensis isolates, a reported pathogen to mice and also of concern as a potential pathogen to humans. Our results suggest a wide distribution of FLA, including potential pathogenic species, in tap-water sources of western Japan.

Detection and genotyping of Cryptosporidium from brown rats (Rattus norvegicus) captured in an urban area of Japan.

We investigated the prevalence and genotypes of Cryptosporidium parasite in 50 brown rats (Rattus norvegicus) inhabiting an urban area of Japan. Fecal samples collected from the animals were examined by an immuno-fluorescence assay (IFA). Genomic DNA was extracted directly from fecal sample of each animal and nested PCR was performed to amplify part of the 18S ribosomal RNA (18SrRNA) of the Cryptosporidium species. The detection rate was 8% by IFA and 38% by nested PCR. The sequence and phylogenetic analyses of 13 PCR products showed that the Cryptosporidium from brown rats were clustered into four distinct genotypes. Interestingly, one of the four genotypes was significantly distinct from the C. parvum and C. hominis genotypes. Our results suggest the existence of a new genotype of Cryptosporidium in brown rats.