Masafumi Fukuyama

Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum.

The relationships between Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis were examined by means of carbohydrate fermentation, DNA-DNA hybridization, ribotyping and random amplified polymorphic DNA-PCR (RAPD-PCR). The levels of DNA-DNA hybridization among the strains of B. infantis, B. longum and B. suis used in this study were 67-81% under optimal conditions (42 degrees C) and 63-85% under stringent conditions (52 degrees C). Although the strains showed varied carbohydrate-fermentation patterns, the three species were divided into three types, namely the infantis type, the longum type and the suis type, by ribotyping and RAPD-PCR. On the basis of these results, strains of B. infantis, B. longum and B. suis were recognized as distinct groups within a single species. It is concluded that B. infantis and B. suis should be unified as B. longum, the latter species being divided into three biotypes, the infantis type, the longum type and the suis type, by molecular methods.

Isolation and Identification of Methylobacterium Species from the Tap Water in Hospitals in Japan and Their Antibiotic Susceptibility

Contamination of tap water by Methylobacterium species has become a serious concern in hospitals. This study was planned to examine the distribution of Methylobacterium species inhabiting tap water used in Japanese hospitals and antibiotic sensitivity of the isolates in 2004. Species identification of 58 isolates was performed based on the homology of a partial sequence of 16S rDNA. The dominant Methylobacterium species in hospital water were M. aquaticum and M. fujisawaense. To examine the biochemical properties of these isolates, a carbon source utilization was tested using an API50CH kit. The phenotypic character varied widely, and was not necessarily consistent with the results of phylogenic analysis based on the partial 16S rDNA sequence, suggesting that the biochemical properties are not suitable for identification of Methylobacterium species. The isolates were also subjected to antibiotic sensitivity tests. They were resistant to 8 antibiotics, but highly sensitive to imipenem (MIC90=1 μg/ml) and tetracycline (MIC90=8 μg/ml). These findings concerning the isolates revealed the presence of Methylobacterium species with resistance to multiple antibiotics in hospital tap water.

First isolation of urease-positive thermophilic Campylobacter (UPTC) from crows (Corvus levaillantii) in Japan

Two strains of urease-positive thermophilic Campylobacter (UPTC), designated YC98-1 and YC98-2, were identified by biochemical characterization after isolation from the intestinal contents of crows around Yokohama City, Japan, in 1998. The biochemical characteristics of these strains were identical to those of strains described previously. Pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI, SalI, and SmaI of the genomic DNA from the two strains indicated that respective PFGE profiles were distinctly different and distinguishable from each other. This is the first report of the isolation of UPTC from crows (Corvus levaillantii).

Characterization of the Genus Bifidobacterium by Automated Ribotyping and 16S rRNA Gene Sequences

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I–IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis‐longum‐suis type group, (ii) the B. catenulatum‐pseudocatenulatum group, (iii) the B. gallinarum‐saeculare‐pullorum group, and (iv) the B. coryneforme‐indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using EcoRI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.

Characteristics of a Pink‐Pigmented Bacterium Isolated from Biofilm in a Cooling Tower in Tokyo, Japan

Strain K‐20, a Gram‐negative, non‐motile, non‐spore‐forming and strictly aerobic rod, which produces a pale pink pigment, was isolated from biofilm in a cooling tower in Tokyo, Japan. The taxonomic feature of the strain was studied using phenotypic tests and phylogenetic analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain was related to Roseomonas gilardii subsp. rosea, Roseomonas gilardii subsp. gilardii, Roseomonas cervicalis and Roseomonas mucosa at 94.3–94.6 sequence similarities. Growth occurred at 25–40 C and pH 5.0–10.0, optimal at 35 C and pH 7.0. Growth did not occur in the presence of ≧2% NaCl. The API 20NE identification system gave a positive result for urease, L‐arabinose, potassium gluconate, adipic acid, malic acid and trisodium citrate (API code number 0201465). The predominant fatty acids of strain K‐20 were C18:1Δ11 (50.8%) and C16:1 (17.2%). Cells contained ubiquinone 10 (Q‐10) as the major quinone and the G+C content was 72.0 mol%. Based on phenotypic, chemotaxonomic and phylogenetic data, it was assumed that strain K‐20 (=JCM 14634) is a novel species of the genus Roseomonas.

ハトおよびカラスからのVero毒素産生性大腸菌 (VTEC) の分離および血清型

To clarify the source and route of infection with Vero toxin-producing Escherichia coli (VTEC) in humans, we sampled gastrointestinal contents and isolated VTEC from wild birds captured to exterminate harmful birds between August 1997 and January 1998. Pigeons were caught in Sagamihara-shi and crows were caught in Sagamihara-shi, Kawasaki-shi, Yokohama-shi, and the Tokyo metropolitan area. The following results were obtained. 1) VTEC was isolated from 32 of 521 birds (6.1%) examined. Among pigeons, VTEC was isolated from 25 of 262 birds (9.5%) captured in Sagamihara-shi. Among crows, VTEC was isolated from 7 of 184 birds (3.8%) captured in Sagamihara-shi, but not isolated from any bird of 11.4, and 60 birds captured in Yokohama-shi, Kawasaki-shi, and the Tokyo metropolitan area, respectively. 2) Toxin was typed in 33 isolates. There were four VT1-producing isolates (6.5%), 27 VT2-producing isolates (88.7%), and two VT1, VT2-producing isolates (4.8%). 3) The serotypes of the isolates were: O78: H-, 10; O152: H-, 7; O153: H19.2; O164: H-, 1; O128: H-, 1; O164/143: H-, and O1: HUT, 1. The serotype was unknown in 10 isolates. Among 10 isolates for which the serotype could not be determined, auto-aggregation was observed in one isolate. 4) EaeA was investigated in the 33 isolates, and 31 isolates (93.9%) possessed eaeA. The above findings showed that strains with same toxin types and serotypes of human diarrhea-derived VTEC were isolated from pigeons and crows, and the isolates frequently possessed eaeA, which is considered to have an important association with its pathology, suggesting that birds are involved in VTEC infection in humans as a source of infection.

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

Strain L‐47T 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‐47T 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‐47T (JCM 30970T = KCTC 42799T).

乳牛におけるVero毒素産生性大腸菌(VTEC)の汚染状況および分離菌株の血清型

To clarify the source of infection and route of transmission of Verocytotoxin-producing Escherichia coli (VTEC) in humans, we collected fresh feces from healthy dairy cattle reared in Hokkaido, Fukushima, Kanagawa and Okinawa prefectures between June 1996 and March 1997, and attempted to isolate VTEC. The results are described below. 1) VTEC was isolated from 68 (27.1%) of 251 fecal samples tested. VTEC was isolated from 14 (28.0%) of 50 in Hokkaido, 13 (26.0%) of 50 in Fukushima, 20 (39.2%) of 51 in Kanagawa and 21 (21.0%) of 100 in Okinawa. There were no difference in the prevalence among the prefectures. 2) Toxin type and serotype of 85 isolates were determined. Thirty-three isolaties (38.8%) were classified into VT1 toxin and VT2 toxin, respectively, and 19 isolates (22.4%) were classified as the strain that produces both VT1 and VT2 toxins. The toxin types of these isolates were divided by serotypes. The VT1-producing isolates were the most frequent among O111:H-. The VT2-producing isolates included O2:H12, O2:H29, O2:H-, O82:H8, O82:HUT, O153:H19, O153:H42 and O153:H-. Among the isolates producing both VT1 and VT2 toxins, O153:H19 was relatively frequent. Based on findings that many bacterial strains coinciding with toxin types and serotypes of human-derived VTEC isolated from dairy cattle, it was suggested that dairy cattle are closely related to VTEC infection in human as a source of infection.

Isolation, Identification and Antibacterial Susceptibility of Staphylococcus spp. Associated with the Mobile Phones of University Students

From May 2014 to February 2015, 319 university students (male, n=173; female n=146) of 18 to 24 years of age who carried mobile phones or computer tablets were selected as subjects. Staphylococcus spp. were detected in 101 of 319 samples (31.7%). In the present study, 11 strains of S. aureus were isolated and identified, not all of which were methicillin-resistant Staphylococcus aureus (MRSA). Overall, 14 species were identified, with 11 strains (10.9%) of S. xylosus being isolated at the highest frequency. Following this were eight strains (7.9%) of S. cohnii and seven strains (6.9%) each of S. capitis and S. haemolyticus. Staphylococcus spp. isolation was performed with bacterial samples obtained from the mobile phones of 22 specific subjects (males, n=12; females, n=10). Staphylococcus spp. isolation was performed on days -1, 7 and 30 of the experiment. Staphylococcus spp. were positively detected one or more times in 12 subjects (54.5%). In one subject (8.3%), all three tests were positive. Furthermore, two tests were positive in three (25.0%). In the eight remaining subjects (66.7%) Staphylococcus spp. were detected only once. For the three abovementioned tests, we investigated the pulsed-field gel electrophoresis (PFGE) patterns of the strains derived from the mobile phone and from the fingers of three subjects in whom the same bacterial species were isolated twice. From the cases with similarities between strains derived from the fingers and the mobile phones and cases, with consistency in the strains derived from the mobile phone at different times, commonality was observed in the strains derived from the fingers and mobile phones along with chronological uniformity in the strains derived from the mobile phones. A total of 101 Staphylococcus spp. strains were isolated from mobile phones. According to drug susceptibility tests, 99 strains (98.0%) were found to have some degree of resistance to drugs (excluding one strain each of S. aureus and S. haemolyticus). Among these, the strain that showed the highest level of drug resistance was one strain (1.0%) of Staphylococcus spp., which showed resistance to nine drugs. The strain that showed the second highest level of drug resistance was one strain (1.0%) of S. caprea, which showed resistance to seven drugs. In this manner, the drug-resistant tendencies of Staphylococcus spp. isolated from mobile phones were observed.

Bacterial Contamination in Cold Water Samples Obtained from Water Dispensers

We carried out a basic study in order to evaluate the bacterial contamination in water dispensers. Water samples were obtained from water dispensers from October 2012 to November 2013, and standard plate counts (at 36˚C, 24 h) of the samples, as well as heterotrophic plate counts (at 25˚C, 7 d), were estimated with the standard methods for the examination of drinking water in Japan. Standard plate counts exceeding the water-quality standard (1.0×10(2) CFU/ml) were observed in 42 of the 140 samples (30.0%), with a maximum detected bacterial count of 2.1×10(5) CFU/ml. The rate of the standard plate counts exceeding the water quality standard tended to be higher when using a one-way type method or water dispensers with natural water. Ralstonia spp. was most commonly isolated, and Pseudomonas aeruginosa was isolated in a few cases. Some opportunistic pathogens were also isolated, suggesting that we should be more concerned about bacterial contamination in cold water supplied from water dispensers.