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Group A streptococcus is a bacterium that resides in the throat and skin and causes respiratory infection and occasionally glomerulonephritis and rheumatic fever. Streptolysin O (SLO) produced by Streptococcus pyogenes (S. pyogenes) binds to the cell membrane, particularly to that of white and red blood cells, and is toxic to the cells and tissue. In this study, we evaluated the inhibitory activity of water-soluble polyphenols in olives (Olea europaea) against SLO-induced hemolysis. Hydroxytyrosol inhibited SLO-induced hemolytic activity, and the amount required for 50% inhibition of hemolysis was 1.30 µg. These findings suggest that the water-soluble polyphenols contained in olives have inhibitory activity against SLO-induced hemolysis.

We report a facile method for preparing monodisperse hybrid smart gel particles with various morphologies by using microfluidic techniques and the swelling–shrinking phenomenon of thermosensitive poly(N-isopropylacrylamide) (PNIPAM) gel particles. We demonstrate that PNIPAM–polyacrylamide snowman-like, raspberry-like, and dumbbell-like hybrid gel particles can be prepared by controlling the flow rate and temperature.

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)).

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.

Background/Purpose Whether absorption of verotoxin (VT) 2 from the intestine in mice is inhibited by administration bovine immune colostral antibody against VT2 was investigated. Methods Three-week-old mice were administered VT2 solution at 477.8 ng/mL or 955.6 ng/mL, and bovine immune colostral antibody against VT2 was then administered three times. Whey without antibody against VT2 was administered to control mice. Serum levels of VT2 were measured by fluorescence enzyme immunoassay. Results Serum levels of VT2 in mice administered VT2 solution at 477.8 ng/mL and bovine immune colostral antibody against VT2 scarcely changed. By contrast, serum levels of VT2 in control mice increased and peaked 12 hours after administration. Peak values were 15.4 ± 5.04 ng/mL. Furthermore, serum levels of VT2 at 12 hours and 16 hours in control mice were significantly higher than in mice administered bovine colostral antibody against VT2. Serum levels of VT2 in mice administered antibody at 955.6 ng/mL showed no significant differences between repeated administration of bovine immune colostral antibody and controls. Conclusion These results suggest that absorption of VT2 from the intestine was inhibited by repeated administration of bovine immune colostral antibody against VT2 at early stages of Escherichia coli O157:H7 infection, whereas VT2 in the intestine remained at low levels.

Drug susceptibility testing was carried out using 14 antibiotics in order to identify trends in the antibiotic tolerance of 142 strains of Enterobacteriaceae isolated from horsemeat commercially available for raw consumption (basashi). A comparison of the sensitivity to the 14 antibiotics using the 90% MIC (minimum inhibitory concentration) values (MIC90) showed the strongest tolerance to ampicillin (ABPC) at a concentration of > 128 μg/mL, followed by that to fosfomycin (FOM) at a concentration of 128 μg/mL. When the sensitivity to these antibiotics was examined for each individual genus of tested bacteria, Hafnia spp. exhibited relative tolerance to ceftazidime (CAZ) and ceftriaxone (CTRX) at a concentration of 4 μg/mL and 2 μg/mL, respectively, which was high in comparison to that observed for the other strains. Furthermore, Raoultella spp. and Serratia spp. were found to be highly resistant to tetracycline (TC) at a concentration of 128 μg/mL and 64 μg/mL, respectively. Of the 142 strains of test bacteria, 140 (98.6%) demonstrated resistance to ABPC, with the exception of Hafnia alvei and Klebsiella pneumonia. In addition, a total of eight strains (5.6%), seven Serratia marcescens strains and one Raoultella terrigena strain, were found to be resistant to TC. Furthermore, one strain of Citrobacter freundii exhibited resistance to nalidixic acid (NA), while another displayed resistance to ofloxacin (OFLX) (0.7% each), and one strain (0.7%) each of Enterobacter cloacae, Serratia marcescens and Citrobacter youngae demonstrated resistance to fosfomycin (FOM), streptomycin (SM) and kanamycin (KM), respectively. A single strain of C. freundii was found to be resistant to three antibiotics, ABPC, NA and OFLX. Resistance to two antibiotics was confirmed in 11 strains, including seven strains of S. marcescens and one strain of R. terrigena (a total of eight strains) resistant to ABPC and TC, and one strain each of C. youngae, S. marcescens and E. cloacae resistant to ABPC and KM, ABPC and SM, and ABPC and FOM, respectively. In addition, 128 strains were resistant to the single antibiotic of ABPC alone. Of the 140 strains demonstrating antibiotic resistance, 137 (97.9%) retained the conjugative R-plasmid transfer factor, excluding three strains of S. marcescens. All transfer factors were ABPC and retained by a high proportion of the bacterial groups, with one strain (100%) being resistant to three antibiotics, nine (81.8%) of the 11 strains being resistant to two antibiotics, and 127 (99.2%) of the 128 strains being resistant to a single antibiotic. In addition, we examined ESBL productivity in the 140 strains of bacteria demonstrating drug tolerance; however, no strains exhibited this characteristic. Therefore, further observation is required to ascertain trends in antibiotic-tolerant bacteria.

For a microbial ecological analysis, 20 strains of Legionella pneumophila isolated from both unchlorinated Noyu (unattended natural hot spring) samples and chlorinated hot spring bath water samples collected throughout Japan were subjected to a clustering analysis on the basis of a Pulsed-Field Gel Electrophoresis (PFGE) pattern analysis. The PFGE patterns obtained from 19 strains of L. pneumophila after digestion with SfiI were used to divide the strains into two groups (Groups A and B), although the similarity level was very low among the groups. Group A consisted of 8 strains, and all of these strains were isolated from hot spring bath water samples. Group B consisted of 11 strains, and all but two of these strains were isolated from Noyu samples. The chlorine resistance (99.9% CT values) of these isolates was experimentally confirmed, and we attempted to define the relationship between chlorine resistance and the geno-cluster. The average CT value of group A (8 strains from hot spring bath water) was 0.49 mg･min/l and the average of group B (9 strains from Noyu samples) was 0.29 mg･min/l. No remarkable differences in the CT values for the groups were found. A chlorine-sensitive Noyu strain (0.14 mg･min/l) and a chlorine-resistant strain (0.62 mg･min/l) from hot spring bath water were then compared to identify any differences in their lipid composition. There was no notable difference in the ratio of saturated to unsaturated fatty acids between the chlorine-sensitive and chlorine-resistant bacteria. However, the chlorine-sensitive and chlorine-resistant bacteria demonstrated differences in the relative percentages of cell wall and cell membrane fatty acids.

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.

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.

In order to obtain an understanding of the inhabitation of Legionella spp. in hot spring bath water, the isolation of Legionella spp. was attempted in 30 prefectures nationwide in 2009-2010, and the following results have been obtained. 1) Isolation of the bacteria was achieved for 89 out of 366 samples (24.3%). When this was broken down into individual regions, the isolation rate in Kyushu and Okinawa regions was the highest at 58.6%, followed by Hokkaido at 42.4%. With regard to isolation by facility, isolation was achieved at 49 out of 124 facilities (39.5%). 2) In the 89 samples form which genus Legionella were isolated, counts of Legionella spp. in 100 ml of hot spring water were less than 102 CFU in 45 samples (50.6%), followed by 28 samples within the 102 CFU range (31.5%). 3) Of the bacterial species of the 111 strains isolated from the 89 samples of hot spring water collected from all over the country, the bacterial strain that was most frequently identified was Legionella pneumophila, and it accounted for 78 strains (69.6%). Among these strains by serogroup, group 1 was the most common with 25 strains (32.1%), followed by group 4 with 17 strains (21.8%). Moreover, out of the bacterial strains that were identified other than Legionella pneumophila, Legionella londiniensis was the second most common, with 20 strains (17.9%), followed by Legionella micdadei with 5 strains (4.5%). 4) The 90% MIC rate (MIC90) of Legionella pneumophila isolates (82 strains) was tested with 10 drugs. Out of the 10 drugs, rifampicin showed the best antimicrobial activity of 0.016 μg/ml, followed by imipenem at 0.5 μg/ml. However, isolates showed high resistance to minocycline and piperacillin with the MIC90 of minocycline at 16 μg/ml, and that of piperacillin at >256 μg/ml.

A microfluidic device with three-dimensional flow channels is fabricated by stereolithography according to a computer-aided design (CAD) model. By injecting water and oil phases into the device, a monodisperse water-in-oil emulsion is formed. We show that monodisperse thermosensitive poly(-isopropylacrylamide) gel particles can be prepared by photopolymerization of a gelation reagent dissolved in the water phase.

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%.

Heterotrophic bacteria constituting the biofilm produced in a kitchen sink drain were analyzed, and the biofilm formation abilities and the hydrophobicity of the cell surface layer were measured for the isolates. When the biofilm sample was cultured at 36 degrees C and 25 degrees C for 7 days, there were about 10 times more colonies on oligotrophic R2A agar medium than on eutrophic BHI agar medium. From isolates from the biofilm sample, 13 bacterial species were detected. To examine the biofilm formation ability of these strains, we measured the absorbance (OD570) by crystal violet staining. The absorbance of Brevibacterium casei 7-R-36-1 was the highest (3.029). In the comparison of the absorbance values between genera, Brevibacterium spp. (4 strains) showed the highest absorbance (mean: 2.056), followed by K. pneumoniae (4 strains) with a mean of 1.111. Regarding the hydrophobicity of the isolates, the values ranged from 0.002 for P. nitroreducens (strain 1-B-36-2) to 0.096 for M. lacticum (strain 5-R-25-2). The hydrophobicity values were generally low, and the cell surface layer of all tested strains was highly hydrophilic. The diversity of species of bacteria in the biofilm sample produced in the kitchen sink drain was recognized, and all the isolates had biofilm formation abilities.

Enterobacter sakazakii感染症に関する基礎的研究の一環として感染経路や感染源を解明するため，乳幼児への感染源として密接に関連性が考えられる育児用調整粉乳（以下粉乳）における本菌の汚染状況と各種抗菌剤の薬剤感受性試験を行ったところ，以下の成績が得られた。1. 粉乳から本菌の分離を試みたところ，149例9例（6.6％）から本菌が分離された。その内訳において，国産では61例中4例（6.6％）から，外国産では88例中5例（5.7％）からそれぞれ分離された。2. 本菌が分離された粉乳の推定菌量は，国産では4例とも3.6MPN/Kgであった。外国産では5例中4例が国産と同様に3.6MPN/Kgであったが，残りの1例は9.1MPN/Kgと高い菌量であった。3. 薬剤感受性試験におけるMIC_90値の比較では，CTX，CTRX，CPZ，CAZ，CPR，CZOP，GM，MEPMおよびCPFXの9薬剤に高い感受性を，PIPC，EM，MINO，CPおよびRFPの5薬剤に中間の感受性をそれぞれ示したが，ABPCとLCMの2薬剤は耐性を示すことが明らかになった。, To clarify the route and source of infection as a basic study of Enterobacter sakazakii infection, we investigated the state of contamination with this type of bacteria in modified milk powder for infants, which may be closely associated with infantile infection, and conducted drug sensitivity tests using various antimicrobial agents. The following results were obtained:1. In 9 (6.6%) of 149 milk powder samples, this type of bacteria was isolated. They consisted of 4 manufactured in Japan (6.6%, 4/61) and 5 manufactured in other countries (5.7%, 5/88).2. The estimated bacterial amount in milk powder ranged from less than 3 to 3.6 MPN/Kg in the Japanese 4 samples. In the foreign 5 samples, it ranged from less than 3 to 9.1 MPN/Kg.3. The MIC_90 values on drug sensitivity tests were compared. Enterobacter sakazakii was highly susceptible to CTX, CTRX, CPZ, CAZ, CPR, CZOP, GM, MEPM, and CPFX. It showed an intermediate susceptibility to PIPC, EM, MINO, CP, and RFP. However, it was resistance to ABPC and LCM.

Resistance to intestinal proteases and efficacy against verotoxin (VT) 2 of a colostral antibody administered orally via catheter were investigated in beagle dogs. Bovine colostral antibody remained in the small intestine for 2 hours, whereas little serum antibody remained at 1.5 hours after administration. The bovine colostral antibody was not inactivated by proteases in the small intestine. Furthermore, the antibody activity of S-IgA did not change until 2 hours after administration; however, the activity of IgG and IgM antibodies decreased by two-thirds and two-fifths at 2 hours after administration, respectively. Seven beagle dogs inoculated with Escherichia coli (E. coli) O157:H7 producing VT2 were administered bovine colostral antibody or bovine colostral whey without antibody. With administration of bovine colostral whey without antibody, the amount of VT2 in feces decreased gradually after administration and increased again at 5 days after inoculation, while bovine colostral antibody significantly reduced the amount of VT2 in feces the day after administration. In addition, 9 beagle dogs were administered bovine colostral antibody, bovine plasma antibody or saline. The amount of VT2 in feces also significantly reduced rapidly after administration of bovine colostral antibody than administration of bovine plasma antibody or saline.

Forty-five strains of Pseudomonas aeruginosa isolated from cockroaches captured in hospitals were investigated with regard to their biofilm-forming ability and resistance to various disinfectants in various cellular states. The hydrophobicity value varied among the test strains from 0.001 to 0.241, and the mean +/- standard deviation (SD) was 0.101 +/- 0.074. The relative viscosity of the extracellular product was measured. All test strains produced a mucous substance, and the value was 1.05-1.30 (mean +/- SD: 1.11 +/- 0.06), showing that all test strains had a biofilm-forming ability. Bactericidal experiments were performed using 6 disinfectants: ethanol, Hibitane, Isojin, Osvan, Tego 51, and Welpas. When bacteria in suspension were exposed to the disinfectants for 1 min (suspension test), 5 of the 6 disinfectants excluding Tego 51 completely killed all test strains, showing high bactericidal effects. In contrast, when adherent bacteria were exposed to the disinfectants for 1 min (adhesion test), the killing rate by Welpas was 100%, but those by Isojin and ethanol were lower (88.9 and 60.0%, respectively), that by Osvan was only 4.4%, and no bacteria were killed by Hibitane or Tego 51. These findings showed that the bactericidal effects markedly decreased in the case of adherent P. aeruginosa.

Pseudomonas aeruginosa strains were isolated from 45 of 370 (12.2%) cockroaches captured in hospitals. By cockroach species, the bacterial strains were isolated from 39 of 181 (21.5%) Periplaneta fuliginosa and 6 of 183 (3.3%) Blattella germanica, showing a significant difference (p

Bifidobacterium属は,高いプロバイオティクス効果および細菌の分類学のモデルケースとして興味深い状況にある。(独)理化学研究所バイオリソースセンター微生物材料開発室(JCM)に保存されているBifidobacterium属およびその近縁種で,ヒトを分解源とする基準株を検索したところ,9菌種(菌株)が候補としてあげられ,その全ゲノム解析を開始した。その菌種は,Table 1に示したとおりであるが,B. bifidum, B. breveおよびB. catenulatumの3菌種は全ゲノム配列を決定し,B. dentium, P. denticolens, S. inopinataおよびG. vaginalisの3菌種は,コンティグが15前後になっており,B. longumおよびb. scarcoviiについては,ショットガンライブラリの作製を完了した。各菌株について引き続きシークエンス作業を進め,全ゲノム解析後は,大規模なBifidobacterium属の比較ゲノム解析を行う予定である。, It is interested in Genus Bifidobacterium shows the probiotic effects and as the model case of taxonomy. The nine species of Bifidobacterium and the closest related species of the type strains isolated from human origin were obtained from the Microbe Division, Japan Collection of Microorganisms (JCM), RIKEN BioResource Center. We have started sequencing of the complete genome of nine species. We have done the complete genome sequencing of B. bifidum, B. breve and B. catenulatum. The contigs of Parascardovia denticolens, Scardovia inopinata and Gardnerella vaginalis is less than fifteen. The draft sequences of B. dentium genomes have been obtained, and the shotgun library of the B. scardovii and B. longum genomes were prepared. We are going to widely comparative genomics in the Family Bifidobacteriaceae, when all the genomes will be finished.

Campylobacter感染症に関する基礎的研究の一環として,2002～2006年にかけてヒト下痢便および鶏肉,鶏糞便から分解した株を用いて,血清型別,RAPD法による分子疫学的解析,薬剤感受性試験およびキノロン系薬剤に対する耐性株の遺伝子変異について検討を行ったところ,以下の成績が得られた。1)ヒト臨床由来53株と鶏由来102株の計155株を用いて血清型別を行ったところ,85株(54.8%)が16菌型に型別されたが,残りの70株(45.2%)は型別不能であった。型別された85株の内訳はY群に20株(12.9%)と最も多く,次にB群18株(11.6%),D群8株(5.2%)であった。由来別において,ヒト臨床由来ではB群に,鶏由来ではY群にそれぞれ多く型別された。2)RAPD法による分子疫学的検討を行ったところ,供試した155株中124株(80.0%)が類似度89%≦において1～30群に分類され,22群に22株(14.8%)と最も多く,次に18群10株価7%),16群8株(5.4%)などであった。由来別においては同一由来別に分類される傾向が強く認められ,由来の異なる株が共通した群へ分類される株は少数であった。3)ヒト下痢症由来53株の各種薬剤に対するMICの分布状況をMIC_で比較したところ,GMが0.5μg/mlで最も感受性が高く,次にSMとLMが各2μg/ml,EMが4μg/ml,KMとCPが各8μg/ml,RXMが16μg/ml,MINOとCPFXが各32μg/ml,NAとNFLXが各128μg/ml,ABPC,PIPC,CEXおよびTCが各128, As part of a basic study on campylobacteriosis, we performed serotyping, molecular-epidemiological analysis using the RAPD method, and drug sensitivity tests of strains isolated from human diarrheal feces, chicken meat, and chicken feces between 2002 and 2006, and investigated gene mutations in quinolone-resistant strains. 1) On serotyping of 53 human clinical isolates and 102 chicken meat-derived isolates, a total of 155 isolates, 16 types were identified in 85 isolates (54.8 %), but the remaining 70 isolates (45.2 %) could not be typed. Of the 85 typed isolates, 20 isolates (12.9 %) were typed Group Y, which was the most frequent, followed by 18 isolates typed Group B (11.6 %), and 8 isolates typed Group D (5.2 %). Regarding the origin, Groups B and Y were most frequently identified in human clinical and chicken meat-derived isolates, respectively. 2) On molecular-epidemiological analysis by RAPD, 124 of the 155 isolates (80.0 %) were classified into Groups 1-30 at a similarity of 89 % ≦, and 22 isolates were classified into Group 22 (14.8 %), which was the most frequent, followed by 10 isolates into Group 18 (6.7 %), and 8 isolates into Group 16 (5.4 %). Regarding the origin, isolates with the same origin tended to be classified into the same group, and only a few isolates were classified into groups consisting of isolates with a different origin. 3) MIC distribution in 53 human diarrhea-derived strains was compared among various drugs based on MIC_. The MIC_ value of GM was 0.5 mg/ml, showing the highest sensitivity, followed by: SM and LM, 2 mg/ml; EM, 4 mg/ml; KM and CP, 8 mg/ml; RXM, 16 mg/ml; MINO and CPFX, 32 mg/ml; NA and NFLX, 128 mg/ml; and ABPC, PIPC, CEX, and TC, 128 mg/ml

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 ofor=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 C18:1Delta11 (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.

我々は,Lactobacillus reuteri JCM 1112^T, Lactobacillus fermentum IFO 3956, Lactococcus garvieae(ブリに対する毒性株), Lactococcus garvieae(ブリに対する無毒株)およびLactobacillus rhamnosusの全ゲノム解析を完了した。そして,Bifidobacterium breve, Bifidobacterium bifidumおよびBifidobacterium catenulatumについては,全ゲノム配列を決定中である。プロバイオティクスとは,"宿主の健康に対して貢献する生きた微生物"と定義付けられている。本プロジェクトでは,L.reuteriのプロバイオティクス効果を分子レベルで解明することを目的としている。そこで,分類学的にL.reuteriと近縁であるにもかかわらず,プロバイオティクス効果のほとんど認められないL.fermentumについても全ゲノム解析を行った。両菌種の比較ゲノム解析の結果,L.reuteriのゲノムには,抗菌物質であるロイテリン合成系にかかわる遺伝子の48kbの"genomicisland"を有しており,哺乳動物の腸内フローラバランスに良い影響を及ぼしていると推察された。我々は,glycerol dehydratase subunit motif(PFAM PFO2286-PFO2288)として,L.reuteriゲノムに3つの遺伝子(LR1633-LR1635)を検出し,gupCDE(glycerol utilizing enzymes)と命名した。本研究では,そのgupCDE遺伝子破壊候補株(L.reuteri Δ gupCDE)を得て,PCR解析やglycerol dehydratase酵素活性などから,L.reuteri Δ gupCDEを認めた。今後,親株(L.reuteriJCM1112^T株)とL.reuteri Δ gupCDEのそれぞれノトバイオート(モノアイソレート)マウスを解析して,プロバイオティクス効果を明らかにしていく予定である。, We determined the complete genome sequences of Lactobacillus reuteri JCM 1112^T, Lactobacillus fermentum IFO3956, Lactococcus garvieae (a toxic strain for yellowtail), Lactococcus garvieae (a non-toxic strain for yellowtail) and Lactobacillus rhamnosus.The genome analysis of Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium catenulatum is on going. Probiotics are live microorganisms that confer health benefits on the host. The probiotic L. reuteri and non-probiotic L.fermentum are phylogenetically closest to L.reuteri in the Lactobacillus. To elucidate the molecular basis on the probiotic property exhibited by L.reuteri, comparative analysis of both genomes showed that L.reuteri harbored the unique 48kb genomic island containing the genes necessary for the biosynthesis of a reactive 3-hydroxypropionaldehyde (reuterin), which may influence the microflora balance in the gut. We identified three genes (LR1633-LR1635) with a glycerol dehydratase subunit motif (PFAM PF02286-PF02288) in the L.reuteri genome. These genes are designated gupCDE (glycerol utilizing enzymes) in this study. The gupCDE knock-out L.reuteri (L.reuteri Δ gupCDE) was obtained, and the reuterin synthesis will be conducted by glycerol dehydratase in the glycerol metabolism in vivo by the gnotobiotic mice mono-associated with L.reuteri and L.reuteri Δ gupCDE.

To clarify the route and source of Vibrio vulnificus infection, we conducted molecular epidemiological investigation by DNA analysis of 355 environmental isolates (seawater-derived strain: 86, sea mud-derived strain:36, and oyster-derived strain: 233) and 65 human clinical isolates, for a total of 420 isolates, using pulse field gel electrophoresis (PFGE), with the following results. 1. When DNA was cleaved with 2 enzymes, Not I and Sfi I, and subjected to PFGE, Not I DNA interpretation was 76.9%, and Sfi I cleavage was 97.9%, showing that Sfi I was superior in cleaving DNA of this bacteria. 2. Sfi I-interpreted strains were subjected to PFGE and migration patterns were analyzed by UPGMA, but close classification was not possible because similarity was low, this infectious disease clearly originated from multiple rather than a single-clone. In this cluster, we concluded that this infectious disease was acquired through contact between the environment and human beings and viceversa. We identified an assortment of clinical isolates and environment-derived strains among more than 89% of strain groups tested, none of which could be expected to have the same origin. We conclued DNA analysis on these two types of restriction enzymes using PFGE, but were unable to classify test results in detail due to the proliferation of migration patterns and low degree of similarity.

In basic studies on campylobacteriosis, we tested 53 strains from human diarrhea stools and 102 strains from chicken meat and feces obtained between 2002 and 2006 for drug sensitivity to different drugs and gene mutation in quinolone-resistant strains. 1) Of 15 drugs tested, all were resistant to one or more of the following 10 drugs: CEX, 99.4%: ABPC, 59.4%; NA, 40.6%; NFLX, 40.0%; TC and CPFX, 39.4%; PIPC, 38.1%; MINO, 30.3%; KM, 3.2%; and SM, 2.6%. 2) Of 155 drug-resistant strains, 28 (18.1%) were resistant to single drugs and 127 (81.9%) were resistant to multiple drugs. The most frequent pattern of multipledrug resistance was ABPC/PIPC/CEX, followed by ABPC/PIPC/CEX/TC/MINO/NA/NFLX/CPFX. 3) Mutation of GyrA (Thr86 --> Ile) was detected in 43 (97.7%) of 44 quinolone-resistant strains. We found that resistance to beta-lactams, quinolones, and tetracycline antibiotics was high, and most resistant strains were resistant to multiple drugs. We also found that most quinolone-resistant strains had GyrA mutation.

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)).

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.

Drug susceptibility testing was carried out using 14 antibiotics in order to identify trends in the antibiotic tolerance of 142 strains of Enterobacteriaceae isolated from horsemeat commercially available for raw consumption (basashi). A comparison of the sensitivity to the 14 antibiotics using the 90% MIC (minimum inhibitory concentration) values (MIC90) showed the strongest tolerance to ampicillin (ABPC) at a concentration of128 μg/mL, followed by that to fosfomycin (FOM) at a concentration of 128 μg/mL. When the sensitivity to these antibiotics was examined for each individual genus of tested bacteria, Hafnia spp. exhibited relative tolerance to ceftazidime (CAZ) and ceftriaxone (CTRX) at a concentration of 4 μg/mL and 2 μg/mL, respectively, which was high in comparison to that observed for the other strains. Furthermore, Raoultella spp. and Serratia spp. were found to be highly resistant to tetracycline (TC) at a concentration of 128 μg/mL and 64 μg/mL, respectively. Of the 142 strains of test bacteria, 140 (98.6%) demonstrated resistance to ABPC, with the exception of Hafnia alvei and Klebsiella pneumonia. In addition, a total of eight strains (5.6%), seven Serratia marcescens strains and one Raoultella terrigena strain, were found to be resistant to TC. Furthermore, one strain of Citrobacter freundii exhibited resistance to nalidixic acid (NA), while another displayed resistance to ofloxacin (OFLX) (0.7% each), and one strain (0.7%) each of Enterobacter cloacae, Serratia marcescens and Citrobacter youngae demonstrated resistance to fosfomycin (FOM), streptomycin (SM) and kanamycin (KM), respectively. A single strain of C. freundii was found to be resistant to three antibiotics, ABPC, NA and OFLX. Resistance to two antibiotics was confirmed in 11 strains, including seven strains of S. marcescens and one strain of R. terrigena (a total of eight strains) resistant to ABPC and TC, and one strain each of C. youngae, S. marcescens and E. cloacae resistant to ABPC and KM, ABPC and SM, and ABPC and FOM, respectively. In addition, 128 strains were resistant to the single antibiotic of ABPC alone. Of the 140 strains demonstrating antibiotic resistance, 137 (97.9%) retained the conjugative R-plasmid transfer factor, excluding three strains of S. marcescens. All transfer factors were ABPC and retained by a high proportion of the bacterial groups, with one strain (100%) being resistant to three antibiotics, nine (81.8%) of the 11 strains being resistant to two antibiotics, and 127 (99.2%) of the 128 strains being resistant to a single antibiotic. In addition, we examined ESBL productivity in the 140 strains of bacteria demonstrating drug tolerance; however, no strains exhibited this characteristic. Therefore, further observation is required to ascertain trends in antibiotic-tolerant bacteria.

プロバイオティクス乳酸菌としてLactobacillus reunteri (2, 039, 414 bp),そしてその知見のほとんどみられないLactobacillus fermentum(2, 098, 685 bp)の全ゲノム配列を決定した。両菌種は絶対ヘテロ発酵型の乳酸菌としては初めての全ゲノム配列決定となった。Lactobacillus属には,他にホモ発酵型と条件的ヘテロ発酵型の乳酸菌があるが,上記両菌種では,解糖(EMP)経路では必須の6-phosphofructokinase遺伝子の欠損が明らかとなり,そのためペントース・リン酸経路しか機能しなかった。各ORF内で3つ目の塩基の置換が非常に多く,そのことが遺伝子の機能は変わらず,G+C含量に10%もの違いが生じることが認められた。L. reuteriのゲノム中で多くのmobile elementsを発見したが,それはLactobacillus属が環境への適合性を高くし,生存性を有利にするのに貢献していることが示唆された。, We present the complete genome sequences of two lactic acid bacteria (LAB), probiotic Lactobacillus reuteri (2, 039, 414 bp) and non-probiotic Lactobacillus fermentum (2, 098, 685 bp), which share obligately the heterofermentative property in glucose metabolism and have a phylogenetically closest relation. The two genomes are compared with each other and with other lactobacilli exhibiting facultatively heterofermentative and obligately homofermentative properties. A striking common feature of the two lactobacilli is a lack of a gene encoding 6-phosphofructokinase essential for the Embden-Meyerhof-Pamas (EMP) pathway, so that the pentose phosphate pathway is utilized in them. Furthermore, numerous mobile elements in L. reuteri is found in the genomes. These data imply that a genetic plasticity-environment relationship may account for the diversity between these lactobacilli.

The neutralization efficacy of bovine colostral antibody against verotoxin (VT) 1 and 2 was investigated. Cows were immunized with VT1 or VT2 fourteen times at 7-day intervals. A colostral antibody exhibiting high titers was obtained from immunized cows. Survival rates were evaluated in mice administered VT1 or VT2, and those infected with Escherichia coli (E. coli) O157:H7 producing VT1 or VT2. Survival rates after VT1 administration were 100% in the single-administration group, 90% in the repeat-administration group, and 78.6% in the control group. Survival rates after VT2 were 75.0% in the single-administration group, and 100% in the repeat-administration group. All mice in the control group died. Colostral antibody and fosfomycin (FOM) in the colostral antibody group and FOM and skim milk in the control group were administered three times per day for 5 days to mice infected with E. coli O157:H7 producing VT1 or VT2. Survival rates after inoculation with E. coli O157:H7 producing VT1 were 80.0% in the colostral antibody group, and 63.6% in the control group. Survival rates after inoculation with E. coli O157:H7 producing VT2 were 83.3% in the colostral antibody group, and 20.0% in the control group. The survival rate in mice without treatment following inoculation with E. coli O157:H7 producing VT2 was 88.2%. The survival rates in mice infected with E. coli O157:H7 strains producing VT1 or VT2 improved after administration of this colostral antibody, which exhibited neutralization efficacy against VT.

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 Eco RI. 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.

一般的に雄は仔猫に興味を示すことはなく,仔猫の世話をするのは専ら雌猫であるが,今回は,雄成猫が感染源となり,仔猫へ感染するという結果となった。, A multicat household experienced an epidemic of feline calicivirus (FCV) infection. We analyzed molecular evolution of isolated FCVs by a phylogenic tree reconstructed around the genotype of FCV that was isolated from the cat of infectious origin. FCV was isolated from eight of 34 cats: two 1-year-old, male littermates that became latent carriers (Ao198-1 and Ao199-1); two 3-month-old littermates (Ao222-1 and Ao224-1); three 4-month-old littermates (Ao210-1, Ao212-1 and Ao213-1) and a 5-month old (Ao210-1). All the isolates belonged to the genogroup II, and their nucleotide sequences showed >94% homology. They were subdivided into six distinct clusters by phylogenetic analysis, and Ao198-1, the source of infection, was most closely related to Ao199-1, then Ao212-1, Ao210-1, Ao214-1, Ao213-1, Ao222-1and Ao224-1 in this order. The similar result was obtained by phylogenic analysis of amino acid sequences. Sequence alignments of the isolates showed that the nonsynonymous substitution/total number of nucleotides ratio was 60% in the regions C and E. The nucleotide substitution/total number of nucleotides ratio was 33%, 12% and 15.6% at the region C and two hypervariable regions (5'and 3'HVRs) of the region E, respectively, and less than 10% at the other regions. For amino acids, the ratio was 50%, 24%, 10.3% and 25% at 5' and 3'HVRs of the regions C and E, respectively, especially high at the 5'HVR of the region C. The other regions were less than 10%. Our result suggested that the virus, while its transmission to the newborn cats, underwent frequent mutation especially at the regions C and E, suggesting these regions were most often involved in evolution of the FCV genome.

L. reuteriは, glycerol dehydratase (GD; EC 4.2.1.30)によってグリセロールからロイテリンを産生している。本菌株のグリセロール代謝は,グリセロールをGDによってロイテリンに変換し,1,3-propanediol dehydrogenase(EC 1.1.1.202)によって1,3-プロパンジオールに還元する還元反応系と,グリセロールをglycerol dehydrogenase(EC 1.1.1.6)によってジヒドロキシアセトンに変換する酸化反応系の2つの酸化還元反応系で構成されていると考えられる。従来,L. reuteriはGDによってグリセロールからロイテリンを産生していると報告されてきたが,ゲノム解析の結果から,GDではなくdiol dehydratase(DD; EC 4.2.1.28)に近い遺伝子構成であると言える。この遺伝子構造はdehydratase遺伝子を有する他のlactobacilliにおいても共通にみられた。特に, L. reuteriにおいて特徴的だったのは,L. collinoidesとL. brevisでは保存されているNADH: flavin oxidoreductase/NADH oxidase familyのドメイン構造を有する構造遺伝子が欠損している点であった。また,ロイテリン産生を考える上で重要な遺伝子であるglycerol dehydrogenaseと1,3-propanediol dehydrogenaseは, L. reuteriではpdu clusterとは異なるゲノム上の離れた位置に存在していた。つまり,dehydrataseとは別の制御系によって調節されている1,3-propanediol dehydrogenaseが,環境要因を感知し転写が抑制され,その結果,L. reuteriにおいてロイテリンが多量に産生されるものと考えられる。L. reuteriのpdu cluster下流でアデノシルコバラミン(AdoCbl)生合成系を検出した。L. brevisからは,AdoCbl生合成に関与する遺伝子群を検出することはできなかった。すなわち,L. reuteri以外の乳酸菌でAdoCbl生合成は確認されていないことから,他のlactobacilliでは,細胞外のコバラミンが存在している場合のみGDが機能するのに対し,L. reuteriは細胞外にコバラミンが存在しない場合でも,AdoCblを生合成し,ロイテリン産生が可能であると考えられる。以上,L. reuteriのpdu clusterの遺伝子構造,dehydrataseの特徴およびAdoCbl生合成系の存在が,L. reuteriのみでロイテリンが多量に産生される原因であり,L. reuteriの示す高いプロパイオテイクス効果の分子機構を解明する上で重要な知見である。, We attempted to determine the complete genome sequences of Lactobacillus reuteri JCM1112 (type strain) and Lactobacillus fermentum IFO3956. Production of antibacterial substances and adhesion factors for attachment to human intestinal cells largely influences contributed to the probiotic effects of lactic acid bacteria on the physiology. L. reuteri is known to produce a non-peptidic antibacterial substance, reuterin. Different from unlike bacteriocins, reuterin shows has an the antimicrobial activity against not only Grampositive bacteria but also Gram-negative bacteria, yeasts, fungi and protozoans. An operon in L. reuteri JCM1112 was found to encode the reuterin production system, whereas no orthologous counterpart was found in L. fermentum IFO3956. We analyzed a group set of genes involved in the reuterin synthesis in L. reuteri JCM1112, and found that the pdu cluster contained a class of genes responsible for polyhedral organelle formation, which were not present in the dha regulon, but lacked structural genes with the domain structure common to the NADH: flavin oxidoreductase/NADH oxidase family. In other bacteria that possess the dha regulon, the expressions of glycerol dehydratase and 1,3-propanediol dehydrogenase are subjected to synchronous transcriptional regulation to prevent the excessive production of cytotoxic reuterin. L. reuteri was shown to have a different regulatory system. Glycerol dehydrogenase conserved in L. reuteri might contribute to the maintenance of the intracellular oxidation-reduction balance when 1,3-propanediol dehydrogenase is being expressed. The pdu cluster of L. reuteri contained the structural genes homologous to propanol dehydrogenase (pduQ), propionaldehyde dehydrogenase (pduP) and propionate kinase (PduW) of S. typhimurium. This indicates that L. reuteri possesses an oxidation pathway to produce 3-hydroxypropionic acid from glycerol as well as a reduction pathway from glycerol to 1,3-propanediol via reuterin. A group of structural genes highly homologous to the genes involved in the biosynthesis of adenosylcobalamin (AdoCbl), a coenzyme of dehydratase was found in the downstream of the pdu cluster of L. reuteri. This is the first discovery for of the genes for the presence of AdoCbl biosynthesis in lactic acid bacteria. The hemABCL, cobACD and cysG genes were all found within the cob/cbi cluster in L. reuteri, while they are distantly located at different genomic loci in other bacterial species. The G+C content of the cob/cbi cluster, as well as the pdu cluster, was markedly lower than that of the other regions, which suggests that the gene cluster might have been acquired from other organisms through horizontal transmission. Thus, L. reuteri is able to transcribe almost all enzymes essential for the biosynthesis of AdoCbl from L-glutamate by a single transcription unit, indicating suggesting that L. reuteri could may produce AdoCbl more efficiently than any other bacteria. Our results also suggest that AdoCbl produced by L. reuteri might act may serve as a source of vitamin B_ in the intestine of humans incapable of synthesizing the substance. These genetic characteristics are specific only to L. reuteri in Lactobacillus, and are the causes for the production of reuterin in large quantities. In addition, it is considered that the efficient energy-production system and the inhibitory activity on the growth of other bacteria are importantly required for the contribution of probiotic lactic acid bacteria should contribute to the survivability in mammalian intestines, which is an important requirement for probiotic lactic acid bacteria.

Previously, we have performed T typing of Streptococcus pyogenes strains isolated from patients with streptococcal toxic shock syndrome (STSS) in Japan, and streptococcal pyrogenic exotoxin (SPE) typing for epidemiological examination. In this study, we conducted a drug sensitivity test using these strains, and investigated the results of gene analysis by pulse-field gel electrophoresis (PFGE) of S. pyogenes strains derived from patients with STSS, the patient's family, and patients other than those with STSS. To clarify the relationship between the host and bacterial factors, we investigated the association between clinical symptoms and T typing of the isolated strains/production of streptococcal pyrogenic exotoxin. There were no strains resistant to beta-lactams, and only 1 strain was resistant to multiple agents other than beta-lactams. The PFGE pattern of T1 type strains was classified into 2 ; the pattern was consistent between the strains derived from patients with STSS and those derived from the patient's family. The PFGE pattern of T3 type strains was classified into 5 (IV) ; Pattern I, which was most frequently observed, was detected in both the strains derived from patients with STSS/non-STSS. However, Patterns II and III were detected only in the strains derived from patients with non-STSS. Patterns IV and V were detected only in the strains derived from patients with STSS. When examining the association between clinical symptoms and bacterial factors, disseminated intravascular coagulation (DIC) was associated with T1-SPE B-producing strains, and pharyngitis was associated with T3-SPE A-producing strains. In the future, the relationship between the host and bacterial factors should be further investigated.

As a part of studies on the source of infection of Vero toxin-producing Escherichia coli (VTEC), O157:H7 strains isolated from human infectious enteritis between 1986 and 1995 and O157:H7 strains isolated from feces of milk cows between 2001 and 2003 were subjected to drug sensitivity test with drugs widely used as therapeutic drugs for various infectious diseases in humans and animals, and the following results were obtained. 1) Drug sensitivity tests with 20 drugs were performed in 52 strains derived human from diarrhea and 100 strains derived from milk cows, and resistance was noted in 115 strains (75.7%): 36 of the 52 human diarrhea-derived strains (69.2%) and 79 of the 100 milk cow-derived strains (79.0%). 2) The human diarrhea-derived strains and milk cow-derived strains were compared with regard to MIC90 of each drug. The antibacterial activity of the drugs was generally higher against the human diarrhea-derived strains than against the milk cow-derived strains. 3) In the 115 strains exhibiting resistance, the most frequent pattern of drug resistance was single drug resistance noted in 80 strains (68.4%), and multidrug resistance was noted in 35 strains (30.4%) consisting of 17 strains with resistance to 3 drugs, 14 strains with resistance to 2 drugs, and 2 strains each with resistance to 4 drugs and 5 drugs. More strains were multidrug-resistant in the milk cow-derived strains.

In attempt to elucidate the route and source of Vibrio vulnificus infection. serotyping and drug sensitivity tests of environment-derived strains and human clinical isolates were performed. 1) Serotyping of isolates from the two types of source were determined. Of environment-derived strains, 72.5% were classified into 18 types, and O7 was the most frequent type, accounting for 73.1%, and the second frequent type was O4, accounting for 6.1%. Of human clinical isolates, 87.1% were classified into eight types, and O4 was the most frequent, accounting for 73.5%, and O7 was the secondly most frequent, accounting for 12.9%. 2) Serotypes were investigated by regions. In eastern Japan, 69.2% were classified into 18 types, and O7 and O4 accounting for 44.6% and 5.7%, respectively. In western Japan, 64.8% were classified into eight types, and O7 was the most frequent, accounting for 20.4%, and secondly frequent type was O4, accounting for 11.1%. 3) Regarding the relationship between biotypes and serotypes, environment-derived biotype-I strains were widely distributed in the serotypes, but most biotype-I human clinical isolates were distributed in serotypes O1-O7, showing a difference between the two types of sources. However, many biotype-II strains from the two types of sources included in the serotype O7 group. 4) Drug sensitivity was compared based on MIC90 between strains from the two types of sources. Environment-derived strains were sensitive to ABPC, PIPC, CPZ, CTX, LMOX, MEPM, GM, EM, TC, DOXY, MINO, CP, NA and CPFX, but some strains were resistant to CER, CET, CTX, CMZ, KM and LCM. Human clinical isolates were sensitive to EM, TC, DOXY, MINO, CP, NA and CPFX, but some strains were resistant to ABPC, PIPC, CER, CET, CPZ, CTX, CMZ, LMOX, MEPM, KM, GM, AMK and LCM.

L.fermentumとL.reuteriは,分類学上の生理生化学的諸性状が一致することから同一菌種とされていたが,L.reuteriが独立する分類がなされた。分類学上の諸性状が一致するにもかかわらず,プロバイオティクス効果の知見はL.renteriに集中している。そこで,プロバイオティクス効果と遺伝的な背景を考察するために,両菌種の全ゲノム塩基配列を決定し比較ゲノムによる両菌種の遺伝子な特徴づけを行った。パルスフィールドゲル電気泳動法により両菌株のゲノムサイズを推定した。プラスミドは両菌株共に存在しなかった。染色体DNAの平均2.0kbと約10kb断片のゲノムライブラリーを構築した。各クローンに対してDYEnamic ET Terminator(Amersham Biosciences社)によるシークエンス反応を行い,キャピラリーDNAシークエンサーで解析した。得られたデータは,Phred/Phrapによるアッセンブルと,アノテーションはGenome Gambler(MKI社)により行った。ゲノムサイズは両菌株とも約1.8Mbでほぼ同様であったが,両菌株のゲノムの制限酵素地図的にはかなり異なっていた。ORFの数はそれぞれ1,600ぐらいで,輸送,エネルギー代謝に分類される遺伝子が主であった。両菌株よりβ-グルクロニダーゼ遺伝子は見つからなかった。ロイテリン(抗菌性物質)産生系は,L.reuteriにおいてオペロンとしてコードされていたが,L.fermentumには存在しなかった。プロバイオティクス効果においてヒト腸管付着因子の存在は重要であり,L.reuteriでいくつかの細胞付着性遺伝子が確認された。, L. fermentum and L. reuteri were separated from L. fermentum by polyacrylamide gel electrophoresis but not physicochemical phenotypes in 1980. On the other hand, L. fermentum is generally isolated from plant origin fermented foods and silage, whereas L. reuteri is typically isolated from mammalian intestinal tract and feces. Probiotics have recently been the focus of intense lactic acid bacterial researches interest. Although L. fermentum is phenotypically similar to L. reuteri, many research data of probiotics concentrate on L. reuteri not but on L. fermentum. The present studies are aimed at sequencing complete genomes of L. fermentum and L. reuteri, and analyzing those comparative genomes. We have sequenced the complete genomes of these species, and each genome contains approximate 1.8-Mb. The circular chromosomes of L. fermentum and L. reuteri have the average G+C contents of 52% and 39%, respectively. Five 23S rDNAs and six 23S rDNAs exist in the chromosomes of L. fermentum and L. reuteri, respectively. The numbers of insertion sequence (IS) elements and transposons (Tn) of L. fermentum are more than those of L. reuteri. L. reuteri possesses the structural genes determining reuterin (3-hydroxypropionaldehyde), an antimicrobial substance, production, but L. Fermentum does not possess this operon.

ヒトのVero毒素産生性大腸菌(VTEC)感染症における感染源や感染経路を明らかにするため,1997年8月から1998年1月までの期間にハトは相模原市で,カラスは相模原市,川崎市,横浜市および東京都内で害鳥駆除のために捕獲した野鳥の腸管内容物を採材し,VTECの分離を試みたところ,以下の成績が得られた。1)供試した521例中32例(6.1%)からVTECが分離された。その内訳において,ハトでは相模原市262例中25例(9.5%)から,カラスでは相模原市184例中7例(3.8%)からそれぞれ分離されたが,横浜市11例,川崎市4例および東京都内60例からは1例も分離されなかった。2)分離された33株について毒素型別を行ったところ,VT1産生株が4株(6.5%),VT2産生株が27株(88.7%),VT1とVT2両毒素産生株が2株(4.8%)であった。3)分離株の血清型では,O78:H-に10株,次にO152:H-に7株,O153:H19に2株,O164:H-,O128:H-,O164/O143:H-およびO1:HUTに各1株が型別されたが,残り10株は型別不能であった。また,型別不能10株のうち,1株は自家凝集が認められた。4)供試した33株についてeacAを確認したところ,31株(93.9%)が保有していた。以上のことから,ヒト下痢症由来VTECの毒素型や血清型と一致する菌株がハトやカラスから分離されたことや病原性の発現に重要な関与が考えられるeacAを高率に保有していたことから,ヒトVTEC感染症の感染源の一つとしてこれらの鳥類も関与する可能性が考えられた。, 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, respect ively. 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 Ol: 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 diar rhea-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.

As part of an epidemiological study of legionellosis, we investigated the growth within Acanthamoeba sp. and antibiotic susceptibility of 62 strains of Legionella spp. isolated from surface soils nationwide in 2001. 1) All strains tested grew in Acanthamoeba sp., suggesting that the strains were pathogenic. The minimum bacterial number required for the growth in the amoeba was 10(3)-10(8) CFU/ml and there were differences between the strains. 2) Susceptibility to 10 drugs was investigated using the Etest. The MIC90 values of imipenem, as a beta-lactam, and rifampicin, as an antitubercular agent, were 0.047 microgram/ml and 0.064 microgram/ml, respectively, showing high sensitivity. In contrast, sensitivity to minocycline, as a tetracycline, and piperacillin, as a beta-lactam, was low and the MIC90 values were 12 micrograms/ml and 16 micrograms/ml, respectively. Sensitivity to minocycline was particularly low, with a MIC value of 32 micrograms/ml, in two strains. The above findings suggested that all soil-derived strains were pathogenic, and susceptibility of the strains tended to be slightly lower than that of clinical isolates.

As a part of basic studies to elucidate the source of infection of Verotoxin-producing Escherichia coli (VTEC) infectious disease, fresh feces were collected from pigs raised in Kanto District (A and B Prefectures) and Kyushu District (C and D Prefectures) between April and October in 2000, and isolation, serotyping, toxin typing, and drug sensitivity test of VTEC were performed. 1) Of 411 fecal samples tested, VTEC was isolated from 44 samples (10.7%), consisting of 12 of 112 samples (10.7%) from A Prefecture, nine of 100 samples (9.0%) from B Prefecture, 18 of 99 samples (18.2%) from C Prefecture, and five of 100 samples (5.0%) from D Prefecture. 2) Forty-five isolates were serotyped. Four isolates (8.9%) were typed as type 3, but the remaining 41 isolates (91.1%) could not be typed. The four typed isolates consisted of two O112ac:H- isolates and one each of O126:H- and O157:H7. 3) Toxin was typed in 45 isolates. Twenty-seven (60.0%) and 17 isolates (37.8%) produced VT 2 and VT1, respectively, and one isolate (2.2%) produced both VT1 and VT2. 4) Drug sensitivity tests of 45 isolates were performed. All 45 isolates (100%) were multidrug-resistant that were resistant to multiple drugs. Nineteen, nine, four, four, seven, one, and one isolates were resistant to five, six, two, three, four eight, and nine drugs, respectively. The above findings confirmed contamination in all districts, although the VTEC isolation rate varied among the sampling districts. Serotyping clarified the presence of O157:H7 and O112ac:H- that are detected in human VTEC infectious disease. The drug sensitivity tests clarified the presence of many multidrug-resistant strains.

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.

We previously reported that certain constituents in brewed coffee exhibited antibacterial activities against a strain of Legionella pneumophila. The constituents showing antibacterial activities were included only in extracts cold with water or hot water. To determine the antibacterial substances in coffee extract, the extract was fractionated by HPLC using a UV/photodiode array detector. The optimum HPLC conditions for analysis were UV wavelength of 250 nm and eluents of methanol/acetic acid (10/90), pH 3.0. When several fractions separated by HPLC were investigated for antibacterial activities against L. pneumophila, it was found that three peak fractions exhibited strong antibacterial activities. Each product from these fractions was analyzed by NMR and LC-mass spectrometry, and the chemical structure of each was determined. It was shown that the antibacterial substances was were protocatechuic acid (3,4-dihydroxy benzoic acid), chlorogenic acid, and caffeic acid.

As a result of screening 380 strains of Staphylococcus spp. isolated from the nasal cavity for growth inhibition of MRSA, two strains were shown to inhibit the growth of MRSA. The biochemical properties of these strains were investigated. The API codes of the strains were 266022210 and 166032210, and both were identified to be S.epidermidis. Both strains showed alkaline phosphatase and α-glucosidase activities, but no constitutive enzymes specific to MRSA growth-inhibiting bacteria were detected. In a susceptibility test of 15 drugs for MRSA growth-inhibiting bacteria, neither bacterium showed marked resistance to any of the drugs, showing that these MRSA growth-inhibiting bacteria are not resistant strains. MRSA was inoculated into the filtrate of the MRSA growth-inhibiting bacteria, and the time-course MRSA counts largely differed depending on the inoculation count, and the filtrate of the MRSA growth-inhibiting bacteria had antibacterial effects on MRSA at low bacteria concentration.

Minimum Inhibitory Concentration (MIC) of 4 new-quinolone antibacterial reagents and 4 macrolide antibiotics against periodontopathic bacteria 21 species 24 strains of standard strains including oral related bacteria and clinical isolated (1993-1999) 20 strains of Porphyromonas gingivalis, 7 strains of Actinobacillus actinomycetemcomitans were determined. Minocycline HCl was used as the comparative reagent. Macrolide showed moderate antibacterial activity against standard strains of oral related bacteria except Fusobacterium nucleatum. New-quinolone showed excellent activity against Eikenella corrodens and Actinobacillus actinomycetemcomitans. Concerning to the clinical isolated strains, Clarithromycin effected P. gingivalis (MIC90 0.1 microgram/ml) and Levofloxacin and Ciprofloxacin showed high-potency antibacterial activity against clinical isolated Actinobacillus actinomycetemcomitans (MIC90 0.013-0.025 microgram/ml).

To elucidate the source and route of VTEC infection, we performed pulse field gel electrophoresis (PFGE) an 50 isolates from human diarrhea typed as serotypes O157, O111, and O26, which were very frequently isolated from patients with VTEC infection between 1986 and 1997, and 32 isolates from dairy cattle, a total of 82 isolates. The isolates were genetically analyzed based on the electrophoresis patterns of DNA, and a phylogenetic tree was prepared. The isolates were classified based on similarity > or = 89. The following results of the molecular epidemiological investigation were obtained. 1) Based on the electrophoresis patterns of DNA obtained by PFGE, 34 of the 49 O157 isolates (69.4%) were divided into groups 1-9, 15 of the 18 O111 isolates (83.3%) were divided into groups 1-3, and 12 of the 15 O26 isolates (80%) were divided into groups 1-3. Of the grouped isolates, group 8 of O157, groups 2 and 3 of O111, and group 3 of O26 included isolates from human diarrhea and dairy cattle, but the other groups included isolates from only one of the two sources. 2) With regard to regional investigation, groups 6 and 9 of O157 included human diarrhea-derived isolates from Yokohama and Ehime, and group 8 included a human diarrhea-derived isolate from Yokohama and a dairy cattle-derived isolate from Tokushima. Group 3 of O111 included a human diarrhea-derived isolate from Ehime and a dairy cattle-derived isolate from Hokkaido. Group 3 of O26 included human diarrhea-derived isolate from Ehime and dairy cattle-derived isolate from Sagamihara and Hokkaido. Since the above findings showed that although the frequency was low, isolates from human diarrhea and dairy cattle were included in the same groups, it was demonstrated that dairy cattle are closely related to the human infectious disease of the intestinal tract as a source of infection. However, classification using the PFGE method is difficult due to diversity of the electrophoresis pattern of DNA. It is necessary to investigate the classification by a combination of the PFGE method with phage typing, ribotyping, and RAPD-PCR, and to investigate more numbers of patient-derived and animal-derived isolates.

The use of dried herbs has frequently been incorporated into daily life in Japan in recent years. The antibacterial activity of dried herbs on Legionella pneumophila, which has been attracting attention as an emerging infectious disease, was investigated, and the following were obtained. 1) The antibacterial activity of extract solutions of 28 dried herbs on L.pneumophila was investigated by the disc method, and zone inhibition of the bacteria was observed with 22 herbs (78.6%) including raspberry leaf, common sage, lemon balm, and peppermint, showing an antibacterial activity of herbs on L.pneumophila. Herbs of the perilla family in particular had a strong activity. 2) Bactericidal experiments were performed with various concentrations of the herb extracts. The herbs had a bactericidal effect on L.pneumophila, and the activity was concentration dependent. Among the herbs with a 100% bactericidal rate, the maximal dilution factor was ≧16-fold that of savory and hibiscus extracts, and the second highest was 8-fold that of common thyme extract. 3) Time-course changes in the bacterial count of L.pneumophila were measured in the extract solutions of peppermint, common sage, and common thyme of the perilla family, which had strong antibacterial activity. In all these herbs, the inoculated bacterial count of 10^6 CFU/ml became undetectable after three to 12 hours. Therefore, it was shown that although the action is not immediate, herbs have a bactericidal activity on L.pneumophila.

The colony hybridization (CH) method as a rapid method for detecting Legionellaspp. was compared with the conventional culture method. In the comparison of colony count in which a suspension ofL. pneumophila was examined, a positive correlation was shown between the results by both methods. The regression line was Y=0.85+1.34Xand the correlation coefficient was 0.95. The maximum number of detectable colonies on the plate was 144 colony forming units, although there was a tendency for the CH data to give slightly lower counts than those of the culture method. The detection time in the CH method was half of that in the culture method, and the final result was obtained within 4 or 5 d. In addition the results by the CH method were rarely influenced by other contaminating microorganisms, since the transfer of colonies was performed at the initial stage of culture.