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Supplementary Table 2 from Dietary Tricin Suppresses Inflammation-Related Colon Carcinogenesis in Male Crj: CD-1 Mice

Supplementary Table 1 from Dietary Tricin Suppresses Inflammation-Related Colon Carcinogenesis in Male Crj: CD-1 Mice

Atypical Aeromonas salmonicida ( i.e. subsp. achromogenes and subsp. masoucida) are one of the major opportunistic pathogens that cause ulcer diseases in a variety of fishes, in which this pathogen has become a worldwide economic threat in sectors that handle of particular high-priced ornamental fishes like varicolored carp and goldfish due to appearance damages. Here we reported that the kuma bamboo grass (Sasa veitchii) extracts (KBGE) that contained a variety of fatty acids, exhibited antibacterial activity against nine Aeromonas strains including 5 atypical A. salmonicida strains. Experimental challenges with four atypical A. salmonicida strains revealed that supplementation with 375 to 750 μg/ml of the KBGE restored the survival of goldfish in coincidence of inhibition of both bacterial replication and superoxide dismutase (SOD) activity upon infection, compared with those of untreated control. Together, our data demonstrating the antibacterial effects of the plant extracts proposes its possible implication for prevention of Aeromonas infection in the ornamental fish.

Atypical Aeromonas salmonicida ( i.e. subsp. achromogenes and subsp. masoucida) are one of the major opportunistic pathogens that cause ulcer diseases in a variety of fishes, in which this pathogen has become a worldwide economic threat in sectors that handle of particular high-priced ornamental fishes like varicolored carp and goldfish due to appearance damages. Here we reported that the kuma bamboo grass (Sasa veitchii) extracts (KBGE) that contained a variety of fatty acids, exhibited antibacterial activity against nine Aeromonas strains including 5 atypical A. salmonicida strains. Experimental challenges with four atypical A. salmonicida strains revealed that supplementation with 375 to 750 μg/ml of the KBGE restored the survival of goldfish in coincidence of inhibition of both bacterial replication and superoxide dismutase (SOD) activity upon infection, compared with those of untreated control. Together, our data demonstrating the antibacterial effects of the plant extracts proposes its possible implication for prevention of Aeromonas infection in the ornamental fish.

We investigated the susceptibility to antibacterial agents of 186 clinical isolates of Pseudomonas aeruginosa isolated from medical facilities in Gifu, Aichi, Toyama, and Fukui prefectures from October 2013 to February 2014. MIC₅₀/₉₀ of piperacillin (PIPC), tazobactam/piperacillin (TAZ/PIPC), ceftazidime (CAZ), cefepime (CFPM), imipenem (IPM), meropenem (MEPM), doripenem (DRPM), aztreonam (AZT), ciprofloxacin (CPFX), levofloxacin (LVFX), amikacin (AMK) and colistin (CL) against P aeruginosa was 8/32, 4/32, 2/8, 2/16, 1/32, 0.5/8, 0.25/4, 8/32, 0.25/8, 0.5/16, 4/8 and 1/1pg/mLrespectively. Two strains of multidrug resistant P aeruginosa were isolated (1.1%). They were isolated from the respiratory tract, intra-abdominal, and urinary infection. The susceptible ratio against P aeruginosa derived from intra-abdominal infection for carbapenem was lower than those from respiratory tract and urinary infection. The susceptible ratio against P aeruginosa derived from urinary infection for penicillin, cephem, monobactam, and fluoroquinolone was lower than those from respiratory and intra-abdominal infection. It is meaningful to pay attention to the susceptibility to antibacterial agents in each clinical specimen from infected organ.

Little information is available on the distribution of antimicrobial resistance genes in anaerobes in Japan. To understand the background of antimicrobial resistance in anaerobes involved in intra-abdominal infections, we investigated the distribution of eight antimicrobial resistance genes (cepA, cfiA, cfxA, ermF, ermB, mefA, tetQ, and nim) and a mutation in the gyrA gene in a total of 152 organisms (Bacteroides spp., Prevotella spp., Fusobacterium spp., Porphyromonas spp., Bilophila wadsworthia, Desulfovibrio desulfuricans, Veillonella spp., gram-positive cocci, and non-spore-forming gram-positive bacilli) isolated between 2003 and 2004 in Japan. The cepA gene was distributed primarily in Bacteroides fragilis. Gene cfxA was detected in about 9 % of the Bacteroides isolates and 75 % of the Prevotella spp. isolates and did not appear to contribute to cephamycin resistance. Two strains of B. fragilis contained the metallo-β-lactamase gene cfiA, but they did not produce the protein product. Gene tetQ was detected in about 81, 44, and 63 % of B. fragilis isolates, other Bacteroides spp., and Prevotella spp. isolates, respectively. The ermF gene was detected in 25, 13, 56, 64, and 16 % of Bacteroides spp., Prevotella spp., Fusobacterium spp., B. wadsworthia, and anaerobic cocci, respectively. Gene mefA was found in only 10 % of the B. fragilis strains and 3 % of the non-B. fragilis strains. Genes nim and ermB were not detected in any isolate. Substitution at position 82 (Ser to Phe) in gyrA was detected in B. fragilis isolates that were less susceptible or resistant to moxifloxacin. This study is the first report on the distribution of resistance genes in anaerobes isolated from intra-abdominal infections in Japan. We expect that the results might help in understanding the resistance mechanisms of specific anaerobes.

Furonaphthoquinones are promising skeletons for anticancer drug molecules. In particular, methoxylated furonaphthoquinones are characteristic constituents of Tabebuia plants. In this research, we synthesized the furonaphthoquinones by effective one-pot cascade reactions of 3-phenyliodonio-1,2,4-trioxo-1,2,3,4-tetrahydronaphthalenides with 3-butyn-2-ol in the presence of palladium and cuprous catalysts via Sonogashira coupling and intramolecular cyclization. Furthermore, we demonstrated that the synthetic furonaphthoquinones showed moderate cytotoxicity against human leukemia U937 and HL-60 cells. Our work highlights the importance of furonaphthoquinones as antileukemic agents.

Gene-microbiota interactions are now proposed to be a special case of gene-environmental interaction. Preclinical and clinical data summarized in this article reveal that a specific serum metabolite, associated with alterations in gut microbiome composition, might have an emerging role in the onset and pathogenesis of autism. Altered level of this specified metabolite may induce perturbations in the epigenome and modulate the expression of key disease susceptible genes in neurons and their associated cells during critical periods of neurodevelopment. The gut microbiota itself is now regarded as a reservoir for environmental epigenetic factors.

Flavonoids are widely occurring polyphenols that are found in plants. The aim of this study was to investigate the structure-activity relationships of 5,7-dihydroxyflavones, with a focus on the effect of B ring structure substitution on the antiproliferative effects of the compounds in human leukemia HL-60 cells. We prepared a series of 5,7-dihydroxyflavones and evaluated their ability to inhibit the proliferation of HL-60 cells by using the MTT assay. The apoptosis- and cell differentiation-inducing ability of the most potent flavones were investigated using staining and morphological analyses. This study explored the antileukemic and chemopreventive potency of 5,7-dihydroxyflavones, particularly diosmetin and chrysoeriol, which have both hydroxy and methoxy groups on the B ring.

Background: We examined the anti-influenza virus activity of tricin, 4′,5,7-trihydroxy-3′,5′-dimethoxyflavone, against five viruses: A/Solomon islands/3/2006 (H1N1), A/Hiroshima/52/2005 (H3N2), A/California/07/2009 (H1N1pdm), A/Narita/1/2009 (H1N1pdm) and B/Malaysia/2506/2004 strains in vitro and against A/PR/8/34 virus in vivo. Methods: The effect of tricin was studied by an infectious virus yield reduction assay. The anti-influenza virus mechanism of the tricin was examined by western blot analysis, real-time reverse transcriptase PCR analysis, haemagglutination inhibition (HI) assay and neuraminidase (NA) inhibition assay. The anti-influenza virus efficacy of tricin was further examined in a murine influenza virus infection model. Results: Tricin of 3.3 to 30 μM significantly reduced seasonal A (H1N1), (H3N2) viruses, novel A (H1N1pdm) virus, as well as B virus in a dose-dependent manner. The 50% effective concentrations of tricin were 3.4 μM for seasonal A (H3N2) virus, 4.9 μM for B virus and 8.2 μM for A/Narita (H1N1pdm) virus. Tricin decreased the expression of haemagglutinin (HA) protein and matrix (M) protein, and messenger RNA expression of HA and M of influenza virus in the infected cells. Tricin exhibited little or no effects on influenza virus HI and NA activities. In the mouse infection model, tricin was significantly effective in reducing body weight loss, and also effective in prolonging survival times of infected mice. Conclusions: Tricin was indicated to possess anti-influenza virus activity and to ameliorate body weight loss and survival rate of influenza-A-virus-infected mice. Tricin is a novel compound with potential anti-influenza virus activity in vitro and in vivo.

The anti-human cytomegalovirus (HCMV) activity of tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), a derivative from Sasa albo-marginata, was studied in the human embryonic fibroblast cell line MRC-5. In a plaque assay, tricin and ganciclovir (GCV) showed concentration-dependent inhibitory properties from 0.05 to 3.6 μM and 0.01 to 1.0 μM, respectively. Tricin had no virucidal effects on cell-free HCMV. Treatment with tricin 1 h before, or 1 h or 3 h after viral infection significantly suppressed HCMV replication. Moreover, tricin inhibited the expression of immediate early (IE) 2 mRNA and DNA polymerase (UL54) mRNA in HCMV-infected cells. Western blot analysis also demonstrated that tricin decreased the expression of IE antigen (especially IE2) and cyclooxygenase 2 (COX-2) expression in HCMV-infected cells. In the presence of tricin, prostaglandin E2 (PGE2) accumulation by HCMV infection was completely inhibited. These results suggest that tricin is a novel compound with potential COX inhibitor-dependent anti-HCMV activity.

We evaluated the in vitro antianaerobic activity of razupenem (SMP-601, PTZ601), a new parenterally administered carbapenem, against 70 reference strains and 323 clinical isolates. Razupenem exhibited broad-spectrum activity against anaerobes, inhibiting most of the reference strains when used at a concentration of ≤1 μg/ml. Furthermore, it exhibited strong activity, comparable to those of other carbapenems (meropenem and doripenem), against clinically isolated non -fragilis Bacteroides spp. (MIC 90 s of 2 μg/ml), with MIC 90 values of 0.06, 0.03, and 0.5 μg/ml against Prevotella spp., Porphyromonas spp., and Fusobacterium spp., respectively. Clinical isolates of anaerobic Gram-positive cocci, Eggerthella spp., and Clostridium spp. were highly susceptible to razupenem (MIC 90 s, 0.03 to 1 μg/ml).

現在我々は，代替医療薬を中心に抗 HCMV 薬の探索を行っており，防腐作用（抗菌作用）を含む種々の生理活性をもつクマザサに着目し，その熱水抽出液に抗 HCMV 作用があることを明らかにしてきた．今回，クマザサ熱水抽出液の成分の一部が分離・精製・同定され，その中の 1 つである tricin がウイルス粒子産生に最も高い抑制効果を示すことがわかった．また，その抑制効果は，クマザサ抽出液同様のウイルス感染後の処理だけでなく，感染前処理した場合にも見られることがわかった．さらにリアルタイム RT-PCR 法および，ウエスタンブロット法による解析の結果，tricin がウイルスの複製・増殖に重要な major immediate early (IE) 遺伝子の発現を抑制することがわかり，tricin が現在使用されている GCV とは異なる作用機序により抗 HCMV 効果をもつことが示され，新たな治療薬としての可能性が示唆された．

Antimicrobial susceptibilities of 23 strains of Desulfovibrio spp. were tested by Etest. Generally, Desulfovibrio spp. were highly susceptible to sulbactam-ampicillin, meropenem, clindamycin, metronidazole, and chloramphenicol: MIC 90 s of 6, 4, 0.19, 0.25, and 8 μg/ml, respectively. In addition, these strains generally showed high MICs to piperacillin and piperacillin-tazobactam. D esulfovibrio fairfieldensis (eight strains) was the species least susceptible to most agents, especially β-lactams, and was the only species resistant to fluoroquinolones. D esulfovibrio desulfuricans strain Essex 6 isolates were less susceptible to β-lactams than D. desulfuricans strain MB isolates.

Background Acne vulgaris is a multifactorial inflammatory disease of the sebaceous follicles of the face and torso that frequently occurs in adolescence. Initially, acne starts as a non-inflammatory comedo. Subsequently, inflammatory reactions evolve to pustules, granulomas and cystic lesions. Many pathogenic mechanisms have been proposed including sebum excretion, obstruction of hair follicles, impaired keratinization of hair epithelium, bacterial overgrowth and immunological mechanisms; the role of Propionibacterium acnes ( P. acnes ) is particularly important. Facultative anaerobic gram-positive rods have been implicated in acne pathogenesis. However, the host immune response to P. acnes has not been as yet elucidated. Objectives The aim of the present study is to evaluate the importance of the immune response to P. acnes and the bacteriological factor in the pathogenesis of acne. Methods P. acnes isolated from acne lesions and healthy volunteers skin were cultured. The peripheral blood mononuclear cells (PBMC) from acne patients or healthy volunteers were stimulated with viable P. acnes , and cytokine production was evaluated using RT–PCR and ELISA. Results IFN-γ, IL-12p40, and IL-8 mRNA and protein production were significantly increased in PBMC from acne patients compared to that from normal donors. However, different P. acnes species isolated from acne lesions or normal subjects showed no difference in cytokines production from acne patients and normal subjects PBMC. Conclusions The inflammatory response of acne appears to be attributable to P. acnes -induced host immune response rather than P. acnes strains from normal skin or acne lesions.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. Propionibacterium acnes is the only microorganism so far isolated from sarcoid lesions. To examine whether P. acnes isolates from sarcoid tissues differ from those obtained from non-sarcoid tissues, we studied cell invasiveness, serotype, and polymorphisms of the P. acnes trigger factor protein and the two invasion-associated proteins (named PAmce and PAp60) in 35 P. acnes isolates from sarcoid lymph nodes and 127 isolates from non-sarcoid tissues. Most of the serotype I isolates (79/112; 71%), but none of the serotype II isolates (0/50) were cell-invasive. Two prominent types of trigger factors, one with and one without a 15 amino acid-residue deletion, corresponded to serotype II and serotype I, respectively. Non-invasive isolates had genomic mutations that caused more than one amino acid change in either the PAmce or PAp60 gene, with four exceptional isolates. P. acnes was finally classified into nine isotypes, and isolates obtained from sarcoid and non-sarcoid tissue did not differ. Although the finding did not link P. acnes to sarcoidosis, the present study clarified the cell invasiveness of P. acnes and the close correlation of cell invasiveness to the serotype and genotype of the two invasion-associated P. acnes genes.

Strict anaerobes are highly sensitive to oxygen, but the mutagenicity of oxygen in strict anaerobes has not been well understood. Prevotella melaninogenica, a strict anaerobe, is susceptible to oxygen and shows an increase in oxidative DNA damage upon exposure to oxygen. In this study, we have investigated the mutagenicity of oxygen and the types of mutations induced by oxygen. Exposure to oxygen decreased cell survival and increased the levels of 8-oxo-deoxyguanosine (8-oxodG). The frequency of rifampicin-resistant mutants was markedly increased after exposure to oxygen. After sequencing a 254-bp fragment of the rpoB gene, which encodes the β subunit of bacterial RNA polymerase, a target molecule of rifampicin, we found that most mutants induced by oxygen had GC to TA transversions, a signature of 8-oxodG. In addition, all detected single-nucleotide changes would lead to amino acid changes that confer rifampicin resistance. These results indicate that oxygen is mutagenic in a strict anaerobe, P. melaninogenica, and its mutagenic characteristics could be analyzed with this experimental system.

In 1968, the agar dilution method was developed as an independent Japanese method for measuring the minimal inhibitory concentration (MIC) of antimicrobial agents. As this method differed in a few respects from the MIC measurement methods used in other countries, it was revised in 1981, by a committee headed by Susumu Mitsuhashi, and the revised method (Chemotherapy 29:76-79, 1981) has been used since then.In 1979, an agar dilution method for measuring the MIC of anaerobes was developed by a committee chaired by Nozomu Kosakai (Chemotherapy 27:559-561, 1979). In 1990, a committee headed by Sachiko Goto approved a broth microdilution method for nonfastidious bacteria (Chemotherapy 38:102-105, 1990). Later, a committee headed by Atsushi Saito examined media that would be suitable for nonfastidious bacteria and fastidious bacteria, and they endeavored to prepare a broth microdilution method for anaerobic bacteria. In this context, a new broth microdilution method was proposed at the 40th Annual Meeting of the Japanese Society of Chemotherapy (JSC) in Nagoya in 1992, and the proposal was adopted as the standard JSC method after some modification (Chemotherapy 41: 183-189, 1993).The agar dilution method has remained unrevised for approximately 20 years. A proposal to review this method was recently made, and the 2007 Committee on Antimicrobial Susceptibility Testing was formed, comprising the JSC members listed below. Under the auspices of this committee, the method revised in 1981 was reviewed in comparison to the international standard method (Clinical and Laboratory Standards Institute [CLSI] method).

We investigated the susceptibility to antibacterial agents, genotype of penicillin-binding protein (PBP) genes and macrolide resistant genes, and the serotypes against 270 strains of Streptococcus pneumoniae isolated from medical facilities in Gifu and Aichi prefectures between October 2011 and April 2012. These results were compared with those against S. pneumoniae isolated in 2008-2009 and 2010-2011. The number of gPSSP with 3 normal PBP genes, gPISP with 1 or 2 normal PBP genes and gPRSP with 3 abnormal genes isolated in 2011-2012 was 15 (5.6%), 162 (60.0%) and 93 (34.4%) strains, respectively. Compared with those isolated in 2008-2009 and 2010-2011, the numbers of gPRSP were decreasing. On the other hand, the isolates with no macrolide-resistant gene, only mefA, only ermB, and both mefA and ermB were 16 (5.9%), 75 (27.8%), 153 (56.7%) and 26 (9.6%). Compared with those isolated in 2008-2009 and 2010-2011, the numbers of isolates with ermB, which was usually associated with high-level resistance, were increasing. The prevalent pneumococcal serotypes in children were type 3 (14.4%), following by type 15 and 19F (9.3%). The coverages of 7-valent pneumococcal conjugate vaccine (PCV7) and 13-valent pneumococcal conjugate vaccine (PCV13) were calculated as 22.9% and 49.2%, respectively. The coverages of PCV7 and PCV13 in gPRSP isolated from children were 47.7% (21/44 strains) and 72.7% (32/44 strains). The MIC90 of each antibacterial agent was as follows; 0.125pg/mL for imipenem, panipenem and garenoxacin, 0.25 μg/mL for meropenem and doripenem, 0.5 μg/mL for cefditoren, moxifloxacin and tosufloxacin, 1 μg/mL for amoxicillin, clavulanic acid/amoxicillin, cefteram, cefcapene and ceftriaxone, 2 μg/mL for benzylpenicillin, ampicillin, sulbactam/ampicillin, piperacillin, tazobactam/piperacillin and levofloxacin, 4 μg/mL for cefdinir, flomoxef and pazufloxacin, 16 μg/mL for minocycline,64 μg/mL for clarithromycin and azithromycin, and these MIC90s were about the same as those in 2010-2011.

Considerable information has been accumulated in the field of anaerobic bacteria and anaerobic infections in the last ten years. Here we tried to briefly introduce several selected topics of clinical importance in this field: Proposal of the term "Nanaerobe", Changes of classification and nomenclature of anaerobes, Anaerobic bacteremia, Lemierre's syndrome as a revival anaerobic infection, Atopobium vaginae as Bacterial Vaginosis-associated bacteria, and new actions of the Clostridium perfringens toxins.

Although vitamin C is considered to act both as pro-oxidant and antioxidant, the mechanisms underlying these actions are still unclear. Using the oxygen-sensitive system of a strict anaerobe, Prevotella melaninogenica, we investigated both the pro-oxidant and antioxidant mechanisms of vitamin C. In the presence of vitamin C, the 8-hydroxydeoxyguanosine (8OHdG) formation induced by oxygen exposure was enhanced, probably due to the action of vitamin C on hydrogen peroxide generated during oxygen exposure: while catalase almost completely suppressed the enhancing effect of vitamin C, 8OHdG formation induced by hydrogen peroxide was enhanced by vitamin C. By contrast, the presence of vitamin C inhibited bacterial cell death, membrane damage, and lipid peroxidation induced by oxygen exposure. Sodium azide showed similar effects to vitamin C, thus the antioxidant action of vitamin C may be due to its quenching of the singlet oxygen generated in this system. Both the pro-oxidant and antioxidant effects of vitamin C were observed only in acidic conditions.

Bacteroides fragilis ATCC25285から得られたアルカリ安定脂質は赤外吸収スペクトルでアミド結合に由来する特徴的な吸収がみられることや,スフィンゴシンを有することなどからスフィンゴリン脂質であることが示された。このスフィンゴリン脂質の薄層クロマトグラフィーによる分析からセラミドホスホリルエタノールアミン(CPE)とセラミドホスホリルグリセロール(CPG)の2種類のスフィンゴリン脂質が検出された。また,Bacteroides属の全10菌種についてスフインゴリン脂質の組成を分析して菌種間の分布を調べたところ,CPEとCPGは7菌種から見いだされた。さらに,薄層クロマトグラフ上にはスフィンゴリン脂質と考えられる未知スポットが見いだされた。各菌種で見いだされたスフィンゴリン脂質組成は,菌種について特徴的であり,菌株間での変異は認められなかった。

Bacteroides and related species formerly classified in the genus Bacteroides have phosphosphingolipids as their membrane lipid. To evaluate the possible involvement of bacterial sphingolipids in the infection process, the effects of Bacteroides sphingolipids on murine neutrophils were examined in this study. Observation by differential interference contrast microscopy showed morphological changes in murine neutrophils in the presence of the sphingolipids, indicating biological activity by the Bacteroides sphingolipids. The lipids dose-dependently inhibited superoxide release from the neutrophils stimulated with phorbol myristate acetate. This observation suggests that bactericidal activity of the neutrophils may be affected by Bacteroides sphingolipids. Taken together, these findings provide evidence that Bacteroides sphingolipids may contribute to the pathogenesis of mixed infections by direct inhibition of neutrophil function.

We report a case of severe oral infection with a high fever due to Lactobacillus rhamnosus during induction chemotherapy for acute myeloid leukemia. The patient did not improve on treatment with meropenem, clindamycin, or vancomycin until neutrophil recovery. Since L. rhamnosus GG is used in dairy products, and the patient ingested dairy products daily before starting chemotherapy, we suspected an association between the ingestion of dairy products and the development of infection. Pulsed-field gel electrophoresis using two different restriction enzymes showed that the strain isolated from the patient was identical to the L. rhamnosus GG strain isolated from dairy products and ATCC #53103. This was confirmed by a PCR assay with species-specific L. rhamnosus GG primers. Since Lactobacillus infection, particularly L. rhamnosus infection, can be fatal in immunocompromised hosts, we should consider Lactobacillus as a causative organism when Gram-positive rods are detected during treatment with broad-spectrum antibiotics and vancomycin. The causal association between the ingestion of dairy products containing Lactobacillus and Lactobacillus infection in immunocompromised hosts warrants further study.

The in vitro activity of SCH27899, a novel oligosaccharide antimicrobial agent, was compared with those of representatives of six classes of antimicrobial agents (piperacillin, clarithromycin, clindamycin, vancomycin, sitafloxacin and metronidazole) against clinical isolates of anaerobic bacteria and Propionibacterium acnes. Against Peptostreptococcus: spp. and Clostridium difficile, SCH27899 was the most potent (MIC(90) < 0.125 mg/L) of the agents examined. Besides these Gram-positive anaerobes, SCH27899 showed a moderate level of activity against Prevotella bivia, Prevotella intermedia and Porphyromonas: spp. (MIC(90)< or = 4 mg/L).

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

The in vitro activity of R-95867, the active metabolite of a new oral carbapenem, CS-834, was compared with those of DU-6859a, cefditoren, ampicillin/sulbactam and clindamycin against a variety of anaerobic bacteria. R-95867 inhibited 90% of anaerobic strains at

The bft gene encoding Bacteroides fragilis toxin (BFT) has been devided into two subtypes, bft-1 and bft-2. We found a novel subtype by sequencing a segment of the bft gene from 64 enterotoxigenic B. fragilis (ETBF) strains isolated in Japan. The 1548-bp nucleotide sequences of the new bft, the bft-1, and bft-2 genes were determined for five, four, and four ETBF strains, respectively; the nucleotide sequence was identical among each bft subtype and the degree of identity between each subtype was between 89 and 94%. Most of the variations between the three subtypes were detected in the region encoding mature toxin. A multiplex PCR was developed with a four-primer mix to subtype the bft sequences. The subtyping of 143 ETBF isolates from extraintestinal and stool specimens of humans and cows showed that the bft-1 was the most prevalent subtype, followed by bft-2 and a new bft subtype. No other subtype was found among the strains tested.

We established a typing system for Prevotella intermedia and Prevotella nigrescens using the combination of PCR ribotyping and arbitrarily primed PCR (AP-PCR) fingerprinting and applied this system to the study of intrafamilial incidence of these species in the oral cavity. PCR ribotyping followed by subtyping by AP-PCR fingerprinting was applied to each type strain of P. intermedia and P. nigrescens and 54 isolates (32 isolates of P. intermedia and 24 isolates of P. nigrescens ) from extraoral infections, resulting in an excellent discriminatory power (discrimination index, 0.99) for both species. A total of 18 subjects from six families, with the subjects from each family comprising the mother, the father, and a child who had subclinical early-stage to moderate adult periodontitis or simple gingivitis and who carried P. intermedia or P. nigrescens , or both, were enrolled in the study of intrafamilial carriage. When 20 colonies per specimen of subgingival plaque, if available, were picked from primary culture, 115 P. intermedia and 178 P. nigrescens isolates were recovered from the 18 subjects. Among the subjects studied, family members shared the same subtype strain(s) but non-family members did not. Multiple subtypes were found in 8 (57%) of the 14 P. nigrescens -positive subjects but in only 3 (27%) of the 11 P. intermedia -positive subjects; the difference was, however, not statistically significant ( P = 0.14). These results suggest that the combination of PCR ribotyping and AP-PCR fingerprinting is well suited for the epidemiological study of P. intermedia and P. nigrescens and that each family seems to carry a distinct subtype(s) of these species.

Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.

The regression coefficients affecting the pharmacokinetic parameters of the breakpoints of antimicrobial agents (ie, empiric breakpoints) obtained retrospectively from clinical trial data were considered, and the pharmacokinetic parameters that were thought to strongly correlate to the empiric breakpoints were selected. The regression coefficients of the selected pharmacokinetic parameters were estimated, based on the categoric regression analysis. Estimated regression coefficients were rounded, and breakpoints were constructed using these rounded values. The breakpoint for complicated cystitis was determined, and it was decided that to obtain the breakpoint of complicated pyelonephritis, one tube (ie, one-half value) would be subtracted from the breakpoint of complicated cystitis, as calculated using the formula.

OBJECTIVE: Prevotella bivia is one of the anaerobic bacteria that resides in the flora of the female genital tract. We studied the pathogenicity of P. bivia in a rat pyometra model. METHODS: The experimental animal (rat) model of pyometra was developed to investigate the pathogenicity of P. bivia in a rat pyometra model. RESULTS: In the groups inoculated with aerobes alone, the infection rate was 10% (1/10) in the Staphylococcus aureus- or Staphylococcus agalactiae-inoculated group and 20% (2/10) in the Escherichia coli-inoculated group. Infection was not established in the groups inoculated with anaerobes alone. High infection rates were observed in all the mixed-infection groups. In the S. agalactiae- and Bacteroides fragilis-, S. agalactiae- and P. bivia-, F. coli- and B. fragilis-, and E. coli- and P. bivia-inoculated groups, an infection rate of 100% (10/10) was demonstrated. The efficacy of antibiotics such as flomoxef (FMOX) could be determined using a rat pyometra model. In relation to the alteration of vaginal microbial flora during the menstrual cycle, estrogen increased the growth of P. bivia. CONCLUSION: Mixture of aerobic bacteria and P. bivia increased the pathogenicity of P. bivia. Estrogen would be useful for raising up the inflammatory change of the uterus in experimental models of genital tract infection due to P. bivia.

We investigated genotype of penicillin-binding protein (PBP) genes and macrolide resistant genes, the serotypes and the susceptibility to antibacterial agents against 258 strains of Streptococcus pneumoniae isolated from medical facilities in Gifu and Aichi prefectures between January 2010 and March 2011. These results were compared with those against 377 strains of S. pneumoniae isolated in 2008-2009. The number of genotype penicillin-susceptible S. pneumoniae (gPSSP) with 3 normal PBP genes, genotype penicillin-intermediate S. pneumoniae (gPISP) with 1 or 2 normal PBP genes and genotype penicillin-resistant S. pneumoniae (gPRSP) with 3 abnormal genes was 11 (4.3%), 135 (52.3%) and 112 (43.4%) strains, respectively. The isolates with no macrolide-resistant gene, only mefA, only ermB, and both mefA and ermB were 17 (6.6%), 65 (25.2%), 143 (55.4%) and 33 (12.8%). The prevalent pneumococcal serotypes isolated from children were type 19F (18.2%), following by type 6A and 15 (11.7%). The potential coverage of pneumococcal conjugate vaccine (PCV7) was 43.8%. The prevalent pneumococcal serotypes isolated from adults were high in order of type 19F (12.8%), type 6A, 3 and 11 (10.3%), excepting non-typable strains (17.9%), and from elderly persons were type 6B (23.2%) and type 3 (13.4%). The MIC90 of each antibacterial agents was as follows; 0.0625 microg/mL for garenoxacin, 0.125 microg/mL for panipenem, 0.25 microg/mL for imipenem, doripenem, tosufloxacin, 0.5 microg/mL for cefditoren, meropenem, moxifloxacin, 1 microg/mL for amoxicillin, clavulanic acid/amoxicillin, cefteram, cefcapene, ceftriaxone, 2 microg/mL for benzylpenicillin, piperacillin, tazobactam/ piperacillin, pazufloxacin, levofloxacin, 4 microg/mL for cefdinir, flomoxef, 16 microg/mL for minocycline,64 microg/mL for clarithromycin, azithromycin and these MIC90s were about the same as those in 2008-2009.

We investigated the antimicrobial activity of several drugs against 131 extended-spectrum beta-lactamase (ESBL) positive clinical isolates in Gifu and Aichi prefecture from 2007 to 2011. Meropenem (MEPM) and doripenem (DRPM) gave the lowest MIC50 at 0.0313 microg/mL. MEPM gave the lowest MIC90 at 0.0625 microg/mL. According to the Clinical and Laboratory Standards Institute breakpoints, the susceptible rates of carbapenems, tazobactam/piperacillin (TAZ/PIPC) and cefmetazole (CMZ) were higher than 90%. The susceptible rates of MEPM, DRPM, imipenem (IPM), TAZ/PIPC and CMZ were 98.5%, 98.5%, 94.7%, 94.7% and 92.4%. We used the PCR method and identified the molecular types of the ESBL positive isolates. Seventy-two strains had CTX-M-9 group gene and CTX-M-9 group gene is the most frequently detected. Against the CTX-M-9 group gene harboring strains which were the most common in our investigation, the susceptible rates of TAZ/PIPC, MEPM, DRPM and IPM were 100%. It is suggested that not only carbapenems but also TAZ/PIPC and CMZ are useful against infections caused by ESBL positive isolates.

The study was conducted to elucidate the possibility of hospital infection of methicillin-resistant Staphylococcus aureaus (MRSA) through feces. Fecal cultures of 12 inpatients positive for MRSA in the respiratory tract and 11 inpatients negative for MRSA in the respiratory tract were performed from April to October in 1993. Fecal cultures of inpatients positive for MRSA in the respiratory tract were continued until September in 1996, finally a total of 50 cases were enrolled. MRSA was isolated from feces of 7 patients (58.3%) of the 12 patients positive for MRSA in the respiratory tract, while no MRSA was isolated from feces of 11 patients negative for MRSA in the respiratory tract. Twenty-seven patients (54.0%) of 50 patients positive for MRSA in the respiratory tract yielded MRSA in the feces. Twenty-three patients (85.2%) of the 27 patients positive for MRSA in the fecal flora had received H2-blocker, while 11 patients (47.8%) of the 23 patients negative for MRSA in the fecal flora received H2-blocker; these differences were statistically significantly (p < 0.01). These findings suggest the possible role of feces in hospital infection with MRSA and the necessity for careful administration of H2-blocker to patients positive for MRSA in the respiratory tract.

The bacteriological epidemiology of bacterial vaginosis (BV) and the efficacy of vaginal or oral treatment of BV with clindamycin (CLDM) were investigated. The epidemiology of BV was investigated in 100 symptomatic women before CLDM therapy. Two groups consisting of 50 patients each with the diagnosis of symptomatic BV were treated with either oral administration of 450 mg CLDM three times daily or 2% CLDM phosphate in vaginal cream (self-made) 5 g once a day, for 7 days. There was no significant difference in efficacy among vaginal and oral therapies with CLDM. Vulvovaginal irritation occurred in 3 patients orally treated and in 1 patient vaginally treated. Gastrointestinal disturbances were observed in 4 orally treated patients. A slight abnormal elevation of the glutamine-oxaloacetic transaminase level was also found in 1 patient orally treated. Since there were no statistically significant differences in efficacy rates between vaginal and oral CLDM treatments, we favor vaginal treatment of BV, based on less adverse effects.

Several methoxylated furonaphthoquinones, isolated from Tabebuia plants, are prepared via a direct one-pot cascade reaction via Sonogashira coupling and intramolecular cyclization.

Furonaphthoquinones are promising skeletons for anticancer drug molecules. In particular, methoxylated furonaphthoquinones are characteristic constituents of Tabebuia plants. In this research, we synthesized the furonaphthoquinones by effective one-pot cascade reactions of 3-phenyliodonio-1,2,4-trioxo-1,2,3,4-tetrahydronaphthalenides with 3-butyn-2-ol in the presence of palladium and cuprous catalysts via Sonogashira coupling and intramolecular cyclization. Furthermore, we demonstrated that the synthetic furonaphthoquinones showed moderate cytotoxicity against human leukemia U937 and HL-60 cells. Our work highlights the importance of furonaphthoquinones as antileukemic agents.

Since we had experienced a case of misidentification for group B Streptococcus (GBS) (Streptococcus agalactiae) by automated identification system RAISUS, we have investigated the identification accuracy for GBS by automated identification system RAISUS system using our stock isolated of GBS. Among 31 strains including standard strain ATCC13813 of GBS, 30 strains were identified as GBS, while only 1 strain was misidentified as Streptococcus constellatus. We have needed 3 hours to identify 29 true GBS strains, while it has taken 6 hours for misidentified strain as S. constellatus. Since we have needed more than 4 hours for our misidentified strain of GBS, the tendency for identification time to become long has been recognized. Longer identification time might be associated with the misidentification. The improvement for galactose and glycerol tests of RAISUS by the manufacturer has resulted in better identification. As for the strains to be identified as S. constellatus by RAISUS, we might re-check it with other identification kits when there is higher clinical necessity.

Polymerase chain reaction (PCR) assay was compared with cell culture assay performed with use of HT29/C1 (human colonic epithelial) cells for identifying strains of enterotoxin-producing Bacteroides fragilis (ETBF) isolated from extraintestinal specimens. A total of 188 unselected strains obtained over 2 years at a central clinical laboratory in Tokyo were tested. Overall, 35 strains (18.6%) were positive by cell culture and PCR assay, 152 strains were negative by both assays, and 1 strain was negative by cell culture assay but positive by the PCR assay; the same results were obtained in repeated assays. Among 64 strains from blood, 18 (28.1%) were ETBF, a rate that was significantly higher (P < .05) than the 17 ETBF (13.7%) among 124 strains from other sites. These results suggest that PCR assay is a simple and reliable tool for detecting ETBF and that enterotoxin may be a virulence factor in bacteremia caused by B. fragilis.

The in vitro activity of DU-6859a, a new fluoroquinolone, was compared with those of other fluoroquinolones against clinical isolates of anaerobic bacteria and Gardnerella vaginalis. DU-6859a was the most active agent; it inhibited 90% of isolates of almost all species tested, including Bacteroides fragilis ator = 0.39 micrograms/mL. Although the other quinolones tested were active against gram-positive anaerobes, inhibiting their growth ator = 1.56 micrograms/mL, these agents were less active against the B. fragilis group and Prevotella bivia (90% of which were inhibited ator = 6.25 micrograms/mL). Mobiluncus species and G. vaginalis, which are well associated with bacterial vaginosis, were inhibited by DU-6859a at 0.1 microgram/mL. These results suggest that DU-6859a is a promising oral agent for the treatment of bacterial infections due to anaerobic bacteria; however, further studies, including determination of vaginal levels of this compound, should be performed to study the role of DU-6859a in the treatment of bacterial vaginosis.

The vaginal flora of 118 Japanese and 208 Thai pregnant women were investigated for the presence of bacterial vaginosis (BV), BV-associated organisms, and BV-associated enzyme. A similar prevalence of BV was found among the Japanese (13.6%) and Thai women (15.9%). The microbial flora of women with BV were complex; the mean number of isolates recovered in the BV group was approximately 2 times more than that in a group of healthy women. Prevotella species, Porphyromonas species, Peptostreptococcus species, Mobiluncus species, Gardnerella vaginalis, and H2O2-nonproducing lactobacilli were significantly associated with BV. These organisms were less associated with H2O2-producing lactobacilli, which were predominant in women with normal flora, suggesting that H2O2-producing lactobacilli have antibacterial activity against BV-associated organisms. The vaginal sialidase assay by means of a filter-paper spot test was not proved to be a useful screening aid for diagnosis of BV because of the low sensitivity (69.4%) of this test.

Antigen (Ag)-stimulated phospholipase D (PLD) activation and secretion were almost abolished by pretreatment of rat basophilic leukemia (RBL)-2H3 cells for 4 h with 5 ng/ml Clostridium difficile Toxin B which is known to inhibit Rho family proteins (Rho, Cdc42, Rac). The concentration-dependent inhibition of PLD activation was well correlated with the level of glucosylation of Rho family proteins. In streptolysin O-permeabilized RBL cells, Toxin B suppressed [3H] phosphatidylbutanol (PBut) formation in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) by 67 and 43%, respectively. The synergistic PLD activation by GTP gamma S and PMA was also reduced by Toxin B by 67%. These results suggest that the IgE receptor-coupled PLD activation is largely mediated by Rho proteins.

In vitro antimicrobial activity of NM394 (6-fluoro-1-methyl-7-(1-piperazinyl)-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid) against clinical isolates collected from samples (uterine contents, ascites, pus, etc) obtained from patients with obstetric and gynecologic infections was investigated and compared with that of four other quinolones: norfloxacin, ofloxacin, ciprofloxacin, and tosufloxacin. Among 80 strains of aerobes and 60 strains of anaerobes determined, the minimum inhibitory concentrations (MICs) of NM394 for 50% inhibition against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S aureus (MRSA), Streptococcus agalactiae, Escherichia coli, Peptostreptococcus magnus, Bacteroides fragilis , and Prevotella bivia were 0.39, 25, 0.78, 0.0125, 0.2, 3.13, and 0.78 μg/mL, respectively, and the MICs for 90% inhibition against MSSA, MRSA, S agalactiae, E coli, P magnus, B fragilis , and P bivia were 1.56, > 100, 1.56, 0.025, 0.39, 6.25, and 1.56 μg/mL, respectively. The antimicrobial activity of NM394 against gram-positive bacteria was less than that of tosufloxacin but was comparable to that of ofloxacin and ciprofloxacin. Judging from its antibacterial activity, the efficacy of NM394 against infections caused by MRSA might be expected to be similar to that of the other four quinolones. The antibacterial activity of NM394 against aerobic gram-negative bacteria, especially E coli , was greater than that of the other new quinolones. However, against anaerobic gram-negative bacteria, especially B fragilis , the antibacterial activity of NM394 appeared to be less than that of the other agents. The clinical implications of these results, if any, remain unknown until further clinical therapeutic trials are conducted.