bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Ganglioside Content Affect the Binding of Clostridium perfringens Epsilon‐Toxin to Detergent‐Resistant Membranes of Madin‐Darby Canine Kidney Cells
Authors: Shimamoto Seiko , Tamai Eiji , Matsushita Osamu , Minami Junzaburo , Okabe Akinobu , Miyata Shigeru
Published: November 2013

Epsilon‐toxin (ET) of Clostridium perfringens, which causes fatal enterotoxemia in ungulates, was previously shown to bind to and form a heptameric pore within the detergent‐resistant membranes (DRMs) of MDCK cells. Depletion of cholesterol has also been shown to decrease the cytotoxicity of ET and its heptamerization. In this study, we investigated the effects of changes in sphingolipids, other DRM components of MDCK cells, on the cells' susceptibility to ET. Treatment with fumonisin B1 and PDMP, inhibitors of sphingolipid and glycosphingolipid syntheses, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside GM1 dramatically decreased the ET binding, heptamerization and cytotoxicity. These effects were shown not to be due to ET binding to GM1 or to denaturation of ET. We also found that the ET cytotoxicity towards MDCK cells decreased with an increase in culture time. In accordance with the resistance observed for prolonged cultured cells, GM3, a major ganglioside component, increased and sialidase treatment increased their susceptibility. These results suggest that membrane‐anchored sialic acid of GM3 within DRMs inhibits ET binding, leading to prevention of the heptamerization of ET and cell death. It is also suggested that sialidase produced by this organism aids the targeting of ET to MDCK cells.
Analysis of Cellular Fatty Acids in Orientia tsutsugamushi as Taxonomic Markers
Authors: Shim Soo‐Kyoung , Shin Yong‐Kook , Choi Eun‐Na , Han Bok‐Ghee , Lee Hae‐Kyung , Choi Young‐Sill , Lee Byung‐Chul , Yoon Kwon‐Sang , Park Mi‐Yeoun
Published: November 2013

Six Orientia strains including 3 prototype strains such as Gilliam, Karp, and Kato, and 3 strains (Boryong, Pajoo, and Yongworl) isolated in Korea, were studied for the profiles of their cellular fatty acids. All tested strains contained octadecenoic acid C18:1 ω3±3.5%), octadecanoic acid C18:0 (15.3±1.5%), and hexadecanoic acid C16:0 (12.7±1.7%) as major components; however, interestingly, eicosenoic acid C20:1 ω9c (2.6 ±0.6%) was found in all strains except the Yongworl strain. Furthermore none of the strains contained 3‐hydroxy fatty acids. The ratios of total saturated fatty acid (SFA) to total unsaturated fatty acid (UFA) were within the range of 0.34 to 0.54. These results showed that the cellular fatty acid profile should provide more reliable information for the identification of these bacteria.
Human Papillomavirus Genotypes and the p53 Codon 72 Polymorphism in Cervical Cancer of Northeastern Thailand
Authors: Settheetham‐Ishida Wannapa , Kanjanavirojkul Nipa , Kularbkaew Churairat , Ishida Takafumi
Published: November 2013

Human papillomavirus (HPV) infection including sub‐strain identification was studied in patients with squamous cell cervical cancer (SCCA) in Northeastern Thailand. Subjects were 90 cases of SCCA and 100 healthy controls. Prevalence of high‐risk group of HPV infection in the controls and the SCCA patients were 13.0% and 86.7%, respectively. The HPV infection significantly increased the risk for cervical cancer 43.5‐fold (95% confidential interval: 17.5–110.6; P < 0.00001). Among HPV carrier patients with SCCA (n=78), HPV‐16 was also prominent (70.5%) followed by HPV‐18 (23.1%). There was no statistical difference in the subtype distribution between the SCCA and the control groups. There was no significant association between genotype distribution of the p53 codon 72 polymorphism and HPV infection. HPV infection was confirmed as a critical risk factor for cervical cancer development in Northeast Thailand. Since polymorphism of the p53 itself as well as in combination with HPV infection may not be a genetic risk for cervical cancer, much attention should be paid to other risk factors such as sexual behavior and smoking.
Rapid Categorization of Pathogenic Escherichia coli by Multiplex PCR
Authors: Kimata Keiko , Shima Tomoko , Shimizu Miwako , Tanaka Daisuke , Isobe Junko , Gyobu Yotaku , Watahiki Masanori , Nagai Yoshiyuki
Published: November 2013

A one‐shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli (EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli (EPEC); invE for enteroinvasive E. coli (EIEC); elt, estp, and esth for enterotoxigenic E. coli (ETEC); CVD432 and aggR for enteroaggregative E. coli (EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.
CCR7 Ligand‐Enhanced Phagocytosis of Various Antigens in Mature Dendritic Cells—Time Course and Antigen Distribution Different from Phagocytosis in Immature Dendritic Cells
Authors: Kikuchi Kazuhiro , Yanagawa Yoshiki , Onoé Kazunori
Published: November 2013

CC chemokine receptor 7 (CCR7) is selectively expressed on mature dendritic cells (DC). The CCR7 ligands, CC chemokine ligand (CCL) 19 and CCL21, facilitate migration of mature DC from the peripheral tissues to regional lymph nodes. We previously demonstrated that CCR7 ligands induced rapid receptor‐mediated endocytosis of dextran in mature DC. In the present study, we further examined the effects of CCR7 ligands on endocytosis of other kinds of antigen, mannosilated bovine serum albumin (Mann‐BSA), Escherichia coli (E. coli), or ovalbumin‐containing immune complex (OVA‐IC), by mature DC. We found that CCR7 ligands enhanced the endocytosis of Mann BSA, E. coli, and OVA‐IC in mature DC but not in immature DC. The endocytosis of BSA was not enhanced by CCR7 ligands. Furthermore, the phagocytosis of OVA‐IC was significantly inhibited by anti‐FcY receptor III/II antibody. These results demonstrate that CCR7 ligands enhance only receptor‐mediated endocytosis by mature DC. When rapidly phagocytosed E. coli were traced in CCL19‐treated mature DC, most of the phagocytosed E. coli did not colocalize with the lysosomal marker: lysosome‐associated membrane protein‐1 (Lamp‐1), whereas most of E. coli taken up relatively slowly by immature DC colocalized with Lamp‐1. These results suggest that phagocytosis of antigens by immature and mature DC plays different functional roles.
Rapid and Simple Identification of Orientia tsutsugamushi from Other Group Rickettsiae by Duplex PCR Assay Using groEL Gene
Authors: Park Hyo‐Soon , Lee Jung‐Hee , Jeong Eun‐Ju , Kim Jung‐Eun , Hong Sung‐Jin , Park Tae‐Kyu , Kim Tae‐Yung , Jang Won‐Jong , Park Kyung‐Hee , Kim Bum‐Joon , Kook Yoon‐Hoh , Lee Seung‐Hyun
Published: November 2013

In this study, two new duplex PCR methods based on the groEL gene were developed and investigated for the diagnosis of rickettsiae. The first duplex PCR assay amplified the 229‐bp and the 366‐bp DNAs of 6 strains including typhus group (TG) and spotted fever group (SFG) rickettsiae, and 5 scrub typhus group (STG) rickettsiae, respectively. The second duplex PCR assay amplified the 397‐bp and the 213‐bp DNAs of 6 Rickettsia strains and 5 STG strains. These duplex PCR methods could simultaneously perform the rapid identification of rickettsiae and the differential diagnosis of STG and other group rickettsiae in a single reaction.
Chemokine and Cytokine Production in Susceptible C3H/HeN Mice and Resistant BALB/c Mice during Orientia tsutsugamushi Infection
Authors: Yun Ji‐Hyun , Koh Young‐Sang , Lee Keun‐Hwa , Hyun Jin‐Won , Choi Yeon‐Joo , Jang Won‐Jong , Park Kyung‐Hee , Cho Nam‐Hyuk , Seong Seung‐Yong , Choi Myung‐Sik , Kim Ik‐Sang
Published: November 2013

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1α (MIP‐1α), MIP‐2, monocyte chemoattractant protein 1 (MCP‐1), and cytokines gamma‐interferon (IFN‐γ), interleukin‐12 (IL‐12), IL‐10, and tumor necrosis factor alpha (TNF‐α) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible‐infection model (BALB/c‐Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.
Homologous Recombination with Linear DNA to Insert Antigenic Protein in the Flagellin: Improvement of the Th1 Immune Response
Authors: Moigne Vincent Le , Robreau Georges , Mahana Wahib
Published: November 2013

Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non‐functional, chromosomal flagellin gene (fliC) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non‐motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non‐motile, non‐transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N‐terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN‐γ production and the lack of IL‐4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.
Capsule of Streptococcus pyogenes Is Essential for Delayed Death of Mice in a Model of Streptococcal Toxic Shock Syndrome
Authors: Iida Ken‐ichiro , Seki Masanori , Saito Mitsumasa , Kawamura Yoshiaki , Kajiwara Hideko , Yoshida Shin‐ichi
Published: November 2013

We have previously reported a mouse model of severe group A streptococcal infection (Microbiol. Immunol. 45: 777–786, 2001). When we injected Streptococcus pyogenes strains intramuscularly, the mice suffered from acute phase of infection for a few days but recovered from the illness and gained body weight. These mice, however, began to die after 3 weeks of infection, which we called ‘delayed death.’ Bacterial strains isolated from organs of the dead mice showed thick capsules. We, therefore, constructed a hyaluronic acid capsule gene, hasA, knockout mutant by homologous recombination and the effect of capsule on the death was observed. hasA knockout strain did not cause delayed death, though it caused acute death at high doses of infection. According to this result, the capsule is a critical pathogenic factor for causing the delayed death in our mouse model.
Adjuvant Effect of the Propionibacterium acnes and Its Purified Soluble Polysaccharide on the Immunization with Plasmidial DNA Containing a Trypanosoma cruzi Gene
Authors: Mussalem Juliana Sekeres , Vasconcelos José Ronnie C. , Squaiella Carla Cristina , Ananias Renata Zeigler , Braga Eleni Gonçalves , Rodrigues Maurício M. , Longo‐Maugéri Ieda Maria
Published: November 2013

In the present work we investigated the role of killed Propionibacterium acnes or a soluble polysaccharide extracted from bacterium cell wall in modulated experimental immunization with plasmidial DNA. We used a plasmid, p154/13, containing a gene‐encoding catalytic domain of Trypanosoma cruzi (T. cruzi) trans‐sialidase. As previously described, immunization of BALB/c mice with p154/13 elicited humoral, cell‐mediated and protective immune responses against T. cruzi infection. In this study we describe that both P. acnes and its soluble polysaccharide fraction have the ability to modulate the immune response elicited by p154/13. Treatment with these adjuvants enhanced specific trans‐sialidase Th1 immune response, as revealed by a lower IgG1/IgG2a ratio and stronger in vitro IFN‐γ synthesis by CD4+ T cells. The most important fact was that treatment with P. acnes or its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzi challenge. These data suggest that P. acnes or its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruzi infection.
Analysis of Amphotericin B‐Induced Cell Signaling with Chemical Inhibitors of Signaling Molecules
Authors: Matsuo Kenichiro , Hotokezaka Hitoshi , Ohara Naoya , Fujimura Yuji , Yoshimura Atsutoshi , Okada Yukio , Hara Yoshitaka , Yoshida Noriaki , Nakayama Koji
Published: November 2013

Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor‐κB (NF‐κB), a well‐known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte‐like cell line. Among chemical inhibitors of signaling molecules, U‐73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA‐AM (calcium chelator), LFM‐A13 (Bruton's tyrosine kinase (Btk)‐specific inhibitor), and PP2 (c‐Src kinase inhibitor) suppressed AmB‐induced phosphorylation of p65 and translocation of p65 into the nucleus. U‐73122 and Gö6976 reduced AmB‐mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)‐α and interleukin (IL)‐6) in RAW264.7 cells. Furthermore, AmB‐induced activation of NF‐κB was observed in toll‐like receptor (TLR) 2‐expressed cells, and the activation of NF‐κB was inhibited by U‐73122, whereas peptidoglycan‐induced NF‐κB activation, which was also dependent on TLR2, was not inhibited by U‐73122. Finally, U‐73122 partially suppressed in vivo production of TNF‐α and IL‐6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c‐Src and NF‐κB. These signaling molecules may become a target for chemotherapy suppressing AmB‐induced proinflammatory cytokine production.
Analysis of a Bacterial Lipopolysaccharide‐Activated Serine Kinase That Phosphorylates p65/L‐Plastin in Macrophages
Authors: Hagi Akifumi , Hirata Hajime , Shinomiya Hiroto
Published: November 2013

We previously identified p65/L‐plastin as a phosphorylated protein in LPS‐stimulated macrophages and determined its phosphorylation site. In vitro kinase assay using peptide substrates revealed that LPS‐stimulated kinase activity selectively phosphorylated their serine‐5 (Ser‐5) residue. Kinase inhibitors for cAMP‐dependent kinase such as H‐89 inhibited the Ser‐5 phosphorylation, but cAMP was not essential for the kinase activity. The LPS‐stimulated kinase activity in cytosol fractions of macrophages was recovered as a sharp peak by anion exchange chromatography. These findings suggest that an as yet unknown H‐89‐sensitive serine kinase is rapidly activated by LPS stimulation and then phosphorylates p65/L‐plastin, playing a vital role in macrophage activation.
Phenotypic and Molecular Characteristics of Escherichia coli Isolated from Aquatic Environment of Bangladesh
Authors: Alam Munirul , Nur‐A‐Hasan  , Ahsan Sunjukta , Pazhani Gururaja Perumal , Tamura Kazumichi , Ramamurthy Thadavarayan , Gomes Donald James , Rahman Sabita R. , Islam Atiqul , Akhtar Fahmina , Shinoda Sumio , Watanabe Haruo , Faruque Shah M. , Nair Gopinath Balakrish
Published: November 2013

Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype‐based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid profiling showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat‐stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat‐labile enterotoxin (LT), serotype‐based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype‐based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.
First Detection of Rickettsia in Soft‐Bodied Ticks Associated with Seabirds, Japan
Authors: Kawabata Hiroki , Ando Shuji , Kishimoto Toshio , Kurane Ichiro , Takano Ai , Nogami Sadao , Fujita Hiromi , Tsurumi Miyako , Nakamura Noboru , Sato Fumio , Takahashi Mamoru , Ushijima Yoko , Fukunaga Masahito , Watanabe Haruo
Published: November 2013

Rickettsia was first detected in seabird soft‐bodied ticks, Carios capensis and C. sawaii in Japan. According to sequence analysis, Rickettsia in Japan was identical to Rickettsia scc31 in C. capensis in the U.S.A. This suggested that an environmental circulation had consisted among microorganisms, ticks and long distance migratory seabirds around the Pacific Ocean.
Evaluation of Antifungal Pharmacodynamic Characteristics of AmBisome against Candida albicans
Authors: Takemoto Koji , Yamamoto Yutaka , Ueda Yutaka
Published: November 2013

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time‐kill method and murine systemic infection model. A time‐kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8‐, 12‐, 24‐hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration‐dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.
Identification and Molecular Characterization of an N‐Acetylmuraminidase, Aml, Involved in Streptococcus mutans Cell Separation
Authors: Yoshimura Goh , Komatsuzawa Hitoshi , Hayashi Ikue , Fujiwara Tamaki , Yamada Sakuo , Nakano Yoshio , Tomita Yuko , Kozai Katsuyuki , Sugai Motoyuki
Published: November 2013

We previously demonstrated Streptococcus mutans produces two bacteriolytic enzymes of 100 kDa and 80 kDa (G. Yoshimura et al. Microbiol. Immunol. 48, 465–469, 2004). Here, we identified the protein sequence of these enzymes and found they come from a single gene product designated as automutanolysin (Aml). Aml has a modular design where the N‐terminus contains five 13‐amino‐acid repeats and a C‐terminal enzyme active domain. Aml selectively lyses S. mutans and S. sobrinus but no other oral streptococci. This suggests Aml possesses strong substrate specificity towards cariogenic bacteria present in the human oral cavity. Analysis of S. mutans peptidoglycan fragments released by Aml shows the enzyme is an N‐acetylmuraminidase. We found Ca2+ enhances the activity; and EGTA, EDTA and iodoacetic acid inhibit the activity. The optimum pH range for lytic activity was 6 to 7. Disruption of the aml gene in S. mutans results in the formation of a longer bacterial cell chain length that was dispersed by the addition of a low concentration of Aml. This suggests Aml is involved in S. mutans cell separation.
Listeria monocytogenes Induces the Expression of Retinoic Acid‐Inducible Gene‐I
Authors: Imaizumi Tadaatsu , Sashinami Hiroshi , Mori Fumiaki , Matsumiya Tomoh , Yoshida Hidemi , Nakane Akio , Wakabayashi Koichi , Oyama Chikara , Satoh Kei
Published: November 2013

Retinoic acid‐inducible gene‐I (RIG‐I) is considered to play a role in innate immunity against virus infections. We showed by immunohistochemical study that RIG‐I expression is upregulated in vivo in hepatic Kupffer cells and in splenic reticular cells of mice infected with Listeria monocytogenes. Both heat‐killed L. monocytogenes and live L. monocytogenes induced the expression of RIG‐I in cultured RAW264.7 murine macrophage‐like cells in vitro. RIG‐I may also be involved in innate immunity against Listeria infection.
High Efficiency Cross‐Reactive Monoclonal Antibody Production by Oral Immunization with Recombinant Norwalk Virus‐Like Particles
Authors: Tanaka Tomoyuki , Kitamoto Noritoshi , Jiang Xi , Estes Mary K.
Published: November 2013

Monoclonal antibodies (MAbs) are important for in‐depth antigenic characterization and diagnosis of infections with human caliciviruses that cause almost all outbreaks of nonbacterial gastroenteritis. We compared different routes of immunization with nonreplicating virus‐like particles (VLPs) from recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX) administered to BALB/c mice to determine the efficiency of hybridoma production. Oral immunization with VLPs without adjuvant resulted in high yields of MAb‐secreting hybridomas (90%) to these VLPs of IgG (61%), IgM (29%) and IgA (10%) isotypes. Fusions with mesenteric lymph node lymphocytes yielded MAbs of various subclasses including IgG2a, IgG3, IgM and IgA. These results suggest that an immunization route that mimics the natural route of viral infection pathway may facilitate MAb technology by increasing the yields of antibody secreting hybridoma cells.
Mycobacterium kumamotonense Sp. Nov. Recovered from Clinical Specimen and the First Isolation Report of Mycobacterium arupense in Japan: Novel Slowly Growing, Nonchromogenic Clinical Isolates Related to Mycobacterium terrae Complex
Authors: Masaki Takayuki , Ohkusu Kiyofumi , Hata Hiroyuki , Fujiwara Nagatoshi , Iihara Hirotoshi , Yamada‐Noda Makiko , Nhung Pham Hong , Hayashi Masahiro , Asano Yuko , Kawamura Yoshiaki , Ezaki Takayuki
Published: November 2013

Three mycobacterium strains isolated from clinical specimens in Japan were provisionally assigned to the genus Mycobacterium based on their phenotypical characteristics. These isolates were further investigated to determine their specific taxonomic statuses. Mycolic acid analysis and 16S rRNA gene, rpoB, and hsp65 sequence data for the isolates showed that they are most similar to M. terrae complex. DNA‐DNA hybridization studies indicated that the three strains were of two species and were distinguishable from M. terrae, M. nonchromogenicum, and M. hiberniae. Therefore, these strains represent two novel species within the genus Mycobacterium. However, one potential new species should have been considered as M. arupense with the 16S rRNA gene and hsp65 sequences similarities of 99.8% and 100% respectively; it was isolated from human specimens in the United States and was proposed in June 2006 as a new species. This report describes the first isolation of M. arupense in Japan, suggesting that the organism is clinically relevant. In addition, we propose the novel species designation Mycobacterium kumamotonense sp. nov. The type strain is CST 7247T (=GTC 2729T, =JCM 13453T, =CCUG 51961T).
Prolonged Viral RNA Detection in Blood and Lymphoid Tissues from Coxsackievirus B4 E2 Orally‐Inoculated Swiss Mice
Authors: Jaïdane Hela , Gharbi Jawhar , Lobert Pierre‐Emmanuel , Lucas Bernadette , Hiar Raïda , M'Hadheb Manel Ben , Brilot Fabienne , Geenen Vincent , Aouni Mahjoub , Hober Didier
Published: November 2013

The spreading of viral RNA within Swiss Albino mice orally inoculated with coxsackievirus B4 E2 strain (CVB4 E2) was studied by using RT‐PCR and semi‐nested‐RT‐PCR methods. Viral RNA was detected in various organs: pancreas, heart, small intestine, spleen, thymus, and blood at various postinfectious (p.i.) times ranging from 8 hr to 150 days. Our results show that (i) outbred mice can be infected with CVB4 E2 following an oral inoculation, which results in systemic spreading of viral RNA, (ii) CVB4 E2 infection can be associated with a prolonged detection of viral RNA in spleen, thymus and blood, up to 70 days p.i. and further in other organ tissues.
Long‐Term Prevalence of Antimicrobial‐ResistantSalmonella entericaSubspeciesentericaSerovar Infantis in the Broiler Chicken Industry in Japan
Authors: Asai Tetsuo , Ishihara Kanako , Harada Kazuki , Kojima Akemi , Tamura Yutaka , Sato Shizuo , Takahashi Toshio
Published: November 2013

Eleven broiler isolates ofSalmonellaInfantis obtained between 1989 and 1998 were examined for antimicrobial susceptibility and pulse field gel electrophoresis (PFGE) profiles. Seven strains ofS. Infantis isolated after 1993 harbored similar antimicrobial susceptibilities to the recent isolates between 2001 and 2003. In comparison of PFGE profile with 22 isolates obtained from 22 apparently healthy broiler chickens between 2001 and 2003, the predominant cluster included the seven strains isolated after 1993. We could not clarify the reasons why the serovar has been prevalent in the broiler industry for a long time, but current antimicrobial usage is not always linked to its prevalence.
Tandem Repeats of Lactoferrin‐Derived Anti‐Hepatitis C Virus Peptide Enhance Antiviral Activity in Cultured Human Hepatocytes
Authors: Abe Ken‐ichi , Nozaki Akito , Tamura Kazushi , Ikeda Masanori , Naka Kazuhito , Dansako Hiromichi , Hoshino Hiro‐o , Tanaka Katsuaki , Kato Nobuyuki
Published: November 2013

Previously, we found that bovine and human lactoferrin (LF) specifically inhibited hepatitis C virus (HCV) infection in cultured non‐neoplastic human hepatocyte‐derived PH5CH8 cells, and we identified 33 amino acid residues (termed C‐s3–33; amino acid 600–632) from human LF that were primarily responsible for the binding activity to the HCV E2 envelope protein and for the inhibiting activity against HCV infection. Since the anti‐HCV activity of C‐s3–33 was weaker than that of human LF, we speculated that an increase of E2 protein‐binding activity might contribute to the enhancement of anti‐HCV activity. To test this possibility, we made two repeats [(C‐s3–33)2] and three repeats [(C‐s3–33)3] of C‐s3–33 and characterized them. Far‐Western blot analysis revealed that the E2 protein‐binding activities of (C‐s3–33)2 and (C‐s3–33)3 became stronger than that of the C‐s3–33, and that the binding activity of (C‐s3–33)3 was stronger than that of (C‐s3–33)2. Using an HCV infection system in PH5CH8 cells, we demonstrated that the anti‐HCV activities of (C‐s3–33)2 and (C‐s3–33)3 became stronger than that of the C‐s3–33. Furthermore, using a recently developed infection system with a VSV pseudotype harboring the green fluorescent protein gene and the native E1 and E2 genes, we demonstrated that the antiviral activities of (C‐s3–33)2 and (C‐s3–33)3 were stronger than that of C‐s3–33. These results suggest that tandem repeats of LF‐derived anti‐HCV peptide are useful as anti‐HCV reagents.
Survey of Babesia microti Infection in Field Rodents in Japan: Records of the Kobe‐Type in New Foci and Findings of a New Type Related to the Otsu‐Type
Authors: Saito‐Ito Atsuko , Kasahara Midori , Kasai Masatoshi , Dantrakool Anchalee , Kawai Atsuko , Fujita Hiromi , Yano Yasuhiro , Kawabata Hiroki , Takada Nobuhiro
Published: November 2013

Of 247 rodents comprising 5 genera and 7 species collected at 17 sites throughout Japan from 2003 to 2005, Babesia microti was detected microscopically and by polymerase chain reaction (PCR) in 36 rodents comprising 2 genera and 3 species from 12 sites. Based on the analysis of small subunit ribosomal RNA gene (SSUrDNA) sequences, the Kobe‐type, the etiological type of the first Japanese case of human infection was found in Apodemus speciosus and Apodemus argenteus in Aomori, the northernmost prefecture of the Japanese mainland, while the U.S.‐type was found on Hokkaido Island and the Otsu‐type was widely distributed. In addition, a new Otsu‐related type was detected exclusively in Eothenomys andersoni in Nagano, a prefecture in central Japan. The sequences of internal transcribed spacer 1 to 2 (ITS1/2) of the present Kobe‐ and Otsu‐types were almost identical to those of the same types previously identified. The ITS1/2 sequence of the U.S.‐type identified in Hokkaido in this survey was somewhat different from that of the U.S.‐type strain originating from the U.S.A., with approximately 95% identity. This value was similar to the 94% identity found between the ITS1/2 sequences of the Otsu‐type and the new Otsu‐related type. The new Otsu‐related type of B. microti was isolated as the Nagano strain, which was serologically differentiated from the other type strains of B. microti. The divergence and distribution of genotypes are important factors in investigating the epidemiology of human B. microti infection in Japan.
Molecular Detection of Various Rickettsiae in Mites (Acari: Trombiculidae) in Southern Jeolla Province, Korea
Authors: Choi Yeon‐Joo , Lee Eun‐Mi , Park Jin‐Mi , Lee Kyung‐Min , Han Seung‐Hoon , Kim Jae‐Kwon , Lee Seung‐Hyun , Song Hyeon‐Je , Choi Myung‐Sik , Kim Ik‐Sang , Park Kyung‐Hee , Jang Won‐Jong
Published: November 2013

This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission.
Spread of Community‐Acquired Methicillin‐Resistant Staphylococcus aureus (MRSA) in Hospitals in Taipei, Taiwan in 2005, and Comparison of Its Drug Resistance with Previous Hospital‐Acquired MRSA
Authors: Takano Tomomi , Saito Kohei , Teng Lee‐Jene , Yamamoto Tatsuo
Published: November 2013

Panton‐Valentine leucocidin (PVL)‐positive methicillin‐resistant Staphylococcus aureus (PVL+ MRSA) is an emerging pathogen in the community worldwide. The incidence of PVL+ MRSA in Taipei, Taiwan was 23.3% for hospital MRSA. PVL+ MRSA was isolated from both outpatients and inpatients. Some PVL+ (mecA+) strains (36.8%) showed low MIC values (μ2 μg/ml) to oxacillin. A major PVL+ MRSA resistance pattern was oxacillin and clindamycin resistance (81%). There was no multidrug resistance over three drugs, in contrast to patient PVL– MRSA with resistance to five drugs as a major resistance pattern. The majority of PVL+ MRSA belonged to multilocus sequence (ST) type 59, while PVL– MRSA belonged to ST239, ST59 and ST5. The data suggests that although PVL+ CA‐MRSA is isolated at a high incidence from hospitals in Taipei, the drug resistance is mostly selected in the community and less prominent compared with previous PVL– hospital‐acquired MRSA.
In Vitro Th1 Cytokine‐Independent Th2 Suppressive Effects of Bifidobacteria
Authors: Iwabuchi Noriyuki , Takahashi Noritoshi , Xiao Jin‐zhong , Miyaji Kazuhiro , Iwatsuki Keiji
Published: November 2013

A comparison between 17 strains of lactic acid bacteria and 15 strains of bifidobacteria indicated that bifidobacteria induced significantly lower levels of interleukin‐12 (IL‐12) in murine splenic cells. The present study aims to evaluate the effect and mechanism of Bifidobacterium longum BB536, a probiotic strain, in suppressing antigen‐induced Th2 immune response in vitro. BB536 suppressed immunoglobulin (Ig) E and IL‐4 production by ovalbumin‐sensitized splenic cells, but induction of Th1‐inducing cytokine production, such as IL‐12 and gamma interferon (IFN‐γ) tended to be lower compared with lactic acid bacteria. Neutralization with antibodies to IL‐12, IFN‐γ, IL‐10 and transforming growth factor β indicated negative involvement of Th1‐inducing cytokines and regulatory cytokines in the suppression of Th2 immune response by BB536, especially when treated at higher doses of BB536 (>10 μg cells/ml). Furthermore, BB536 induced the maturation of immature bone marrow‐derived dendritic cells (BM‐DCs), and suppressed antigen‐induced IL‐4 production mediated by BM‐DCs. These results suggested that BB536 suppressed Th2 immune responses, partially independent of Th1‐inducing cytokines and independent of regulatory cytokines, mediated by antigen‐presenting cells such as dendritic cells.
A Novel Agar Medium to Detect Hydrogen Peroxide‐Producing Bacteria Based on the Prussian Blue‐Forming Reaction
Authors: Saito Mitsumasa , Seki Masanori , Iida Ken‐ichiro , Nakayama Hiroaki , Yoshida Shin‐ichi
Published: November 2013

The classic method for H2O2 detection involving Prussian blue formation was adapted to formulate a novel agar medium that makes possible in situ detection of H2O2 produced by bacteria. Using this medium, colonies of H2O2‐producing species including Streptococcus pyogenes were easily identified by the appearance of blue halos. The utility of the medium was further illustrated by its successful application to the isolation of H2O2‐producing mutants from a non‐H2O2‐producing stain of S. pyogenes.
In Vitro Susceptibilities of Candida spp. to Panomycocin, a Novel Exo‐β‐1,3‐Glucanase Isolated from Pichia anomala NCYC 434
Authors: İzgü Fatih , Altınbay Demet , Türeli Akif Emre
Published: November 2013

Panomycocin, the killer toxin of Pichia anomala NCYC 434 (K5), is a 49 kDa monomeric glycoprotein with exo‐β–1,3‐glucanase activity (patent pending). In this study we evaluated the in vitro activity of panomycocin against a panel of 109 human isolates of seven different pathogenic Candida spp. using microdilution and time‐kill methods. Panomycocin was most active against C. tropicalis, C. pseudotropicalis and C. glabrata with MIC90 values of 1 μg/ml. It displayed significant activity against C. albicans and C parapsilosis with MIC90 values of 4 and 2 μg/ml, respectively. For C. krusei, the MIC90 value was 8 μg/ml. Panomycocin was fungicidal against all the tested Candida spp. The MFC values were only one or 2 dilutions higher than the MICs with the exception of C. krusei isolates with MFCs greater than or equal to 4 × MIC. Results of this study indicated that panomycocin could be considered as a natural antifungal agent against Candida infections and has significant potential for further investigation.
Small and Rough Colony Pseudomonas aeruginosa with Elevated Biofilm Formation Ability Isolated in Hospitalized Patients
Authors: Ikeno Takako , Fukuda Kazumasa , Ogawa Midori , Honda Masahisa , Tanabe Tadao , Taniguchi Hatsumi
Published: November 2013

Pseudomonas aeruginosa is a key pathogen of nosocomial infection, and causes persistent infection in patients with specific diseases like cystic fibrosis (CF). It has been reported that patients affected with CF discharge, at a high frequency, small colony variants with high adherence ability. In routine laboratory testing, we found atypical small and rough type (SR) colony variants of P. aeruginosa. The SRs and the counterpart wild type (WT) colonies showed similar biochemical features, antimicrobial susceptibilities, pulsed‐field gel electrophoresis (PFGE) profiles, serotypes, and twitching motilities. The biofilm formation abilities of all the SR colonies, however, were extremely elevated as compared to those of the counterpart WT colonies. The frequency of SR‐positive patients was 3.1% of the P. aeruginosa‐positive inpatients (5/160), and that of the SR isolates was 0.6% of the P. aeruginosa strains (6/970) isolated in our laboratory over a period of 6 months. The SR‐positive patients did not have any common disease or particular antibiotics treatment. The PFGE profiles showed that the SRs and the counterpart WTs were identical to each other, and also that three of the five SR/WT pairs were clonally similar. The three pairs were recovered from the feces, urine, and endotracheal secretion, respectively, of three patients hospitalized in two distinct wards. The results suggest that P. aeruginosa spontaneously produced highly adherent SR colonies in hospitalized patients, and these colonies may tend to spread in a hospital.
Demonstration and Characterization of Manganese Superoxide Dismutase of Providencia alcalifaciens
Authors: Chen Xiuhao , Kodama Toshio , Iida Tetsuya , Honda Takeshi
Published: November 2013

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and play a role in the pathogenesis of certain invasive bacteria. In this study, we reported for the first time here that Providencia alcalifaciens, a member of the family Enterobacteriaceae, produces a superoxide dismutase (SOD) as a major protein in culture supernatants. This protein was purified by a series of column chromatographic separations. The N‐terminal amino acid sequence of the protein was determined to be highly homologous to manganese superoxide dismutase of Escherichia coli or Salmonella reported. The gene (sodA) encoding for SOD of P. alcalifaciens was cloned and sequenced. The sodA‐encoded protein has a molecular weight of about 23.5 kDa, and the DNA sequence of P. alcalifaciens sodA gene (627 bp) has about 83% identity to the E. coli SOD gene. We constructed a sodA deletion mutant and its complemented strain of P. alcalifaciens. In J774, a macrophage cell line, the sodA deletion mutant was more susceptible to killing by macrophages than the wildtype strain and its complemented strain. When we injected the mutant strain, its complemented strain and wildtype strain intraperitoneally into DDY strain mice, we found that the sodA deletion mutant proved significantly less virulent while the complemented strain recovered the virulence to the same level of wildtype strain of P. alcalifaciens. These results suggested that manganese superoxide dismutase plays an important role in intracellular survival of P. alcalifaciens.
Human Herpesvirus 6 Infection of CD4+ T‐Cell Subsets
Authors: Otani Naruhito , Okuno Toshiomi
Published: November 2013

The immune system includes CD4+ regulatory T (Treg) cells that play a role in self‐tolerance and demonstrate functional variations that govern immune responses. HHV‐6 is an important immunosuppressive virus that completely replicates in vivo and in vitro in only CD4+ T cells. However, there have been no reports of the specific T‐cell subpopulation that permits the replication of this virus. Here, we evaluated the infectivity of HHV‐6 to specific T‐cell populations such as CD4+CD25high, which includes the majority of Treg cells, and CD4+CD25–. These cells were isolated from peripheral blood and then expanded. The expanded cell fractions were then infected with the HHV‐6 variant B strain, and the spreads of infected cells were evaluated by immunofluorescence. Viral growth was also quantified by real‐time PCR. The effects of virus infection on cytokine production from these T‐cell subsets were examined using ELISA. Our results revealed that both these fractions permitted complete HHV‐6 replication. Virus infection enhanced the production of both Th1‐ and Th2‐type cytokines from CD4+CD25– T cells; however, only Th2‐type cytokine release was augmented from viral‐infected CD4+CD25high T cells. Further, while virus‐infected CD4+CD25high T cells shift their antiviral immunity toward Th2 dominance by producing IL‐10, the role of virus‐infected CD4+CD25– T cells remains obscure.
Small Red Bean (Azuki) Sheds Biologically Active Substances as a Prerequisite Step for Germination, One of Which Displays the Antiviral Activity against the Rabies Virus Infectivity and Infections in Culture
Authors: Kawai Akihiko , Fujita Kimie
Published: November 2013

When small red beans (azuki bean; Vigna angularis Ohwi et Ohashi) were soaked and warmed in water or saline, the beans began to absorb water to swell and exuded kinds of substances probably as a prerequisite step for seed germination. Such exudate fluids displayed strong antiviral activity against the rabies virus infections in culture. On the other hand, little anti‐rabies activity was detected in the aqueous extracts from the red beans when tested soon after the extraction from powdered beans, while low titers of antiviral activity appeared gradually in the extracts during cold storage. In contrast, no antiviral activity was detected in the exudate fluids from non‐colored azuki beans (white azuki), implicating that a certain anthocyanin‐related substance is involved in the antiviral activity of red beans. Production of antiviral and cytotoxic activities were affected differently depending on the bean‐soaking conditions. In addition, the antiviral activity resisted to 10 min‐heating in boiling water, while the cytotoxicity was greatly weakened by the heating, suggesting that different substances are involved in the antiviral and cytotoxic activities. Further studies on the antiviral activity of the exudate fluids demonstrated that anti‐rabies activity of the bean exudates affected not only the very early phase of infection cycle, but the viral infectivity was also affected similarly, implicating a possible application of azuki bean exudate fluids to post‐exposure treatment of rabid dog‐bite injuries in combination with vaccination.
Orally Administrated Lactobacillus gasseri TMC0356 and Lactobacillus GG Alleviated Nasal Blockage of Guinea Pig with Allergic Rhinitis
Authors: Kawase Manabu , He Fang , Kubota Akira , Harata Gaku , Hiramatsu Masaru
Published: November 2013

Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC0356) were investigated for their ability to alleviate nasal blockage associated with allergic rhinitis using a guinea pig model. The increases in sRaw at 10 min and 5 hr after the exposure of the nasal mucosa to OVA were significantly alleviated in the guinea pigs orally administrated with LGG and TMC0356 compared with those of the control (P<0.05 and P<0.01). The total numbers of leukocytes, particularly eosinophils and neutrophils from the nasal cavity lavage fluid, and the OVA‐specific IgE concentration in the serum were also decreased in the guinea pigs orally administrated with LGG and TMC0356, although the decreases were not statistically significant. These results suggest that LGG and TMC0356 can alleviate antigen‐induced nasal blockage in earlyphase and late‐phase inflammatory responses associated with allergic rhinitis.
Comparative Proteome Analysis of the Outer Membrane Proteins of in Vitro‐Induced Multi‐Drug Resistant Neisseria gonorrhoeae
Authors: Yoo Jeong Sik , Seong Won Keun , Kim Tong Soo , Park Yong Keun , Oh Hee‐Bok , Yoo Cheon Kwon
Published: November 2013

Antimicrobial‐resistant gonococcus has been a major problem in sexually transmitted disease control. Outer membrane proteins (OMPs) of Neisseria gonorrhoeae were suggested to have influence on its resistance to antibiotics. So, in this work, we provide a proteomic analysis tool for examining the OMPs of N. gonorrhoeae and also provide a comparative analysis of the OMPs between the susceptible parent strain (92WT) and the resistance‐induced isogenic mutant (92mu13) to determine the OMPs responsible for resistance. The 2‐D gel spots of 92mu13 differed from 92WT particularly in porin, pilus secretion protein (PilQ) and enzymes. PilQ expression in 92mu13 was considerably reduced by abrupt termination at nucleotide 2,112. This made it difficult to form a high molecular mass (HMM) pore at the outer membrane; it is suspected that reduction of PilQ serves a role in antibiotic resistance in N. gonorrhoeae. The amount of porin was not changed but its isoelectric point (pI) shifted to a basic region, which is caused by the alteration of an amino acid of porin and it is suggested to relate to the development of antimicrobial resistance. Differential regulation of the enzymes involved in metabolism was found in 92mu13, believed to represent an adaptation of N. gonorrhoeae to the antibiotic environment.