American Society for Microbiology Acta Medica Okayama 2576-098X 2026 Complete genome sequence of Helicobacter pylori strain NY43 harboring bacteriophage KHP30 e00747-26 EN Michiko Takahashi Department of Surgery, Kochi Medical School, Kochi University Hiromichi Maeda Department of Surgery, Kochi Medical School, Kochi University Satoru Seo Department of Surgery, Kochi Medical School, Kochi University Hiroaki Takeuchi Department of Medical Laboratory Sciences, Health and Science, International University of Health and Welfare Graduate School Jumpei Uchiyama Department of Infectious Diseases, Graduate School of Medicine, Density and Pharmaceutical Sciences, Okayama University Shigenobu Matsuzaki Department of Surgery, Kochi Medical School, Kochi University The Helicobacter pylori NY43 strain spontaneously releases the bacteriophage (phage) KHP30. Here, we report the complete genome sequence of H. pylori NY43. In addition to the chromosome of H. pylori NY43, we successfully obtained the complete sequence of the episomal prophage KHP30. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0099-2240 2026 Interaction with the N-acetylgalactosamine moiety facilitates pore formation by Cry4Aa toxin, thereby increasing its toxicity e00714-26 EN Mohammad Tofazzal Hossain Howlader Department of Entomology, Bangladesh Agricultural University Taiyo Tsutsui Applied Chemistry and Biotechnology Program, School of Engineering, Okayama University Kouichi Saito Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tohru Hayakawa Applied Chemistry and Biotechnology Program, School of Engineering, Okayama University Cry4Aa is a mosquito-larvicidal toxin produced by Bacillus thuringiensis serovar israelensis. Cry4Aa exhibits a characteristic architecture comprising three domains (I, II, and III) and functions as a pore-forming toxin, consistent with many previously reported insecticidal Cry toxins. However, the mechanism preceding pore formation by Cry4Aa, including receptor recognition, remains to be elucidated. The present study examined the potential involvement of sugar moieties in the Cry4Aa mode of action, with a particular focus on the process of pore formation. Cry4Aa and the sugar N-acetylgalactosamine exhibited considerably increased binding affinity. Furthermore, N-acetylgalactosamine treatment significantly enhanced the toxicity of Cry4Aa against Culex pipiens mosquito larvae. In the presence of N-acetylgalactosamine, the binding of Cry4Aa to phosphatidylcholine liposomes increased by 1.4-fold. The presence of this sugar did not affect the single-channel conductance of Cry4Aa pores formed in artificial lipid bilayers; however, it did appear to increase the number of Cry4Aa pores per designated area. In silico molecular docking analysis revealed promising binding sites within the cavity, surrounded by the Cry4Aa domains. These results suggest that the interaction of Cry4Aa with N-acetylgalactosamine moieties is a vital step in the process of pore formation. This interaction may induce a conformational change in the Cry4Aa molecule that facilitates its insertion into the target membrane. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2165-0497 14 8 2026 High-throughput screening system for antimycobacterial compounds using in vitro infected cells: construction and application to compounds in the Ōmura Natural Compound Library e00484-26 EN Takemasa Takii Department of Mycobacterium Reference and Research, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Aoi Kimishima Ōmura Satoshi Memorial Institute, Kitasato University Masato Iwatsuki Ōmura Satoshi Memorial Institute, Kitasato University Yoshihiro Watanabe Ōmura Satoshi Memorial Institute, Kitasato University Naoya Ohara Department of Oral Microbiology, Graduate School of Medicine, Density and Pharmaceutical Sciences, Okayama University Yukihiro Asami Ōmura Satoshi Memorial Institute, Kitasato University The global increase in multidrug-resistant tuberculosis strains poses a major threat; consequently, there is an urgent need to identify and develop novel drugs. We have previously presented a simple fibroblast assay (SFA) screening method that enables the simultaneous assessment of antibacterial activity and cytotoxicity toward host cells. This method was developed based on our finding that live (but not dead) Mycobacterium tuberculosis bacilli exhibit cytotoxic activity against human lung fibroblasts, and that this cytotoxicity, which is proportional to the number of viable bacterial cells, is inhibited by known antimycobacterial drugs. While conventional methods to measure M. tuberculosis growth require 2–4 weeks to detect antibacterial activity; the SFA method enables detection within 2–3 days and can detect compounds that are active within host cells. Here, we applied high-throughput screening, based on assessment of cytotoxicity, to identify candidate antimycobacterial agents. In parallel, by comparing the direct antibacterial activity of compounds in liquid medium (using the broth dilution test) with that observed via SFA, we developed a new SFA-based screening system to select compounds with low cytotoxicity that are active outside or inside the host cells. Using this dual system, we screened 742 compounds from the Ōmura Natural Compound Library (Kitasato University, Japan), with 62 hits. Of these, 19 were excluded owing to their toxicity, and six exhibited antibacterial activity only via SFA screening. Four promising antimycobacterial candidates with no cytotoxicity at the 10 ƒÊg/mL threshold�\aridicin A, lankamycin, streptovaricin, and lariatin A�\were identified. identified.This novel system supports the rapid identification of low-cytotoxicity antimycobacterial compounds that are active inside or outside the host cells. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2165-0497 14 9 2026 Combinations of long terminal repeat and tax protein substitutions influence bovine leukemia virus transmission through altered viral productivity e0086926 EN Hironobu Murakami School of Veterinary Medicine, Azabu University Reiichiro Sato Graduate School of Medicine and Veterinary Medicine, University of Miyazaki Jumpei Uchiyama Department of Infectious Diseases, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Kazuyuki Sogawa School of Life and EnvironmentaScience, Azabu University Hiroho Ishida School of Veterinary Medicine, Azabu University Makoto Nagai School of Veterinary Medicine, Azabu University Bovine leukemia virus (BLV) infection imposes a substantial economic burden on the global cattle industry. Although proviral load (PVL) in blood influences transmission, the effects of viral genetic variations, including the high viral production-associated long terminal repeat (LTR) substitution at nucleotide position 175 (LTR175), on herd-level dynamics remain unclear. This study investigated the effects of specific substitutions on the regulation of viral productivity and transmission. Longitudinal monitoring of a single farm (herd size: 50–68 cows; 438 samples collected) over a 7-year period revealed that viral strains carrying cytosine at LTR175 (LTR175C) significantly increased in frequency, becoming dominant over wild-type strains carrying thymidine. In contrast, amino acid substitutions in the Tax protein at positions 69 (Tax69) and 73 (Tax73) modified viral transactivation independent of LTR175. Structural modeling via AlphaFold2 predicted that Tax69 and Tax73 mutations alter protein properties, suggesting modulation of transactivation activity. Consistent with this prediction, in vitro assays confirmed that the substitutions modified viral production by regulating transactivation independent of LTR175. Moreover, high viral-productivity haplotypes containing LTR175C and Tax73Q (glutamine at amino acid residue 73) exhibited a transmission advantage within the herd, even when host PVL levels were not significantly elevated. These findings suggest that cellular-level viral productivity is one of the key determinants of transmission fitness. Overall, this study revealed a regulatory interaction between the LTR and Tax protein that optimizes viral spread and highlights the need for control strategies that account for viral genetic substitutions. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2165-0497 2026 Escalation of CTX-M-producing extensively drug-resistant Shigella spp. in Kolkata, India, following the COVID-19 pandemic e02824-25 EN Puja Bose Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Gourab Halder Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Pratanu Kayet Division of Bioinformatics, ICMR-National Institute for Research in Bacterial Infections Goutam Chowdhury Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Surajit Basak Division of Bioinformatics, ICMR-National Institute for Research in Bacterial Infections Deboleena Roy Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Daisuke Imamura Research Center for Intestinal Health Science, Okayama University Shin-ichi Miyoshi Research Center for Intestinal Health Science, Okayama University Masatomo Morita Department of Bacteriology I, National Institute of Infectious Diseases Thandavarayan Ramamurthy Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Hemanta Koley Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Santasabuj Das Division of Clinical Medicine, ICMR-National Institute for Research in Bacterial Infections Asish Kumar Mukhopadhyay Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections Shigella spp. is recognized by the World Health Organization as a high-priority pathogen due to its global prevalence, unique pathogenic mechanisms, and growing antimicrobial resistance (AMR). Nearly half of all Shigella strains worldwide are now multidrug-resistant (MDR), and the emergence of extensively drug-resistant (XDR) variants�\resistant to ciprofloxacin, ceftriaxone, and azithromycin�\has severely limited effective treatment options. The present study is based on prospective laboratory surveillance involving 323 Shigella isolates collected during 2021–2023, with pre-COVID-19 pandemic data included from a previously published study solely for historical comparison. The presence of antibiotic resistance genes (ARGs) was investigated, and whole-genome sequencing (WGS) was performed on representative isolates to assess phylogenetic relatedness with global isolates. Approximately 10% of isolates exhibited resistance to third-generation cephalosporins. While only 3% of Shigella isolates carried the blaCTX-M-15 gene from 2013 to 2019, its prevalence increased to 26% by 2022–2023. Among 38 ceftriaxone-resistant S. sonnei isolates, 33 were also resistant to azithromycin, categorizing them as XDR. These isolates showed 48% clonal similarity and high phylogenetic resemblance to the isolates reported from England. Hybrid genome assembly revealed a plasmid harboring both the blaCTX-M-15 and mphA ARGs. Conjugation experiments and plasmid profiling confirmed the plasmid�fs transferability. We report a rising trend in third-generation cephalosporin resistance among Shigella spp., primarily driven by the spread of extended-spectrum ƒÀ-lactamase-producing S. flexneri and the emergence of XDR S. sonnei. These findings underscore the urgent need for strengthened national AMR containment strategies and enhanced international surveillance of cephalosporin-resistant Shigella to mitigate this growing public health threat. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0066-4804 69 1 2025 Genomic epidemiology and genetic characteristics of clinical Campylobacter species cocirculating in West Bengal, India, 2019, using whole genome analysis e01108-24 EN Daichi Morita Department of Microbiology, Graduate School of Biomedical and Health Sciences, Hiroshima University Asish Kumar Mukhopadhyay Division of Bacteriology, ICMR - National Institute of Cholera and Enteric Diseases Goutam Chowdhury Division of Bacteriology, ICMR - National Institute of Cholera and Enteric Diseases Fumito Maruyama Section of Microbial Genomics and Ecology, The IDEC Institute, Hiroshima University Miyuki Kanda Department of Cellular and Molecular Biology, Graduate School of Biomedical and Health Sciences, Hiroshima University Yuki Yamamoto Department of Cellular and Molecular Biology, Graduate School of Biomedical and Health Sciences, Hiroshima University Hidetoshi Tahara Department of Cellular and Molecular Biology, Graduate School of Biomedical and Health Sciences, Hiroshima University Piyali Mukherjee Division of Bacteriology, ICMR - National Institute of Cholera and Enteric Diseases Mainak Bardhan Division of Bacteriology, ICMR - National Institute of Cholera and Enteric Diseases Takanori Kumagai Department of Microbiology, Graduate School of Biomedical and Health Sciences, Hiroshima University Kei Kitahara Collaborative Research Centre of Okayama University for Infectious Diseases at ICMR-NICED Shin-Ichi Miyoshi Collaborative Research Centre of Okayama University for Infectious Diseases at ICMR-NICED Shanta Dutta Division of Bacteriology, ICMR - National Institute of Cholera and Enteric Diseases Teruo Kuroda Department of Microbiology, Graduate School of Biomedical and Health Sciences, Hiroshima University Campylobacter species are the most common pathogens responsible for foodborne gastroenteritis worldwide. India is a region with frequent diarrheal infections and a high level of Campylobacter infection incidence, but the detailed genomic information is limited. This study aimed to characterize 112 isolates of Campylobacter from diarrhea patients at two hospitals in Kolkata, West Bengal, by whole genome analysis. The Campylobacter isolates consisted of 90 C. jejuni, 20 C. coli, and 2 C. lari isolates. Multilocus sequence typing analysis revealed that the largest sequence type (ST) populations were ST-2131 in C. jejuni and ST-830 in C. coli and seven novel STs were found in C. jejuni and one in C. coli. Notably, ST-2131, which is rarely seen elsewhere, was positive for a sialylated LOS-related gene (wlaN +neuA + cstIII) associated with Guillain-Barré syndrome. Antibiotic resistance factors predicted from the genome sequence included blaOXA variants (58.9%), tet(O) (54.5%), tet(W) (0.9%), ant(6)-Ia (0.9%), mutation in GyrA (T86I, T86I+D90N, T86I+P104S, T86I+D90N+P104S) (79.5%), and mutation in 23S rRNA (A2075G) (12.5%). In addition to the high drug resistance of Campylobacter in Kolkata, Campylobacter pathogens were circulating that may be associated with Guillain-Barré syndrome. This study indicates the importance of genomic analysis in the surveillance of pathogens, which provides genomic information on genetic diversity, virulence mechanisms, and determinants of antimicrobial resistance. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0019-9567 93 1 2025 A newly developed oral infection mouse model of shigellosis for immunogenicity and protective efficacy studies of a candidate vaccine e00346-2 EN Risha Haldar Division of Clinical Medicine, ICMR-National Institute of Cholera and Enteric Diseases Prolay Halder Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases Hemanta Koley Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases Shin-ichi Miyoshi Division of Medicine, Dentistry and Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Santasabuj Das Division of Clinical Medicine, ICMR-National Institute of Cholera and Enteric Diseases Shigella infection poses a significant public health challenge in the developing world. However, lack of a widely available mouse model that replicates human shigellosis creates a major bottleneck to better understanding of disease pathogenesis and development of newer drugs and vaccines. BALB/c mice pre-treated with streptomycin and iron (FeCl3) plus desferrioxamine intraperitoneally followed by oral infection with virulent Shigella flexneri 2a resulted in diarrhea, loss of body weight, bacterial colonization and progressive colitis characterized by disruption of epithelial lining, loss of crypt architecture with goblet cell depletion, increased polymorphonuclear infiltration into the mucosa, submucosal swelling (edema), and raised proinflammatory cytokines and chemokines in the large intestine. To evaluate the usefulness of the model for vaccine efficacy studies, mice were immunized intranasally with a recombinant protein vaccine containing Shigella invasion protein invasion plasmid antigen B (IpaB). Vaccinated mice conferred protection against Shigella, indicating that the model is suitable for testing of vaccine candidates. To protect both Shigella and Salmonella, a chimeric recombinant vaccine (rIpaB–T2544) was developed by fusing IpaB with Salmonella outer membrane protein T2544. Vaccinated mice developed antigen-specific serum IgG and IgA antibodies and a balanced Th1/Th2 response and were protected against oral challenge with Shigella (S. flexneri 2a, Shigella dysenteriae, and Shigella sonnei) using our present mouse model and Salmonella (Salmonella Typhi and Paratyphi) using an iron overload mouse model. We describe here the development of an oral Shigella infection model in wild-type mouse. This model was successfully used to demonstrate the immunogenicity and protective efficacy of a candidate protein subunit vaccine against Shigella. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0021-9193 206 10 2024 Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis e00307-24 EN Ryogo Yamamoto Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuya Ishikawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yusuke Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuyuki Furuta Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shin-Ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chikara Kaito Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in Bacillus subtilis, a model Gram-positive bacterium. Our findings demonstrated that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis. Specifically, overexpression of ugtP resulted in increased levels of diglucosyldiacylglycerol (Glc2DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, ugtP overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying ugtP mutations. These mutants showed increased levels of Glc2DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc2DAG levels, driven by ugtP overexpression, modify the phospholipid composition and confer daptomycin resistance in B. subtilis without altering the cell surface charge of the bacteria. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0021-9193 208 5 2026 Knockout of tusA confers cationic antimicrobial resistance via fur and omp genes in Escherichia coli e00103-26 EN Kazuya Ishikawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kiho Nakata Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Koichiro Yamada Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mio Uneme Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuyuki Furuta Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chikara Kaito Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University tRNA 2-thiouridine synthesizing protein A (TusA), a sulfur-carrier protein, plays a crucial role in tRNA sulfur modification. Several studies have reported that tusA deficiency affects iron–sulfur (Fe–S) homeostasis in addition to tRNA sulfur modification, resulting in pleiotropic phenotypes. In this study, we analyzed the phenotype of tusA-deficient Escherichia coli and its underlying mechanisms. Although the Keio tusA knockout strain showed increased swimming motility and flagellar biosynthesis, these phenotypes were not restored by tusA complementation. Genome resequencing of the original Keio tusA knockout strain identified an unintended secondary mutation in lrhA, a transcriptional regulator of flagellar and chemotaxis genes. This secondary mutation impaired lrhA function, contributing to enhanced flagellar synthesis and swimming motility, as well as altered global gene expression. A tusA knockout strain in which the secondary mutation was corrected (ƒ¢tusA) exhibited reduced swimming motility compared with the wild-type strain, despite showing no abnormalities in flagellar formation. Furthermore, ƒ¢tusA displayed increased resistance to cationic antibacterial agents, including cetyltrimethylammonium bromide, cetylpyridinium chloride, and protamine sulfate. The reduced motility caused by tusA deletion was observed independently of Fur, a global regulator of iron homeostasis, whereas resistance to cationic antibacterial agents was abolished in the fur knockout background. In addition, altered expression of outer membrane protein (omp) genes was observed in ƒ¢tusA, and deletion of these omp genes abolished resistance to cationic antibacterial agents. Together, these results indicate that, by disentangling the phenotypic effects of tusA deletion from those of the unintended secondary mutation, loss of tusA confers resistance to cationic antibacterial agents through a Fur-dependent and Omp-dependent mechanism. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2576-098X 15 4 2026 Draft genome sequence of blaIMP-1-carrying Phytobacter ursingii OUH-01, isolated from the feces of an acute lymphoblastic leukemia patient e0087625 EN Shuma Tsuji Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital Shinnosuke Fukushima Department of Infectious Diseases, Okayama University Hospital Iori Urakami Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Kazuyoshi Gotoh Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences We report the 5.7 Mbp draft genome of carbapenem-resistant Phytobacter ursingii strain OUH-01, recovered from the feces of a patient with acute lymphoblastic leukemia. The genome comprises a chromosome (G+C content of 53.5%), three plasmids, and four linear contigs, including the 222 kb plasmid pOUH-phyto-IMP-01 carrying blaIMP-1. Nanopore/Illumina sequencing achieved 235�~ coverage. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2379-5042 10 5 2025 Sodium butyrate inhibits the expression of virulence factors in Vibrio cholerae by targeting ToxT protein e00824-24 EN Sushmita Kundu Division of Biochemistry, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Suman Das Division of Biochemistry, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Priyanka Maitra Division of Biochemistry, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Prolay Halder Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Hemanta Koley Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Asish K. Mukhopadhyay Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Shin-ichi Miyoshi Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shanta Dutta Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Nabendu Sekhar Chatterjee Division of Biochemistry, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Sushmita Bhattacharya Division of Biochemistry, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases) Cholera, a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, remains a global health threat in developing countries due to its high transmissibility and increased antibiotic resistance. There is a pressing need for alternative strategies, with an emphasis on anti-virulent approaches to alter the outcome of bacterial infections, given the increase in antimicrobial-resistant strains. V. cholerae causes cholera by secreting virulence factors in the intestinal epithelial cells. These virulence factors facilitate bacterial colonization and cholera toxin production during infection. Here, we demonstrate that sodium butyrate (SB), a small molecule, had no effect on bacterial viability but was effective in suppressing the virulence attributes of V. cholerae. The production of cholera toxin (CT) was significantly reduced in a standard V. cholerae El Tor strain and two clinical isolates when grown in the presence of SB. Analysis of mRNA and protein levels further revealed that SB reduced the expression of the ToxT-dependent virulence genes like tcpA and ctxAB. DNA-protein interaction assays, conducted at cellular (ChIP) and in vitro conditions (EMSA), indicated that SB weakens the binding between ToxT and its downstream promoter DNA, likely by blocking DNA binding. Furthermore, the anti-virulence efficacy of SB was confirmed in animal models. These findings suggest that SB could be developed as an anti-virulence agent against V. cholerae, serving as a potential alternative to conventional antibiotics or as an adjunctive therapy to combat cholera. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0099-2240 2025 Efficient resuscitation of early-stage viable but non-culturable cells of Vibrio cholerae using treatment with proteolytic enzymes EN Shin-ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mona Ogasawara Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shiho Niwaki Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Rena Sugihara Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Basilua Andre Muzembo Research Institute of Nursing Care for People and Community, University of Hyogo Daisuke Imamura Research Center for Intestinal Health Science, Okayama University Vibrio cholerae, the etiological agent of cholera, is ubiquitous in environmental brackish waters. Exposure to low water temperatures induces the bacterium to enter a viable but non-culturable (VBNC) state. In this study, a stepwise decrease in water temperature to 4�‹C was found to delay the transition to the non-culturable state compared to an abrupt temperature drop, suggesting that V. cholerae cells partially adapt to low temperatures. V. cholerae VBNC cells maintained at 4�‹C gradually lost their ability to revert to a culturable state. However, VBNC cells in the early stage of dormancy were efficiently resuscitated following treatment with proteolytic enzymes, including proteinase K. The abundance of culturable V. cholerae cells in brackish estuarine waters was quantified using the most probable number (MPN)–quantitative polymerase chain reaction (qPCR) method. Although culturable cells were undetectable in samples treated with bovine serum albumin, they were estimated at 93 and 1,500 MPN/mL in two water samples collected on different days and pre-incubated with proteinase K. Similarly, the abundance of Vibrio species increased markedly following treatment with this enzyme. Additionally, cells of Vibrio species were enumerated by the plating method using CHROMagar Vibrio plates. Consistent with the results of the MPN–qPCR method, treatment with proteinase K resulted in over a 100-fold increase in colony formation. Collectively, these findings suggest that treatment with proteinase K is effective for resuscitating and quantifying V. cholerae VBNC cells in environmental water samples. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0066-4804 69 12 2025 Genomic portrayal of emerging carbapenem-resistant El Tor variant Vibrio cholerae O1 e00740-25 EN Sreeja Shaw ICMR-National Institute for Research in Bacterial Infections Agila Kumari Pragasam V. Ramalingaswami Bhawan, Indian Council of Medical Research Goutam Chowdhury ICMR-National Institute for Research in Bacterial Infections Prosenjit Samanta ICMR-National Institute for Research in Bacterial Infections Deboleena Roy ICMR-National Institute for Research in Bacterial Infections Debjani Ghosh ICMR-National Institute for Research in Bacterial Infections Thandavarayan Ramamurthy ICMR-National Institute for Research in Bacterial Infections Jigna Karia Medical College Baroda Govind Ninama Medical College Baroda Shin-ichi Miyoshi Okayama University Yukihiro Akeda National Institute of Infectious Diseases Hemanta Koley ICMR-National Institute for Research in Bacterial Infections Asish Kumar Mukhopadhyay ICMR-National Institute for Research in Bacterial Infections The escalating prevalence of carbapenem-resistant (CR) enteric pathogens elicits significant challenges to public health management and effective antimicrobial therapy. While carbapenem resistance is rare in Vibrio cholerae O1 (VC), the recent emergence of CR strains reveals a concerning shift in their antimicrobial resistance (AMR) landscape. This study aims to characterize the resistance mechanisms in newly identified El Tor CRVC isolated from cholera patients in Gujarat, India during 2019. Fifty VC isolates were screened for major virulence-associated genes along with the determination of their antibiotic resistance profiles using Kirby-Bauer disk diffusion and MIC assays. Whole-genome sequencing (WGS) was employed to investigate the underlying mechanisms of CR. All the isolates exhibited hypervirulent Haitian alleles of major virulence genes and AMR profiles of typical multidrug resistance (MDR). Strikingly, 12% (6/50) of them were resistant to carbapenems and other antibiotics. Molecular analysis revealed that these CR isolates were clonally related and harbored a 142 kbp IncA/C type conjugative mega-plasmid with several AMR encoding genes, including blaNDM-1, that can be easily transferred to other bacterial species and confer donor AMR patterns. The plasmid�fs competence for horizontal gene transfer presents a significant risk of dissemination to other enteric pathogens and thereby may complicate the treatment. This finding emphasizes the urgent need for enhanced genomic surveillance and robust antimicrobial stewardship programs aimed at curbing the spread of CRVC strains and mitigating their impact on cholera treatment and containment strategies. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2379-5042 2025 Analysis of the drug target of the anti-tuberculosis compound OCT313: phosphotransacetylase is a potential drug target for anti-mycobacterial agents e00463-25 EN Takemasa Takii Department of Mycobacterium Reference and Research, the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Tomohiro Hasegawa Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University Saotomo Itoh Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University Shinji Maeda Graduate School of Pharmaceutical Sciences, Hokkaido University of Sciences Takayuki Wada Department of Microbiology, Graduate School of Human Life and Ecology, Osaka Metropolitan University Yasuhiro Horita Department of Clinical Pharmaceutics, Graduate School of Medical Sciences, Nagoya City University Akihito Nishiyama Department of Bacteriology, Niigata University School of Medicine Sohkichi Matsumoto Department of Bacteriology, Niigata University School of Medicine Naoya Ohara Department of Oral Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Aoi Kimishima Laboratory of Applied Microbial Chemistry, Ōmura Satoshi Memorial Institute, Kitasato University Yukihiro Asami Laboratory of Applied Microbial Chemistry, Ōmura Satoshi Memorial Institute, Kitasato University Shigeaki Hida Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University Kikuo Onozaki Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University Tuberculosis (TB) is one of the most common infectious diseases caused by bacteria worldwide. The increasing prevalence of multidrug-resistant TB (MDR-TB) and latent TB infection (LTBI) has intensified the global TB burden. Therefore, the development of new drugs for MDR-TB and LTBI is urgently required. We have reported that the derivative of dithiocarbamate sugar derivative, 2-acetamido-2-deoxy-ƒÀ-D-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313), exhibits anti-mycobacterial activity against MDR-MTB. Here, we identified the target of OCT313. In experimentally generated OCT313-resistant bacteria, adenine at position 1,092 in the metabolic enzyme phosphotransacetylase (PTA) gene was replaced with cytosine. This mutation is a nonsynonymous mutation that converts methionine to leucine at position 365 in the PTA protein. OCT313 inhibited the enzymatic activity of recombinant wild-type PTA, but not of the mutant PTA (M365L). PTA is an enzyme that produces acetyl-coenzyme A (acetyl-CoA) from acetyl phosphate and CoA and is involved in metabolic pathways; therefore, it was expected to also be active against dormant Mycobacterium tuberculosis bacilli. OCT313 exhibits antibacterial activity in the Wayne model of dormancy using Mycobacterium bovis BCG, and overexpression of PTA in OCT313-resistant bacilli restored sensitivity to OCT313. Collectively, the target of OCT313 is PTA, and OCT313 is a promising antimicrobial candidate for MDR-TB and LTBI. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0022-538X 99 10 2025 Human herpesvirus 6B U65 binds to histone proteins and suppresses interferon production e00984-25 EN Haokun Li Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirohito Ogawa Department of Virology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Da Teng Department of Virology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Okame Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hikaru Namba Department of Virology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoyuki Honda Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Human herpesvirus 6B (HHV-6B), a member of the Betaherpesvirinae subfamily, is a T-lymphotropic virus that causes exanthem subitum and has been implicated in neuroinflammatory conditions such as multiple sclerosis. The tegument proteins, which are characteristic of herpesviruses, play a crucial role in the envelopment of virions and evasion of host immune defenses, such as the interferon ƒÀ (IFNƒÀ) signaling pathway. However, the precise mechanisms through which the HHV-6B tegument proteins modulate the IFNƒÀ pathway are not yet fully understood. In this study, we identified a novel function of the HHV-6B tegument protein U65 as an inhibitor of IFNƒÀ production. Additionally, two host histone proteins, hCG_2039566 (H2ACG) and H2AC7, were identified as positive regulators of innate immune pathways. U65 interacts with H2ACG and H2AC7, impairing their ability to promote the IFNƒÀ pathway. Furthermore, we demonstrated that U65 plays critical roles during HHV-6B infection. This study highlights a critical strategy employed by HHV-6B to evade immune defenses, shedding light on its mechanisms for counteracting host responses. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2379-5042 10 6 2025 Mycobacterium tuberculosis bacillus induces pyroptosis in human lung fibroblasts e00110-25 EN Takemasa Takii Department of Mycobacterium Reference and Research, the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Hiroyuki Yamada Department of Mycobacterium Reference and Research, the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Chihiro Motozono Department of Molecular Immunology, Research Institute for Microbial Diseases, The University of Osaka Sho Yamasaki Department of Molecular Immunology, Research Institute for Microbial Diseases, The University of Osaka Jordi B. Torrelles Texas Biomedical Research Institute and International Center for the Advancement of Research & Education (I•CARE) Joanne Turner Texas Biomedical Research Institute and International Center for the Advancement of Research & Education (I•CARE) Aoi Kimishima Laboratory of Applied Microbial Chemistry, Ōmura Satoshi Memorial Institute, Kitasato University Yukihiro Asami Laboratory of Applied Microbial Chemistry, Ōmura Satoshi Memorial Institute, Kitasato University Naoya Ohara Department of Oral Microbiology, Graduate School of Medicine, Density and Pharmaceutical Sciences, Okayama University Shigeaki Hida Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University Hidetoshi Hayashi Department of Cell Signaling, Graduate School of Pharmaceutical Sciences, Nagoya City University Kikuo Onozaki Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University We previously reported that live, but not dead, virulent Mycobacterium tuberculosis (Mtb) H37Rv bacilli induce cell death in human lung fibroblast cell lines, MRC-5, MRC-9, and TIG-1. Here, using two distinct Mtb strains from two different lineages (HN878 lineage 2 and H37Rv lineage 4), we confirmed cell death at day 2 after infection with a device that measures cell growth/cytotoxicity in real time (Maestro-Z [AXION]). Mtb bacilli uptake by the fibroblast was confirmed with a transmission electron microscope on day 2. Expressions of inflammatory cytokines and interleukin (IL)�|1ƒÀ, IL-6, and IL-8 were observed when exposed to live, but not dead bacteria. The cell death of fibroblasts induced by both Mtb strains tested was prevented by caspase-1/4 and NLRP3 inflammasome inhibitors, but not by caspase-3 and caspase-9 inhibitors. Therefore, we classified the fibroblast cell death by Mtb infection as pyroptosis. To investigate the biological and pathological relevance of fibroblast cell death by Mtb infection, we performed dual RNA-Seq analysis on Mtb within fibroblasts and Mtb-infected fibroblasts at day 2. In Mtb bacilli tcrR, secE2, ahpD, and mazF8 genes were highly induced during infection. These genes play roles in survival in a hypoxic environment, production of a calcium-binding protein-inducing cytokine, and regulation of transcription in a toxin-antitoxin system. The gene expressions of IL-1ƒÀ, IL-6, and IL-8, caspase-4, and NLRP3, but not of caspase-3 and caspase-9, were augmented in Mtb bacilli-infected fibroblasts. Taken together, our study suggests that Mtb bacilli attempt to survive in lung fibroblasts and that pyroptosis of the host fibroblasts activates the immune system against the infection. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0019-9567 2025 Xenopus laevis as an infection model for human pathogenic bacteria EN Ayano Kuriu Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kazuya Ishikawa Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kohsuke Tsuchiya Division of Immunology and Molecular Biology, Cancer Research Institute, Kanazawa University Kazuyuki Furuta Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Chikara Kaito Division of Molecular Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Animal infection models are essential for understanding bacterial pathogenicity and corresponding host immune responses. In this study, we investigated whether juvenile Xenopus laevis could be used as an infection model for human pathogenic bacteria. Xenopus frogs succumbed to intraperitoneal injection containing the human pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. In contrast, non-pathogenic bacteria Bacillus subtilis and Escherichia coli did not induce mortality in Xenopus frogs. The administration of appropriate antibiotics suppressed mortality caused by S. aureus and P. aeruginosa. Strains lacking the agr locus, cvfA (rny) gene, or hemolysin genes in S. aureus, LIPI-1-deleted mutant of L. monocytogenes, which attenuate virulence within mammals, exhibited reduced virulence in Xenopus frogs compared with their respective wild-type counterparts. Bacterial distribution analysis revealed that S. aureus persisted in the blood, liver, heart, and muscles of Xenopus frogs until death. These results suggested that intraperitoneal injection of human pathogenic bacteria induces sepsis-like symptoms in Xenopus frogs, supporting their use as a valuable animal model for evaluating antimicrobial efficacy and identifying virulence genes in various human pathogenic bacteria. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0099-2240 2025 Enterobacterial common antigen repeat-unit flippase WzxE is required for Escherichia coli growth under acidic conditions, low temperature, and high osmotic stress conditions EN Saki Yamaguchi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuya Ishikawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuyuki Furuta Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chikara Kaito Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Colanic acid and enterobacterial common antigen (ECA) are cell-surface polysaccharides that are produced by many Escherichia coli isolates. Colanic acid is induced under acidic, low temperature, and high-salt conditions and is important for E. coli resistance to these stresses; however, the role of ECA in these stresses is less clear. Here, we observed that knockout of flippase wzxE, which translocates lipid-linked ECA repeat units from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in the sensitivity of E. coli BW25113 to acidic conditions. The wzxE-knockout mutant showed reduced growth potential and viable counts in vegetable extracts with acidic environments, including cherry tomatoes, carrots, celery, lettuce, and spinach. A double-knockout strain of wzxE and wecF (glycosyltransferase that adds the third-and-final sugar of the lipid-linked ECA repeat unit) was not sensitive to acidic conditions, with similar results obtained for a double-knockout strain of wzxE and wcaJ (glycosyltransferase that initiates colanic acid lipid-linked repeat-unit biosynthesis). The wzxE-knockout mutant was sensitive to low temperatures or high-salt conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of wcaJ. These results suggest that lipid-linked ECA repeat units confer E. coli susceptibility to acidic, low temperatures, and high-salt conditions in a colanic acid-dependent manner and that wzxE suppresses this negative effect. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0022-538X 99 3 2025 A capsidless (+)RNA yadokarivirus hosted by a dsRNA virus is infectious as particles, cDNA, and dsRNA e02166-24 EN Muhammad Fadli Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Sakae Hisano Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Guy Novoa Department of Structure of Macromolecules, Centro Nacional Biotecnología (CNB-CSIC), Campus de Cantoblanco José R. Castón Department of Structure of Macromolecules, Centro Nacional Biotecnología (CNB-CSIC), Campus de Cantoblanco Hideki Kondo Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Nobuhiro Suzuki Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Capsidless yadokariviruses (members of the order Yadokarivirales) with (+)RNA genomes divert the capsid of their partner icosahedral double-stranded RNA (dsRNA) viruses in different families of the order Ghabrivirales into the replication site. A yadokarivirus, AfSV2, has been reported from a German strain of the ascomycete fungus Aspergillus foetidus coinfected by two dsRNA viruses, a victorivirus (AfSV1, family Pseudototiviridae) and an alternavirus (AfFV, family Alternaviridae). Here, we identified AfSV1 as the partner of AfSV2 in a Japanese A. foetidus strain after showing the infectiousness of AfSV2 in three forms: virus particles (heterocapsid), transforming full-length complementary DNA (cDNA), and purified replicated form (RF) dsRNA that is believed to be inactive as a translational template. Virion transfection of virus-free A. foetidus protoplasts resulted in the generation of two strains infected either by AfSV1 alone or by both AfSV1 and AfSV2. Transformants with AfSV2 full-length cDNA launched AfSV2 infection only in the presence of AfSV1, but not those with AfSV2 RNA-directed RNA polymerase mutant cDNA. The purified fractions containing AfSV2 RF dsRNA also launched infection when transfected into protoplasts infected by AfSV1. Treatment with dsRNA-specific RNase III, but not with proteinase K, S1 nuclease, or DNase I, abolished the infectivity of AfSV2 RF dsRNA. Furthermore, we confirmed the infectiousness of gel-purified AfSV2 RF dsRNA in the presence of AfSV1. Taken together, our results show the unique infectious entity of AfSV2 and the expansion of yadokarivirus partners in the family Pseudototiviridae and provide interesting evolutionary insights. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2379-5042 9 8 2024 New lineages of RNA viruses from clinical isolates of Rhizopus microsporus revealed by fragmented and primer-ligated dsRNA sequencing (FLDS) analysis EN Wasiatus Sa'diyah Institute of Plant Science and Resources, Okayama University Yan-Jie Zhao Department of Life and Environmental Sciences, Laboratory of Fungal Interaction and Molecular Biology (Donated by IFO), University of Tsukuba Yuto Chiba Department of Life and Environmental Sciences, Laboratory of Fungal Interaction and Molecular Biology (Donated by IFO), University of Tsukuba Hideki Kondo Institute of Plant Science and Resources, Okayama University Nobuhiro Suzuki Institute of Plant Science and Resources, Okayama University Sayaka Ban Medical Mycology Research Center, Chiba University Takashi Yaguchi Medical Mycology Research Center, Chiba University Syun-Ichi Urayama Department of Life and Environmental Sciences, Laboratory of Fungal Interaction and Molecular Biology (Donated by IFO), University of Tsukuba Daisuke Hagiwara Department of Life and Environmental Sciences, Laboratory of Fungal Interaction and Molecular Biology (Donated by IFO), University of Tsukuba Rhizopus microsporus is a species in the order Mucorales that is known to cause mucormycosis, but it is poorly understood as a host of viruses. Here, we examined 25 clinical strains of R. microsporus for viral infection with a conventional double-stranded RNA (dsRNA) assay using agarose gel electrophoresis (AGE) and the recently established fragmented and primer-ligated dsRNA sequencing (FLDS) protocol. By AGE, five virus-infected strains were detected. Then, full-length genomic sequences of 12 novel RNA viruses were revealed by FLDS, which were related to the families Mitoviridae, Narnaviridae, and Endornaviridae, ill-defined groups of single-stranded RNA (ssRNA) viruses with similarity to the established families Virgaviridae and Phasmaviridae, and the proposed family "Ambiguiviridae." All the characterized viruses, except a potential phasmavirid with a negative-sense RNA genome, had positive-sense RNA genomes. One virus belonged to a previously established species within the family Mitoviridae, whereas the other 11 viruses represented new species or even new genera. These results show that the fungal pathogen R. microsporus harbors diverse RNA viruses and extend our understanding of the diversity of RNA viruses in the fungal order Mucorales, division Mucoromycota. Identifying RNA viruses from clinical isolates of R. microsporus may expand the repertoire of natural therapeutic agents for mucormycosis in the future. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2576-098X 12 12 2023 Complete genomic sequence of Vibrio fluvialis strain IDH5335 isolated from a patient with diarrhea in Kolkata, India EN Goutam Chowdhury Collaborative Research Center of Okayama University for Infectious Diseases in India at ICMR-NICED Kei Kitahara Collaborative Research Center of Okayama University for Infectious Diseases in India at ICMR-NICED Makoto Taniguchi Oral Microbiome Center, Taniguchi Dental Clinic Kazuma Uesaka Graduate School of Bioagricultural Sciences, Nagoya University Basilua Andre Muzembo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Debmalya Mitra Collaborative Research Center of Okayama University for Infectious Diseases in India at ICMR-NICED Ayumu Ohno Collaborative Research Center of Okayama University for Infectious Diseases in India at ICMR-NICED Thandavarayan Ramamurthy ICMR-National Institute of Cholera and Enteric Diseases Shanta Dutta ICMR-National Institute of Cholera and Enteric Diseases Shin-ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Asish Kumar Mukhopadhyay ICMR-National Institute of Cholera and Enteric Diseases We isolated a Vibrio fluvialis strain (IDH5335) from a stool sample collected from a patient with diarrhea. In this announcement, we report the complete genomic sequence of this organism, which was obtained by combining Illumina and Oxford Nanopore sequencing data. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2165-0497 11 3 2023 Metataxonomic Analysis of the Uterine Microbiota Associated with Low Fertility in Dairy Cows Using Endometrial Tissues Prior to First Artificial Insemination e04764-22 EN Takuya Yagisawa Hokkaido Agriculture Mutual Aid Association Jumpei Uchiyama Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Iyo Takemura-Uchiyama Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Shun Ando Hokkaido Agriculture Mutual Aid Association Osamu Ichii Laboratory of Anatomy, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University Hironobu Murakami Laboratory of Infectious Diseases, School of Veterinary Medicine, Azabu University Osamu Matsushita Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Seiji Katagiri Laboratory of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University The deterioration in reproductive performance in association with low fertility leads to significant economic losses on dairy farms. The uterine microbiota has begun to attract attention as a possible cause of unexplained low fertility. We analyzed the uterine microbiota associated with fertility by 16S rRNA gene amplicon sequencing in dairy cows. First, the alpha (Chao1 and Shannon) and beta (unweighted and weighted UniFrac) diversities of 69 cows at four dairy farms that had passed the voluntary waiting period before the first artificial insemination (AI) were analyzed with respect to factors including farm, housing style, feeding management, parity, and AI frequency to conception. Significant differences were observed in the farm, housing style, and feeding management, except parity and AI frequency to conception. The other diversity metrics did not show significant differences in the tested factors. Similar results were obtained for the predicted functional profile. Next, the microbial diversity analysis of 31 cows at a single farm using weighted UniFrac distance matrices revealed a correlation with AI frequency to conception but not with parity. In correlation with AI frequency to conception, the predicted function profile appeared to be slightly modified and a single bacterial taxon, Arcobacter, was detected. The bacterial associations related to fertility were estimated. Considering these, the uterine microbiota in dairy cows can be varied depending on the farm management practices and may become one of the measures for low fertility. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2150-7511 2022 Three-Layered Complex Interactions among Capsidless (+)ssRNA Yadokariviruses, dsRNA Viruses, and a Fungus EN Yukiyo Sato Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Sakae Hisano Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Carlos Jose Lopez-Herrera Instituto de Agricultura Sostenible C.S.I.C., Alameda del Obispo Hideki Kondo Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Nobuhiro Suzuki Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University We have previously discovered a virus neo-lifestyle exhibited by a capsidless positive-sense (+), single-stranded (ss) RNA virus YkV1 (family Yadokariviridae) and an unrelated double-stranded (ds) RNA virus YnV1 (proposed family "Yadonushiviridae") in a phytopathogenic ascomycete, Rosellinia necatrix. YkV1 has been proposed to replicate in the capsid provided by YnV1 as if it were a dsRNA virus and enhance YnV1 replication in return. Recently, viruses related to YkV1 (yadokariviruses) have been isolated from diverse ascomycetous fungi. However, it remains obscure whether such viruses generally show the YkV1-like lifestyle. Here, we identified partner viruses for three distinct yadokariviruses, YkV3, YkV4a, and YkV4b, isolated from R. necatrix that were coinfected with multiple dsRNA viruses phylogenetically distantly related to YnV1. We first established transformants of R. necatrix carrying single yadokarivirus cDNAs and fused them with infectants by single partner candidate dsRNA viruses. Consequently, YkV3 and YkV4s replicated only in the presence of RnMBV3 (family Megabirnaviridae) and RnMTV1 (proposed family "Megatotiviridae"), respectively. The partners were mutually interchangeable between the two YkV4 strains and three RnMTV1 strains but not between other combinations involving YkV1 or YkV3. In contrast to YkV1 enhancing YnV1 accumulation, YkV4s reduced RnMTV1 accumulation to different degrees according to strains. Interestingly, YkV4 rescued the host R. necatrix from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3) or host fungus. Overall, we revealed that while yadokariviruses generally require partner dsRNA viruses for replication, each yadokarivirus partners with a different dsRNA virus species in the three diverse families and shows a distinct symbiotic relation in a fungus. IMPORTANCE A capsidless (+)ssRNA virus YkV1 (family Yadokariviridae) highjacks the capsid of an unrelated dsRNA virus YnV1 (proposed family "Yadonushiviridae") in a phytopathogenic ascomycete, while YkV1 trans-enhances YnV1 replication. Herein, we identified the dsRNA virus partners of three yadokariviruses (YkV3, YkV4a, and YkV4b) with genome organization different from YkV1 as being different from YnV1 at the suborder level. Their partners were mutually interchangeable between the two YkV4 strains and three strains of the partner virus RnMTV1 (proposed family "Megatotiviridae") but not between other combinations involving YkV1 or YkV3. Unlike YkV1, YkV4s reduced RnMTV1 accumulation and rescued the host fungus from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3, family Megabirnaviridae) or host fungus. These revealed that while each yadokarivirus has a species-specific partnership with a dsRNA virus, yadokariviruses collectively partner extremely diverse dsRNA viruses and show three-layered complex mutualistic/antagonistic interactions in a fungus. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0022-538X 95 17 2021 Proof of Concept of the Yadokari Nature: a Capsidless Replicase-Encoding but Replication-Dependent Positive-Sense Single-Stranded RNA Virus Hosted by an Unrelated Double-Stranded RNA Virus e00467-21 EN Subha Das Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Md Mahfuz Alam Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Rui Zhang Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Sakae Hisano Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Nobuhiro Suzuki Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Viruses typically encode their own capsids that encase their genomes. However, a capsidless positive-sense single stranded RNA [(+)ssRNA] virus, YkV1, depends on an unrelated double-stranded RNA (dsRNA) virus, YnV1, for encapsidation and replication. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2165-0497 9 1 2021 Use of Recombinant Endolysin to Improve Accuracy of Group B Streptococcus Tests e00077-21 EN Hidehito Matsui Ōmura Satoshi Memorial Institute, Kitasato University Jumpei Uchiyama Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Masaya Ogata School of Veterinary Medicine, Azabu University, Sagamihara Tadahiro Nasukawa School of Veterinary Medicine, Azabu University, Sagamihara Iyo Takemura-Uchiyama School of Veterinary Medicine, Azabu University, Sagamihara Shin-ichiro Kato Kochi University Hironobu Murakami School of Veterinary Medicine, Azabu University, Sagamihara Masato Higashide Kotobiken Medical Laboratories, Inc., Tsukuba Hideaki Hanaki Ōmura Satoshi Memorial Institute, Kitasato University Group B Streptococcus (GBS) causes serious neonatal infection via vertical transmission. The prenatal GBS screening test is performed at the late stage of pregnancy to avoid risks of infection. In this test, enrichment culture is performed, followed by GBS identification. Selective medium is used for the enrichment; however, Enterococcus faecalis, which is a potential contaminant in swab samples, can interfere with the growth of GBS. Such bacterial contamination can lead to false-negative results. Endolysin, a bacteriophage-derived enzyme, degrades peptidoglycan in the bacterial cell wall; it is a promising antimicrobial agent for selectively eliminating specific bacterial genera/species. In this study, we used the recombinant endolysin EG-LYS, which is specific to E. faecalis; the endolysin potentially enriched GBS in the selective culture. First, in the false-negative model (coculture of GBS and E. faecalis, which disabled GBS detection in the subsequent GBS identification test), EG-LYS treatment at 0.1 mg/ml improved GBS detection. Next, we used 548 vaginal swabs to test the efficacy of EG-LYS treatment in improving GBS detection. EG-LYS treatment (0.1 mg/ml) increased the GBS-positive ratio to 17.9%, compared to 15.7% in the control (phosphate-buffered saline [PBS] treatment). In addition, there were an increased number of GBS colonies under EG-LYS treatment in some samples. The results were supported by the microbiota analysis of the enriched cultures. In conclusion, EG-LYS treatment of the enrichment culture potentially improves the accuracy of the prenatal GBS screening test. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2576-098X 10 28 2021 Complete Genome Sequence of Pseudomonas amygdali pv. tabaci Strain 6605, a Causal Agent of Tobacco Wildfire Disease e00405-21 EN Hidenori Matsui Graduate School of Environmental and Life Sciences, Okayama University Takafumi Nishimura Graduate School of Environmental and Life Sciences, Okayama University Shuta Asai Center for Sustainable Resource Science, RIKEN Sachiko Masuda Center for Sustainable Resource Science, RIKEN Ken Shirasu Center for Sustainable Resource Science, RIKEN Mikihiro Yamamoto Graduate School of Environmental and Life Sciences, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Sciences, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Sciences, Okayama University Yuki Ichinose Graduate School of Environmental and Life Sciences, Okayama University Pseudomonas amygdali pv. tabaci strain 6605 is the bacterial pathogen causing tobacco wildfire disease that has been used as a model for elucidating virulence mechanisms. Here, we present the complete genome sequence of P. amygdali pv. tabaci 6605 as a circular chromosome from reads using a PacBio sequencer. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2150-7511 11 3 2020 Hadaka Virus 1: a Capsidless Eleven-Segmented Positive-Sense Single-Stranded RNA Virus from a Phytopathogenic Fungus, Fusarium oxysporum e00450-20 EN Yukiyo Sato Institute of Plant Science and Resources, Okayama University Wajeeha Shamsi Institute of Plant Science and Resources, Okayama University Atif Jamal Crop Diseases Research Institute, National Agricultural Research Centre Muhammad Faraz Bhatti Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST) Hideki Kondo Institute of Plant Science and Resources, Okayama University Nobuhiro Suzuki Institute of Plant Science and Resources, Okayama University The search for viruses infecting fungi, or mycoviruses, has extended our knowledge about the diversity of RNA viruses, as exemplified by the discovery of polymycoviruses, a phylogenetic group of multisegmented RNA viruses with unusual forms. The genomic RNAs of known polymycoviruses, which show a phylogenetic affinity for animal positive-sense single-stranded RNA [(+)RNA] viruses such as caliciviruses, are comprised of four conserved segments with an additional zero to four segments. The double-stranded form of polymycovirus genomic RNA is assumed to be associated with a virally encoded protein (proline-alanine-serine-rich protein [PASrp]) in either of two manners: a capsidless colloidal form or a filamentous encapsidated form. Detailed molecular characterizations of polymycoviruses, however, have been conducted for only a few strains. Here, a novel polymyco-related virus named Hadaka virus 1 (HadV1), from the phytopathogenic fungus Fusarium oxysporum, was characterized. The genomic RNA of HadV1 consisted of an 11-segmented positive-sense RNA with highly conserved terminal nucleotide sequences. HadV1 shared the three conserved segments with known polymycoviruses but lacked the PASrp-encoding segment. Unlike the known polymycoviruses and encapsidated viruses, HadV1 was not pelleted by conventional ultracentrifugation, possibly due to the lack of PASrp. This result implied that HadV1 exists only as a soluble form with naked RNA. Nevertheless, the 11 genomic segments of HadV1 have been stably maintained through host subculturing and conidiation. Taken together, the results of this study revealed a virus with a potential novel virus lifestyle, carrying many genomic segments without typical capsids or PASrp-associated forms. IMPORTANCE Fungi collectively host various RNA viruses. Examples include encapsidated double-stranded RNA (dsRNA) viruses with diverse numbers of genomic segments (from 1 to 12) and capsidless viruses with nonsegmented (+)RNA genomes. Recently, viruses with unusual intermediate features of an infectious entity between encapsidated dsRNA viruses and capsidless (+)RNA viruses were found. They are called polymycoviruses, which typically have four to eight dsRNA genomic segments associated with one of the virus-encoded proteins and are phylogenetically distantly related to animal (+)RNA caliciviruses. Here, we identified a novel virus phylogenetically related to polymycoviruses, from the phytopathogenic fungus Fusarium oxysporum. The virus, termed Hadaka virus 1 (HadV1), has 11 (+)RNA genomic segments, the largest number in known (+)RNA viruses. Nevertheless, HadV1 lacked a typical structural protein of polymycoviruses and was not pelleted by standard ultracentrifugation, implying an unusual capsidless nature of HadV1. This study reveals a potential novel lifestyle of multisegmented RNA viruses. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 00951137 52 3 2014 Haitian variant tcpA in Vibrio cholerae O1 El Tor strains in Kolkata, India 1020 1021 EN Priyanka Ghosh Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED) Arindam Naha Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED) Surajit Basak Department of Molecular Biology & Bioinformatics, Tripura University Santanu Ghosh Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED) T. Ramamurthy Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED) Hemanta Koley Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED) Ranjan K Nandy Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED) Sumio Shinoda Collaborative Research Center of Okayama University for Infectious Diseases at NICED Haruo Watanabe National Institute of Infectious Diseases Asish K. Mukhopadhyay Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED) No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 00951137 51 3 2013 Molecular Characterization of High-Level-Cholera-Toxin-Producing El Tor Variant Vibrio cholerae Strains in the Zanzibar Archipelago of Tanzania 1040 1045 EN A. Naha 1National Institute of Cholera and Enteric Diseases G. Chowdhury 1National Institute of Cholera and Enteric Diseases J. Ghosh-Banerjee 1National Institute of Cholera and Enteric Diseases M. Senoh Collaborative Research Center of Okayama University for Infectious Diseases at NICED, T. Takahashi Collaborative Research Center of Okayama University for Infectious Diseases at NICED, B. Ley The International Vaccine Institute K. Thriemer The International Vaccine Institute J. Deen The International Vaccine Institute L. V. Seidlein Menzies School of Health Research S. M. Ali Ministry of Health and Social Welfare A. Khatib Ministry of Health and Social Welfare T. Ramamurthy 1National Institute of Cholera and Enteric Diseases R. K. Nandy 1National Institute of Cholera and Enteric Diseases G. B. Nair 1National Institute of Cholera and Enteric Diseases Y. Takeda Collaborative Research Center of Okayama University for Infectious Diseases at NICED, A. K. Mukhopadhyay 1National Institute of Cholera and Enteric Diseases Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0270-7306 28 7 2008 Novel transcription-factor-like function of human matrix metalloproteinase 3 regulating the CTGF/CCN2 gene 2391 2413 EN Takanori Eguchi Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazumi Kawata Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiki Mukudai Bio-Dental Research Center, Okayama University Dental School Junji Uehara Department of Oral & Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshihiro Ohgawara Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Soichiro Ibaragi Department of Oral & Maxillofacial Surgery & Biopathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Sasaki Department of Oral & Maxillofacial Surgery & Biopathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuo Kuboki Department of Oral & Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaharu Takigawa Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences �@Matrix metalloproteinase 3 (MMP3) is well known as a secretory endopeptidase that degrades extracellular matrices. Recent reports indicated the presence of MMPs in the nucleus (A. J. Kwon et al., FASEB J. 18:690-692, 2004); however, its function has not been well investigated. Here, we report a novel function of human nuclear MMP3 as a trans regulator of connective tissue growth factor (CCN2/CTGF). Initially, we cloned MMP3 cDNA as a DNA-binding factor for the CCN2/CTGF gene. An interaction between MMP3 and transcription enhancer dominant in chondrocytes (TRENDIC) in the CCN2/CTGF promoter was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by overexpressed MMP3, whereas a TRENDIC mutant promoter lost the response. Also, the knocking down of MMP3 suppressed CCN2/CTGF expression. By cytochemical and histochemical analyses, MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 and six putative nuclear localization signals in MMP3 also were shown. Furthermore, we determined that heterochromatin protein gamma coordinately regulates CCN2/CTGF by interacting with MMP3. The involvement of this novel role of MMP3 in the development, tissue remodeling, and pathology of arthritic diseases through CCN2/CTGF regulation thus is suggested. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2169-8287 5 41 2017 Chloroplast genome sequences of seven strains of bloom-forming raphidophyte, Heterosigma akashiwo e01030-17 EN Sergio Seoane Department of Plant Biology and Ecology, University of the Basque Country (UPV/EHU) Kiwamu Hyodo Institute of Plant Science and Resources, Okayama University Shoko Ueki Institute of Plant Science and Resources, Okayama University We report here the complete chloroplast genome sequences of seven strains of the bloom-forming raphidophyte Heterosigma akashiwo. These ~160-kb sequences contain 124 protein-, 6 rRNA-, and 34 tRNA-coding sequences. Notable sequence variations were observed among these seven sequenced and two previously characterized strains. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0095-1137 52 2 2014 Necessity for Reassessment of Patients with Serogroup 2 Hepatitis C Virus (HCV) and Undetectable Serum HCV RNA 544 548 EN Yuki Moritou Fusao Ikeda Yasuto Takeuchi Hiroyuki Seki Shintaro Nanba Yoshiaki Iwasaki Kazuhide Yamamoto We encountered a patient positive for anti-hepatitis C virus (HCV) whose serum HCV RNA was undetectable with the Roche AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM) version 1 but showed a high viral load with the Abbott RealTime HCV assay (ART). Discrepancies in the detectability of serum HCV RNA were investigated among 891 consecutive patients who were positive for anti-HCV. Specific nucleotide variations causing the undetectability of HCV RNA were determined and confirmed by synthesizing RNA coding those variations. Serum samples with the discrepancies were also reassessed by CAP/CTM version 2. Among the 891 anti-HCV-positive patients, 4 patients had serum HCV RNA levels that were undetectable by CAP/CTM version 1 despite having levels of > 5 log IU/ml that were detected by ART. All four patients had HCV genotype 2a and high titers of anti-HCV. Sequencing of the HCV 5' noncoding regions revealed 2 common variations, A at nucleotide (nt) 145 and T at nt 151. Synthesized RNAs of the HCV 5' noncoding region with standard (NCR145G151C) and variant nucleotides at nt 145 and nt 151 were quantified with CAP/CTM. RNAs of NCR145G151C and NCR145G151T were quantifiable with CAP/CTM version 1, while those of NCR145A151T and NCR145A151C went undetected. The substitution from G to A at nt 145 specifically conferred this undetectability, while this undetectability was reverted in synthesized HCV RNA with correction of this variation. Reassessment of these samples by CAP/CTM version 2 resulted in similar levels of HCV RNA being detected by ART. We conclude that HCV patients with undetectable HCV RNA by CAP/CTM version 1 should be reassessed for viral quantification. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0095-1137 50 5 2012 Development and Evaluation of a PCR Assay for Tracking the Emergence and Dissemination of Haitian Variant ctxB in Vibrio cholerae O1 Strains Isolated from Kolkata, India 1733 1736 EN Arindam Naha G. P. Pazhani Mou Ganguly Santanu Ghosh T. Ramamuranthy Ranjan K. Nandy G. Balakrish Nair Yoshifumi Takeda Asish K. Mukhopadhyay A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata during April 2006, and 93.3% of strains isolated during 2011 carried the new allele. Dendrogram analysis showed a pulsed-field gel electrophoresis (PFGE) pattern of the new variant strains isolated recently that was distinct from the PFGE pattern of the strains carrying classical ctxB that closely matched the 2006 to 2007 variant strains. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0095-1137 50 4 2012 Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls 1308 1312 EN Subrata Sabui Sanjucta Dutta Anusuya Debnath Avishek Ghosh T. Hamabata K. Rajendran T. Ramamurthy James P. Nataro Dipika Sur Myron M. Levine Nabendu Sekhar Chatterjee Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0095-1137 48 11 2010 Cholera Toxin Production by the El Tor Variant of Vibrio cholerae O1 Compared to Prototype El Tor and Classical Biotypes 4283 4286 EN J Ghosh-Banerjee M Senoh T Takahashi T Hamabata S Barman H Koley A. K Mukhopadhyay T Ramamurthy S Chatterjee M Asakura S Yamasaki G. B Nair Y Takeda Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0021-9193 188 24 2006 A homologue of the 3-oxoacyl-(acyl carrier protein) synthase III gene located in the glycosylation island of Pseudomonas syringae pv. tabaci regulates virulence factors via N-acyl homoserine lactone and fatty acid synthesis 8376 8384 EN Fumiko Taguchi Yujiro Ogawa Kasumi Takeuchi Tomoko Suzuki Kazuhiro Toyoda Tomonori Shiraishi Yuki Ichinose Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Delta orf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Delta orf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Delta orf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Delta orf3 mutant. The phenotypes of the Delta orf3 mutant and an AHL synthesis (Delta psyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Delta orf3 mutant. The swarming motility of the Delta orf3 mutant was greater than that of the Delta psyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Delta orf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0021-9193 189 19 2007 Flagellin Glycans from two pathovars of Pseudomonas syringae contain rhamnose in D and L configurations in different ratios and modified 4-amino-4,6-dideoxyglucose 6945 6956 EN Kasumi Takeuchi Hiroshi Ono Mitsuru Yoshida Tadashi Ishii Etsuko Katoh Fumiko Taguchi Ryuji Miki Katsuyoshi Murata Hanae Kaku Yuki Ichinose Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and H-1 and C-13 nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, beta-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1 -> 3)-alpha-L-Rhap-(1 -> 2)-alpha-L-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely Of L-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both L-Rha and D-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae. No potential conflict of interest relevant to this article was reported.