‰ªŽRˆãŠw‰ï Acta Medica Okayama 0030-1558 138 2 2026 �׋Û�«ƒRƒ‰ƒQƒi�[ƒ[‚Ì�\‘¢Šˆ�«‘ŠŠÖ‚ƈã—Éž—p 50 57 EN Osamu Matsushita Professor Emeritus, Okayama University No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2041-1723 17 1 2026 Bacterial collagenase harnesses collagen geometry for processive cleavage 5485 EN Hiroya Oki Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, The University of Osaka Katsuki Takebe Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Adjoa Bonsu Department of Chemistry and Biochemistry, University of Arkansas Kazunori Fujii Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University Ryo Masuda Waseda Research Institute for Science and Engineering, Waseda University Nicholas Henderson Department of Chemistry and Biochemistry, University of Arkansas Takehiko Mima Department of Medical Technology, Faculty of Health Sciences, Ehime Prefectural University of Health Sciences Takaki Koide Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University Mahmoud Moradi Department of Chemistry and Biochemistry, University of Arkansas Osamu Matsushita Department of Bacteriology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Joshua Sakon Department of Chemistry and Biochemistry, University of Arkansas Kazuki Kawahara Graduate School of Pharmaceutical Sciences, The University of Osaka Collagen, the major structural protein in the animal extracellular matrix, forms a triple helix that resists proteolysis and requires specialised enzymes for degradation. Flesh-eating bacteria secrete collagenases that unwind the collagen triple helix and processively trim Gly–X–Y triplet repeats, yet the molecular basis of this process has remained obscure. Here, cryo-electron microscopy reveals how Hathewaya histolytica collagenase ColH engages its substrate and exploits the helix�fs architecture for catalysis. ColH encircles a single collagen triple helix in a closed-ring conformation and, through dynamic domain motions, dehydrates and destabilises it. The enzyme undergoes substrate-assisted twisting to adopt a rigid ratcheted conformation, in which one chain is bent into a tripeptide-long �ebight�f and threaded into the active site for cleavage, while two uncut strands are partitioned to non-catalytic sites. Release of the bight appears to reset the enzyme, with the uncut strands serving as guiding tracks. Repeated cycling between dynamic and rigid states likely enables triplet-by-triplet translocation, allowing ColH to harness collagen�fs geometry for processive degradation. These findings reveal a bacterial strategy for collagen unwinding and cleavage distinct from that of mammalian collagenases, highlighting divergent evolutionary solutions for degrading one of nature�fs most intractable substrates. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 2772-7076 14 2025 Genetic variability in Neisseria meningitidis strains isolated in a Japanese hospital 100511 EN Kazuyoshi Gotoh Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Shinnosuke Fukushima Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital Shuma Tsuji Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Objectives: Neisseria meningitidis is a significant pathogen causing invasive meningococcal disease, posing clinical and public health concerns worldwide. This study aimed to investigate the genetic characteristics of N. meningitidis clinical isolates at Okayama University Hospital in Japan.<br> Methods: Between 2018 and 2023, five clinical strains were isolated, of which three were subjected to the antimicrobial susceptibility testing and whole genetic analysis using MiSeq platform (Illumina, San Diego, CA, USA).<br> Results: One non-groupable isolate, belonging to sequence types (STs)-11026 (ST-32 complex), exhibited non-susceptibility to penicillin G, with a five-mutation pattern (F504L, A510V, I515V, H541N, and I566V) in the penA amino acid sequence and additional mutations (XXXIV and N513Y) characteristic of a mosaic penA gene. The other two isolates, ST-1655 (ST-23 complex) with serogroup Y and ST-2057 with serogroup B, were susceptible to penicillin G, neither of which contained the five-mutation pattern. Levofloxacin resistance was observed in two isolates carrying the T91I mutation in the gyrA protein.<br> Conclusion: Our findings suggest the presence of antimicrobial-resistant N. meningitidis in Japan, underscoring the necessity for continuous local surveillance. Additional research is crucial for clarifying the ongoing spread of resistance mechanisms and for establishing effective countermeasures to reduce the clinical burden of invasive meningococcal disease. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 1365-2672 136 10 2025 Gut dysbiosis allows foodborne salmonella colonization in edible crickets: a probiotic strategy for enhanced food safety lxaf217 EN Shuma Tsuji Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jumpei Uchiyama Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kenji Yokota Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuyoshi Gotoh Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Aims: Edible insects, including crickets, represent a promising protein source, yet concerns over foodborne pathogens limit consumer acceptance. This study investigated whether gut microbiota modulates colonization by Salmonella enterica subsp. enterica serovar Enteritidis (SE) in the two-spotted cricket (Gryllus bimaculatus).<br> Methods and Results: Under standard conditions, SE was undetectable in crickets despite prolonged exposure; however, antibiotic-induced dysbiosis enabled stable SE colonization. Long-read 16S rRNA sequencing revealed significant microbiota shifts, notably a reduction in Lactococcus garvieae. In vitro assays showed strong inhibitory effects of L. garvieae against SE, and supplementation of dysbiotic crickets with L. garvieae reduced SE colonization by ∼1000-fold.<br> Conclusions: The native cricket gut microbiota, especially L. garvieae, plays a protective role against SE colonization. Enhancing beneficial gut bacteria could mitigate pathogen risks and promote edible insects as a sustainable protein. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1341-321X 31 2 2024 Clinical and microbiological characteristics of high-level daptomycin-resistant Corynebacterium species: A systematic scoping review 102575 EN Shinnosuke Fukushima Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital Kazuyoshi Gotoh Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Shuma Tsuji Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Hidemasa Akazawa Department of Infectious Diseases, Okayama University Hospital Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fumio Otsuka Department of Infectious Diseases, Okayama University Hospital Introduction: Corynebacterium species potentially develop high-level daptomycin resistance (HLDR) shortly after daptomycin (DAP) administration. We aimed to investigate the clinical and microbiological characteristics of HLDR Corynebacterium infections.<br> Methods: We first presented a clinical case accompanied by the results of a comprehensive genetic analysis of the isolate, and then performed a systematic scoping review. Based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews, we searched for articles with related keywords, including �gCorynebacterium�h, �gDaptomycin", and "Resistance�h, in the MEDLINE and Web of Science databases from the database inception to October 25, 2024. Clinical case reports and research articles documenting the isolation of HLDR Corynebacterium species, defined by a minimum inhibitory concentration of DAP at ≥256 ƒÊg/mL, were deemed eligible for this review.<br> Results: Of 80 articles screened, seven case reports detailing eight cases of HLDR Corynebacterium infections, as well as five research articles, were included. C. striatum was the most common species (7/9 cases, 77.8 %), and prosthetic device-associated infections accounted for 66.7 % of the cases. Duration of DAP administration before the emergence of HLDR isolates ranged from 5 days to 3 months; three-quarters of the cases developed within 17 days. Three HLDR isolates were genetically confirmed to have an alteration in pgsA2. The majority of the patients were treated with either glycopeptides or linezolid, with favorable outcomes. In vitro experiments confirmed that C. striatum strains acquire the HLDR phenotype at higher rates (71 %–100 %) within 24 h of incubation, compared to other Corynebacterium strains.<br> Conclusion: DAP monotherapy, especially for prosthetic device-associated infections, can result in the development of HLDR Corynebacterium. Additional research is warranted to investigate the clinical implications of this potentially proliferating antimicrobial resistant pathogen. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 19 10 2024 Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6 e0310962 EN Sosuke Munetomo Department of Public Health, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University 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 Thamonwan Wanganuttara Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Yumiko Yamamoto Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Toshihiro Tsukui Nippon Zenyaku Kogyo Co. Ltd. Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital Shuji Kanamaru School of Life Science and Technology, Tokyo Institute of Technology Hideyuki Kanda Department of Public Health, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Osamu Matsushita Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Methicillin-resistant Staphylococcus spp. present challenges in clinical and veterinary settings because effective antimicrobial agents are limited. Phage-encoded peptidoglycan-degrading enzyme, endolysin, is expected to be a novel antimicrobial agent. The enzymatic activity has recently been shown to be influenced by the linker between functional domains in the enzyme. S6_ORF93 (ORF93) is one of the endolysins derived from previously isolated Staphylococcus giant phage S6. The ORF93 was speculated to have a catalytic and peptidoglycan-binding domain with a long linker. In this study, we examined the influence of linker shortening on the characteristics of ORF93. We produce wild-type ORF93 and the linker deletion mutants using an Escherichia coli expression system. These mutants were designated as ORF93-Delta 05, ORF93-Delta 10, ORF93-Delta 15, and ORF93-Delta 20, from which 5, 10, 15, and 20 amino acids were removed from the linker, respectively. Except for the ORF93-Delta 20, ORF93 and its mutants were expressed as soluble proteins. Moreover, ORF93-Delta 15 showed the highest yield and bacteriolytic activity, while the antimicrobial spectrum was homologous. The cold storage experiment showed a slight effect by the linker deletion. According to our results and other studies, linker investigations are crucial in endolysin development. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 78 5 2024 Phenotypic and Genetic Characteristics of Carbapenemase-Producing Enterobacterales Isolates at Okayama University Hospital 371 376 EN Kazuyoshi Gotoh Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Makoto Miyoshi Department of Medical Laboratory Science, Graduate School of Health Sciences, Okayama University I Putu Bayu Mayura Department of Clinical Microbiology, Faculty of Medicine, Udayana University Shuma Tsuji Department of Medical Laboratory Science, Graduate School of Health Sciences, Okayama University Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Shinnosuke Fukushima Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital Original Article 10.18926/AMO/67657 Spread of carbapenemase-producing Enterobacterales (CPE) is an ongoing public health issue worldwide, including in Japan. In this study, we investigated the phenotypic and genetic characteristics of CPE isolates at Okayama University Hospital over the 5 years (2013-2018) prior to the outbreak of the 2019 coronavirus pandemic. Of 24 carbapenem-resistant Enterobacterales isolated during the study period, we identified 8 CPE isolates harboring blaIMP-1 (5 isolates) and blaIMP-6 genes (3 isolates). Bacterial species and carbapenem susceptibility patterns exhibited diversity. Minimum inhibitory concentrations (MICs) of meropenem were generally higher than those of imipenem and biapenem. Results of pulsed-field gel electrophoresis demonstrated that neither clonal nor plasmid-mediated outbreaks of blaIMP-harboring CPE isolates have developed at our hospital. One Klebsiella oxytoca isolate showed a high MIC (128 ƒÊg/mL) of meropenem, which could be explained by the high plasmid copy number. Subsequent analysis of this isolate may elucidate the intricacies of carbapenem resistance profiles among CPE isolates. Collectively, our findings underscore the necessity for ongoing genetic surveillance of CPE, complemented by tailored approaches for infection prevention and control. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0732-8893 110 1 2024 Cefazolin inoculum effect in methicillin-susceptible Staphylococcus aureus clinical isolates 116399 EN Shuma Tsuji Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuyoshi Gotoh Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Tadahiro Manabe Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Shinnosuke Fukushima Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital We investigated the prevalence and characteristics of Cefazolin inoculum effect (CInE) among clinical MSSA isolates in Japan. Although 35.5 % (39 isolates) were positive for the blaZ gene, none met the phenotypic criteria for CInE. Our findings suggested a very low prevalence of CInE among MSSA isolates in our clinical setting. 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.

Okayama University Medical School Acta Medica Okayama 0386-300X 77 1 2023 Prevalence of Inducible Macrolide, Lincosamide, and Streptogramin B (inducible MLSB) Resistance in Clindamycin-Susceptible Staphylococcus aureus at Okayama University Hospital 1 9 EN Lutfun Nahar Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takahiro Nada Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Kazuyoshi Gotoh Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fumio Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/64355 Inducible resistance to the macrolide, lincosamide, and streptogramin B (iMLSB) antibiotic family is a latent mechanism for antimicrobial resistance in Staphylococcus aureus. We here investigated the frequency and genotypic profiles of iMLSB resistance in clindamycin (CLDM)-susceptible S. aureus isolated in Okayama University Hospital from June 2020 to June 2021. We phenotypically screened the iMLSB resistance via D-zone test and performed PCR testing for the erythromycin ribosomal methylase (erm) genes: ermA and ermC. Among 432 CLDM-susceptible S. aureus isolates, 138 (31.9%) exhibited an iMLSB-resistance phenotype, with methicillinresistant S. aureus isolates (MRSA; 61 isolates: 58.6%) exhibiting higher positivity than methicillin-sensitive S. aureus isolates (MSSA; 77 isolates: 23.5%) (p<0.001). Male patients had a higher frequency of iMLSB resistance than females (OR [95%CI]: 1.8 [1.2-2.8]; p=0.007). Genotypically, ermA predominated in both MSSA (70.1%) and MRSA (86.9%) compared to ermC (14.3% in MSSA and 11.5% in MRSA). A single strain of MRSA possessed both ermA and ermC, while 12 (15.6%) MSSA isolates were negative for both ermA and ermC, suggesting the presence of other genetic mechanisms. Collectively, these results show that approximately 33% of CLDM-susceptible S. aureus isolates at our university hospital exhibited iMLSB resistance, predominantly caused by ermA in both MSSA and MRSA. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2076-2607 10 12 2022 Effects of Helicobacter pylori and Nitrate-Reducing Bacteria Coculture on Cells 2495 EN Hinako Ojima Department of Bacteriology, Academic Field of Health Sciences, Okayama University Sakiko Kuraoka Department of Gastroenterology and Hepatology, Academic Field of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Shyoutarou Okanoue Department of Gastroenterology and Hepatology, Academic Field of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Hiroyuki Okada Himeji Red Cross Hospital Kazuyoshi Gotoh Department of Bacteriology, Academic Field of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Osamu Matsushita Department of Bacteriology, Academic Field of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Akari Watanabe Department of Oral Health Care and Rehabilitation, Institute of Biomedical Sciences, Graduate School, Tokushima University Kenji Yokota Department of Bacteriology, Academic Field of Health Sciences, Okayama University Helicobacter pylori infection is an important risk factor for developing gastric cancer. However, only a few H. pylori-infected people develop gastric cancer. Thus, other risk factors aside from H. pylori infection may be involved in gastric cancer development. This study aimed to investigate whether the nitrate-reducing bacteria isolated from patients with atrophic gastritis caused by H. pylori infection are risk factors for developing atrophic gastritis and gastric neoplasia. Nitrate-reducing bacteria were isolated from patients with atrophic gastritis caused by H. pylori infection. Among the isolated bacteria, Actinomyces oris, Actinomyces odontolyticus, Rothia dentocariosa, and Rothia mucilaginosa were used in the subsequent experiments. Cytokine inducibility was evaluated in monocytic cells, and mitogen-activated protein kinase (MAPK) activity and cell cycle were assessed in the gastric epithelial cells. The cytotoxicities and neutrophil-inducing abilities of the Actinomyces and Rothia species were enhanced when cocultured with H. pylori. Th1/Th2-related cytokines were also expressed, but their expression levels differed depending on the bacterial species. Moreover, H. pylori and Actinomyces activated MAPK (ERK and p38) and affected cell cycle progression. Some nitrate-reducing bacteria cocultured with H. pylori may promote inflammation and atrophy by inducing cytokine production. In addition, the MAPK activation and cell cycle progression caused by these bacteria can contribute to gastric cancer development. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 1341-321X 28 12 2022 Detection of Enterobacter cloacae complex strain with a blaNDM-1-harboring plasmid from an elderly resident at a long-term care facility in Okayama, Japan 1697 1699 EN Kazuyoshi Gotoh Department of Bacteriology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of General Medicine, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Haruto Yamada Department of Clinical Laboratory, Okayama City Hospital Osamu Matsushita Department of Bacteriology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fumio Otsuka Department of General Medicine, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Amidst the global spread of antimicrobial resistance, New Delhi metallo-beta-lactamase (NDM)-type carbapenemase-producing Enterobacterales (CPE) remain uncommon in Japan, and the detection of such highly drug-resistant organisms is limited to inbound cases. There is little evidence regarding the prevalence of NDM beta-lactamase gene (blaNDM)-harboring CPE in the domestic community, especially in the provincial cities of Japan. Herein, we report the isolation of a blaNDM-1-harboring plasmid in Enterobacter cloacae complex strain isolated from an elderly woman without a history of traveling abroad who had resided in a long-term care facility in Okayama, Japan. The multidrug-resistant blaNDM-harboring CPE isolate was detected in a stool sample of the patient during routine screening at admission. We performed whole-genome sequencing analysis of the isolate using MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) platforms. The isolate was identified as sequence type 171, which has predominantly been reported in the United States and China. The blaNDM-1 gene was encoded on the 46,161 bp IncX3 plasmid, with sequence similarity to plasmids of similar size isolated from individuals in China. Collectively, the genomic data suggest that an imported CPE isolate may have spread among healthy individuals in the regional area of Japan. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0168-1702 319 2022 Phylogenic analysis of new viral cluster of large phages with unusual DNA genomes containing uracil in place of thymine in gene-sharing network, using phages S6 and PBS1 and relevant uncultured phages derived from sewage metagenomics 198881 EN 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 Kazuyoshi Gotoh Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Shin-ichiro Kato Research Institute of Molecular Genetics, Kochi University Yoshihiko Sakaguchi Department of Microbiology, Kitasato University School of Medicine Hironobu Murakami School of Veterinary Medicine, Azabu University Tomoki Fukuyama School of Veterinary Medicine, Azabu University Mao Kaneki School of Veterinary Medicine, Azabu University Osamu Matsushita Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Shigenobu Matsuzaki Department of Medical Laboratory Science, Faculty of Health Sciences, Kochi Gakuen University Bacteriophages (phages) are the most diverse and abundant life-form on Earth. Jumbophages are phages with double-stranded DNA genomes longer than 200 kbp. Among these, some jumbophages with uracil in place of thymine as a nucleic acid base, which we have tentatively termed "dU jumbophages" in this study, have been reported. Because the dU jumbophages are considered to be a living fossil from the RNA world, the evolutionary traits of dU jumbophages are of interest. In this study, we examined the phylogeny of dU jumbophages. First, tBLASTx analysis of newly sequenced dU jumbophages such as Bacillus phage PBS1 and previously isolated Staphylococcus phage S6 showed similarity to the other dU jumbophages. Second, we detected the two partial genome sequences of uncultured phages possibly relevant to dU jumbophages, scaffold_002 and scaffold_007, from wastewater metagenomics. Third, according to the gene-sharing network analysis, the dU jumbophages, including phages PBS1 and S6, and uncultured phage scaffold_002 formed a cluster, which suggested a new viral subfamily/family. Finally, analyses of the phylogenetic relationship with other phages showed that the dU jumbophage cluster, which had two clades of phages infecting Gram-negative and Gram-positive bacteria, diverged from the single ancestral phage. These findings together with previous reports may imply that dU jumbophages evolved from the same origin before divergence of Gram-negative and Gram-positive bacteria. No potential conflict of interest relevant to this article was reported.

Microbiology Society Acta Medica Okayama 0022-2615 70 10 2021 In vitro effectiveness of biapenem against IMP-producing Enterobacteriaceae EN Kazuyoshi Gotoh Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Makoto Miyoshi Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan I Putu Bayu Mayura Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fumio Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The options available for treating infections with carbapenemase-producing Enterobacteriaceae (CPE) are limited; with the increasing threat of these infections, new treatments are urgently needed. Biapenem (BIPM) is a carbapenem, and limited data confirming its in vitro killing effect against CPE are available. In this study, we examined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of BIPM for 14 IMP-1-producing Enterobacteriaceae strains isolated from the Okayama region in Japan. The MICs against almost all the isolates were lower than 0.5 µg ml�|1, indicating susceptibility to BIPM, while approximately half of the isolates were confirmed to be bacteriostatic to BIPM. However, initial killing to a 99.9 % reduction was observed in seven out of eight strains in a time–kill assay. Despite the small data set, we concluded that the in vitro efficacy of BIPM suggests that the drug could be a new therapeutic option against infection with IMP-producing CPE. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0934-9723 41 2 2021 Detection of in-frame mutation by IS30-family insertion sequence in the phospholipid phosphatidylglycerol synthase gene (pgsA2) of high-level daptomycin-resistant Corynebacterium striatum 331 333 EN Kazuyoshi Gotoh Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Putu Bayu Mayura Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusaku Enomoto Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fumio Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The emergence of high-level daptomycin (DAP)-resistant (HLDR) Corynebacterium striatum has been reported as a result of loss-of-function point mutations or premature stop codon mutations in a responsible gene, pgsA2. We herein describe the novel detection of an HLDR C. striatum clinical isolate, in which IS30-insertion was corroborated to cause destruction of pgsA2 gene. We isolated an HLDR C. striatum from a critically ill patient with underlying mycosis fungoides who had been treated with DAP for ten days. With a sequence investigation, IS30-insertion was discovered to split pgsA2 in the HLDR C. striatum strain, which may cause disrupted phospholipid phosphatidylglycerols (PG) production. Future studies should survey the prevalence of IS-mediated gene inactivation among HLDR C. striatum clinical isolates. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6651 13 7 2021 Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection 467 EN Aina Ichihara Department of Bacteriology, Academic Field of Health Science Okayama University Hinako Ojima Department of Bacteriology, Academic Field of Health Science Okayama University Kazuyoshi Gotoh Department of Bacteriology, Academic Field of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Osamu Matsushita Department of Bacteriology, Academic Field of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Susumu Take Department of Gastroenterology and Hepatology, Kurashiki Central Hospital Hiroyuki Okada Department of Gastroenterology and Hepatology, Academic Field of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Akari Watanabe Department of Oral Health Care and Rehabilitation, Institute of Biomedical Sciences, Tokushima University Graduate School Kenji Yokota Department of Bacteriology, Academic Field of Health Science Okayama University The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori. No potential conflict of interest relevant to this article was reported.

Dove Medical Press Ltd Acta Medica Okayama 1178-6973 14 2021 Antibacterial Effects of Disulfiram in Helicobacter pylori 1757 1764 EN Tomomi Kobatake Graduate School of Health Science Okayama University Keiki Ogino Department of Environmental Medicine, Koch Medical School Hiroyuki Sakae Department of Gastroenterology and Hepatology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences Kazuyoshi Gotoh Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences Akari Watanabe Department of Oral Health Care and Rehabilitation, Institute of Biomedical Sciences, Tokushima University Graduate School Osamu Matsushita Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences Hiroyuki Okada Department of Gastroenterology and Hepatology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University Kenji Yokota Graduate School of Health Science Okayama University Background: Helicobacter pylori infection poses a risk of the occurrence of gastrointestinal diseases, such as gastric cancer. Its incidence rate is significantly reduced by eradication, and thereby, eradication therapy is generally performed. Disulfiram is an oral prescription drug mainly used for the treatment of alcohol dependence. In recent years, reports have been made on its anticancer and antibacterial effects, and thus, it has recently become an interesting subject. This study aimed to examine the antibacterial activity of disulfiram, investigate the presence or absence of its antibacterial activity on H. pylori, and determine whether it could be a new bactericidal drug against drug-resistant H. pylori.<br> <br> Materials and Methods: Drug-sensitive strains of H. pylori and amoxicillin-resistant, clarithromycin-resistant, and metronidazole-resistant strains were used, and a growth inhibition test of H. pylori using disulfiram was performed. Furthermore, the expression of urease, vacuolating cytotoxin A (VacA), and CagA, the virulence proteins of H. pylori, was quantitatively analyzed using the Western blotting method. In addition, for H. pylori used in this study, the 16SrDNA sequence, a ribosomal gene involved in protein production, was analyzed to examine the presence or absence of gene mutation.<br> <br> Results: Disulfiram suppressed the growth of 7 out of 12 H. pylori strains at 1 mu g/mL, and no correlation was observed between their susceptibility/resistance to current eradication antimicrobial drugs and disulfiram resistance. Disulfiram reduced the expression levels of urease, VacA, and CagA proteins. H. pylori, which showed resistance to disulfiram, tended to have fewer gene deletions/insertions in the 16S rDNA sequence; however, no specific mutation was detected. Conclusion: Disulfiram has a bactericidal effect on H. pylori at low concentrations, suggesting that it can be used as a supplement for current H. pylori eradication drugs. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0022-3492 90 9 2019 Acceleration of bone regeneration of horizontal bone defect in rats using collagen�]binding basic fibroblast growth factor combined with collagen scaffolds 1043 1052 EN Shin Nakamura Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Takashi Ito Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Kentaro Okamoto Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Takehiko Mima Department of Bacteriology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Kentaro Uchida Department of Orthopedic Surgery, Kitasato University School of Medicine Yasir D. Siddiqui Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Masahiro Ito Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Masako Tai Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Keisuke Okubo Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Keisuke Yamashiro Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Kazuhiro Omori Department of Periodontics and Endodontics, Okayama University Hospital Tadashi Yamamoto Department of Periodontics and Endodontics, Okayama University Hospital Osamu Matsushita Department of Bacteriology, Dentistry and Pharmaceutical Sciences Shogo Takashiba Department of Pathophysiology�]Periodontal Science, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Background</br> Basic fibroblast growth factor (bFGF) has been applied for periodontal regeneration. However, the application depends on bone defect morphology because bFGF diffuses rapidly from defect sites. In a previous study, collagen�]binding bFGF (CB�]bFGF) has been shown to enhance bone formation by collagen�]anchoring in the orthopedic field. The aim of this study is to demonstrate the efficacy of CB�]bFGF with collagen scaffolds in bone regeneration of horizontal bone defect.</br> Methods</br> Cell proliferation activity and collagen binding activity of CB�]bFGF was confirmed by WST�]8 assay and collagen binding assay, respectively. The retention of CB�]bFGF in the collagen sheet (CS) was measured by fluorescence imaging. The rat horizontal alveolar bone defect model was employed to investigate the efficacy of CB�]bFGF with collagen powder (CP). After 4 and 8 weeks, the regenerative efficacy was evaluated by microcomputed tomography, histological, and immunohistochemical analyses.</br> Results</br> CB�]bFGF had a comparable proliferation activity to bFGF and a collagen binding activity. CB�]bFGF was retained in CS longer than bFGF. At 8 weeks postoperation, bone volume, bone mineral content, and new bone area in CB�]bFGF/CP group were significantly increased compared with those in other groups. Furthermore, epithelial downgrowth was significantly suppressed in CB�]bFGF/CP group. At 4 weeks, the numbers of osteocalcin, proliferating cell nuclear antigen, and osteopontin�]positive cells at the regeneration site in CB�]bFGF/CP group were greater than those in other groups.</br> Conclusions</br> CB�]bFGF/CP effectively promoted bone regeneration of horizontal bone defect possibly by sustained release of bFGF. The potential of CB�]bFGF composite material for improved periodontal regeneration in vertical axis was shown. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 67 2 2013 The Genetic Diversity of Helicobacter pylori Virulence Genes Is Not Associated with Gastric Atrophy Progression 93 98 EN Masahide Kita Kenji Yokota Hiroyuki Okada Susumu Take Ryuta Takenaka Yoshiro Kawahara Keiji Oguma Osamu Matsushita Kazuhide Yamamoto Original Article 10.18926/AMO/49667 Atrophy of the gastric mucosa is a precursor of intestinal-type gastric cancer, and Helicobacter pylori infection causes atrophic gastritis. The aim of this study was to determine whether the genetic diversity of H. pylori virulence genes is associated with the development and progression of gastric atrophy in humans. We isolated and cultured H. pylori strains from patients with gastric ulcer and duodenal ulcer accompanied by atrophic gastritis in background mucosa. H. pylori strains were stored at �|80�Ž prior to the experiments being carried out. We analyzed iceA, babA, vacA, cagA, and cagE genes by PCR. The cagA gene was analyzed through sequencing of the C-terminal region containing the EPIYA motif, which is related to tyrosine phosphorylation. Severe atrophy was observed in patients with gastric ulcer. The major phenotype of the vacA gene was s1c/m1 (93µ). The cagA gene was detected in all strains. The cagE gene was not detected in 2 and 5 strains from the mild cases and severe cases, respectively. The major cagA EPIYA motif, which is amino acids repeat in the C terminus, was the A-B-D type (44 of 58 strains). The virulence genes were not statistically associated with the severity of atrophy in the background gastric mucosa in humans. Not only identification of bacterial virulence factors but also studies of the host response will be necessary to investigate the progression of gastric atrophy and subsequent cancer development in humans. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 67 1 2013 Phospholipase C Produced by Clostridium botulinum Types C and D:Comparison of Gene, Enzymatic, and Biological Activities with Those of Clostridium perfringens Alpha-toxin 9 18 EN Ni Nengah Dwi Fatmawati Yoshihiko Sakaguchi Tomonori Suzuki Masataka Oda Kenta Shimizu Yumiko Yamamoto Jun Sakurai Osamu Matsushita Keiji Oguma Original Article 10.18926/AMO/49252 Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival. No potential conflict of interest relevant to this article was reported.