Aug. 2017, 第84回日本細菌学会北海道支部学術総会, 優秀賞　　　　　　　　　　　　　　　
札幌地下歩行空間での空気中浮遊細菌の菌叢解析
大久保寅彦;松尾淳司;大崎敬子;神谷茂;山口博之

Sep. 2016, 第83回日本細菌学会北海道支部学術総会, 優秀賞　　　　　　　　　　　　　　　
北海道大学病院にて分離されたニューデリー・メタロβラクタマーゼNDM-5産生大腸菌の性状解析
大久保寅彦;松尾淳司;山口博之

Sep. 2015, 第82回日本細菌学会北海道支部学術総会, 最優秀賞　　　　　　　　　　　　　　　
繊毛虫との共培養で大腸菌から漏れ出す クオラムセンシング分子 autoinducer-2 について
大久保寅彦;松尾淳司;山﨑智拡;花輪智子;中村眞二;神谷茂;山口博之

Chlamydia trachomatis L2 434/Bu readily activates glycolysis under hypoxia for efficient metabolism
Ruiyu Li, Saicheng Zhang, Satoko Otsuguro, Manabu Nagao, Akira Matsuda, Jeewan Thapa, Torahiko Okubo, Katsumi Maenaka, Hideaki Higashi, Hiroyuki Yamaguchi
Biochemical and Biophysical Research Communications, 150461, 150461, Elsevier BV, Aug. 2024
Scientific journal

Differential impact of environmental factors on airborne live bacteria and inorganic particles in an underground walkway.
Hiroyuki Yamaguchi, Torahiko Okubo, Eriko Nozaki, Takako Osaki
PloS one, 19, 3, e0300920, 2024, [International Magazine]
English, Scientific journal, We previously reported that variations in the number and type of bacteria found in public spaces are influenced by environmental factors. However, based on field survey data alone, whether the dynamics of bacteria in the air change as a result of a single environmental factor or multiple factors working together remains unclear. To address this, mathematical modeling may be applied. We therefore conducted a reanalysis of the previously acquired data using principal component analysis (PCA) in conjunction with a generalized linear model (Glm2) and a statistical analysis of variance (ANOVA) test employing the χ2 distribution. The data used for the analysis were reused from a previous public environmental survey conducted at 8:00-20:00 on May 2, June 1, and July 5, 2016 (regular sampling) and at 5:50-7:50 and 20:15-24:15 on July 17, 2017 (baseline sampling) in the Sapporo underground walking space, a 520-meter-long underground walkway. The dataset consisted of 60 samples (22 samples for "bacterial flora"), including variables such as "temperature (T)," "humidity (H)," "atmospheric pressure (A)," "traffic pedestrians (TP)," "number of inorganic particles (Δ5: 1-5 μm)," "number of live airborne bacteria," and "bacterial flora." Our PCA with these environmental factors (T, H, A, and TP) revealed that the 60 samples could be categorized into four groups (G1 to G4), primarily based on variations in PC1 [Loadings: T(-0.62), H(-0.647), TP(0.399), A(0.196)] and PC2 [Loadings: A(-0.825), TP(0.501), H(0.209), T(-0.155)]. Notably, the number of inorganic particles significantly increased from G4 to G1, but the count of live bacteria was highest in G2, with no other clear pattern. Further analysis with Glm2 indicated that changes in inorganic particles could largely be explained by two variables (H/TP), while live bacteria levels were influenced by all explanatory variables (TP/A/H/T). ANOVA tests confirmed that inorganic particles and live bacteria were influenced by different factors. Moreover, there were minimal changes in bacterial flora observed among the groups (G1-G4). In conclusion, our findings suggest that the dynamics of live bacteria in the underground walkway differ from those of inorganic particles and are regulated in a complex manner by multiple environmental factors. This discovery may contribute to improving public health in urban settings.

Mastitis on selected farms in Wakiso district, Uganda: Burden, pathogens and predictors of infectivity of antimicrobial resistant bacteria in dairy herds.
Steven Kakooza, Francis Mutebi, Paul Ssajjakambwe, Eddie Wampande, Esther Nabatta, Collins Atuheire, Sayaka Tsuchida, Torahiko Okubo, Kazunari Ushida, John Baligwamunsi Kaneene
Veterinary medicine and science, 9, 5, 2376, 2385, Sep. 2023, [International Magazine]
English, Scientific journal, BACKGROUND: Mastitis and associated antimicrobial resistance (AMR) are major challenges to the dairy industry worldwide. OBJECTIVE: This study aimed to expose the mastitis burden, causative bacteria and drivers for mastitis-causing multi-drug-resistant (MDR) Staphylococci infectivity in cows on dairy farms in Wakiso district, Uganda. METHODS: On 22 farms, practices were documented using questionnaires, and 175 cows were screened by the California mastitis test. Composite milk samples from the positive reactors were submitted to the laboratory for bacterial culture testing. Antimicrobial sensitivity testing by the Kirby Bauer disc diffusion method was done only on Staphylococci with a panel of 10 antimicrobials of clinical relevance. RESULTS: Mastitis was detected in 80.6% (n = 141) of the 175 sampled cows, of which sub-clinical mastitis (76.0%: n = 133) was predominant. The Chi-squared analysis hypothesized that cow age (p = 0.017), sub-county (p = 0.013), parity (p < 0.0001), sex of farm owner (p = 0.003), farm duration in dairy production (p = 0.048) and the use of milking salve (p = 0.006) were associated with mastitis. Coagulase-negative Staphylococci were the most prevalent (71.4%; n = 95), followed by Staphylococcus aureus (30.1%, n = 40). Staphylococci (76.3%; n = 135) were majorly resistant to penicillin and tetracycline. Only one isolate was phenotyped as a methicillin-resistant Staphylococcus specie (MRSS). The prevalences of MDR strains at cow and isolate level were 6.3% and 8.3%. The major MDR phenotype identified was penicillin-tetracycline-trimethoprim-sulphamethoxazole. The isolate detected as an MRSS exhibited the broadest MDR pattern. Cow parity was identified as a predictor of infectivity of mastitis-causing MDR Staphylococci in dairy herds. CONCLUSION: The high prevalence of mastitis and associated pathogen AMR found exposes possibilities of economic losses for the dairy sector warranting the need for farmer sensitization on the institution of proper mastitis prevention and control programs, with emphasis on milking hygiene practices and routine disease monitoring.

Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole
Saicheng Zhang, Yuki Funahashi, Satoho Tanaka, Torahiko Okubo, Jeewan Thapa, Shinji Nakamura, Hideaki Higashi, Hiroyuki Yamaguchi
Microbes and Infection, 25, 5, 105097, 105097, Elsevier BV, Jun. 2023
Scientific journal

The fecal microbiomes analysis of Marabou storks (Leptoptilos crumenifer) reveals their acclimatization to the feeding environment in the Kampala urban areas, Uganda.
Sayaka Tsuchida, Atsushi Ueda, Steven Kakooza, Torahiko Okubo, Eddie M Wampande, Takuji Yamada, Kazunari Ushida
The Journal of veterinary medical science, 85, 4, 450, 458, 30 Mar. 2023, [Domestic magazines]
English, Scientific journal, The Marabou stork (Leptoptilos crumenifer) is a typical scavenging bird and adapted to the Savannah environment, where they show a carnivorous feeding style. However, Marabou stork recently penetrated into the city areas and acclimatized to the urban environment, where they modified their feeding habits to an omnivorous type toward more carbohydrate. To reveal their adaptation to the variable feeding customs, this study compared the gut microbiomes and chemical compositions of feces of Marabou storks inhabiting two different locations in peri urban Kampala: one is a slaughter house floc that predicted their original carnivorous feeding, and the other is a landfill floc that adapted more to the omnivorous feeding. 16S rRNA gene sequencing analysis revealed more diverse gut microbiome, more enriched Lactobacilli, and less abundant Peptostreptococci in the landfill flock comparing to the slaughter house flock. Isolation work and predicted metagenome analysis confirmed more diverse Lactobacilli and more enriched functions for carbohydrate metabolism in the landfill flock. In addition, chemical composition of feces revealed higher ammonia in the former, which is consisting with higher Peptostreptococci and their practice of carnivorous feeding. These results highlighted their adaptation to the variable feeding environment, which presumably protects their health and ensure survival of species.

Human pathogenic bacteria on high-touch dry surfaces can be controlled by warming to human-skin temperature under moderate humidity.
Ayano Konno, Torahiko Okubo, Yoshiaki Enoeda, Tomoko Uno, Toyotaka Sato, Shin-Ichi Yokota, Rika Yano, Hiroyuki Yamaguchi
PloS one, 18, 9, e0291765, 2023, [International Magazine]
English, Scientific journal, Healthcare-associated infections have become a major health issue worldwide. One route of transmission of pathogenic bacteria is through contact with "high-touch" dry surfaces, such as handrails. Regular cleaning of surfaces with disinfectant chemicals is insufficient against pathogenic bacteria and alternative control methods are therefore required. We previously showed that warming to human-skin temperature affected the survival of pathogenic bacteria on dry surfaces, but humidity was not considered in that study. Here, we investigated environmental factors that affect the number of live bacteria on dry surfaces in hospitals by principal component analysis of previously-collected data (n = 576, for CFU counts), and experimentally verified the effect of warming to human-skin temperature on the survival of pathogenic bacteria on dry surfaces under humidity control. The results revealed that PCA divided hospital dry surfaces into four groups (Group 1~4) and hospital dry surfaces at low temperature and low humidity (Group 3) had much higher bacterial counts as compared to the others (Group 1 and 4) (p<0.05). Experimentally, warming to human-skin temperature (37°C with 90% humidity) for 18~72h significantly suppressed the survival of pathogenic bacteria on dry surfaces, such as plastic surfaces [p<0.05 vs. 15°C (Escherichia coli DH5α, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and blaNDM-5 E. coli)] or handrails [p<0.05 vs. 15~25°C (E. coli DH5α, S. aureus, P. aeruginosa, A. baumannii)], under moderate 55% humidity. Furthermore, intermittent heating to human-skin temperature reduced the survival of spore-forming bacteria (Bacillus subtilis) (p<0.01 vs. continuous heating to human-skin temperature). NhaA, an Na+/H+ antiporter, was found to regulate the survival of bacteria on dry surfaces, and the inhibitor 2-aminoperimidine enhanced the effect of warming at human-skin temperature on the survival of pathogenic bacteria (E. coli DH5α, S. aureus, A. baumannii) on dry surfaces. Thus, warming to human-skin temperature under moderate humidity is a useful method for impairing live pathogenic bacteria on high-touch surfaces, thereby helping to prevent the spread of healthcare-associated infections.

One Health Approachに基づいたフルオロキノロン耐性大腸菌の市中内拡散・定着様式の解明　　　　　　　　　　　　　　　
佐藤 豊孝, 立花 徹, 玉井 聡, 星野 祐治, 鳥越 慎吾, 榊原 啓一郎, 前谷 茂樹, 福田 昭, 大久保 寅彦, 臼井 優, 高橋 聡, 横田 伸一, 田村 豊, 堀内 基広
日本獣医学会学術集会講演要旨集, 165回, [F2A, 10], (公社)日本獣医学会, Sep. 2022
Japanese

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines
Ryoya Tsujikawa, Jeewan Thapa, Torahiko Okubo, Shinji Nakamura, Saicheng Zhang, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi
Current Microbiology, 79, 9, Springer Science and Business Media LLC, Sep. 2022
Scientific journal

Chlamydia trachomatis Requires Functional Host-Cell Mitochondria and NADPH Oxidase 4/p38MAPK Signaling for Growth in Normoxia
Jeewan Thapa, Gen Yoshiiri, Koki Ito, Torahiko Okubo, Shinji Nakamura, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi
Frontiers in Cellular and Infection Microbiology, 12, Frontiers Media SA, 26 May 2022
Scientific journal, Chlamydia trachomatis (Ct) is an intracellular energy-parasitic bacterium that requires ATP derived from infected cells for its growth. Meanwhile, depending on the O₂ concentration, the host cells change their mode of ATP production between oxidative phosphorylation in mitochondria (Mt) and glycolysis; this change depends on signaling via reactive oxygen species (ROS) produced by NADPH oxidases (NOXs) as well as Mt. It has been proposed that Ct correspondingly switches its source of acquisition of ATP between host-cell Mt and glycolysis, but this has not been verified experimentally. In the present study, we assessed the roles of host-cell NOXs and Mt in the intracellular growth of CtL2 (L2 434/Bu) under normoxia (21% O₂) and hypoxia (2% O₂) by using several inhibitors of NOXs (or the downstream molecule) and Mt-dysfunctional (Mt^d) HEp-2 cells. Under normoxia, diphenyleneiodonium, an inhibitor of ROS diffusion, abolished the growth of CtL2 and other Chlamydiae (CtD and C. pneumoniae). Both ML171 (a pan-NOX inhibitor) and GLX351322 (a NOX4-specific inhibitor) impaired the growth of CtL2 under normoxia, but not hypoxia. NOX4-knockdown cells diminished the bacterial growth. SB203580, an inhibitor of the NOX4-downstream molecule p38MAPK, also inhibited the growth of CtL2 under normoxia but not hypoxia. Furthermore, CtL2 failed to grow in Mt^d cells under normoxia, but no effect was observed under hypoxia. We conclude that under normoxia, Ct requires functional Mt in its host cells as an ATP source, and that this process requires NOX4/p38MAPK signaling in the host cells. In contrast to hypoxia, crosstalk between NOX4 and Mt via p38MAPK may be crucial for the growth of Ct under normoxia.

アメーバによる共生細菌依存的な細菌運搬現象のメカニズムの検証　　　　　　　　　　　　　　　
大久保 寅彦, 佐藤 豊孝, 横田 伸一, 中村 眞二, 山口 博之, 田中 菜那
日本細菌学雑誌, 77, 1, 64, 64, 日本細菌学会, Feb. 2022
Japanese

Wild ciliates differ in susceptibility to Legionella pneumophila JR32.
Airi Kawashiro, Torahiko Okubo, Shinji Nakamura, Jeewan Thapa, Masaki Miyake, Hiroyuki Yamaguchi
Microbiology (Reading, England), 167, 8, Aug. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, We investigated how Legionella pneumophila (Lp) JR32 interacts with Anteglaucoma CS11A and Colpoda E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 18S rDNA-based phylogenetic analysis showed that Anteglaucoma CS11A and Colpoda E6 formed distinct clades. Scanning electron microscopy showed that Anteglaucoma CS11A had a bigger-sized body than Colpoda E6 and, unlike Tetrahymena IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the icm/dot virulence genes, indicating that they all interacted with LpJR32. In co-cultures with Anteglaucoma CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 icm/dot deletion mutant instead. Anteglaucoma CS11A died within 2 days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with Colpoda E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the Colpoda E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the Tetrahymena IB co-cultures with LpJR32 or Δicm/dot, the Lp levels were maintained, albeit with temporal decreases, and the Tetrahymena IB levels were also maintained. We conclude that unlike Tetrahymena IB with pellet production, Anteglaucoma CS11A can be killed by LpJR32 infection, and Colpoda E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection.

Usefulness of a 3D-printing air sampler for capturing live airborne bacteria and exploring the environmental factors that can influence bacterial dynamics.
Saaya Mori, Sakura Ishiguro, Satoru Miyazaki, Torahiko Okubo, Ryosuke Omori, Ayako Kai, Kyohei Sugiyama, Airi Kawashiro, Masato Sumi, Jeewan Thapa, Shinji Nakamura, Chietsugu Katoh, Hiroyuki Yamaguchi
Research in microbiology, 172, 6, 103864, 103864, 14 Jul. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, We created a handmade 3D-printed air sampler to effectively collect live airborne bacteria, and determined which environmental factors influenced the bacteria. Bacterial colony forming units (CFUs) in the air samples (n=37) were monitored by recording the environmental changes occurring over time, then determining the presence/absence of correlations among such changes. The bacterial CFUs changed sharply and were significantly correlated with the DNA concentrations, indicating that the captured bacteria made up most of the airborne bacteria. Spearman's rank correlation analysis revealed significant correlations between the bacterial CFU values and some environmental factors (humidity, wind speed, insolation, and 24-h rainfall). Similarly the significant associations of CFU with humidity and wind speed were also found by multiple regression analysis with box-cox transformation. Among our panel of airborne bacteria (952 strains), 70 strains were identified as soil-derived Bacillus via the production of Escherichia coli- and Staphylococcus aureus-growth inhibiting antibiotics and by 16S rDNA typing. Soil-derived protozoa were also isolated from the air samples. We conclude that the airborne bacteria mainly derived from soil can alter in number according to environmental changes. Our sampler, which was created by easy-to-customize 3D printing, is a useful device for understanding the dynamics of live airborne bacteria.

A retrospective analysis of antimicrobial resistance in pathogenic Escherichia coli and Salmonella spp. isolates from poultry in Uganda.
Steven Kakooza, Adrian Muwonge, Esther Nabatta, Wilfred Eneku, Dickson Ndoboli, Eddie Wampande, Damian Munyiirwa, Edrine Kayaga, Maria Agnes Tumwebaze, Mathias Afayoa, Paul Ssajjakambwe, Dickson Stuart Tayebwa, Sayaka Tsuchida, Torahiko Okubo, Kazunari Ushida, Ken'ichi Sakurai, Francis Mutebi
International journal of veterinary science and medicine, 9, 1, 11, 21, 19 May 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, There are increasing reports of antimicrobial treatment failures for bacterial diseases of poultry in Uganda. The paucity of data on antimicrobial resistance (AMR) of pathogenic bacteria in Uganda is a major setback to AMR control. This study investigated the occurrence of fowl typhoid, colibacillosis, and AMR in associated pathogens from 2012 to 2018. Laboratory records from the Central Diagnostic Laboratory (CDL), a National Veterinary Diagnostic Facility located at Makerere University, were reviewed. Archived isolates of the causative bacteria for the two diseases were also evaluated for AMR. The frequencies of the two disease conditions, their clinical and necropsy presentations and the demographic data of the diagnostic samples were summarized from the records. Archived bacterial isolates were revived before antimicrobial susceptibility testing. This was done on Mueller Hinton agar using the disk diffusion method, against 16 antimicrobials of medical and veterinary importance according to the Clinical Laboratory Standards Institute guidelines. A total of 697 poultry cases were presented for bacteriological investigations in the review period. Colibacillosis and salmonellosis had prevalence rates of 39.7% (277/697) and 16.2% (113/697), respectively. A total of 63 and 92 isolates of Escherichia coli and Salmonella spp., respectively, were archived but 43 (68.3%) E. coli and 47 (51.1%) Salmonella spp. isolates were recovered and evaluated for AMR. Multidrug resistance was more frequent in E. coli (38; 88.4%) than salmonellae (25; 53.2%), (p < 0.001). The high prevalence of colibacillosis, salmonellosis and the AMR of associated pathogens warrants immediate institution of appropriate disease control measures.

Distribution of amoebal endosymbiotic environmental chlamydia Neochlamydia S13 via amoebal cytokinesis.
Miho Okude, Junji Matsuo, Tomohiro Yamazaki, Kentaro Saito, Yoshikazu Furuta, Shinji Nakamura, Jeewan Thapa, Torahiko Okubo, Hideaki Higashi, Hiroyuki Yamaguchi
Microbiology and immunology, 65, 3, 115, 124, Mar. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, We previously isolated a symbiotic environmental amoeba, harboring an environmental chlamydia, Neochlamydia S13. Interestingly, this bacterium failed to survive outside of host cells and was immediately digested inside other amoebae, indicating bacterial distribution via cytokinesis. This may provide a model for understanding organelle development and chlamydial pathogenesis and evolution; therefore, we assessed our hypothesis of Neochlamydia S13 distribution via cytokinesis by comparative analysis with other environmental Chlamydiae (Protochlamydia R18 and Parachlamydia Bn9 ). Dual staining with 4',6-diamidino-2-phenylindole and phalloidin revealed that the progeny of Neochlamydia S13 and Protochlamydia R18 existed in both daughter cells with a contractile ring on the verge of separation. However, in contrast to other environmental Chlamydiae, little Neochlamydia S13 16S ribosomal DNA was amplified from the culture supernatant. Interestingly, Neochlamydia S13 failed to infect aposymbiotic amoebae, indicating an intimate interaction with the host cells. Furthermore, its infectious rates in cultures expanded from a single amoeba were always maintained at 100%, indicating distribution via cytokinesis. We concluded that unlike other environmental Chlamydiae, Neochlamydia S13 has a unique ability to divide its progeny only via host amoebal cytokinesis. This may be a suitable model to elucidate the mechanism of cell organelle distribution and of chlamydial pathogenesis and evolution.

共生細菌依存的にアメーバに起こる細菌運搬現象には運ばれる細菌のNa+/H+アンチポーターNhaAが関与する　　　　　　　　　　　　　　　
田中 菜那, 大久保 寅彦, 佐藤 豊孝, 横田 伸一, タパ・ジーワン, 山口 博之
日本細菌学雑誌, 76, 1, 68, 68, 日本細菌学会, Feb. 2021
Japanese

Na+/H+アンチポーター(NhaA)は大腸菌の乾燥抵抗性を規定する　　　　　　　　　　　　　　　
榎枝 秀朗, 田中 菜那, 大久保 寅彦, 佐藤 豊孝, 横田 伸一, タパ・ジーワン, 山口 博之
日本細菌学雑誌, 76, 1, 71, 71, 日本細菌学会, Feb. 2021
Japanese

Isolation and Characterization of Antimicrobial-Resistant Escherichia coli from Retail Meats from Roadside Butcheries in Uganda.
Torahiko Okubo, Montira Yossapol, Shiori Ikushima, Steven Kakooza, Eddie M Wampande, Tetsuo Asai, Sayaka Tsuchida, Kenji Ohya, Fumito Maruyama, John D Kabasa, Kazunari Ushida
Foodborne pathogens and disease, 17, 11, 666, 671, Nov. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, Retail meats are one of the main routes for spreading antimicrobial-resistant bacteria (ARB) from livestock to humans through the food chain. In African countries, retail meats are often sold at roadside butcheries without chilling or refrigeration. Retail meats in those butcheries are suspected to be contaminated by ARB, but it was not clear. In this study, we tested for the presence of antimicrobial-resistant Escherichia coli from retail meats (n = 64) from roadside butcheries in Kampala, Uganda. The meat surfaces were swabbed and inoculated on PetriFilm SEC agar to isolate E. coli. We successfully isolated E. coli from 90.6% of these retail meat samples. We identified the phylogenetic type, antimicrobial susceptibility, and antimicrobial resistance genes prevalence between retail meat isolates (n = 89). Phylogenetic type B1 was identified from 70.8% of the retail meat isolates, suggesting that the isolates originated primarily from fecal contamination during meat processing. Tetracycline (TET)-resistant isolates with tetA and/or tetB gene(s) were the most frequently detected (28.1%), followed by ampicillin (AMP) resistance genes with blaTEM (15.7%,) and sulfamethoxazole-trimethoprim (SXT) resistance genes with sul2 (15.7%). No extended-spectrum beta-lactamase-producing isolates were detected. A conjugation assay showed that resistance to AMP, TET, and SXT could be simultaneously transferred to recipients. These findings suggest that antimicrobial-resistant E. coli can easily be transferred from farms to tables from retail meats obtained from roadside butcheries.

Hypoxia promotes Chlamydia trachomatis L2/434/Bu growth in immortal human epithelial cells via activation of the PI3K-AKT pathway and maintenance of a balanced NAD+/NADH ratio.
Jeewan Thapa, Kent Hashimoto, Saori Sugawara, Ryoya Tsujikawa, Torahiko Okubo, Shinji Nakamura, Hiroyuki Yamaguchi
Microbes and infection, 22, 9, 441, 450, 19 May 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, Chlamydia trachomatis LGV (CtL2) causes systemic infection and proliferates in lymph nodes as well as genital tract or rectum producing a robust inflammatory response, presumably leading to a low oxygen environment. We therefore assessed how CtL2 growth in immortal human epithelial cells adapts to hypoxic conditions. Assessment of inclusion forming units, the quantity of chlamydial 16S rDNA, and inclusion size showed that hypoxia promotes CtL2 growth. Under hypoxia, HIF-1α was stabilized and p53 was degraded in infected cells. Moreover, AKT was strongly phosphorylated at S473 by CtL2 infection. This activation was significantly diminished by LY-294002, a PI3K-AKT inhibitor, which decreased the number of CtL2 progeny. HIF-1α stabilizers (CoCl2, desferrioxamine) had no effect on increasing CtL2 growth, indicating no autocrine impact of growth factors produced by HIF-1α stabilization. Furthermore, in normoxia, CtL2 infection changed the NAD+/NADH ratio of cells with increased gapdh expression; in contrast, under hypoxia, the NAD+/NADH ratio was the same in infected and uninfected cells with high and stable expression of gapdh, suggesting that CtL2-infected cells adapted better to hypoxia. Together, these data indicate that hypoxia promotes CtL2 growth in immortal human epithelial cells by activating the PI3K-AKT pathway and maintaining the NAD+/NADH ratio with stably activated glycolysis.

Chlamydia trachomatis isolated from cervicovaginal samples in Sapporo, Japan, reveals the circulation of genetically diverse strains.
Jeewan Thapa, Takanori Watanabe, Mana Isoba, Torahiko Okubo, Kiyotake Abe, Kunihiro Minami, Hiroyuki Yamaguchi
BMC infectious diseases, 20, 1, 53, 53, 16 Jan. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, BACKGROUND: This study was conducted to understand the molecular epidemiology of circulating Chlamydia trachomatis (Ct) strains in Sapporo, Japan. METHODS: A total of 713 endocervical samples collected from April 2016 to March 2019 were screened for Ct. The obtained Ct positive samples were analyzed by ompA genotyping and multilocus sequence analysis (MLSA). RESULTS: Eighty-three (11.6%) samples were positive for Ct plasmid DNA. Sequence analysis of the ompA gene from the 61 positive cases revealed eight genotypes: F (40.9%), E (19.6%), D (14.7%), G (9.8%), H (6.5%), I (3.2%), K (3.2%), and J (1.6%). The globally dominant genotype E and F strains were highly conserved with 13 ompA genetic variants being detected, whereas genotype D strains were the most diverse. Genetic characterization of D strains revealed that D1 genetic variants may be potentially specific to Sapporo. MLSA revealed 13 unique sequence types (STs) including four novel STs from 53 positive samples, with the globally dominant STs 39 and 19 being predominant. STs 39, 34, and 21 were exclusively associated with genotypes E and F indicating their global dominance. Novel ST70 and ST30 were specifically associated with genotype D. CONCLUSION: Our study has revealed the circulation of genetically diverse Ct strains in the women population of Sapporo, Japan. We suggest identifying a transmission network of those successful strains and implementing public health prevention strategies to control the spread of Ct in Sapporo.

Effect of thermal control of dry fomites on regulating the survival of human pathogenic bacteria responsible for nosocomial infections.
Tomoko Shimoda, Torahiko Okubo, Yoshiki Enoeda, Rika Yano, Shinji Nakamura, Jeewan Thapa, Hiroyuki Yamaguchi
PloS one, e0226952, 12, e0226952, Dec. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, We monitored the survival of human pathogenic bacteria [Escherichia coli (ATCC), extended-spectrum β-lactamase-producing E. coli (Clinical isolate), New Delhi metallo-β-lactamase-producing E. coli (clinical isolate), Staphylococcus aureus (ATCC)] on dry materials (vinyl chloride, aluminum, plastic, stainless steel) at distinct temperatures (room temperature or 15°C-37°C). These bacteria favored a lower temperature for their prolonged survival on the dry fomites, regardless of the material type. Interestingly, when mixed with S. aureus, E. coli survived for a longer time at a lower temperature. Cardiolipin, which can promote the survival of S. aureus in harsh environments, had no effect on maintaining the survival of E. coli. Although the trends remained unchanged, adjusting the humidity from 40% to 60% affected the survival of bacteria on dry surfaces. Scanning electron microscopic analysis revealed no morphological differences in these bacteria immediately before or after one day of dry conditions. In addition, ATP assessment, a method used to visualize high-touch surfaces in hospitals, was not effective at monitoring bacterial dynamics. A specialized handrail device fitted with a heater, which was maintained at normal human body core temperature, successfully prohibited the prolonged survival of bacteria [Enterococcus faecalis (ATCC), E. coli (ATCC), Pseudomonas aeruginosa (ATCC), S. aureus (ATCC), Acinetobacter baumannii (clinical isolate), and Serratia marcescens (clinical isolate)], with the exception of spore-forming Bacillus subtilis (from our laboratory collection) and the yeast-like fungus Candida albicans (from our laboratory collection)] on dry surfaces. Taken together, we concluded that the tested bacteria favor lower temperatures for their survival in dry environments. Therefore, the thermal control of dry fomites has the potential to control bacterial survival on high-touch surfaces in hospitals.

Screening of hospital-manhole sewages using MacConkey agar with cefotaxime reveals extended-spectrum β-lactamase-producing Escherichia coli
Torahiko Okubo, Takaki Hasegawa, Akira Fukuda, Jeewan Thapa, Masaru Usui, Yutaka Tamura, Hiroyuki Yamaguchi
International Journal of Antimicrobial Agents, 54, 6, 831, 833, Dec. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal

Complete genome and bimodal genomic structure of the amoebal symbiont Neochlamydia strain S13 revealed by ultra-long reads obtained from MinION.
Yamagishi J, Hayashida K, Matsuo J, Okubo T, Kuroda M, Nagai H, Sekizuka T, Yamaguchi H, Sugimoto C
Journal of human genetics, 65, 1, 41, 48, Nov. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Neochlamydia strain S13 is an amoebal symbiont of an Acanthamoeba sp. The symbiont confers resistance to Legionella pneumophila on its host; however, the molecular mechanism underlying this resistance is not completely understood. Genome analyses have been crucial for understanding the complicated host-symbiont relationship but segregating the host's genome DNA from the symbiont's DNA is often challenging. In this study, we successfully identified a bimodal genomic structure in Neochlamydia strain S13 using PacBio RS II supported by ultra-long reads derived from MinION. One mode consisted of circular sequences of 2,586,667 and 231,307 bp; the other was an integrated sequence of the two via long homologous regions. They encoded 2175 protein-coding regions, some of which were implied to be acquired via horizontal gene transfer. They were specifically conserved in the genus Neochlamydia and formed a cluster in the genome, presumably by multiplication through genome replication. Moreover, it was notable that the sequenced DNA was obtained without segregating the symbiont DNA from the host. This is an easy and versatile technique that facilitates the characterization of diverse hosts and symbionts in nature.

Detection of the sul2-strA-strB gene cluster in an ice core from Dome Fuji Station, East Antarctica.
Torahiko Okubo, Rieko Ae, Jun Noda, Yoshinori Iizuka, Masaru Usui, Yutaka Tamura
Journal of global antimicrobial resistance, 17, 72, 78, Jun. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, OBJECTIVES: Bacteria harbouring antimicrobial resistance genes (ARGs) have been isolated from various locations, including ancient microbiomes, indicating that these genes pre-date the discovery of antibiotics. To gain further information regarding ARGs in the pre-antibiotic era, ice samples derived from Dome Fuji Station, Eastern Antarctica, were examined. METHODS: DNA was extracted from firn or ice core samples (n=3; 1200-1400ybp, 1700-2100ybp and 2200-2800ybp, respectively) under sterile conditions. Whole-genome amplification and PCR analyses were utilised to detect ARGs. RESULTS: A 2764-bp gene cluster containing the type II dihydropteroate synthase gene sul2 and the aminoglycoside phosphotransferase genes strA and strB was detected in the 1200-1400-year-old Antarctic ice core (DF-63.5). The sul2-strA-strB gene cluster is frequently associated with plasmid RSF1010 and transposon Tn5393; however, these elements were not detected in sample DF-63.5. The gene cluster exhibited a high level of sequence identity to sequences harboured in present-day bacteria, although there were sequence polymorphisms in the strA gene. Furthermore, expression of this gene cluster in Escherichia coli resulted in reduced susceptibility to dihydrostreptomycin and sulfamethoxazole. CONCLUSION: The results of this study provide further evidence that certain ARGs existed in the pre-antibiotic era. Because the sul2 gene confers resistance to the synthetic compound sulfamethoxazole, these findings suggest that ARGs against synthetic antimicrobials emerged in bacteria during the pre-antibiotic era.

A simple and short microbiology practical improves undergraduate nursing students' awareness of bacterial traits and ability to avoid spreading infections
RIKA YANO, Torahiko Okubo, Tomoko Shimoda, Junji Matsuo, Hiroyuki Yamaguchi
BMC Medical Education, 19, 53, https://doi.org/10.1186/s12909, 53, Feb. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, BACKGROUND: Nurses are responsible for implementing appropriate measures to reduce hospital infections, especially with multidrug resistant bacteria, so nursing students should learn about microbiology. This helps them to understand bacterial dissemination and infectious disease control. Because of tight schedules, however, its teaching is limited in undergraduate nursing classes in Japan. We therefore tested whether a simple short practical session in a microbiology class could help to improve undergraduate nursing students' awareness of bacterial traits and how to prevent infections. METHODS: This study involved second-grade nursing students (n = 76). Two short practical sessions (a total of 3 h, across 2 days) were used to assess the effectiveness of washing or disinfection on hand bacteria in a 16-class microbiology course (total class time was 24 h, plus an exam). Hand bacteria were sampled on LB agar plates with orientation during the first half-day, and the plates examined for colonies with distinct color or morphological traits, and discussed, in the second session, a week later. Questionnaires before and after the exercise were used to assess changes in awareness of unseen bacteria inhabiting around us connecting bacterial traits and how to prevent infections. RESULTS: The results showed that the practical increased the nursing students' awareness of fomites (utensils) (p = 0.0115), fomites (contact-based) (p = 0.0016), habitats (body surface) (p = 0.0127), action facilitating hospital infection (p = 0.0166), and changes in physical condition caused by bacterial infections (p = 0.0136). There were no changes in word associations (p = 0.627) or habitats (inside body) (p = 0.308). Difficulty score, which is an element in questionnaire psychometric properties, tended to be close to the expected score through the practical, but not statistical significant. In addition, regardless of before or after practical, Cronbach α score, which is an indicator of the reliability among items of multi-choice questions, showed > 0.8, indicating validity of evaluation items. Thus, the student's awareness of unseen bacteria inhabiting around us was significantly increased as compared to those before practical in microbiology class. CONCLUSIONS: The simple short practical effectively improved nursing students' awareness of unseen bacteria inhabiting around us in microbiology course, useful for even tight teaching schedules.

Activation of caspase-3 during Chlamydia trachomatis-induced apoptosis at a late stage.
Matsuo J, Haga S, Hashimoto K, Okubo T, Ozawa T, Ozaki M, Yamaguchi H
Canadian journal of microbiology, 65, 2, 135, 143, Feb. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, The obligate intracellular bacterium Chlamydia trachomatis activates the host cell apoptosis pathway at a late stage of its developmental cycle. However, whether caspase-3, which is a key enzyme of apoptosis, is activated in Chlamydia-infected cells remains unknown. Here, we established HEp-2 cells stably expressing cFluc-DEVD, which is a caspase-3 substrate sequence inserted into cyclic firefly luciferase, and then monitored the dynamics of caspase-3 activity in cells infected with Chlamydia. Transfected cells without infection showed a significant increase in luciferase activity due to stimulation with staurosporine, an inducer of apoptosis. Activation was significantly blocked by addition of caspase inhibitor z-VAD-fmk. Furthermore, as expected, Chlamydia infection caused a significant increase in luciferase activation at 36-48 h postinfection with a contrastive decrease at 24 h postinfection, which is already well known. Such activation caused by the infection was much stronger when the amount of bacteria was increased. Thus, caspase-3 activation was accurately monitored by the luciferase activity in HEp-2 cells constitutively expressing the cFluc-DEVD probe. Furthermore, our data showed that C. trachomatis activates caspase-3 in host cells at a late stage of infection.

Phenotypic and genotypic analyses of antimicrobial resistant bacteria in livestock in Uganda
Torahiko Okubo, Montira Yossapol, Fumito Maruyama, Eddie M. Wampande, Steven Kakooza, Kenji Ohya, Sayaka Tsuchida, Tetsuo Asai, John D. Kabasa, Kazunari Ushida
Transboundary and Emerging Diseases, 66, 1, 317, 326, Jan. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, © 2018 Blackwell Verlag GmbH Antimicrobial resistant bacteria (ARB) in livestock are a global public health concern, not only because they prolong infectious diseases but also they can be transferred from animals to humans via the food chain. Here, we studied ARB in livestock at commercial and subsistence farms (n = 13) in Wakiso and Mpigi districts, Uganda. We enquired from the farmers about the type and the purpose of antimicrobial agents they have used to treat their livestock. After collecting faeces, we isolated antimicrobial resistant Escherichia coli from livestock faeces (n = 134) as an indicator bacterium. These strains showed resistance to ampicillin (44.8%), tetracycline (97.0%), and sulfamethoxazole-trimethoprim (56.7%). The frequency of ampicillin-resistance was significantly correlated with the usage of penicillins to livestock in the farms (p = 0.04). The metagenomics data detected 911 antimicrobial resistant genes that were classified into 16 categories. Genes for multidrug efflux pumps were the most prevalent category in all except in one sample. Interestingly, the genes encoding third-generation cephalosporins (bla CTX-M ), carbapenems (bla ACT ), and colistin (arnA) were detected by metagenomics analysis although these phenotypes were not detected in our E. coli strains. Our results suggest that the emergence and transmission of cephalosporin, carbapenem, and/or colistin-resistant bacteria among livestock can occur in future if these antimicrobial agents are used.

Chlamydia pneumoniae enhances IL-8 production with reduced azithromycin sensitivity under hypoxia
Junji Matsuo, Kohei Sakai, Torahiko Okubo, Hiroyuki Yamaguchi
APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, in press, 3, 131, 138, Dec. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Obligate intracellular bacterium Chlamydia pneumoniae causes respiratory tract infections such as community-acquired pneumonia. During infection, C. pneumoniae induces inflammatory responses in host cells and the oxygen concentration at the infection sites subsequently decreases. Because hypoxic conditions influence further inflammatory responses and reduced antibacterial effects, this may exacerbate the C. pneumoniae infection. Here, we show inflammatory responses and drug sensitivity in C. pneumoniae-infected cells under hypoxic conditions. First, we confirmed the enhanced growth of C. pneumoniae under hypoxia, which indicates that the hypoxic condition we used could adequately reproduce past reports. We then demonstrated a significant increase in production of the pro-inflammatory cytokine Interleukin 8 (IL-8) in C. pneumoniae-infected cells under hypoxic conditions. Furthermore, hypoxia decreased the antibacterial effects of azithromycin against C. pneumoniae compared with normoxic conditions. Together, our data suggest that inflammatory responses and drug sensitivity may have been underestimated in C. pneumoniae infection in previous studies. Thus, to accurately understand the Chlamydia infection, it may be necessary to perform in vitro experiments under hypoxic conditions.

Tetrahymena promotes interactive transfer of carbapenemase gene encoded in plasmid between fecal Escherichia coli and environmental Aeromonas caviae.
Matsushita M, Okubo T, Hasegawa T, Matsuo J, Watanabe T, Iwasaki S, Fukumoto T, Hayasaka K, Akizawa K, Shimizu C, Yamaguchi H
Microbiology and immunology, 62, 11, 720, 728, Oct. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Tetrahymena can facilitate plasmid transfer among Escherichia coli or from E. coli to Salmonella Enteritidis via vesicle accumulation. In this study, whether ciliates promote the interactive transfer of plasmids encoding blaIMP-1 between fecal E. coli and environmental Aeromonas caviae was investigated. Both bacteria were mixed with or without ciliates and incubated overnight at 30°C. The frequency of plasmid-acquired bacteria was estimated by colony counts using an agar plate containing ceftazidim (CAZ) followed by determination of the minimum inhibitory concentration (MIC). Cultures containing ciliates interactively transferred the plasmid between E. coli and Aeromonas with a frequency of 10-4 to 10-5 . All plasmid-acquired bacteria showed a MIC against CAZ of >128 μg/mL and the plasmid transfer was confirmed by PCR amplification of the blaIMP-1 gene. Fluorescent observation showed that both bacteria accumulated in the same vesicle and that transwell sequestering significantly decreased the transfer frequency. Although ciliates preferentially ingested E. coli rather than A. caviae, both bacteria were co-localized into the same vesicles of ciliates, indicating that their meeting is associated with the gene transfer. Thus, ciliates interactively promote plasmid transfer between E. coli and A. caviae. The results of this study will facilitate control of the spread of multiple-antibiotic resistant bacteria.

Co-harboring of cephalosporin (bla)/colistin (mcr) resistance genes among Enterobacteriaceae from flies in Thailand
Akira Fukuda, Masaru Usui, Torahiko Okubo, Chie Tagaki, Nop Sukpanyatham, Yutaka Tamura
FEMS Microbiology Letters, 365, 16, fny178, Aug. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, The spreading of antimicrobial-resistant Enterobacteriaceae, especially those co-harboring plasmid-mediated cephalosporin (bla) and colistin (mcr) resistance genes, is becoming increasingly problematic. As a vector, flies carry antimicrobial-resistant bacteria (ARB) into human and livestock habitats. To investigate ARB in flies, we collected 235 flies from 27 sites (18 urban areas, five pig farms and four chicken farms) in Thailand during 2013-2015. Cefotaxime-resistant Enterobacteriaceae (CtxRE) and bla-positive CtxRE were isolated from 70 (29.8%) and 48 (20.4%) flies, respectively. In 93 bla-positive CtxRE isolates that included Escherichia coli, Enterobacter spp., and Klebsiella pneumoniae from 48 flies, the most frequent bla gene was TEM (n = 62), followed by CTX-M-55 (n = 31), CTX-M-14 (n = 26), CMY-2 (n = 24) and SHV (n = 10), and 58 isolates harbored multiple types of these genes. In addition, we detected the mcr-1 (n = 1) and mcr-3 (n = 19) genes in bla-positive CtxRE isolates from 16 flies. In conjugation experiments, 10 mcr-3- and bla-positive isolates exhibited co-transfer of mcr-3 and blaTEM-1 genes. These results suggest that a relatively high proportion of flies in Thailand carries cephalosporin-resistant Enterobacteriaceae harboring co-transmissible cephalosporin and colistin resistance genes.

Acanthamoeba S13WT relies on its bacterial endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid media
Torahiko Okubo, Mizue Matsushita, Shinji Nakamura, Junji Matsuo, Hiroki Nagai, Hiroyuki Yamaguchi
Environmental Microbiology Reports, 10, 3, 344, 354, Wiley-Blackwell, 01 Jun. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Soil-borne amoeba Acanthamoeba S13WT has an endosymbiotic relationship with an environmental Neochlamydia bacterial strain. However, regardless of extensive experiments in liquid media, the biological advantage of the symbiosis remained elusive. We therefore explored the role of the endosymbiont in predator-prey interactions on solid media. A mixed culture of the symbiotic or aposymbiotic amoebae and GFP-expressing Escherichia coli or Salmonella Enteritidis was spotted onto the centre of a LB or B-CYE agar plate preinoculated with a ring of mCherry-expressing Legionella pneumophila (Legionella ‘wall’). The spread of the amoebae on the plate was assessed using a fluorescence imaging system or scanning electron microscopy. As a result, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacked these GFP-expressing bacteria and formed flower-like fluorescence patterns in an anticlockwise direction. Other bacteria (Pseudomonas aeruginosa and Stenotrophomonas maltophilia), but not Staphylococcus aureus, were also backpacked by the symbiotic amoebae on LB agar, although lacked the movement to anticlockwise direction. Furthermore, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacking the E. coli broke through the Legionella ‘wall’ on B-CYE agar plates. Thus, we concluded that Acanthamoeba S13WT required the Neochlamydia endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid agar.

Amoebal endosymbiont Neochlamydia protects host amoebae against Legionella pneumophila infection by preventing Legionella entry
Chinatsu Maita, Mizue Matsushita, Masahiro Miyoshi, Torahiko Okubo, Shinji Nakamura, Junji Matsuo, Masaharu Takemura, Masaki Miyake, Hiroki Nagai, Hiroyuki Yamaguchi
Microbes and Infection, 20, 4, 236, 244, Elsevier Masson SAS, 01 Apr. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Acanthamoeba isolated from environmental soil harbors the obligate intracellular symbiont Neochlamydia, which has a critical role in host amoebal defense against Legionella pneumophila infection. Here, by using morphological analysis with confocal laser scanning fluorescence microscopy and transmission electron microscopy, proteome analyses with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and liquid chromatography–mass spectrometry (LC/MS), and transcriptome analysis with DNA microarray, we explored the mechanism by which the Neochlamydia affected this defense. We observed that when rare uptake did occur, the symbiotic amoebae allowed Legionella to grow normally. However, the symbiotic amoebae had severely reduced uptake of Legionella when compared with the aposymbiotic amoebae. Also, in contrast to amoebae carrying the endosymbiont, the actin cytoskeleton was significantly disrupted by Legionella infection in aposymbiotic amoebae. Furthermore, despite Legionella exposure, there was little change in Neochlamydia gene expression. Taken together, we concluded that the endosymbiont, Neochlamydia prevents Legionella entry to the host amoeba, resulting in the host defense against Legionella infection.

Long-term survival of Naegleria polaris from Antarctica after 10 years of storage at 4 A degrees C
Junji Matsuo, Shinji Nakamura, Torahiko Okubo, Manabu Fukui, Hiroyuki Yamaguchi
PARASITOLOGY RESEARCH, 117, 3, 937, 941, SPRINGER, Mar. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, A free-living amoeba, Naegleria is ubiquitously distributed in various natural environments. Since some Naegleria spp. are exclusively distributed in the Arctic and sub-Antarctic regions, we hypothesized that the amoeba may be useful to determine long-term survival of Naegleria in laboratory conditions at 4 A degrees C. The main objective of the study is to determine that a species of an environmental amoebal isolated can live at low temperatures after a long time. Here, we therefore show long-term survival of an amoeba, Naegleria polaris isolated from a sediment sample, which was collected from Antarctica 10 years ago, and since stored at 4 A degrees C. The sample was put on non-nutrient agar plates with heat-killed Escherichia coli, and then the plate was incubated at 4, 15, or 30 A degrees C. Motile amoebae were seen only when the plate was incubated at 15 A degrees C. The sequencing of ribosomal DNA including internal transcribed spacers (ITS) 1, 5.8S rDNA, and ITS2 region revealed the amoebae to be N. polaris, which is exclusively distributed in the Arctic and sub-Antarctic regions. Scanning electron microscopic observation showed that no typical sucker-like structure was seen on the surface of N. polaris, but the cysts were similar to those of Naegleria fowleri. Thus, our result shows, for the first time, that N. polaris can survive after 10 years of storage at 4 A degrees C. This finding may help us understand the still undescribed effects of environmental samples on viability of amoebae.

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth (Chlamydia trachomatis and Chlamydia pneumoniae) in immortal human epithelial HeLa cells
Kazuya Yamakawa, Junji Matsuo, Torahiko Okubo, Shinji Nakamura, Hiroyuki Yamaguchi
Journal of Infection and Chemotherapy, 24, 2, 83, 87, Elsevier B.V., 01 Feb. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPARγ (peroxisome proliferator-activated receptor-γ) and LXRα (liver X receptor α), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist
5CPPSS-50, an LXRα inhibitor
and T0070907, a PPARγ inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method
Sakai K, Matsuo J, Watanabe T, Okubo T, Nakamura S, Yamaguchi H
Journal of Microbiological Methods, 153, 84, 91, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Impact of bacterial traces belonging to the Enterobacteriaceae on the prevalence of Chlamydia trachomatis in women visiting a community hospital in Japan
Taki K, Watanabe T, Matsuo J, Sakai K, Okubo T, Matsushita M, Abe K, Minami K, Yamaguchi H
Journal of Infection and Chemotherapy, 24, 10, 815, 821, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, We explored the bacteria present in the vaginal microbiota facilitating the prevalence of Chlamydia trachomatis in women visiting a community hospital in Sapporo, Japan, by amplicon sequencing. A total of 273 cervical swab samples were collected, and bacterial vaginosis was evaluated in all specimens by assessment of the Nugent score. In 16 of the samples, bacterial 16S rDNA could not be detected and they were therefore omitted from subsequent experiments (n = 257). A significant negative correlation was observed between the Nugent scores and the amount of Lactobacillus 16S rDNA. Among the 257 samples, chlamydial plasmid was detected in 20 samples and was used for amplicon sequencing. No significant association between the Nugent score and the prevalence of C. trachomatis was detected. Based on the results of chlamydial plasmid detection and the Nugent score, chlamydia-negative samples (n = 27) were randomly selected. Finally, the number of operational taxonomic units (OTUs) obtained from amplicon sequencing was compared between chlamydia-positive (n = 20) and -negative samples (n = 27), revealing that a significant difference was only detected for the OTU numbers of Enterobacteriaceae between the C. trachomatis-positive and -negative groups. However, almost all of the samples utilized for amplicon sequencing failed to grow on MacConkey agar plates and produce indole. Taken together, we concluded that traces of bacteria, not live bacteria, belonging to the Enterobacteriaceae indicated the flow of bacteria through the anogenital route along with gut indole, and the resulting impact on the prevalence of C. trachomatis in the cervicogenital tract of women in Japan.

Analysis of adult damselfly fecal material aids in the estimation of antibiotic-resistant Enterobacterales contamination of the local environment.
Yamaguchi Y, Okubo T, Matsushita M, Wataji M, Iwasaki S, Hayasaka K, Akizawa K, Matsuo J, Shimizu C, Yamaguchi H
PeerJ, 6, e5755, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Because damselflies are ubiquitously but focally present in natural environments and play a critical role as predators of other insect species, the fecal matter of damselflies may be useful for investigating antibiotic-resistant bacterial populations, including human pathogens, in local environments. We therefore examined the prevalence of antibiotic-resistant bacteria, including Enterobacterales, in fecal material from 383 damselflies (adults and larvae) collected from seven locations around Sapporo City, Japan, in 2016 and 2017. Fecal samples were plated on soybean casein digest (SCD) agar plates with and without antibiotics (SCD-A and SCD-w/o, respectively) to identify environmental bacteria and gut bacteria, respectively, and on MacConkey agar plates with antibiotics (MacConkey-A) to select for Gram-negative bacteria, including human pathogenic Enterobacterales species. The prevalence of colonies on each of the plates was compared, and representative colonies on MacConkey-A plates were identified to the species level using an API 20E kit and the MALDI Biotyper system. Overall, SCD-w/o plates showed a gut bacterial load of approximately 108 colony-forming units per adult damselfly or larva. There was a significant difference between the prevalence of colonies on the SCD-A and MacConkey-A plates, and a significantly increased prevalence of antibiotic-resistant bacteria on MacConkey-A plates was observed in samples collected from Shinoroshinkawa. Cluster analysis based on minimum inhibitory concentration values of 59 representative isolates from MacConkey-A agar plates revealed that samples from Shinoroshinkawa contained a higher prevalence of Enterobacterales than those from other sampling locations. Thus, fecal materials discharged by adult damselflies could be used in future studies as a simple tool for estimating antibiotic-resistant bacteria, including Enterobacterales species, in the local environment.

Lateral Gene Transfer Between Protozoa-Related Giant Viruses of Family Mimiviridae and Chlamydiae.
Watanabe T, Yamazaki S, Maita C, Matushita M, Matsuo J, Okubo T, Yamaguchi H
Evolutionary bioinformatics online, 14, 1176934318788337, 1176934318788337, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Obligate intracellular chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. While pathogenic chlamydiae have adapted to a wide range of vertebrates, environmental chlamydiae inhabit unicellular amoebae, the free-living Acanthamoeba. However, how and why this divergence occurred remains unclear. Meanwhile, giant viruses consisting of protozoa-related and protozoa-unrelated viruses have been discovered, with the former group being suggested to have more influenced environmental chlamydiae during their evolution while cohabiting host amoebae. Against this background, we attempted to visualize genes of giant viruses in chlamydial genomes by bioinformatic analysis mainly with comparative genome and phylogenic analysis, seeking genes present in chlamydiae that are specifically shared with protozoa-related giant viruses. As a result, in contrast to protozoa-unrelated giant viruses, the genes of protozoa-related giant viruses were significantly shared in both the chlamydia genomes depending on the giant virus type. In particular, the prevalence of Mimiviridae genes among the protozoa-related giant virus genes in chlamydial genomes was significantly high. Meanwhile, the prevalence of protozoa-related giant virus genes in pathogenic chlamydia genomes was consistently higher than those of environmental chlamydiae; the actual number of sequences similar to giant virus was also significantly predominant compared with those in the environmental chlamydial genomes. Among them, the most prevalent of giant virus was in the case of chlamydiae with Megavirus chiliensis; total of 1338 genes of the chlamydiae were found to be shared with the virus (444 genes specific to environmental chlamydiae, 892 genes shared between both chlamydiae, only two genes in the pathogenic chlamydiae). Phylogenic analysis with most prevalent sets (Megavirus chiliensis and Protochlamydia EI2 or Chlamydia trachomatis L2 434Bu) showed the presence of orthologs between these with several clustered. In addition, Pearson's single regression analysis revealed that almost the prevalence of the genes from the giant viruses in chlamydial genomes was negatively and specifically correlated with the number of chlamydial open reading frames (ORFs). Thus, these results indicated the trace of lateral gene transfer between protozoa-related giant viruses of family Mimiviridae and chlamydiae. This is the first demonstration of a putative linkage between chlamydiae and the giant viruses, providing us with a hint to understand chlamydial evolution.

Walker occupancy has an impact on changing airborne bacterial communities in an underground pedestrian space, as small-dust particles increased with raising both temperature and humidity
Torahiko Okubo, Takako Osaki, Eriko Nozaki, Akira Uemura, Kouhei Sakai, Mizue Matushita, Junji Matsuo, Shinji Nakamura, Shigeru Kamiya, Hiroyuki Yamaguchi
PLOS ONE, 12, 9, e0184980, PUBLIC LIBRARY SCIENCE, Sep. 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal, Although human occupancy is a source of airborne bacteria, the role of walkers on bacterial communities in built environments is poorly understood. Therefore, we visualized the impact of walker occupancy combined with other factors (temperature, humidity, atmospheric pressure, dust particles) on airborne bacterial features in the Sapporo underground pedestrian space in Sapporo, Japan. Air samples (n = 18; 4,800L/each sample) were collected at 8: 00 h to 20: 00 h on 3 days (regular sampling) and at early morning/late night (5: 50 h to 7: 50 h/22: 15 h to 24: 45 h) on a day (baseline sampling), and the number of CFUs (colony forming units) OTUs (operational taxonomic units) and other factors were determined. The results revealed that temperature, humidity, and atmospheric pressure changed with weather. The number of walkers increased greatly in the morning and evening on each regular sampling day, although total walker numbers did not differ significantly among regular sampling days. A slight increase in small dust particles (0.3-0.5 mu m) was observed on the days with higher temperature regardless of regular or baseline sampling. At the period on regular sampling, CFU levels varied irregularly among days, and the OTUs of 22-phylum types were observed, with the majority being from Firmicutes or Proteobacteria (gamma-), including Staphylococcus sp. derived from human individuals. The data obtained from regular samplings reveled that although no direct interaction of walker occupancy and airborne CFU and OTU features was observed upon Pearson's correlation analysis, cluster analysis indicated an obvious lineage consisting of walker occupancy, CFU numbers, OTU types, small dust particles, and seasonal factors (including temperature and humidity). Meanwhile, at the period on baseline sampling both walker and CFU numbers were similarly minimal. Taken together, the results revealed a positive correlation of walker occupancy with airborne bacteria that increased with increases in temperature and humidity in the presence of airborne small particles. Moreover, the results indicated that small dust particles at high temperature and humidity may be a crucial factor responsible for stabilizing the bacteria released from walkers in built environments. The findings presented herein advance our knowledge and understanding of the relationship between humans and bacterial communities in built environments, and will help improve public health in urban communities.

Diversity changes of microbial communities into hospital surface environments
Rika Yano, Tomoko Shimoda, Reina Watanabe, Yasutoshi Kuroki, Torahiko Okubo, Shinji Nakamura, Junji Matsuo, Sadako Yoshimura, Hiroyuki Yamaguchi
JOURNAL OF INFECTION AND CHEMOTHERAPY, 23, 7, 439, 445, ELSEVIER SCIENCE BV, Jul. 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal, Previous works have demonstrated considerable variability in hospital cleanliness in Japan, suggesting that contamination is driven by factors that are currently poorly controlled. We undertook 16S rRNA sequence analysis to study population structures of hospital environmental microbiomes to see which factor(s) impacted contamination. One hundred forty-four samples were collected from surfaces of three hospitals with distinct sizes ("A": > 500 beds, "B": 100-500 beds, "C": < 100 beds). Sample locations of two ward types (Surgical and Internal) included patient room bed table (multiple) (4BT), patient overbed table (multiple) (4OT), patient room sink (multiple) (4S), patient room bed table (single) (SBT), patient overbed table (single) (SOT), patient room sink (single) (SS), nurse desk (ND), and nurse wagon (NW). Total DNA was extracted from each sample, and the 50 samples that yielded sufficient DNA were used for further 16S rRNA sequencing of hospital microbiome populations with cluster analysis. The number of assigned bacterial OTU populations was significantly decreased in hospital "C" compared to the other hospitals. Cluster analysis of sampling locations revealed that the population structure in almost all locations of hospital "C" and some locations in the other hospitals was very similar and unusually skewed with a family, Enterobacteriaceae. Interestingly, locations included patient area (4OT, 4BT, SBT) and nurse area (ND), with a device (NW) bridging the two and a place (4S and SS) shared between patients or visitors. We demonstrated diversity changes of hospital environmental microbiomes with a skewed population, presumably by medical staff pushing NWs or sinks shared by patients or visitors. (C) 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Ciliates promote the transfer of a plasmid encoding bla(NDM)-5 from Escherichia coli, isolated from a hospital in Japan, to other human pathogens
Torahiko Okubo, Mizue Matushita, Yukiko Ohara, Junji Matsuo, Satoshi Oguri, Tatsuya Fukumoto, Kasumi Hayasaka, Kouzi Akizawa, Hitoshi Shibuya, Chikara Shimizu, Hiroyuki Yamaguchi
INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, 49, 3, 387, 388, ELSEVIER SCIENCE BV, Mar. 2017, [Peer-reviewed], [International Magazine]
English

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation
Tatsuya Fukumoto, Junji Matsuo, Torahiko Okubo, Shinji Nakamura, Kentaro Miyamoto, Kentaro Oka, Motomichi Takahashi, Kouji Akizawa, Hitoshi Shibuya, Chikara Shimizu, Hiroyuki Yamaguchi
BMC MICROBIOLOGY, 16, 1, 292, 8, BIOMED CENTRAL LTD, Dec. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, Background: Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments.
Results: One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heatkilled Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were identified.
Conclusion: These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen.

Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica)
Akira Fukuda, Masaru Usui, Torahiko Okubo, Yutaka Tamura
MICROBIAL DRUG RESISTANCE, 22, 4, 336, 341, MARY ANN LIEBERT, INC, Jun. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/ Donor) was >= 1.3x10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0x10(-4) to 5.7x10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

Use of Aeromonas spp. as General Indicators of Antimicrobial Susceptibility among Bacteria in Aquatic Environments in Thailand
Masaru Usui, Chie Tagaki, Akira Fukuda, Torahiko Okubo, Chanchai Boonla, Satoru Suzuki, Kanako Seki, Hideshige Takada, Yutaka Tamura
FRONTIERS IN MICROBIOLOGY, 7, 710, 710, FRONTIERS MEDIA SA, May 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, Antimicrobials are widely used, not only for treating human infections, but also for treatment of livestock and in fish farms. Human habitats in Southeastern Asian countries are located in close proximity to aquatic environments. As such, the human populations within these regions are at risk of exposure to antimicrobial resistant bacteria, and thereby disseminating antimicrobial resistance genes (ARGs). In this study, we collected water samples from 15 sites (5 sites in Chao Phraya River, 2 sites at the mouth of Chao Phraya River, 3 sites in Ta Chin River, and 5 sites at city canals) and 12 sites (6 sites at city canals; 2 sites at chicken farms; 2 sites at pig farms; and 2 samples from sites at pig farms, which were subsequently treated at a biogas plant) in Thailand in 2013 and 2014, respectively. In total, 117 Aeromonas spp. were isolated from the water samples, and these organisms exhibited various antimicrobial susceptibility profiles. Notably, there was a significant correlation between the environmental concentration of tetracyclines and the rates of tetracycline resistance in the isolated Aeromonas spp.; however, both the concentration and rates of tetracycline resistance in samples derived from pig farms were higher than those of samples harvested from other aquatic environments. These findings suggest that the high concentrations of antimicrobials observed in these aquatic environments likely select for ARGs. Furthermore, they indicate that Aeromonas spp. comprise an effective marker for monitoring antimicrobial resistance in aquatic environments.

犬由来菌に対するフルオロキノロン系抗菌薬の殺菌能力評価
大久保 寅彦, 臼井 優, 福田 昭, 佐藤 友美, 田村 豊
Journal of the veterinary medicine, 69, 1, 39, 42, 文永堂出版, Jan. 2016, [Peer-reviewed]
Japanese, Scientific journal

Draft genome sequences of Legionella pneumophila JR32 and Lp01 laboratory strains domesticated in Japan
Chinatsu Maita, Mizue Matushita, Torahiko Okubo, Junji Matsuo, Masaki Miyake, Hiroki Nagai, Hiroyuki Yamaguchi
Genome Announcements, 4, 4, American Society for Microbiology, 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, We report here the draft genome sequences of two Legionella pneumophila variant strains (JR32 and Lp01_666) originally derived from a Philadelphia-1 clinical isolate, domesticated in Japan, with distinct susceptibility to amoebae. Detailed genomic analysis will allow us to better understand Legionella adaptation and survival mechanisms in host cells.

Isolation of multiple drug-resistant enteric bacteria from feces of wild Western Lowland Gorilla (Gorilla gorilla gorilla) in Gabon
Pierre Philippe Mbehang Nguema, Torahiko Okubo, Sayaka Tsuchida, Shiho Fujita, Juichi Yamagiwa, Yutaka Tamura, Kazunari Ushida
JOURNAL OF VETERINARY MEDICAL SCIENCE, 77, 5, 619, 623, JAPAN SOC VET SCI, May 2015, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, Prevalence of drug-resistant bacteria in wildlife can reveal the actual level of anthropological burden on the wildlife. In this study, we isolated two multiple drug-resistant strains, GG6-2 and GG6-1-1, from 27 fresh feces of wild western lowland gorillas in Moukalaba-Doudou National Park, Gabon. Isolates were identified as Achromobacter xylosoxidans and Providencia sp., respectively. Minimum inhibitory concentrations of the following 12 drugs-ampicillin (ABPC), cefazolin (CEZ), cefotaxime (CTX), streptomycin (SM), gentamicin (GM), kanamycin (KM), tetracycline (TC), nalidixic acid (NA), ciprofloxacin (CPFX), colistin (CL), chloramphenicol (CP) and trimethoprim (TMP)-were determined. Isolate GG6-2 was resistant to all antimicrobials tested and highly resistant to CTX, SM, TC, NA and TMP. Isolate GG6-1-1 was resistant to ABPC, CEZ, TC, CL, CP and TMP.

Draft genome sequence of Chlamydia trachomatis strain 54, isolated from the urogenital tract of a male in Japan
Tomohiro Yamazaki, Junji Matsuo, Momoka Kikuchi, Kentaro Miyamoto, Kentaro Oka, Motomichi Takahashi, Satoshi Takahashi, Torahiko Okubo, Hiroyuki Yamaguchi
Genome Announcements, 3, 5, American Society for Microbiology, 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal, We report the draft genome sequence of Chlamydia trachomatis strain 54, isolated from the urogenital tract of a male in Japan, with unique polymorphic membrane proteins. Detailed genomic analysis will aid our understanding of the selective pressures that lead to sexual differentiation in chlamydial adaptive evolution.

Protozoal ciliate promotes bacterial autoinducer-2 accumulation in mixed culture with Escherichia coli
Satoshi Oguri, Tomoko Hanawa, Junji Matsuo, Kasumi Ishida, Tomohiro Yamazaki, Shinji Nakamura, Torahiko Okubo, Tatsuya Fukumoto, Kouzi Akizawa, Chikara Shimizu, Shigeru Kamiya, Hiroyuki Yamaguchi
JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 61, 5, 203, 210, MICROBIOL RES FOUNDATION, 2015, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, We have previously demonstrated conjugation of Escherichia coli into vacuoles of the protozoal ciliate (Tetrahymena thermophila). This indicated a possible role of ciliates in evoking bacterial quorum sensing, directly connecting bacterial survival via accumulation in the ciliate vacuoles. We therefore assessed if ciliates promoted bacterial autoinducer (AI)-2 accumulation with vacuole formation, which controls quorum sensing. E. coli AI-2 accumulation was significantly enhanced in the supernatants of a mixed culture of ciliates and bacteria, likely depending on ciliate density rather than bacterial concentration. As expected, AI-2 production was significantly correlated with vacuole formation. The experiment with E. coli luxS mutants showed that ciliates failed to enhance bacterial AI-2 accumulation, denying a nonspecific phenomenon. Fluorescence microscopy revealed accumulation of fragmented bacteria in ciliate vacuoles, and, more importantly, expulsion of the vacuoles containing disrupted bacteria into the culture supernatant. There was no increase in the expression of luxS (encoding AI-2) or ydgG (a transporter for controlling bacterial export of AI-2). We conclude that ciliates promote bacterial AI-2 accumulation in a mixed culture, via accumulation of disrupted bacteria in ciliate vacuoles followed by ex-pulsion of the vacuoles, independently of luxS or ydgG gene induction. This is believed to be the first demonstration of a relationship between E. coli AI-2 dynamics and ciliates. In the natural environment, ciliate biotopes may provide a survival advantage to bacteria inhabiting such biotopes, via evoking quorum sensing.

Bacterial Diversity in Sea Ice from the Southern Ocean and the Sea of Okhotsk　　　　　　　　　　　　　　　
Torahiko Okubo, Yuika Tosaka, Toyotaka Sato, Masaru Usui, Chie Nakajima, Yasuhiko Suzuki, Satoshi Imura, Yutaka Tamura
Journal of Applied & Environmental Microbiology, 2, 6, 266, 272, Oct. 2014, [Peer-reviewed]
English, Scientific journal

Isolation of Escherichia coli Strains with AcrAB-TolC Efflux Pump-Associated Intermediate Interpretation or Resistance to Fluoroquinolone, Chloramphenicol and Aminopenicillin from Dogs Admitted to a University Veterinary Hospital
Toyotaka Sato, Shin-ichi Yokota, Risa Ichihashi, Tomoka Miyauchi, Torahiko Okubo, Masaru Usui, Nobuhiro Fujii, Yutaka Tamura
JOURNAL OF VETERINARY MEDICAL SCIENCE, 76, 7, 937, 945, JAPAN SOC VET SCI, Jul. 2014, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, Understanding the prevalence of antimicrobial-resistance and the relationship between emergence of resistant bacteria and clinical treatment can facilitate design of effective treatment strategies. We here examined antimicrobial susceptibilities of Escherichia coli isolated from dogs admitted to a university hospital (University hospital) and companion animal clinics (Community clinics) in the same city and investigated underlying multidrug-resistance mechanisms. The prevalence of E. coli with intermediate and resistant interpretations to ampicillin (AMP), enrofloxacin (ENR) and chloramphenicol (CHL) was higher in the University hospital than in the Community clinics cases. Use of antimicrobials, including fluoroquinolone, was also significantly higher in the University hospital than in the Community clinics cases. Upon isolation using EN R-supplemented agar plates, all EN R-resistant isolates had 3-4 nucleotide mutations that accompanied by amino acid substitutions in the quinolone-resistance-determining regions of gyrA, parC and parE, and 94.7% of all isolates derived from the University hospital showed AMP and/or CHL resistance and possessed bla(TEM) and/or catA1. The average niRNA expression levels of acrA, acrB and yo/C and the prevalence of organic solvent tolerance, in isolates derived from ENR-supplemented agar plates were significantly higher in the University hospital than in the Community clinics isolates. Thus, E. coli derived from the University hospital cases more often showed concomitant decreased susceptibilities to aminopenicillins, fluoroquinolones and CH L than did those derived from the Community clinics; this was related to an active AcrAB-ToIC efflux pump, in addition to acquisition of specific resistance genes and genetic mutations.

北海道で分離された大腸菌におけるキノロン、アミノグリコシド、セフェム交叉耐性株に関する検討　　　　　　　　　　　　　　　
横田 伸一, 大越 康雄, 大久保 寅彦, 佐藤 豊孝, 田村 豊, 塚本 尚行, 桑原 理
日本化学療法学会雑誌, 62, Suppl.A, 403, 403, (公社)日本化学療法学会, May 2014
Japanese

Association of Veterinary Third-Generation Cephalosporin Use with the Risk of Emergence of Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Dairy Cattle in Japan
Toyotaka Sato, Torahiko Okubo, Masaru Usui, Shin-ichi Yokota, Satoshi Izumiyama, Yutaka Tamura
PLOS ONE, 9, 4, e96101, PUBLIC LIBRARY SCIENCE, Apr. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, The use of extended-spectrum cephalosporins in food animals has been suggested to increase the risk of spread of Enterobacteriaceae carrying extended-spectrum b-lactamases to humans. However, evidence that selection of extended-spectrum cephalosporin-resistant bacteria owing to the actual veterinary use of these drugs according to criteria established in cattle has not been demonstrated. In this study, we investigated the natural occurrence of cephalosporin-resistant Escherichia coli in dairy cattle following clinical application of ceftiofur. E. coli isolates were obtained from rectal samples of treated and untreated cattle (n = 20/group) cultured on deoxycholate-hydrogen sulfide-lactose agar in the presence or absence of ceftiofur. Eleven cefazoline-resistant isolates were obtained from two of the ceftiofur-treated cattle; no cefazoline-resistant isolates were found in untreated cattle. The cefazoline-resistant isolates had mutations in the chromosomal ampC promoter region and remained susceptible to ceftiofur. Eighteen extended-spectrum cephalosporin-resistant isolates from two ceftiofur-treated cows were obtained on ceftiofur-supplemented agar; no extended-spectrum cephalosporin-resistant isolates were obtained from untreated cattle. These extended-spectrum cephalosporin-resistant isolates possessed plasmid-mediated beta-lactamase genes, including blaC(TX-M-2) (9 isolates), bla(CTX-M-14) (8 isolates), or bla(CMY-2) (1 isolate); isolates possessing bla(CTX-M-2) and bla(CTX-M-14) were clonally related. These genes were located on self-transmissible plasmids. Our results suggest that appropriate veterinary use of ceftiofur did not trigger growth extended-spectrum cephalosporin-resistant E. coli in the bovine rectal flora; however, ceftiofur selection in vitro suggested that additional ceftiofur exposure enhanced selection for specific extended-spectrum cephalosporin-resistant beta-lactamase-expressing E. coli clones

Comparison of broad-spectrum cephalosporin-resistant Escherichia coli isolated from dogs and humans in Hokkaido, Japan
Torahiko Okubo, Toyotaka Sato, Shin-ichi Yokota, Masaru Usui, Yutaka Tamura
JOURNAL OF INFECTION AND CHEMOTHERAPY, 20, 3-4, 243, 249, SPRINGER JAPAN KK, Mar. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, Resistance to broad-spectrum cephalosporins (BSCs) in Enterobacteriaceae in companion animals has become a great concern for public health. To estimate the dissemination of BSC-resistant bacteria between dog and human, we examined the BSC-resistance determinants of and genetic similarities between 69 BSC-resistant Escherichia coli isolates derived from canine rectal swabs (n = 28) and human clinical samples (n = 41). Some E. coli isolates possessed bla(TEM-1b) (14 canine and 16 human isolates), bla(CTx-M-2) (6 human isolates), bla(CTx-M-14) (3 canine and 14 human isolates), bla(CTx-M-27) (1 canine and 15 human isolates), and bla(CMY-2) (11 canine and 3 human isolates). The possession of CTX-M-type beta-lactamases was significantly more frequent in human isolates, whereas CMY-2 was more common in canine isolates. Bacterial typing methods (phylogenetic typing, O-antigen serotyping, and pulsed-field gel electrophoresis) showed little clonal relationship between canine isolates and human isolates. Plasmid analysis and Southern blotting indicated that the plasmids encoding CMY-2 were similar among canine and human isolates. Based on the differences in the major beta-lactamase and the divergence of bacterial types between canine and human isolates, it seems that clonal dissemination of BSC-resistant E. coli between canines and humans is limited. The similarity of the CMY-2-encoding plasmid suggests that plasmid-mediated beta-lactamase gene transmission plays a role in interspecies diffusion of BSC-resistant E. coli between dog and human. (C) 2013, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Phylogenetic association of fluoroquinolone and cephalosporin resistance of D-O1-ST648 Escherichia coli carrying bla(CMY-2) from faecal samples of dogs in Japan
Toyotaka Sato, Shin-ichi Yokota, Torahiko Okubo, Masaru Usui, Nobuhiro Fujii, Yutaka Tamura
JOURNAL OF MEDICAL MICROBIOLOGY, 63, Pt 2, 263, 270, SOC GENERAL MICROBIOLOGY, Feb. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal, This study aimed to investigate the genetic association between fluoroquinolone (FQ) and/or cephalosporin (CEP) resistance in Escherichia coli isolates from dogs, and the risk to human health. We characterized E. coli clinical isolates, derived from faecal samples of dogs attending veterinary hospitals, using phylogenetic grouping, determination of virulence factor (VF) prevalence, multilocus sequence typing (MLST) and O serotyping. The D group was the dominant phylogenetic group among strains resistant to FQ and/or CEP. In contrast, the dominant group among susceptible strains was group B2. Group D strains showed a significantly higher prevalence of VFs than strains belonging to groups A and B1, and were resistant to significantly more antimicrobials than group B2 strains. The phylogenetic distribution of FQ CEP-resistant E. coli groups (FQ CEPRECs) and FQ-resistant groups was significantly correlated (r=0.98), but FQ CEPRECs and CEP-resistant E. coli groups were not correlated (r=0.58). Data from PFGE, 0 serotype and MLST analyses indicated that the majority of FQ-resistant strains derived from a particular lineage of phylogenetic group D: serotype 01 and sequence type (ST) 648. Some D-O1-ST648 strains carried bla(CMY-2), showed multidrug resistance and possessed a higher prevalence of the VFs kspMT, ompT and PAI compared with other group D strains. Our data indicate that the emergence of FQ-CEP-resistant E. coli is based primarily on FQ-resistant E. coli. Moreover, as strains of the D-O1-ST648 lineage have been found in clinical isolates derived from humans at a relatively high frequency, our findings indicate that the spreading of D-O1-ST648 strains may cause serious difficulties in both veterinary and human clinical fields in the future.

The Role of Flies in Spreading the Extended-Spectrum beta-lactamase Gene from Cattle
Masaru Usui, Tomohiro Iwasa, Akira Fukuda, Toyotaka Sato, Torahiko Okubo, Yutaka Tamura
MICROBIAL DRUG RESISTANCE, 19, 5, 415, 420, MARY ANN LIEBERT, INC, Oct. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, The spreading of antimicrobial-resistant bacteria and genes from food-producing animals to humans has been a subject of increasing concern. To clarify the role of flies in spreading the extended-spectrum -lactamase (ESBL) gene from food-producing animals to humans, we isolated and characterized a third-generation cephalosporin-resistant Escherichia coli strain from flies and cattle feces from a cattle barn. Cephalosporin-resistant strains were isolated from 14.3% (13/91) of houseflies, 10.3% (7/68) of false stable flies, and 7.5% (7/93) of cattle feces. Twenty-seven cephalosporin-resistant strains were tested for the presence of antimicrobial resistance genes. Of the 27 samples, 22 isolates from 11 houseflies, 5 false stable flies, and 6 cattle feces samples harbored the bla(CTX-M-15) gene. All bla(CTX-M-15)-harboring isolates belonged to phylogenetic group D and the ST38 clonal group. Analysis of pulsed-field gel electrophoresis showed that these isolates were divided into two clusters, indicating that flies carried several of the same clones that were detected in cattle feces. All bla(CTX-M-15) gene-harboring plasmids were transferable and were members of incompatibility group FIB. These results suggest that transferable plasmids encoding ESBL were prevalent among flies and cattle. As vectors, flies may play an important role in spreading ESBL-producing bacteria from food-producing animals to humans.

豚疣贅性心内膜炎から分離したStreptococcus suisの薬剤感受性及び分子疫学的解析
ONO YUTA, YANAGISAWA RISA, YOKOYAMA MITSUE, FURUSAKI YOJI, OKUBO TORAHIKO, IKEDA TETSUYA, SHIMIZU SHUN'ICHI
獣医公衆衛生研究, 16, 1, 33, 36, Sep. 2013
Japanese

動物病院来院猫からのセファロスポリン耐性およびフルオロキノロン耐性大腸菌の検出　　　　　　　　　　　　　　　
大久保 寅彦, 小野 匡, 佐藤 豊孝, 臼井 優, 田村 豊
北海道獣医師会雑誌, 57, 8, 411, 411, (公社)北海道獣医師会, Aug. 2013
Japanese

犬由来大腸菌におけるフルオロキノロン耐性及びセファロスポリン耐性の関連　　　　　　　　　　　　　　　
佐藤 豊孝, 横田 伸一, 大久保 寅彦, 臼井 優, 藤井 暢弘, 田村 豊
日本獣医師会雑誌, 66, 5, 350, 350, (公社)日本獣医師会, May 2013
Japanese

伴侶動物由来細菌へのフルオロキノロン剤曝露による耐性菌出現リスク
臼井 優, 佐藤 豊孝, 大久保 寅彦, 田村 豊
獣医畜産新報, 66, 4, 279, 282, 文永堂出版(株), Apr. 2013
Japanese, 伴侶動物由来細菌を用いて、エンロフロキサシンおよびオルビフロキサシン耐性菌の出現率を調べ、耐性菌出現頻度試験を行った。エンロフロキサシンおよびオルビフロキサシンは伴侶動物由来細菌に対して同等の有効性を示し、耐性菌出現リスクに関しては大腸菌に対してはオルビフロキサシン、緑膿菌およびStaphylococcus pseudintermediusに対してはエンフロキサシンが高い傾向があることが示唆された。(著者抄録)

Contribution of the AcrAB-TolC efflux pump to high-level fluoroquinolone resistance in Escherichia coli isolated from dogs and humans
Toyotaka Sato, Shin-Ichi Yokota, Torahiko Okubo, Kanako Ishihara, Hiroshi Ueno, Yasukazu Muramatsu, Nobuhiro Fujii, Yutaka Tamura
Journal of Veterinary Medical Science, 75, 4, 407, 414, 2013, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, Fluoroquinolone resistance is mainly caused by mutations in quinolone resistance-determining regions of DNA gyrase and topoisomerase IV in Escherichia coli. The AcrAB-TolC efflux pump contributes to resistance against fluoroquinolone and other antimicrobials. In this study, we investigated a high-level mechanism of fluoroquinolone resistance in E. coli that was isolated from human clinical samples and canine fecal samples. E. coli strains with high levels of fluoroquinolone resistance have been found to be frequently resistant to cephalosporins. Strains with high-level fluoroquinolone resistance exhibited lower intracellular enrofloxacin (ENR) concentrations, higher expression of AcrA, and a greater reduction in the fluoroquinolone minimum inhibitory concentration for treatment with an efflux pump inhibitor. The frequency of strains with enhanced ENR resistance selection and the survival rate of E. coli in the presence of ENR in vitro were correlated well with AcrA protein expression levels in the parental strains. These results suggest that AcrAB-TolC efflux pump over-expression is related to high-level fluoroquinolone resistance and the selection of strains with enhanced fluoroquinolone resistance. © 2013 The Japanese Society of Veterinary Science.

Amino acid substitutions in gyrA and parC are associated with fluoroquinolone resistance in mycoplasma bovis isolates from Japanese dairy calves
Toyotaka Sato, Torahiko Okubo, Masaru Usui, Hidetoshi Higuchi, Yutaka Tamura
Journal of Veterinary Medical Science, 75, 8, 1063, 1065, 2013, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fuoroquinolone (enrofoxa-cin, orbifoxacin and danofoxacin) susceptibility in 58Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fuoroquinolone-MICs≤2 μg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinolone-resistant isolates (fuoroquinolone-MICs≥4 μg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fuoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fuoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fuoroquinolone resistance inM. bovis isolates. © 2013 The Japanese Society of Veterinary Science..

Fluoroquinolone resistance mechanisms in an Escherichia coli isolate, HUE1, without quinolone resistance-determining region mutations
Toyotaka Sato, Shin-Ichi Yokota, Ikuo Uchida, Torahiko Okubo, Masaru Usui, Masahiro Kusumoto, Masato Akiba, Nobuhiro Fujii, Yutaka Tamura
Frontiers in Microbiology, 4, MAY, 125, 125, Frontiers Research Foundation, 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB-TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli. © 2013 Sato, Yokota, Uchida, Okubo, Usui, Kusumoto, Akiba, Fujii and Tamura.

High Prevalence of Cross-Resistance to Aminoglycosides in Fluoroquinolone-Resistant Escherichia coli Clinical Isolates
Naoyuki Tsukamoto, Yasuo Ohkoshi, Torahiko Okubo, Toyotaka Sato, Osamu Kuwahara, Nobuhiro Fujii, Yutaka Tamura, Shin-ichi Yokot
CHEMOTHERAPY, 59, 5, 379, 384, KARGER, 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, Background: Multidrug-resistant Escherichia coli, especially a lineage of 025b:H4-ST131, has increased and spread worldwide. The surveillance of cross-resistance of E. coli is necessary. Methods: Cross-resistance to fluoroquinolones (FQs) and aminoglycosides (AGs) was examined in E. coli isolated in Hokkaido Prefecture, Japan, between 2008 and 2009. Results: Gentamicin (GEN) resistance was more common in FQ-resistant isolates (30/112 strains; 26.8%) than in FQ-susceptible isolates (2/100 strains; 2%). The frequency of GEN resistance was similar in two groups of FQ-resistant strains, 025b:H4-ST131 genotype (22/87 strains; 25.3%) and a group of other FQ-resistant genotypes (8/25 strains; 32.0%). The main AG resistance gene was aac(3)-II (87.5% of GEN-resistant strains). The only amikacin-resistant strain which was FQ resistant carried the aac(6')-Ib-cr gene. CTX-M type extended-spectrum beta-lactamase (ESBL) genes were also found in FQ-resistant strains at a high frequency. However, the number of strains with both ESBL and AG-modifying enzyme genes was relatively low (8 strains). Conclusion: All FQ-resistant strains, not only 025b:H4-ST131, appeared to preferentially acquire ESBL genes and/or genes encoding AG-modifying enzymes; however, the acquisitions of these genes seemed to occur independently. (C) 2014 S. Karger AG, Basel

市販鶏肉由来薬剤耐性サルモネラ属菌における耐性遺伝子の解析　　　　　　　　　　　　　　　
小川 紋, 佐藤 豊孝, 大久保 寅彦, 臼井 優, 田村 豊
北海道獣医師会雑誌, 56, 8, 436, 436, (公社)北海道獣医師会, Aug. 2012
Japanese

薬剤耐性大腸菌の畜舎内伝播におけるハエの役割　　　　　　　　　　　　　　　
臼井 優, 岩佐 友寛, 佐藤 豊孝, 大久保 寅彦, 田村 豊
北海道獣医師会雑誌, 56, 8, 437, 437, (公社)北海道獣医師会, Aug. 2012
Japanese

犬由来大腸菌におけるフルオロキノロン耐性およびセファロスポリン耐性の関連　　　　　　　　　　　　　　　
佐藤 豊孝, 横田 伸一, 大久保 寅彦, 臼井 優, 藤井 暢弘, 田村 豊
北海道獣医師会雑誌, 56, 8, 438, 438, (公社)北海道獣医師会, Aug. 2012
Japanese

牛呼吸器由来Mycoplasma bovisにおけるフルオロキノロン系抗菌薬耐性機構　　　　　　　　　　　　　　　
佐藤 豊孝, 大久保 寅彦, 臼井 優, 樋口 豪紀, 永幡 肇, 田村 豊
日本獣医学会学術集会講演要旨集, 154回, 226, 226, (公社)日本獣医学会, Aug. 2012
Japanese

南極の氷中に含まれていた細菌叢の解析　　　　　　　　　　　　　　　
大久保 寅彦, 佐藤 豊孝, 中村 昇太, 飯田 哲也, 臼井 優, 能田 淳, 萩原 克郎, 田村 豊
日本獣医学会学術集会講演要旨集, 154回, 229, 229, (公社)日本獣医学会, Aug. 2012
Japanese

薬剤耐性大腸菌の畜舎内伝播におけるハエの役割　　　　　　　　　　　　　　　
臼井 優, 岩佐 友寛, 佐藤 豊孝, 大久保 寅彦, 田村 豊
日本獣医学会学術集会講演要旨集, 154回, 273, 273, (公社)日本獣医学会, Aug. 2012
Japanese

市販鶏肉由来薬剤耐性サルモネラ属菌における耐性遺伝子の解析　　　　　　　　　　　　　　　
小川 紋, 佐藤 豊孝, 大久保 寅彦, 臼井 優, 田村 豊
日本獣医学会学術集会講演要旨集, 154回, 276, 276, (公社)日本獣医学会, Aug. 2012
Japanese

Prevalence of Fluoroquinolone-Resistant Escherichia coli O25:H4-ST131 (CTX-M-15-Nonproducing) Strains Isolated in Japan
Shin-ichi Yokota, Toyotaka Sato, Torahiko Okubo, Yasuo Ohkoshi, Tamaki Okabayashi, Osamu Kuwahara, Yutaka Tamura, Nobuhiro Fujii
CHEMOTHERAPY, 58, 1, 52, 59, KARGER, 2012, [Peer-reviewed], [International Magazine]
English, Scientific journal, Background: Fluoroquinolone-resistant and extended-spectrum p-lactamase (ESBL)-carrying multidrug-resistant Escherichia coli have become severely problematic. In particular, a lineage of multilocus sequence-type 51131 which belongs to O25:H4 and carries BBL CTX-M-15 has spread worldwide. Methods: Fluoroquinolone-resistant E. coli strains were isolated from various clinical specimens in a commercial clinical laboratory in 2008 and 2009 in Hokkaido Prefecture, Japan. Results: Among 478 clinical isolates, 112 strains (23.4%) showed levofloxacin (LVX) resistance. About 80% of the fluoroquinolone-resistant strains (88 strains) showed common features, namely O25:H4-ST131, phylogenetic group B and the same mutation pattern in quinolone resistance-determining regions. Pulsed field gel electrophoresis patterns suggested numerous lineages of O25:H4ST131. The fluoroquinolone-resistant strains, including strains of O25:H4-51-131 and other types, more frequently shared CTX-type ESBL genes than did fluoroquinolone-susceptible strains. The ESBL genes fell into the CTX-M-9 and CTX-M-2 groups. CTX-M-15 (CTX-M-1 group) was not found among any of the strains isolated in this study. Sitafloxacin showed markedly potent activity against E. coli isolates compared with LVX, ciprofloxacin and ulifloxacin. Conclusion: The most prevalent fluoroquinolone-resistant strains of E. coli isolated in Hokkaido Prefecture, Japan, are O25:H4ST131. However, similar to other areas of Japan, the 51131 clones represent distinct lineages from the general worldwide dispersal of multidrug-resistant clones which carry CTX-M-15. Copyright (C) 2012 S. Karger AG, Basel

犬及び人由来フルオロキノロン耐性大腸菌におけるプラスミド介在性キノロン耐性遺伝子oqxABの検出　　　　　　　　　　　　　　　
佐藤 豊孝, 横田 伸一, 内田 郁夫, 大久保 寅彦, 石原 加奈子, 岡林 環樹, 藤井 暢弘, 田村 豊
感染症学雑誌, 85, 5, 557, 557, (一社)日本感染症学会, Sep. 2011
Japanese

犬および人由来フルオロキノロン耐性大腸菌の性状比較　　　　　　　　　　　　　　　
佐藤 豊孝, 横田 伸一, 大久保 寅彦, 石原 加奈子, 藤井 暢弘, 田村 豊
北海道獣医師会雑誌, 55, 8, 417, 417, (公社)北海道獣医師会, Aug. 2011
Japanese

ヒト及びイヌ由来大腸菌におけるプラスミド介在性キノロン耐性遺伝子aac(6')-Ib-crの検出　　　　　　　　　　　　　　　
北原 尚英, 佐藤 豊孝, 大久保 寅彦, 石原 加奈子, 田村 豊
北海道獣医師会雑誌, 55, 8, 418, 418, (公社)北海道獣医師会, Aug. 2011
Japanese

牛における第三世代セファロスポリン使用による薬剤耐性菌の発生評価試験　　　　　　　　　　　　　　　
佐藤 豊孝, 大久保 寅彦, 石原 加奈子, 泉山 諭, 田村 豊
日本獣医学会学術集会講演要旨集, 152回, 215, 215, (公社)日本獣医学会, Aug. 2011
Japanese

牛呼吸器由来マイコプラズマにおけるマクロライド系抗菌薬の薬剤感受性調査　　　　　　　　　　　　　　　
佐藤 豊孝, 樋口 豪紀, 大久保 寅彦, 石原 加奈子, 永幡 肇, 田村 豊
日本獣医学会学術集会講演要旨集, 152回, 217, 217, (公社)日本獣医学会, Aug. 2011
Japanese

ヒト及びイヌ由来大腸菌におけるプラスミド介在性キノロン耐性遺伝子aac(6')-Ib-crの検出　　　　　　　　　　　　　　　
北原 尚英, 佐藤 豊孝, 大久保 寅彦, 石原 加奈子, 田村 豊
日本獣医学会学術集会講演要旨集, 152回, 262, 262, (公社)日本獣医学会, Aug. 2011
Japanese

A Fluoroquinolone-Resistant Escherichia coli Clinical Isolate without Quinolone Resistance-Determining Region Mutations Found in Japan
Toyotaka Sato, Shin-ichi Yokota, Ikuo Uchida, Torahiko Okubo, Kanako Ishihara, Nobuhiro Fujii, Yutaka Tamura
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 55, 8, 3964, 3965, AMER SOC MICROBIOLOGY, Aug. 2011, [Peer-reviewed], [International Magazine]
English

犬及び人由来フルオロキノロン耐性大腸菌におけるプラスミド介在性キノロン耐性遺伝子oqxABの検出　　　　　　　　　　　　　　　
佐藤 豊孝, 横田 伸一, 内田 郁夫, 大久保 寅彦, 石原 加奈子, 岡林 環樹, 藤井 暢弘, 田村 豊
感染症学雑誌, 85, 臨増, 191, 191, (一社)日本感染症学会, Mar. 2011
Japanese

犬および人から分離されたセファロスポリン耐性大腸菌における耐性性状の比較　　　　　　　　　　　　　　　
大久保 寅彦, 佐藤 豊孝, 石井 良和, 横田 伸一, 石原 加奈子, 田村 豊
感染症学雑誌, 85, 臨増, 301, 301, (一社)日本感染症学会, Mar. 2011
Japanese

犬由来フルオロキノロン-セファロスポリン多剤耐性大腸菌の系統発生分類による解析　　　　　　　　　　　　　　　
佐藤 豊孝, 横田 伸一, 大久保 寅彦, 石原 加奈子, 岡林 環樹, 藤井 暢弘, 田村 豊
日本獣医学会学術集会講演要旨集, 150回, 253, 253, (公社)日本獣医学会, Sep. 2010
Japanese

犬および人由来セファロスポリン耐性大腸菌における耐性因子の比較　　　　　　　　　　　　　　　
大久保 寅彦, 佐藤 豊孝, 石原 加奈子, 石井 良和, 田村 豊
日本獣医学会学術集会講演要旨集, 150回, 254, 254, (公社)日本獣医学会, Sep. 2010
Japanese

Pathogenic Chlamydia L2 requires aryl hydrocarbon receptors and detyrosinated tubulin for its growth
ZHANG Saicheng, 大久保寅彦, 中村眞二, 東秀明, 山口博之, 日本細菌学雑誌(Web), 78, 1, 2023

Investigation of the mechanism of symbiotic bacteriadependent bacterial transport by amoebae
大久保寅彦, 佐藤豊孝, 佐藤豊孝, 横田伸一, 中村眞二, 山口博之, 日本細菌学雑誌(Web), 77, 1, 2022

One Health Approachに基づいたフルオロキノロン耐性大腸菌の市中内拡散・定着様式の解明
佐藤豊孝, 立花徹, 玉井聡, 星野祐治, 鳥越慎吾, 榊原啓一郎, 前谷茂樹, 福田昭, 大久保寅彦, 臼井優, 高橋聡, 横田伸一, 田村豊, 堀内基広, 日本獣医学会学術集会講演要旨集, 165th (CD-ROM), 2022

Na+/H+ antiporter regulates desiccation tolerance in Escherichia coli
榎枝秀朗, 田中菜那, 大久保寅彦, 佐藤豊孝, 横田伸一, THAPA Jeewan, 山口博之, 日本細菌学雑誌(Web), 76, 1, 2021

Bacterial transport by amoeba that depend on symbiotic bacteria involves the Na⁺/H⁺ antiporter NhaA
田中菜那, 大久保寅彦, 佐藤豊孝, 横田伸一, THAPA Jeewan, 山口博之, 日本細菌学雑誌(Web), 76, 1, 2021

マイクローブトランスファー 身の回りの環境に潜む病原細菌の生態deja vuと健康リスク 病院での環境清浄度評価の盲点と院内細菌叢の実態　　　　　　　　　　　　　　　
矢野 理香, 山口 博之, 松尾 淳司, 大久保 寅彦, 良村 貞子, 下田 智子, 日本細菌学雑誌, 71, 1, 56, 56, Feb. 2016
日本細菌学会, Japanese

O22-04 バンコク周辺の水圏環境における薬剤耐性菌とその耐性遺伝子の解析(口頭発表)
大久保 寅彦, 臼井 優, 鈴木 聡, 高田 秀重, 田村 豊, 日本微生物生態学会講演要旨集, 2014, 98, 98, 2014
日本微生物生態学会, Japanese

O22-05 水圏環境からの薬剤耐性遺伝子伝播におけるハエの役割(口頭発表)
臼井 優, 大久保 寅彦, 福田 昭, 高田 秀重, 鈴木 聡, 田村 豊, 日本微生物生態学会講演要旨集, 2014, 99, 99, 2014
日本微生物生態学会, Japanese

イエネズミ糞便由来大腸菌の薬剤耐性調査
吉沢創太, 大久保寅彦, 佐藤豊孝, 臼井優, 田中和之, 石塚真由美, 田村豊, 日本獣医学会学術集会講演要旨集, 154th, 275, 31 Aug. 2012
Japanese

イエネズミ糞便由来大腸菌の薬剤耐性調査
吉沢創太, 大久保寅彦, 佐藤豊孝, 臼井優, 田中和之, 石塚真由美, 田村豊, 北海道獣医師会雑誌, 56, 8, 437, 437, 10 Aug. 2012
(公社)北海道獣医師会, Japanese

オホーツクと南極海で採取した流氷における重金属濃度と金属耐性遺伝子の検出
能田淳, 登坂唯香, 中島千絵, 大久保寅彦, 石原加奈子, 鈴木定彦, 伊村智, 田村豊, 日本獣医学会学術集会講演要旨集, 151st, 248, 01 Mar. 2011
Japanese

南極およびオホーツク海で採取した流氷における構成菌種の同定と薬剤耐性遺伝子の検出
登坂唯香, 中島千絵, 大久保寅彦, 石原加奈子, 鈴木定彦, 伊村智, 田村豊, 極域科学・宙空圏・気水圏・生物・地学シンポジウム講演予稿集(CD-ROM), 2010, ROMBUNNO.PB23, 2010
Japanese

アメーバによる細菌運搬現象と Na+/H+ アンチポーターNhaA との関係性の検証　　　　　　　　　　　　　　　
大久保 寅彦, 田中 菜那, Thapa Jeewa, 佐藤 豊孝, 横田 伸一, 山口 博之
第86回日本細菌学会北海道支部学術総会, 28 Aug. 2021, Oral presentation

Acanthamoeba S13WT relies on amoebal endosymbiont and bacterial NhaA for its ability to backpack bacteria on solid media.　　　　　　　　　　　　　　　
Okubo T, Tanaka N, Yoshioka C, Nakamura S, Sato T, Yokota S, Yamaguchi H
World Microbe Forum 2021, 22 Jun. 2021, English, Poster presentation
20 Jun. 2021 - 24 Jun. 2021

3D プリンターで作成したエアサンプラーによる空気中浮遊細菌の検出とその変動に関わる環境因子の探索　　　　　　　　　　　　　　　
大久保寅彦, 宮﨑 悟, 鷲見 優斗, タパ ジーワン, 山口 博之
第94回日本細菌学会総会, 24 Mar. 2021, Japanese, Public symposium
23 Mar. 2021 - 25 Mar. 2021

野外水系環境由来の繊毛虫存在下におけるLegionella pneumophilaの生存性の検討　　　　　　　　　　　　　　　
大久保寅彦, 川代愛梨, 中村眞二, Jeewan Thapa, 山口博之
第93回日本細菌学会総会, 20 Feb. 2020, Japanese, Poster presentation
19 Feb. 2020 - 21 Feb. 2020

イトトンボが保有する薬剤耐性菌の検出と分離菌株の性状解析　　　　　　　　　　　　　　　
大久保寅彦, 山口裕々, 綿路昌史, 榎枝秀明, Jeewan Thapa, 山口博之
第85回日本細菌学会北海道支部学術総会, 23 Aug. 2019, Japanese, Oral presentation
23 Aug. 2019 - 23 Aug. 2019

Fecal materials of damselfly can work as an indicator for revealing multidrug-resistant bacteria in local environments　　　　　　　　　　　　　　　
Okubo T, Yamaguchi Y, Wamaji M, Iwasaki S, Hayasaka K, Akizawa K, Yamaguchi H
ASM Microbe 2019, 22 Jun. 2019, English, Poster presentation
20 Jun. 2019 - 24 Jun. 2019

レジオネラの感染に抵抗性を示す環境由来アメーバによる細菌運搬現象　　　　　　　　　　　　　　　
大久保寅彦, 松尾淳司, 中村眞二, 山口博之
第92回日本細菌学会総会, 24 Apr. 2019, Japanese, Nominated symposium
23 Apr. 2019 - 25 Apr. 2019

Staphylococcus aureus prompts Escherichia coli survival under dry conditions: a potential threat from the viewpoint of nosocomial infections　　　　　　　　　　　　　　　
Okubo T, Shimoda T, Nakamura S, Yano R, Matsuo J, Yamaguchi H
ASM Microbe 2018, 10 Jun. 2018, English, Nominated symposium
[International presentation]

病院で用いられる乾燥床材上での細菌間の鬩ぎ合い：残存生菌数と ATP 量のギャップから紐解く細菌の生存戦略　　　　　　　　　　　　　　　
大久保寅彦, 松尾 淳司, 中村 眞二, 山口博之
第91回日本細菌学会総会学術集会, 28 Mar. 2018, Japanese, Oral presentation
[Domestic Conference]

札幌地下歩行空間での空気中浮遊細菌の菌叢解析　　　　　　　　　　　　　　　
大久保寅彦, 松尾淳司, 大崎敬子, 神谷茂, 山口博之
第84回日本細菌学会北海道支部学術総会, 26 Aug. 2017, Japanese, Oral presentation
[Domestic Conference]

Synergistic effect of walkers with other factors on changing airborne bacterial communities in a built environment, Sapporo Underground Pedestrian Space　　　　　　　　　　　　　　　
Okubo T, Matsuo J, Nakamura S, Yamaguchi H
ASM Microbe 2017, 03 Jun. 2017, English, Poster presentation
[International presentation]

北海道大学病院にて分離されたNDM産生大腸菌の性状解析と繊毛虫によるプラスミド伝達の促進　　　　　　　　　　　　　　　
大久保寅彦, 松下瑞江, 松尾淳司, 山口博之
第90回日本細菌学会総会, 20 Mar. 2017, Japanese, Nominated symposium
[Domestic Conference]

北海道大学病院にて分離されたニューデリー・メタロβラクタマーゼNDM-5産生大腸菌の性状解析　　　　　　　　　　　　　　　
大久保寅彦, 松尾淳司, 山口博之
第83回日本細菌学会北海道支部学術総会, 17 Sep. 2016, Japanese, Oral presentation
[Domestic Conference]

繊毛虫との共培養で大腸菌から漏れ出すクオラムセンシング分子 autoinducer-2 について　　　　　　　　　　　　　　　
大久保寅彦, 松尾淳司, 山﨑智拡, 花輪智子, 中村眞二, 神谷茂, 山口博之
第82回日本細菌学会北海道支部学術総会, 05 Sep. 2015, Japanese, Oral presentation
[Domestic Conference]

水圏環境からの薬剤耐性遺伝子伝播におけるハエの役割
USUI YU, OKUBO TORAHIKO, FUKUDA AKIRA, TAKADA HIDESHIGE, SUZUKI AKIRA, TAMURA YUTAKA
日本土壌微生物学会講演要旨集, 16 Oct. 2014, Japanese

バンコク周辺の水圏環境における薬剤耐性菌とその耐性遺伝子の解析
OKUBO TORAHIKO, USUI YU, SUZUKI AKIRA, TAKADA HIDESHIGE, TAMURA YUTAKA
日本土壌微生物学会講演要旨集, 16 Oct. 2014, Japanese

腸球菌の薬剤耐性性状と人為的影響との関係 腸球菌の薬剤耐性性状と人為的影響との関係 腸球菌の薬剤耐性性状と人為的影響との関係　　　　　　　　　　　　　　　
大久保寅彦, 福田昭, 田中和之, 臼井優, 田村豊
第81回日本細菌学会北海道支部学術総会, 29 Aug. 2014, Japanese, Oral presentation
[Domestic Conference]

モンゴル・コスタイ国立公園における遊牧民家畜等の薬剤耐性大腸菌出現状況
HIRANO YUKI, OKUBO TORAHIKO, KUMADA JUN, HAGIWARA KATSURO, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 11 Aug. 2014, Japanese

農場由来耐性菌のベクターとしてのハエの役割
SHIRAKAWA TAKAHIRO, FUKUDA AKIRA, OKUBO TORAHIKO, USUI YU, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 11 Aug. 2014, Japanese

北海道の敗血症豚由来Streptococcus suisに関する分子疫学調査
ONO YUTA, OKUBO TORAHIKO, IKEDA TETSUYA, SHIMIZU SHUN'ICHI, YOKOYAMA MITSUE, FURUSAKI YOJI
北海道獣医師会雑誌, 10 Aug. 2014, Japanese

豚疣贅性心内膜炎から分離したStreptococcus suisの薬剤感受性及び分子疫学的解析
ONO YUTA, YANAGISAWA RISA, OKUBO TORAHIKO, YOKOYAMA MITSUE, IKEDA TETSUYA, SHIMIZU SHUN'ICHI
日本獣医師会雑誌, 20 May 2014, Japanese

北海道で分離された大腸菌におけるキノロン,アミノグリコシド,セフェム交叉耐性株に関する検討
YOKOTA SHIN'ICHI, OKOSHI YASUO, OKUBO TORAHIKO, SATO TOYOTAKA, TAMURA YUTAKA
日本化学療法学会総会プログラム・講演抄録, 19 May 2014, Japanese

Horizontal transmission of antimicrobial resistance genes among Escherichia coli in housefly
FUKUDA AKIRA, USUI MASARU, OKUBO TORAHIKO, TAMURA YUTAKA
日本細菌学雑誌, 25 Feb. 2014, Japanese

春季モンゴルと日本におけるバイオエアロゾルの時空間的変動
NODA JUN, OKUBO TORAHIKO, HAGIWARA KATSURO, NOGUCHI IZUMI, TAMURA YUTAKA
大気環境学会年会講演要旨集, 31 Aug. 2013, Japanese

薬剤耐性遺伝子はイエバエ腸管内で接合伝達する
FUKUDA AKIRA, USUI YU, OKUBO TORAHIKO, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 30 Aug. 2013, Japanese

動物病院来院猫からのセファロスポリン耐性およびフルオロキノロン耐性大腸菌の検出
OKUBO TORAHIKO, ONO TADASHI, SATO TOYOTAKA, USUI YU, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2013, Japanese

犬の膿皮症治療のためのStaphylococcus pseudintermedius特異ファージの分離と抗菌薬によるファージ溶菌活性の増強
MASE KAORI, USUI YU, OKUBO TORAHIKO, IWANO HIDETOMO, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2013, Japanese

豚疣状心内膜炎から分離したStreptococcus suisの薬剤感受性及び分子疫学的解析
YANAGISAWA RISA, ONO YUTA, YOKOYAMA MITSUE, FURUSAKI YOJI, OKUBO TORAHIKO, IKEDA TETSUYA, SHIMIZU SHUN'ICHI
北海道獣医師会雑誌, 10 Aug. 2013, Japanese

犬由来大腸菌におけるフルオロキノロン耐性及びセファロスポリン耐性の関連
SATO TOYOTAKA, YOKOTA SHIN'ICHI, OKUBO TORAHIKO, USUI YU, FUJII NOBUHIRO, TAMURA YUTAKA
日本獣医師会雑誌, 20 May 2013, Japanese

伴侶動物由来細菌へのフルオロキノロン剤曝露による耐性菌出現リスク
USUI MASARU, SATO TOYOTAKA, OKUBO TORAHIKO, TAMURA YUTAKA
獣医畜産新報, 01 Apr. 2013, Japanese

イエネズミ糞便由来大腸菌の薬剤耐性調査
YOSHIZAWA SOTA, OKUBO TORAHIKO, SATO TOYOTAKA, USUI YU, TANAKA KAZUYUKI, ISHIZUKA MAYUMI, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2012, Japanese

市販鶏肉由来薬剤耐性サルモネラ属菌における耐性遺伝子の解析
OGAWA AYA, SATO TOYOTAKA, OKUBO TORAHIKO, USUI YU, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2012, Japanese

牛呼吸器由来Mycoplasma bovisにおけるフルオロキノロン系抗菌薬耐性機構
SATO TOYOTAKA, OKUBO TORAHIKO, USUI YU, HIGUCHI HIDETOSHI, NAGAHATA HAJIME, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2012, Japanese

薬剤耐性大腸菌の畜舎内伝播におけるハエの役割
USUI YU, IWASA TOMONORI, SATO TOYOTAKA, OKUBO TORAHIKO, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2012, Japanese

南極の氷中に含まれていた細菌叢の解析
OKUBO TORAHIKO, SATO TOYOTAKA, NAKAMURA SHOTA, IIDA TETSUNARI, USUI YU, NODA JUN, HAGIWARA KATSURO, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2012, Japanese

犬由来大腸菌におけるフルオロキノロン耐性およびセファロスポリン耐性の関連
SATO TOYOTAKA, YOKOTA SHIN'ICHI, OKUBO TORAHIKO, USUI YU, FUJII NOBUHIRO, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2012, Japanese

薬剤耐性大腸菌の畜舎内伝播におけるハエの役割
USUI YU, IWASA TOMOHIRO, SATO TOYOTAKA, OKUBO TORAHIKO, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2012, Japanese

市販鶏肉由来薬剤耐性サルモネラ属菌における耐性遺伝子の解析
OGAWA AYA, SATO TOYOTAKA, OKUBO TORAHIKO, USUI YU, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2012, Japanese

イエネズミ糞便由来大腸菌の薬剤耐性調査
YOSHIZAWA SOTA, OKUBO TORAHIKO, SATO TOYOTAKA, USUI YU, TANAKA KAZUYUKI, ISHIZUKA MAYUMI, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2012, Japanese

犬および人から分離されたセファロスポリン耐性大腸菌における耐性性状の比較
OKUBO TORAHIKO, SATO TOYOTAKA, ISHII YOSHIKAZU, YOKOTA SHIN'ICHI, ISHIHARA KANAKO, TAMURA YUTAKA
感染症学雑誌, 20 Nov. 2011, Japanese

犬及び人由来フルオロキノロン耐性大腸菌におけるプラスミド介在性キノロン耐性遺伝子oqxABの検出
SATO TOYOTAKA, YOKOTA SHIN'ICHI, UCHIDA IKUO, OKUBO TORAHIKO, ISHIHARA KANAKO, OKABAYASHI TAMAKI, FUJII NOBUHIRO, TAMURA YUTAKA
感染症学雑誌, 20 Sep. 2011, Japanese

牛呼吸器由来マイコプラズマにおけるマクロライド系抗菌薬の薬剤感受性調査
SATO TOYOTAKA, HIGUCHI HIDETOSHI, OKUBO TORAHIKO, ISHIHARA KANAKO, NAGAHATA HAJIME, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2011, Japanese

牛における第三世代セファロスポリン使用による薬剤耐性菌の発生評価試験
SATO TOYOTAKA, OKUBO TORAHIKO, ISHIHARA KANAKO, IZUMIYAMA SATOSHI, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2011, Japanese

ヒト及びイヌ由来大腸菌におけるプラスミド介在性キノロン耐性遺伝子aac(6’)‐Ib‐crの検出
KITAHARA NAOHIDE, SATO TOYOTAKA, OKUBO TORAHIKO, ISHIHARA KANAKO, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 31 Aug. 2011, Japanese

犬および人由来フルオロキノロン耐性大腸菌の性状比較
SATO TOYOTAKA, YOKOTA SHIN'ICHI, OKUBO TORAHIKO, ISHIHARA KANAKO, FUJII NOBUHIRO, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2011, Japanese

ヒト及びイヌ由来大腸菌におけるプラスミド介在性キノロン耐性遺伝子aac(6′)‐Ib‐crの検出
KITAHARA NAOHIDE, SATO TOYOTAKA, OKUBO TORAHIKO, ISHIHARA KANAKO, TAMURA YUTAKA
北海道獣医師会雑誌, 10 Aug. 2011, Japanese

オホーツクと南極海で採取した流氷における重金属濃度と金属耐性遺伝子の検出
NODA JUN, TOSAKA YUIKA, NAKAJIMA CHIE, OKUBO TORAHIKO, ISHIHARA KANAKO, SUZUKI YASUHIKO, IMURA SATOSHI, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 01 Mar. 2011, Japanese

犬および人由来セファロスポリン耐性大腸菌における耐性因子の比較
OKUBO TORAHIKO, SATO TOYOTAKA, ISHIHARA KANAKO, ISHII YOSHIKAZU, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 01 Sep. 2010, Japanese

犬由来フルオロキノロン‐セファロスポリン多剤耐性大腸菌の系統発生分類による解析
SATO TOYOTAKA, YOKOTA SHIN'ICHI, OKUBO TORAHIKO, ISHIHARA KANAKO, OKABAYASHI TAMAKI, FUJII NOBUHIRO, TAMURA YUTAKA
日本獣医学会学術集会講演要旨集, 01 Sep. 2010, Japanese

南極およびオホーツク海で採取した流氷における構成菌種の同定と薬剤耐性遺伝子の検出
TOSAKA YUIKA, NAKAJIMA CHIE, OKUBO TORAHIKO, ISHIHARA KANAKO, SUZUKI YASUHIKO, IMURA SATOSHI, TAMURA YUTAKA
極域科学・宙空圏・気水圏・生物・地学シンポジウム講演予稿集(CD-ROM), 2010, Japanese

医用工学実習　　　　　　　　　　　　　　　
北海道大学医学部保健学科

公衆衛生学実習　　　　　　　　　　　　　　　
北海道大学医学部保健学科

医動物学実習　　　　　　　　　　　　　　　
北海道大学医学部保健学科

医動物学　　　　　　　　　　　　　　　
北海道大学医学部保健学科

臨床微生物学実習　　　　　　　　　　　　　　　
北海道大学医学部保健学科

Functional elucidation of novel effectors of Legionella pneumophila approached through interactions with unique ciliate
Grants-in-Aid for Scientific Research
01 Apr. 2022 - 31 Mar. 2025
大久保 寅彦, 中村 眞二, 山口 博之
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 22K07051

New development of research to elucidate the intracellular adaptation mechanism of genital chlamydia unraveled from its similarity to cancer cells
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2021 - 31 Mar. 2025
山口 博之, 中村 眞二, THAPA JEEWAN, 大久保 寅彦
性感染症の主な原因である細胞内寄生性細菌クラミジア(Chlamydia trachomatis: Ct)の細胞内への適応機構は、未だ遺伝子改変ができないこともあって明らかになっていない。そこでCt感染細胞と癌細胞との類似性[1. PI3K-AKT経路を活性化、2. 通常酸素分圧下でミトコンドリアを要求する、3. 癌抑制因子として知られる芳香族炭素水素受容体転写因子(AHR)のリガンドであるインドールにてその発育が抑制される、4. 北大既存薬(3200剤)/オリジナル(2640剤)ライブラリーのスクリーニングで、癌の進行を促進するG蛋白共役胆汁酸受容体(TGR5)やHIVインテグラーゼの阻害剤などがクラミジアの増殖を完全に阻止した]から、Ctが細胞内で利用している新たな細胞内情報伝達経路とそれら修飾に関わる分泌エフェクター分子を同定すると共に、それら分子の中から癌治療の標的となりうる分子を探ることを目的とする。その結果、初年度の実験にて、以下の成果を得た。
1. Ct感染細胞では低酸素状態にて感染後30分でAKT(Ser473)リン酸化が起こり、持続することを見いだした。
2. インドールがCtが細胞内で増殖を抑制し、その機構として芳香族炭化水素受容体AhR分子が関与する可能性を示唆する結果を得た。
3. Ctの通常酸素下でのミトコンドリアの要求性は、NOX4/p38MAPKと関連していることを明らかにした。4. 北大既存薬ライブラリーでヒットした12薬剤についてその有効性について検証し11薬剤がCtの細胞内発育を有意に抑制することを確認し、KEGG解析にてCtが利用すると予想される新規の22の情報伝達系を同定した。
5. 北大オリジナルライブラリーでヒットした1薬剤についてその有効性について検証し、KEGG解析にてCtが利用すると予想される新規標的分子候補を同定した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 21H02726

次世代の耐性菌対策を考慮した、国際的ハイリスク病原性細菌の市中内定着様式の解明
科学研究費助成事業 基盤研究(B)
01 Apr. 2021 - 31 Mar. 2024
佐藤 豊孝, 小笠原 徳子, 山本 聡, 浦野 恵美子, 青木 弘太郎, 中島 千絵, 臼井 優, 鈴木 仁人, 大久保 寅彦, 福田 昭
本研究では、フルオロキノロン耐性大腸菌ST131をモデルに、『院内』で問題となるAMR感染症を『院外(市中)』からのプローチでその伝播・定着様式を明らかにすることで、現行のAMR対策が報われない国際的なAMR問題の根本的解決に繋がる次世代型(院内-市中一体型)AMR対策に資する科学的知見を提供することが本研究の目的である。本目的達成の為、本年度は、ST131の伝播状況と伝播様式および定着メカニズム(定着因子の同定)を行なった。具体的には、同一地域（札幌市内）から同一時期に院内及び市中の各サンプル群(患者、健常者、動物、環境)から大腸菌を一斉に分離した。その後、シプロフロキサシンに耐性だった株を対象にST131の検出PCRおよび次世代シーケンス解析により行なった。その結果、ヒトの臨床検体(大学病院)からは25%, ヒトの臨床検体(市中クリニック)からは19.2%, ヒトの健常者の便検体からは16.1%, 伴侶動物(犬や猫)の直腸スワブからは18.3%, 家畜の牛、豚、鶏の便検体からはそれぞれ9.5%, 0%, 6.7%のフルオロキノロン耐性率であった。分離大腸菌全体に占めるST131の割合は、ヒトの臨床検体(大学病院)からは12.5%, ヒトの臨床検体(市中クリニック)からは5.7%, ヒトの健常者の便検体からは8.2%, 伴侶動物(犬や猫)の直腸スワブからは15.9%, 家畜の牛、豚、鶏の便検体からは1株も分離されなかった。
日本学術振興会, 基盤研究(B), 札幌医科大学, 21H03622

Understanding and application of microbiota in building environment: creation of pathogen control theory by temperature control
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Pioneering)
30 Jul. 2020 - 31 Mar. 2024
山口 博之, 矢野 理香, 大森 亮介, 大久保 寅彦
消毒剤や抗菌剤に頼らない温度による新たな病原体制御理論を創成し、空間や高頻度接触面の温度を制御することで、感染予防へと応用を目指すために、2年目は以下の研究を実施しいくつかの成果を得た。
1. 乾燥表面の温度調節による大腸菌の生存性の制御に湿度変化が与える影響: 私達は乾燥面を37℃付近に温めることでその乾燥麺に塗抹した細菌の生存性が顕著に低下することを見いだした。その一方で環境温度は、熱を奪う空気中の水蒸気量すなわち湿度の影響も受ける。そこで恒温恒湿機を使用し，温度(25-37℃)と共に湿度(45-90%)が乾燥表面の大腸菌の生存性に与える影響を調査した。その結果湿度と大腸菌の生存率には逆相関関係(r=-0.241)があり，湿度が上がるほど生存率は有意に低下した(p=0.04)。このように、乾燥面での温度制御において湿度によるネガティブな効果は最小限であることを見いだした。
2. 温度制御手摺デバイス上での細菌の生存性の可視化法の開発: バイク用ハンドヒーターを改良し作成したデバイスの効果を振れ幅の大きい培養に頼らず正確かつ簡便に確認する方法を、無蛍光の透明テープとLIVE/DEAD染色による測定系とキーエンス画像解析ソフトを組み合わせることで実現した。具体的には、ヒーターより距離が離れるほど手摺上の生存菌数は有意に低下し、その効果は温度ヒートマップと一致した。
3. 土壌細菌の空間移動に環境要因の変化が及ぼす影響について: 新型コロナ感染症の影響で公共の閉鎖環境での採材ができなかったので、その代替えとして３Dプリンターを用いて空気中に浮遊する細菌を効率よく生け捕りにできるエアサンプラーを用いて北大農場にて実施した。その結果、環境因子(気圧、蒸気圧、湿度、風向き)が連動し変化することにより、空気中に巻き上げられ浮遊し移動することが明らかになった。
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Research (Pioneering), Hokkaido University, 20K20613

入院患者の日常生活動作レベルがベッド周辺環境の微生物汚染に及ぼす影響
科学研究費助成事業 基盤研究(C)
01 Apr. 2020 - 31 Mar. 2024
下田 智子, 矢野 理香, 山口 博之, 大久保 寅彦
本研究は、患者の周辺環境への接触とその接触面での細菌の生存を具体的な患者の日常生活動作レベルを精査し、適切な看護実践（環境整備）へ応用することを目指す。
医療施設において、病原体は手指やその他の媒介物による接触を介して、感受性のある人に移動する可能性があるため病原体で汚染された環境表面の接触伝播経路に着目した。特に患者や医療従事者の手指が接触する機会が多い、ベッド柵やオーバーベッドテーブル等の患者周辺環境に着目した環境の調査研究を計画した。
2020年度は、新型コロナウイルス感染症の影響を受け、患者が療養している環境での調査が困難となった。そこで、2021年度は新型コロナウイルス感染症が発生する前に調査した結果から交絡因子を検討し、患者周辺の環境微生物汚染度と日常生活動作レベルの差異との関連について、準実験研究を用いて検討する計画へ変更した。
準実験研究の目的は、模擬病室内の患者周辺環境において、日常生活動作レベルの差異と模擬患者の手が触れる接触面積やアデノシン３リン酸（ATP）値との関連を検討した。模擬患者（12名）に先行研究で看護師の動作が分析されている車いすへの移乗を依頼した。その際のベッド周辺環境への接触面積やATP値を測定した。日常生活動作レベルの差異と模擬患者の手が触れる接触面積には相関関係がなかった。しかし、ATPで評価した環境微生物汚染度は、日常生活動作レベルが高くなるほど高値を示した。本結果は、模擬患者の日常生活動作レベルの差異とベッド周辺環境の汚染度に関連がある可能性を示唆したと考える。
日本学術振興会, 基盤研究(C), 札幌医科大学, 20K10626

Dissemination of antimicrobial-resistant bacteria from hospital sewage to natural environments
Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists
01 Apr. 2019 - 31 Mar. 2022
Okubo Torahiko
In this study, extended-spectrum β-lactamase-producing E. coli (n=44) were isolated from manhole sewage. Although there was no significant difference in the frequency of resistance between general facility sewage and hospital sewage, the hospital sewage-derived strain was resistant to fluoroquinolones in addition to β-lactam resistance. These results indicate that sewage is an important pathway for the spread of drug-resistant bacteria into the environment, and that hospital sewage in particular contains drug-resistant bacteria that have become highly resistant. In addition, Anteglaucoma harbinensis and nine other strains of ciliates were isolated from sewage. Anteglaucoma showed a unique characterization of Legionella susceptibility, which provided a secondary ciliate useful as a tool in the search for virulence factors of Legionella.
Japan Society for the Promotion of Science, Grant-in-Aid for Early-Career Scientists, Hokkaido University, 19K19403

The collaborative research project for drug resistance bacteria in animal agriculture of tropical Africa and finding the alternatives for drug use.
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
01 Apr. 2016 - 31 Mar. 2020
USHIDA Kazunari
Drug resistance bacteria (DRB) was emerged from human therapies and the animal agriculture. In a recent trend, prevalence of DRB decreased in developed countries. However, it is growing in developing countries. In this study we made a surveillance of DRB in Ugandan animal agriculture, where economic growth pushes the intensive animal production. We also collected potentially probiotic lactic acid bacteria from domestic birds under extensive feeding systems and wild birds for the future reduction of the antimicrobials in animal agriculture. We detected mostly tetracycline-resistant bacteria from feces and meet products of Ugandan livestock. However, we detected several multidrug-resistant ESBL Escherichia coli from feces of scavenging bird in abattoirs and dumping site of Capital city, which indicate the potential risk for transmission of ESBL by these wild birds. We have successfully isolated several potential probiotic lactobacilli from domestic and wild birds.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), 16H02767

熱帯アフリカにおける畜産由来薬剤耐性菌発生の実態把握と対策の提案　　　　　　　　　　　　　　　
KAKENHI
Apr. 2016 - Mar. 2020
牛田一成
JSPS, Competitive research funding

Exploring critical factors responsible for regulating the survival of Chlamydia trachomatis into cervical mucosa
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2016 - 31 Mar. 2019
Yamaguchi Hiroyuki
Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, with an estimated 131 million new cases annually worldwide. Although IFN-γ is a critical defense factor against C. trachomatis genital infection that acts by depleting host cellular tryptophan pool, the presence of indole, which is a precursor for tryptophan, can rescue genital chlamydiae despite depletion of tryptophan in IFN-γ-exposed cells in vitro, presumably, the higher vaginal indole amount prolongs the survival of C. trachomatis in the vaginal tract. Here, using the 554 cervical swabs with the prevalence of C. trachomatis infection, we indicated that the flow of bacteria through the anogenital route along with gut indole, had a strong impact on the prevalence of C. trachomatis in the cervicogenital tract of women. We also established a standardized chlamydial culture method in hypoxia and the detection system of apoptosis with caspase-3 expressing HEp-2 cells into chlamydia-infecte cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 16H05225

The role of amoebal endosymbiont in predator-prey interactions
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2016 - 31 Mar. 2019
Yamaguchi Hiroyuki
We explored the role of the endosymbiont in predator‐prey interactions using symbiotic amoebae (Neochlamydia-symbiotic acanthamoeba: S13 amoebae). As a result, we found here for the first time as follows: 1. the presence of symbiotic Neochlamydia S13 inhabited in the S13 amoebae could be responsible for the restriction of phagocytic activity of Legionella in specific, resulting in the defense of the amoebae against Legionella infection. 2. S13 amoebae required the Neochlamydia endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid agar. These findings might contribute not only to an understanding of the host-parasite relationship, but also to development of a novel strategy against complicated infectious diseases with intracellular parasites.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 16K15270

アメーバ共生細菌原始クラミジアのレジオネラ撃退に関わる分子マシナリーの探索　　　　　　　　　　　　　　　
KAKENHI
Apr. 2016 - Mar. 2019
山口博之
JSPS, Competitive research funding

腟菌叢とメタボローム: クラミジア卵管線維化機構とPID診断バイオマーカーの探索　　　　　　　　　　　　　　　
KAKENHI
Apr. 2016 - Mar. 2019
山口博之
JSPS, Competitive research funding

Effect of bacterial signal molecule AI-2 on the metabolism and pathogenicity of protozoa
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
01 Apr. 2016 - 31 Mar. 2018
Okubo Torahiko, YAMAGUCHI Hiroyuki, MATSUO Junji, NAKAMURA Shinji, MATSUSHITA Mizue, AKIZAWA Kouji, HAYASAKA Kasumi, FUKUMOTO Tatsuya, IWASAKI Sumio
AI-2 is a bacterial signal molecule which is used for bacterial closs talk. In our previous studies, we reported that co-cultivation of bacteria and protozoa causes the raise of AI-2 concentration in the solution. However, the effect of AI-2 on protozoa was not reported. In this study, we compared phenotypic characteristics of protozoa under the presence or absence of AI-2. Our results showed that AI-2 causes the raise of mobility of amoeba cells in a concentraion dependent manner on solid agar surfaces. This suggests that amoeba can detect AI-2 concentration and control its mobility. We showed cross-Kingdom effect of AI-2 on protozoa for the first time.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, 16K19113

細菌由来シグナル分離AI-2が原生生物の代謝及び病原性に与える影響の解明　　　　　　　　　　　　　　　
KAKENHI
Apr. 2016 - Mar. 2018
Torahiko Okubo
JSPS, Principal investigator, Competitive research funding

市販鶏肉の表面で繰り広げられる細菌と原生動物の相互作用の可視化と応用
科学研究費助成事業 研究活動スタート支援
28 Aug. 2015 - 31 Mar. 2016
大久保 寅彦
1. 市販鶏肉上で生じている細菌と原生動物の相互作用を可視化するという当初計画に基づき、予備試験、鶏肉サンプルの採材、微生物叢解析、原生動物の分離培養を実施した。
2. 鶏肉とアメーバまたはアメーバDNAを用いた予備試験により、鶏肉からの原生動物の検出限界は、培養法で1,000cell/g、遺伝子検出法（PCR）で0.1cell/gであると求められた。
3. 市販鶏肉を購入し、(1)当日 (2)4℃ 2days (3)4℃ 7days (4)15℃ 2days (5)15℃ 7daysの条件で静置後にDNAを抽出し、微生物叢解析を行なった。16S rDNAに基づく細菌叢解析の結果、当日サンプルではガンマプロテオバクテリアやバシルスなどが多く、細菌叢に多様性がみられたのに対し、4℃静置サンプルではガンマプロテオバクテリアが圧倒的多数となっていた。属レベルで見た場合、こうしたガンマプロテオバクテリアの多くはエロモナス、シュードモナスなどの水系細菌・低温発育菌であったことから、こうした細菌は冷蔵した鶏肉の上で増殖し続けていたと考えられた。一方、15℃静置サンプルにおいては、2days時点ではガンマプロテオバクテリアが優占化したが、7days時点ではバクテロイデスの大幅な増加がみられた。これは好気性菌の増殖によって鶏肉表面の酸素が消費されたことによるものと考えられる。
4. 当初、18S rDNAにもとづく原生生物叢の解析を実施予定であったが、ニワトリ由来DNAが障害となって解析を実施できなかった。分離培養では、2サンプルから2種類の鞭毛虫(Parabodo sp., Sandona sp.)を得た。これらの鞭毛虫と食中毒菌(E.coli, Salmonella Enteritidis)の共培養を実施したが、鞭毛虫は細菌数の増減に有意な影響を与えなかった。
5. 鶏肉表面で生じている細菌叢構成の劇的な変化、および鶏肉に付着する鞭毛虫を検出した。今回は食中毒菌と相互作用する原生動物は得られなかったが、今後の調査により細菌の生存性や病原性に影響する微生物の検出につなげたい。
日本学術振興会, 研究活動スタート支援, 北海道大学, 15H05997

市販鶏肉表面で繰り広げられる細菌と原生生物の相互作用の可視化と応用　　　　　　　　　　　　　　　
KAKENHI
Sep. 2015 - Mar. 2016
Torahiko Okubo
JSPS, Principal investigator, Competitive research funding

JICA short term volunteer　　　　　　　　　　　　　　　
01 Mar. 2012 - 31 May 2012
Advisor
Research advise
torahiko
短期派遣ボランティアとしてウガンダでの家畜疾病診断ラボ構築プロジェクトに参加。