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| 1.
論文（リポジトリ） |
論文（リポジトリ）
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Sato, Yukuto ; Hermawan, Idam ; Kakita, Tetsuya ; Okano, Sho ; Imai, Hideyuki ; Nagai, Hiroto ; Kimura, Ryosuke ; Yamashiro, Tetsu ; Kajita, Tadashi ; Toma, Claudia
概要:
Background Leptospirosis, a zoonosis caused by species in the spirochete genus Leptospira, is endemic to the Yaeyama reg
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ion in Okinawa, subtropical Japan. Species of the P1 subclade “virulent” group, within the genus Leptospira, are the main etiological agents of leptospirosis in Okinawa. However, their environmental persistence is poorly understood. This study used a combination of bacterial isolation and environmental DNA (eDNA) metabarcoding methods to understand the eco-epidemiology of leptospirosis in this endemic region. Findings Polymerase chain reaction (PCR) characterized twelve human clinical L. interrogans isolates belonging to the P1 subclade “virulent” subgroup and 11 environmental soil isolates of the P1subclade “low virulent” subgroup (genetically related to L. kmetyi, n = 1; L. alstonii, n = 4; L. barantonii, n = 6) from the Yaeyama region targeting four virulence-related genes (lipL32, ligA, ligB and lpxD1). Clinical isolates were PCR positive for at least three targeted genes, while all environmental isolates were positive only for lipL32. Analysis of infected renal epithelial cells with selected clinical and environmental strains, revealed the disassembly of cell-cell junctions for the Hebdomadis clinical strain serogroup. Comparison of leptospiral eDNA during winter and summer identified operational taxonomic units corresponding to the species isolated from soil samples (L. kmetyi and L. barantonii) and additional P2 subclade species (L. licerasiae, L. wolffii-related, among others) that were not detected by soil cultivation. Total Leptospira read counts were higher in summer than in winter and the analysis of leptospiral/animal eDNA relationship suggested Rattus spp. as a potential reservoir animal. Conclusion Our study demonstrated high environmental Leptospira diversity in the Yaeyama region, particularly during summer, when most of the leptospirosis cases are reported. In addition, several Leptospira species with pathogenic potential were identified that have not yet been reported in Yaeyama; however, the environmental persistence of P1 subclade species previously isolated from human clinical cases in this region was absent, suggesting the need of further methodology development and surveillance.
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| 2.
論文（リポジトリ） |
論文（リポジトリ）
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Sebastián, Isabel ; Okura, Nobuhiko ; Humbel, M. Bruno ; Xu, Jun ; Hermawan, Idam ; Matsuura, Chiaki ; Hall, Malgorzata ; Takayama, Chitoshi ; Yamashiro, Tetsu ; Nakamura, Shuichi ; Toma, Claudia
概要:
Bacterial pathogens have evolved multiple strategies to disassemble epithelial cell apical junctional complexes (AJCs) and infect epithelial cells. Leptospirosis is a widespread zoonotic infection, mainly caused by Leptospira
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interrogans, and its dissemination across host cell barriers is essential for its pathogenesis. However, the mechanism of bacterial dissemination across epithelial cell barriers remains poorly characterised. In this study, we analysed the interaction of L. interrogans with renal proximal tubule epithelial cells (RPTECs) and found that at 24 hr post-infection, L. interrogans remain in close contact with the plasma membrane of the RPTEC by extracellularly adhering or crawling. Leptospira interrogans cleaved E-cadherin and induced its endocytosis with release of the soluble N-terminal fragment into the extracellular medium. Concomitantly, a gradual decrease in transepithelial electrical resistance (TEER), mislocalisation of AJC proteins (occludin, claudin-10, ZO-1, and cingulin) and cytoskeletal rearrangement were observed. Inhibition of clathrin-mediated E-cadherin endocytosis prevented the decrease in TEER. We showed that disassembly of AJCs in epithelial cells and transmigration of bacteria through the paracellular route are important for the dissemination of L. interrogans in the host.
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| 3.
論文（リポジトリ） |
論文（リポジトリ）
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Gamage, Chandika D. ; Sato, Yukuto ; Kimura, Ryosuke ; Yamashiro, Tetsu ; Toma, Claudia
概要:
Background:Leptospirosis is one of the most significant zoonoses across the world not only because of its impact on huma
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n and animal health but also because of the economic and social impact on agrarian communities. Leptospirosis is endemic in Sri Lanka where paddy farming activities, the use of draught animals in agriculture, and peridomestic animals in urban and rural areas play important roles in maintaining the infection cycle of pathogenic Leptospira, especially concerning animals as a potential reservoir. In this study, an environmental DNA (eDNA) metabarcoding methodology was applied in two different agro-ecological regions of Sri Lanka to understand the eco-epidemiology of leptospirosis.\nMethodology/Principal findings:Irrigation water samples were collected in Kandy District (wet zone mid-country region 2) and Girandurukotte, Badulla District (intermediate zone low-country region 2); and analysed for the presence of pathogenic Leptospira, associated microbiome and the potential reservoir animals. Briefly, we generated PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. The analysis of eDNA showed different environmental microbiomes in both regions and a higher diversity of Leptospira species circulating in Kandy than in Girandurukotte. Moreover, the number of sequence reads of pathogenic Leptospira species associated with clinical cases such as L. interrogans was higher in Kandy than in Girandurukotte. Kandy also showed more animal species associated with pathogenic bacterial species than Girandurukotte. Finally, several pathogenic bacterial species including Arcobacter cryaerophilus, responsible for abortion in animals, was shown to be associated with pathogenic Leptospira.\nConclusions/Significance:Leptospirosis has been considered to be endemic in wet regions, consistently, leptospiral sequences were detected strongly in Kandy. The great Leptospira species diversity in Kandy observed in this study shows that the etiological agents of leptospirosis in Sri Lanka might be underestimated. Furthermore, our eDNA metabarcoding can be used to discriminate bacterial and animal species diversity in different regions and to explore environmental microbiomes to identify other associated bacterial pathogens in the environment.
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学位論文（リポジトリ） |
学位
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山城, 哲 ; Yamashiro, Tetsu
概要:
報告番号(甲第29号)
現在膿瘍形成性疾患の起炎菌として注目されつつある“Streptococcus milleri group”の呼吸器感染症における臨床的意義について検討した.“S. milleri group”は20歳代の健 … 康成人20人の唾液・咽頭ぬぐい液には10^5CFU/ml以上には存在しなかった.また，全年齢層の120人の健康成人(20歳～80歳)における咽頭ぬぐい液の検討では11.7%(14/120)の頻度に10^5CFU/ml以上の菌量の“S. milleri group”が検出された. 一方起炎菌不明の膿性の喀痰・咽頭ぬぐい液においては24.7%(24/97)と高頻度に分離された.“S. milleri group”が臨床材料から有意に分離された症例32例中10症例において，“S. milleri group”に対する血清抗体価を間接蛍光抗体法を用いて測定したが，全例1:512以上を示し，コントロ一ルとした健康成人18例の抗体価(いずれも1：256以下)に比較し明らかな上昇をみとめた.このうち3症例ではいずれも菌分離2週後で最高を示し，6週以降では下降し，健康人とほぼ同じ値となった.現在市中肺炎の約半数が起炎菌不明とされている.本稿の成績から“S. milleri group”は肺化膿症・膿胸の重要な起炎菌であると同時に，肺炎においてもその起炎菌としての意義が極めて高いことが明らかになった. Streptococcus milleri group" are the part of the indigenous oral flora, and they are proposed to contain three distinct species: Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius. Though not included in the approved lists of bacterial names, "S. milleri group" are regarded as the causative organisms of suppurative infections, such as oral abscess, brain abscess, lung abscess and empyema. I have studied the clinical significance of the "S. milleri group" in respiratory infections. An investigation was performed to confirm the incidence of "S. milleri group" colonization in healthy 120 volunteers' (20 y/o～80 y/o) throats, and it was found that 11.7％ (14/120) were positive. On the other hand, attention should be paid to the fact that the "S. milleri group" was highly isolated, 24 (24.7%) in 97 purulent respiratory specimens (94 sputa and 3 throat swabs) from which no other significant microorganism was recovered. I have measured the serum antibody liters of the "S. milleri group", employing the IFA technique, in 1O patients from whose specimens "S. milleri group" was predominantly isolated, and compared with those of 18 healthy volunteers. Whereas all of the titers of healthy volunleers reveal less than 1:256, those of the patients reveal more than 1:512. And antibody titers to "S. milleri group" showed the highest in two weeks after "S. milleri group" isolation, and came down to healthy adult levels in six weeks or more. Thus far, about half of the causative organisms of acute pneumonia have been reported unknown. In this study I have suggested that the "S. milleri group" plays an important role as the causative organism in respiratory infections including pneumonia. 学位論文 続きを見る |
