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Tsuyoshi Sekizuka, Kentaro Itokawa, Rina Tanaka, Masanori Hashino, Koji Yatsu, Makoto Kuroda Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, 162-8640, JapanCorrespondence: Makoto Kuroda, Pathogen Genomics Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjyuku, Tokyo, 162-8640, Japan, Tel +81-3-5285-1111, Fax +81-3-5285-1166, Email makokuro@niid.go.jpPurpose: Urban wastewater treatment plant (WWTP) effluents, even with proper treatment, may cause antimicrobial resistance (AMR) burden, with a high frequency of acquired antimicrobial resistance genes (ARGs). The dissemination of ARGs into the environment increases the risk of infectious diseases; however, there is little direct evidence regarding their epidemiological effects. This study aimed to assess effluents from urban WWTPs around the Tama River and Tokyo Bay using metagenomic analysis of (AMR) genes (ARGs) and heavy-metal resistance genes.Methods: Metagenomic DNA-seq analysis of water samples and resistome analysis were performed.Results: The most prevalent ARG was the sulfonamide resistance gene, sul1, followed by the quaternary ammonium compound resistance gene, qacE, suggesting that basic gene sets (sul1 and ∆qacE) in the class 1 integrons are the predominant ARGs. The aminoglycoside resistance genes, aadA and aph, and macrolide resistance genes, msr(E) and mph(E), were the predominant ARGs against each antimicrobial. blaOXA and blaGES were frequently detected, whereas the blaCTX-M cluster was faintly detected. Non-metric multidimensional scaling plot analysis and canonical correspondence analysis results suggested that marked differences in ARGs could be involved in the seasonal differences; qnrS2, aac(6’)-Ib, and mef(C) increased markedly in summer, whereas msr(E) was more frequently detected in winter. Heavy-metal (Hg and Cu) resistance genes (HMRGs) were significantly detected in effluents from all WWTPs.Conclusion: We characterized a baseline level of the environmental ARG/HMRG profile in the overall community, suggesting that environmental AMR surveillance, particularly in urban WWTPs, is a valuable first step in monitoring the AMR dissemination of bacteria from predominantly healthy individuals carrying notable ARG/Bs.Keywords: urban sewage, effluent, metagenomics, Enterobacteriaceae, ESBL, carbapenemase, heavy-metal resistance

Miwa Katagiri,1,2 Makoto Kuroda,2 Tsuyoshi Sekizuka,2 Norihide Nakada,3 Yukitaka Ito,4 Masanobu Otsuka,4 Manabu Watanabe,1 Shinya Kusachi1 1Department of Surgery, Toho University Ohashi Medical Center, Meguro-ku, Tokyo, Japan; 2Laboratory of Bacterial Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan; 3Research Center for Environmental Quality Management, Graduate School of Engineering, Kyoto University, Otsu-shi, Shiga, Japan; 4Division of Clinical Microbiology Laboratory, Toho University Ohashi Medical Center, Meguro-ku, Tokyo, JapanCorrespondence: Miwa KatagiriDepartment of Surgery, Toho University Ohashi Medical Center, 2-22-36 Ohashi, Meguro-ku, Tokyo, 153-8515, JapanTel +81354333086Fax +81354333091Email girioyoshikazu0513@gmail.comMakoto KurodaLaboratory of Bacterial Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, JapanTel +81352851111Fax +81352851150Email makokuro@niid.go.jpBackground: Excrement containing antimicrobial-resistant bacteria (ARB) is discharged from the hospital sewage through wastewater treatment plants (WWTP) into rivers, increasing the antimicrobial resistance (AMR) burden on the environment.Purpose: We illustrate the contamination of hospital sewage tanks with ARB harboring antimicrobial resistance genes (ARGs) using comprehensive metagenomic sequencing. During the study period, we moved to a new hospital building constructed for renovation. Therefore, we investigated the difference in bacterial flora in the sewage tanks for each building with different departments, and the change in bacterial flora over time in new sewage tanks. Furthermore, we performed a comparative genome analysis of extended spectrum β-lactamase (ESBL)-producing organisms (EPOs) from hospital sewage and clinical samples. Residual antibiotics in the sewage tank were also measured.Methods: Metagenomic analysis was performed on the hospital sewage samples, followed by whole genome sequencing of EPOs.Results: The bacterial composition of new sewage tanks was comparable with that of old tanks within 1 month after relocation and was instantly affected by excrement. The bacterial composition of sewage tanks in the old and new buildings, containing rooms where seriously ill patients were treated, was similar. Selection on CHROMagar ESBL allowed detection of EPOs harboring blaCTX-M and carbapenemase genes in all sewage tanks. One of the sewage Escherichia coli strain comprising ST393 harboring blaCTX-M-27 corresponded to the clinical isolates based on core genome analysis. Moreover, the levels of levofloxacin and clarithromycin in the hospital sewage were 0.0325 and 0.0135 μg/mL, respectively.Conclusion: Hospital sewage was contaminated with many ARB species, ARGs and residual antibiotics, which can cause a burden on WWTP sewage treatment. The bacterial flora in the sewage tank was rapidly affected, especially by the ward with seriously ill patients. AMR monitoring of hospital sewage may help detect carriers prior to nosocomial ARB-associated outbreaks and control the outbreaks.Keywords: hospital sewage, antimicrobial resistance, antimicrobial resistance gene, metagenome analysis, whole genome sequencing, residual antimicrobial reagents

Yoko Takayama, 1, 2 Tsuyoshi Sekizuka, 3 Hidehito Matsui, 4 Yuzuru Adachi, 4 Ryotaro Eda, 5 Shin Nihonyanagi, 2 Tatsuhiko Wada, 2 Mari Matsui, 6 Satowa Suzuki, 6 Masashi Takaso, 7 Hidero Kitasato, 5 Makoto Kuroda, 3 Hideaki Hanaki 4 1Department of Infection Control and Infectious Diseases, Research and Development Center for New Medical Frontiers, Kitasato University School of Medicine, Kanagawa, Japan; 2Department of Infection Control and Prevention, Kitasato University Hospital, Kanagawa, Japan; 3Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan; 4Infection Control Research Center, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan; 5Department of Microbiology, School of Allied Health Sciences, Kitasato University, Kanagawa, Japan; 6Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Tokyo, Japan; 7Department of Orthopaedic Surgery, Kitasato University School of Medicine, Kanagawa, JapanCorrespondence: Yoko Takayama 1-15-1 Kitasato, Minami-Ku, Sagamihara, Kanagawa 252-0374, JapanTel/Fax +81-42-778-9119Email yoko@med.kitasato-u.ac.jpPurpose: New Delhi metallo-β-lactamase 5 (NDM-5) shows stronger resistance to carbapenems and broad-spectrum cephalosporins than NDM-1 because NDM-5 differs from NDM-1 by two amino acid substitutions. In this study, our aim was to characterize a NDM-5-producing Escherichia coli isolate KY1497 from a patient with urinary tract infection in Japan, who had no recent history of overseas travel.Patients and Methods: NDM-5-producing E. coli isolate KY1497 was detected in the urine sample of a patient hospitalized in a tertiary hospital in Japan. The complete genome sequence of isolate KY1497 was determined by short- and long-read sequencing with hybrid assembly, followed by multilocus sequence typing (MLST), core-genome phylogeny analysis, plasmid analysis, and transconjugation experiments.Results: KY1497 was classified as ST405 by MLST, and core-genome phylogeny exhibited the closest lineage to the clinical isolates in Nepal (IOMTU605) and Canada (FDAARGOS_448). KY1497 harbors blaNDM-5 in the IncFII-IncFIB(pB171) replicon plasmid (pKY1497_1, 123,767 base pairs). Plasmid analysis suggested that the cognate plasmids of pKY1497_1 have a minor plasmid background, rather than the globally disseminated IncX3 plasmid carrying blaNDM-5. Transconjugation analysis revealed that pKY1497_1 is transmissible to the recipient E. coli J53 strain.Conclusion: We characterized a novel Inc replicon plasmid (IncFII-IncFIB[pB171]) carrying blaNDM-5 and its host E. coli strain. NDMs are associated with a high risk of infection worldwide because of their antibiotic resistance and untreatable and hard-to-treat infections. Other patients in the hospital showed negative results for carbapenem-resistant Enterobacteriaceae. As NDM-producing strains are only sporadically detected in Japan, attention should be provided to the community prevalence of NDM-producing E. coli strains to prevent nosocomial infections.Keywords: blaNDM-5, Escherichia coli, sequence type 405, IncFII, IncFIB

Tsuyoshi Sekizuka, Yuba Inamine, Takaya Segawa, Makoto KurodaLaboratory of Bacterial Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Shinjuku, Tokyo, JapanPurpose: New Delhi metallo-β-lactamase (NDM)-5-producing Enterobacteriaceae have been detected in rivers, sewage, and effluents from wastewater treatment plants (WWTPs). Environmental contamination due to discharged effluents is of particular concern as NDM variants may be released into waterways, thereby posing a risk to humans. In this study, we collected effluent samples from a WWTP discharged into a canal in Tokyo Bay, Japan.Methods: Testing included the complete genome sequencing of Escherichia coli GSH8M-2 isolated from the effluent as well as a gene network analysis.Results: The complete genome sequencing of GSH8M-2 revealed that it was an NDM-5-producing E. coli strain sequence type ST542, which carries multiple antimicrobial resistance genes for β-lactams, quinolone, tetracycline, trimethoprim-sulfamethoxazole, florfenicol/chloramphenicol, kanamycin, and fosfomycin. The blaNDM-5 gene was found in the IncX3 replicon plasmid pGSH8M-2-4. Gene network analysis using 142 IncX3 plasmid sequences suggested that pGSH8M-2-4 is related to both clinical isolates of E. coli and Klebsiella species in Eastern Asia. GSH8M-2 also carries the blaCTX-M-55 gene in IncX1 plasmid pGSH8M-2-3.Conclusion: This is the first report of environmental NDM-5-producing E. coli isolated from a WWTP in Japan. NDM-5 detection is markedly increasing in veterinary and clinical settings, suggesting that dual β-lactamases, such as NDM-5 and CTX-M-55, might be acquired through multiple steps in environment settings. Environmental contamination through WWTP effluents that contain producers of NDM variants could be an emerging potential health hazard. Thus, regular monitoring of WWTP effluents is important for the detection of antimicrobial-resistant bacteria that may be released into the waterways and nearby communities.Keywords: sewage, carbapenemase, NDM-5, IncX3, CTX-M-55, IncX1

Masako Kuroda,1 Yasuhiko Hirami,1–3 Masayuki Hata,4 Michiko Mandai,1–3 Masayo Takahashi,1–3 Yasuo Kurimoto1–3 1Department of Ophthalmology, Kobe City Medical Center General Hospital, 2Department of Ophthalmology, Institute of Biomedical Research and Innovation Hospital, 3Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, 4Department of Ophthalmology, Kyoto University Graduate School of Medicine, Kyoto, Japan Background: The purpose of this study was to observe the characteristic findings of spectral-domain optical coherence tomography (SD-OCT) images in the retinas of patients with retinitis pigmentosa and to evaluate their distribution patterns in the early and advanced stages of the disease. Methods: A total of 184 patients (368 eyes) with retinitis pigmentosa were observed using SD-OCT. We studied the presence or absence of continuous inner/outer segment (IS/OS) lines, presence of thinning of the retinal pigment epithelium-Bruch's membrane complex, and distribution patterns of hyperreflective foci in the inner and outer nuclear layers (INL and ONL). Results: The IS/OS junction had partially disappeared in 275 eyes, which were at the early stage of retinitis pigmentosa (group X), whereas the junction had totally disappeared in 93, which were at the advanced stage of retinitis pigmentosa (group Y). Hyperreflective foci in the INL were observed in a significantly larger proportion of the eyes in group X than in group Y (90% versus 61%, P

Masasko Kuroda,1 Akihiro Nishida,1 Masashi Kikuchi,2 Yasuo Kurimoto11Department of Ophthalmology, Kobe City Medical Center General Hospital, Kobe, Hyogo, Japan; 2Kikuchi Eye Clinic, Kobe, Hyogo, JapanAbstract: We report the case of a 66-year-old Japanese man who developed neovascular glaucoma secondary to Purtscher's retinopathy following a head injury. The patient presented at our hospital with blurred vision and a visual field abnormality in his left eye 1 month after suffering from a head injury. Upon initial presentation, his best-corrected visual acuity on a decimal chart was 1.5 oculus dexter and 0.6 oculus sinister. The intraocular pressure (IOP) was 12 mmHg in both eyes. Fundus examination of the left eye revealed multiple white lesions in the posterior pole. Optical coherence tomography demonstrated retinal edema, particularly in the inner retina. On the basis of these findings, a diagnosis of Purtscher's retinopathy was made. One month after the initial examination, the visual acuity in the left eye deteriorated to 0.01 in decimal chart, and the IOP increased to 37 mmHg. Gonioscopy showed angle neovascularization. The patient received an intravitreal bevacizumab injection and panretinal photocoagulation. Subsequently, the IOP normalized and the angle neovascularization regressed.Keywords: blurred vision, visual field, retinal edema, head injury, head trauma

The global epidemic of multidrug-resistant Salmonella Typhimurium DT104 provides an important example, both in terms of the agent and its resistance, of a widely disseminated zoonotic pathogen. Here, with an unprecedented national collection of isolates collected contemporaneously from humans and animals and including a sample of internationally derived isolates, we have used whole-genome sequencing to dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Contrary to current tenets supporting a single homogeneous epidemic, we demonstrate that the bacterium and its resistance genes were largely maintained within animal and human populations separately and that there was limited transmission, in either direction. We also show considerable variation in the resistance profiles, in contrast to the largely stable bacterial core genome, which emphasizes the critical importance of integrated genotypic data sets in understanding the ecology of bacterial zoonoses and antimicrobial resistance.

Silicon carbide (4H) based metal–oxide–semiconductor field-effect transistors provide capabilities in high power and high temperature inaccessible to silicon. However, the performance of thermally grown oxide-based devices remains limited by oxide/semiconductor interface defects. This research employs deposited dielectrics, Al2O3, rather than thermal oxidation. Investigation of various pre-deposition processes reveals different degrees of improvements in the electronic properties. An optimum structure employs the preparation of a nitrided surface via NO annealing, a process known to passivate surface defects, a hydrogen exposure, followed by Al2O3 deposition. Inversion layer field-effect mobilities as high as 52 cm2/V s are reported in the optimum structures. Capacitance–voltage measurements and field-effect mobility characteristics indicate a trapping limited conductivity in Al2O3/4H-SiC inversion channels similar to SiO2/4H-SiC. Leakage currents and interface breakdown are also reported for various Al2O3/4H-SiC MOS structures. [ABSTRACT FROM AUTHOR]