Faculty Profiles - SHIBAYAMA Keigo

Papers - SHIBAYAMA Keigo

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Subtype screening of blaIMP genes using bipartite primers for DNA sequencing.

Ryuji Kawahara, Masanori Watahiki, Yuko Matsumoto, Kaoru Uchida, Makiko Noda, Kanako Masuda, Chiemi Fukuda, Yuki Abe, Yukiko Asano, Kazunori Oishi, Keigo Shibayama, Hiroto Shinomiya

Japanese journal of infectious diseases Vol. 74 ( 6 ) page： 592 - 599 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

DOI： 10.7883/yoken.JJID.2020.926

Isolation and characterization of Helicobacter suis from human stomach. International journal

Emiko Rimbara, Masato Suzuki, Hidenori Matsui, Masahiko Nakamura, Misako Morimoto, Chihiro Sasakawa, Hiroki Masuda, Sachiyo Nomura, Takako Osaki, Noriyo Nagata, Keigo Shibayama, Kengo Tokunaga

Proceedings of the National Academy of Sciences of the United States of America Vol. 118 ( 13 ) page： e2026337118 2021.3

Language：English Publishing type：Research paper (scientific journal)

Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured H. suis directly from human stomachs. Unlike Helicobacter pylori, the viability of H. suis decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated H. suis from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with H. suis clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because H. suis could be isolated from the stomachs of infected mice, our findings satisfied Koch's postulates. Although further prospective clinical studies are needed, H. suis, like H. pylori, is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of H. suis using complete genomes of clinical isolates revealed that the genome of each H. suis isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system-associated genes, and that H. suis isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.

DOI： 10.1073/pnas.2026337118

Japan Nosocomial Infections Surveillance (JANIS): Current Status, International Collaboration, and Future Directions for a Comprehensive Antimicrobial Resistance Surveillance System. Reviewed

Toshiki Kajihara, Koji Yahara, Aki Hirabayashi, Keigo Shibayama, Motoyuki Sugai

Japanese journal of infectious diseases Vol. 74 ( 2 ) page： 87 - 96 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

Japan Nosocomial Infections Surveillance (JANIS) is one of the largest national antimicrobial resistance (AMR) surveillance systems in the world. The JANIS Clinical Laboratory division collects comprehensive specimen-based data from diagnostic microbiology laboratories of participating hospitals to monitor the isolation rate of 11 major bacteria and specific AMR bacteria, and creates antibiograms of approximately 20 bacterial species. Data on the JANIS web database system are also annually tabulated and shared with the WHO Global Antimicrobial Resistance Surveillance System. To create a network of international AMR surveillance systems among Asian countries, Japan is developing an international web database system named ASIan Antimicrobial Resistance Surveillance Network (ASIARS-Net) based on the JANIS system; ASIARS-Net is an open-source database and confidentially available at almost no cost. JANIS continues to evolve in multiple directions; some are discussed at the end of this review.

DOI： 10.7883/yoken.JJID.2020.499

Rapid and simple SNP genotyping for Bordetella pertussis epidemic strain MT27 based on a multiplexed single-base extension assay. Reviewed International journal

Kazunari Kamachi, Shu-Man Yao, Chuen-Sheue Chiang, Kentaro Koide, Nao Otsuka, Keigo Shibayama

Scientific reports Vol. 11 ( 1 ) page： 4823 - 4823 2021.3

Language：English Publishing type：Research paper (scientific journal)

Multilocus variable-number tandem repeat analysis (MLVA) is widely used for genotyping of Bordetella pertussis, the causative bacteria for pertussis. However, MLVA genotyping is losing its discriminate power because prevalence of the epidemic MT27 strain (MLVA-27) is increasing worldwide. To address this, we developed a single nucleotide polymorphism (SNP) genotyping method for MT27 based on multiplexed single-base extension (SBE) assay. A total of 237 MT27 isolates collected in Japan during 1999-2018 were genotyped and classified into ten SNP genotypes (SG1 to SG10) with a Simpson's diversity index (DI) of 0.79 (95% CI 0.76-0.82). Temporal trends showed a marked increase in the genotypic diversity in the 2010s: Simpson's DI was zero in 1999-2004, 0.16 in 2005-2009, 0.83 in 2010-2014, and 0.76 in 2015-2018. This indicates that the SNP genotyping is applicable to the recently circulating MT27 strain. Additionally, almost all outbreak-associated MT27 isolates were classified into the same SNP genotypes for each outbreak. Multiplexed SBE assay allows for rapid and simple genotyping, indicating that the SNP genotyping can potentially be a useful tool for subtyping the B. pertussis MT27 strain in routine surveillance and outbreak investigations.

DOI： 10.1038/s41598-021-84409-0

Emergence of a plasmid-borne tigecycline resistance in Klebsiella pneumoniae in Vietnam. International journal

Aki Hirabayashi, Van Thi Thu Ha, An Van Nguyen, Son Thai Nguyen, Keigo Shibayama, Masato Suzuki

Journal of medical microbiology Vol. 70 ( 3 ) 2021.3

Language：English Publishing type：Research paper (scientific journal)

Tigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, tmexCD1-toprJ1, encoding the resistance-nodulation-cell division (RND) efflux pump was identified on plasmids in Klebsiella pneumoniae isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified K. pneumoniae isolates harbouring the tmexCD1-toprJ1 genes outside of China for the first time. Two tigecycline-resistant K. pneumoniae isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb tmexCD1-toprJ1-carrying plasmid with IncFIB and IncHI1B replicons. The tmexCD1-toprJ1 gene cluster was surrounded by several mobile gene elements, including IS26, and the plasmids had high sequence identity with that of K. pneumoniae isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by tmexCD1-toprJ1-carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.

DOI： 10.1099/jmm.0.001320

Nationwide multicenter questionnaire surveys on countermeasures against antimicrobial resistance and infections in hospitals. Reviewed International journal

Jung-Ho Shin, Seiko Mizuno, Takuya Okuno, Hisashi Itoshima, Noriko Sasaki, Susumu Kunisawa, Mitsuo Kaku, Makiko Yoshida, Yoshiaki Gu, Daiichi Morii, Keigo Shibayama, Norio Ohmagari, Yuichi Imanaka

BMC infectious diseases Vol. 21 ( 1 ) page： 234 - 234 2021.2

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: The goals of the National Action Plan on Antimicrobial Resistance (AMR) of Japan include "implementing appropriate infection prevention and control" and "appropriate use of antimicrobials," which are relevant to healthcare facilities. Specifically, linking efforts between existing infection control teams and antimicrobial stewardship programs was suggested to be important. Previous studies reported that human resources, such as full-time equivalents of infection control practitioners, were related to improvements in antimicrobial stewardship. METHODS: We posted questionnaires to all teaching hospitals (n = 1017) regarding hospital countermeasures against AMR and infections. To evaluate changes over time, surveys were conducted twice (1st survey: Nov 2016, 2nd survey: Feb 2018). A latent transition analysis (LTA) was performed to identify latent statuses, which refer to underlying subgroups of hospitals, and effects of the number of members in infection control teams per bed on being in the better statuses. RESULTS: The number of valid responses was 678 (response rate, 66.7%) for the 1st survey and 559 (55.0%) for the 2nd survey. More than 99% of participating hospitals had infection control teams, with differences in activity among hospitals. Roughly 70% had their own intervention criteria for antibiotics therapies, whereas only about 60 and 50% had criteria established for the use of anti-methicillin-resistant Staphylococcus aureus antibiotics and broad-spectrum antibiotics, respectively. Only 50 and 40% of hospitals conducted surveillance of catheter-associated urinary tract infections and ventilator-associated pneumonia, respectively. Less than 50% of hospitals used maximal barrier precautions for central line catheter insertion. The LTA identified five latent statuses. The membership probability of the most favorable status in the 2nd study period was slightly increased from the 1st study period (23.6 to 25.3%). However, the increase in the least favorable status was higher (26.3 to 31.8%). Results of the LTA did not support a relationship between increasing the number of infection control practitioners per bed, which is reportedly related to improvements in antimicrobial stewardship, and being in more favorable latent statuses. CONCLUSIONS: Our results suggest the need for more comprehensive antimicrobial stewardship programs and increased surveillance activities for healthcare-associated infections to improve antimicrobial stewardship and infection control in hospitals.

DOI： 10.1186/s12879-021-05921-2

Invasive Haemophilus influenzae type a infection and polyarthritis in a 72-year-old Japanese man: A case report. Reviewed International journal

Tsuneaki Kenzaka, Ken Goda, Mayumi Kubota, Kosuke Takayanagi, Tsuyoshi Kenri, Keigo Shibayama, Hozuka Akita

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy Vol. 27 ( 7 ) page： 1084 - 1088 2021.2

Language：English Publishing type：Research paper (scientific journal)

Haemophilus influenzae is a small, nonmotile, non-spore-forming bacterium classified into 6 serotypes (a to f) and non-typeable strains that lack a capsule. Although H. influenzae serotype a (Hia) is prevalent in Canada, the United States, Brazil, Australia, across the African continent, and several other locations, it has not been reported in Japan thus far. Our case was of a 72-year-old Japanese man who sought medical consultation after presenting with chills, fever, and polyarthritis. Cultures of blood and synovial fluid from the left knee revealed H. influenzae infection. Diagnostic imaging showed poor contrast regions in both kidneys, fluid retention around both knee joints, the left shoulder joint, and both elbow joints. Subsequently, the patient was diagnosed with invasive H. influenzae infection accompanied by polyarthritis and renal infarction. 16S ribosomal RNA gene sequencing revealed that the bacterial strain was Hia. The patient was treated with antimicrobial agents and arthroscopic curettage. We present a case of invasive Hia infection accompanied by polyarthritis and renal infarction. To the best of our knowledge, this is the first case of Hia infection in Japan. The case is very rare considering that the disease occurred in an elderly patient who developed polyarthritis.

DOI： 10.1016/j.jiac.2021.01.017

Detection of Bartonella quintana infection among the Homeless Population in Tokyo, Japan, from 2013-2015.

Toshinori Sasaki, Tomohide Adachi, Kazuto Itoh, Mayumi Kubota, Takuya Yamagishi, Maki Hirao, Haruhiko Isawa, Kazunori Oishi, Keigo Shibayama, Kyoko Sawabe

Japanese journal of infectious diseases Vol. 74 ( 5 ) page： 411 - 415 2021.1

Language：English Publishing type：Research paper (scientific journal) Publisher：National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

Several outbreaks of trench fever caused by Bartonella quintana, occurred in soldiers during World Wars I and II. Although the number of trench fever cases has been decreasing worldwide, the disease has been reported among the homeless population in both developing and developed countries. The current prevalence of B. quintana infection in Japan is unclear. We collected blood and body louse (Pediculus humanus humanus) samples from homeless inpatients who had body lice at the time of emergency hospitalization in Tokyo from January 2013 to March 2015. Patients were tested for B. quintana infections using culture method, polymerase chain reaction, and indirect immunofluorescence assay (IFA). Among the 29 patients tested, the presence of Bartonella spp. was confirmed by genomic sequencing of DNA extracted from the samples from 2 patients (blood culture performed for 13 out of 15 patients), and from body louse samples of 20 patients (69%). Immunoglobulin G against B. quintana was detected in 10 patients (34.5%) at a cut-off titer of 1:256 in IFA. B. quintana infection was detected in samples obtained between 2013 and 2015 in Tokyo and needs to be on the list of differential diagnoses performed for febrile homeless individuals.

DOI： 10.7883/yoken.JJID.2020.505

Emergence of DIM-1 and KPC-1 Genes Associated Carbapenem-Resistant Pseudomonas Aeruginosa Isolates in Three Major Hospitals in Hanoi, Vietnam (2010-2015) Reviewed

Tran Hai Anh, Tran Huy Hoang, Vu Thi Ngoc Bich, Trinh Son Tung, Tran Dieu Linh, Pham Ha My, Ngo Thi Hong Hanh, Nguyen Minh Thao, Tran Nhu Duong, Pham Duy Thai, Dang Duc Anh, Keigo Shibayama, Masato Suzuki, Lay-Myint Yoshida, Hong Son Trinh, Le Viet Thanh, Vu Phuong Thom, Luu Thi Vu Nga, Anne-Laure Bañuls, Trinh Khanh Linh, Tran Van Anh, H Rogier van Doorn

2021.1

Language：English Publisher：Research Square

<title>Abstract</title>
<bold>Background:</bold> Multidrug-resistant bacteria including carbapenem resistant <italic>Pseudomonas aeruginosa </italic>are recognised as an important cause of hospital-acquired infections worldwide. To determine the molecular characterisation and antibiotic resistant genes associated with carbapenem-resistant <italic>P. aeruginosa</italic>. <bold>Methods:</bold> we conducted whole-genome sequencing and phylogenetic analysis of 72 carbapenem-resistant <italic>P. aeruginosa </italic>isolated from hospital-acquired infection patients from 2010 to 2015 in three major hospitals in Hanoi, Vietnam. <bold>Results:</bold> We identified three variants of IMP genes, among which IMP-15 gene was the most frequent (n= 34) in comparison to IMP-26 (n= 2) and IMP-51 (n=12). We observed two isolates with imipenem MIC >128mg/L that co-harboured IMP-15 and DIM-1 genes and seven isolates (imipenem MIC> 128mg/L) with KPC-1 gene from the same hospital. MLST data showed that sequence types (ST) of 72 isolates were classified into 18 STs and phylogenetic tree analysis divided these isolates into nine groups. <bold>Conclusion:</bold> Our results provide evidence that not only IMP-26, but other variants of IMPs like IMP-15 and IMP-51 genes and several STs (ST235, ST244, ST277, ST310, ST773 and ST3151) have been disseminated in health care settings in Vietnam. Also, we report the first finding in Vietnam that two isolates belonging to ST1240 and ST3340 harboured two important carbapenemase genes (IMP-15 and, DIM-1) and seven isolates belonging to ST3151 of <italic>P. aeruginosa </italic>carried the KPC-1 gene, which could be a potential cause of seriously restricted available treatment options in healthcare settings.

DOI： 10.21203/rs.3.rs-143498/v1

Other Link： https://www.researchsquare.com/article/rs-143498/v1.html

Plasmid analysis of NDM metallo-β-lactamase-producing Enterobacterales isolated in Vietnam. Reviewed International journal Open Access

Aki Hirabayashi, Koji Yahara, Satomi Mitsuhashi, So Nakagawa, Tadashi Imanishi, Van Thi Thu Ha, An Van Nguyen, Son Thai Nguyen, Keigo Shibayama, Masato Suzuki

PloS one Vol. 16 ( 7 ) page： e0231119 2021

Language：English Publishing type：Research paper (scientific journal)

Carbapenem-resistant Enterobacterales (CRE) represent a serious threat to public health due to the lack of treatment and high mortality. The rate of antimicrobial resistance of Enterobacterales isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene blaNDM, spread via plasmids among Enterobacterales in Vietnam. In this study, we characterized blaNDM-carrying plasmids in Enterobacterales isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-nonsusceptible isolates from a medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 13 blaNDM-positive isolates, including isolates of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Morganella morganii, and Proteus mirabilis, were further sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Almost identical 73 kb IncFII(pSE11)::IncN hybrid plasmids carrying blaNDM-1 were found in a P. mirabilis isolate and an M. morganii isolate. A 112 kb IncFII(pRSB107)::IncN hybrid plasmid carrying blaNDM-1 in an E. coli isolate had partially identical sequences with a 39 kb IncR plasmid carrying blaNDM-1 and an 88 kb IncFII(pHN7A8)::IncN hybrid plasmid in a C. freundii isolate. 148-149 kb IncFIA(Hl1)::IncA/C2 plasmids and 75-76 kb IncFII(Yp) plasmids, both carrying blaNDM-1 were shared among three sequence type 11 (ST11) isolates and three ST395 isolates of K. pneumoniae, respectively. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to blaNDM-1. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.

DOI： 10.1371/journal.pone.0231119

On-Site Genomic Epidemiological Analysis of Antimicrobial-Resistant Bacteria in Cambodia With Portable Laboratory Equipment. Reviewed International journal Open Access

Aki Hirabayashi, Hideji Yanagisawa, Hiromizu Takahashi, Koji Yahara, Philipp Boeing, Bethan Wolfenden, Vandarith Nov, Vichet Lorn, Mom Veng, Vuth Ann, Chau Darapheak, Keigo Shibayama, Masato Suzuki

Frontiers in microbiology Vol. 12 page： 675463 - 675463 2021

Language：English Publishing type：Research paper (scientific journal)

The rapid emergence of carbapenemase-producing gram-negative bacteria (CPGNB) is a global threat due to the high mortality of infection and limited treatment options. Although there have been many reports of CPGNB isolated from Southeast Asian countries, to date there has been no genetic analysis of CPGNB isolated from Cambodia. Sequence-based molecular epidemiological analysis enables a better understanding of the genotypic characteristics and epidemiological significance of antimicrobial-resistant (AMR) bacteria in each country, and allows countries to enact measures related to AMR issues. In this study, we performed on-site genomic epidemiological analysis of CPGNB isolated in Cambodia using a portable laboratory equipment called Bento Lab, which combines a PCR thermal cycler, microcentrifuge, gel electrophoresis apparatus, and LED transilluminator, along with the MinION nanopore sequencer. PCR targeting of major carbapenemase genes using Bento Lab revealed that two Escherichia coli isolates and one Acinetobacter baumannii isolate harbored carbapenemase genes: bla NDM, bla OXA-48, and bla OXA-23, respectively. The results of phenotypic diagnostic tests for CPGNB, such as the carbapenem inactivation method and double-disk diffusion test using a specific inhibitor of metallo-β-lactamases, were consistent with their AMR genotypes. Whole-genome sequencing analysis using MinION revealed that bla NDM-5 gene was carried on a 93.9-kb plasmid with IncFIA/IncFIB/IncFII/IncQ1 replicons, and bla OXA-181 gene was carried on a 51.5-kb plasmid with the IncX3 replicon in E. coli isolates. bla OXA-23 was encoded in two locations on the chromosome of A. baumannii. Plasmids carrying bla NDM-5 or bla OXA-181 in E. coli were highly structurally identical to plasmids prevalent in Enterobacterales in China and other countries, suggesting that they disseminated from a common evolutionary origin. Our findings demonstrate the potential impact of portable laboratory equipment on AMR bacteria research in hospitals and research centers with limited research facilities, and provide the first glimpse into the genomic epidemiology of CPGNB in Cambodia.

DOI： 10.3389/fmicb.2021.675463

Complete Genome Sequence of a Japanese Clinical Isolate of Haemophilus influenzae Type a Strain TAMBA230. Reviewed International journal

Mayumi Kubota, Tsuyoshi Kenri, Yuko Sasaki, Keigo Shibayama, Kosuke Takayanagi, Tsuneaki Kenzaka

Microbiology resource announcements Vol. 9 ( 48 ) 2020.11

Language：English Publishing type：Research paper (scientific journal)

Haemophilus influenzae causes severe infections such as pneumonia and meningitis. Here, we report the complete genome of H. influenzae type a strain TAMBA230, which was isolated in 2019 from a patient exhibiting bacteremia. This represents the first case in Japan of an H. influenzae type a strain associated with invasive infection.

DOI： 10.1128/MRA.01069-20

Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae. Reviewed International journal

David Vizarraga, Akihiro Kawamoto, U Matsumoto, Ramiro Illanes, Rosa Pérez-Luque, Jesús Martín, Rocco Mazzolini, Paula Bierge, Oscar Q Pich, Mateu Espasa, Isabel Sanfeliu, Juliana Esperalba, Miguel Fernández-Huerta, Margot P Scheffer, Jaume Pinyol, Achilleas S Frangakis, Maria Lluch-Senar, Shigetarou Mori, Keigo Shibayama, Tsuyoshi Kenri, Takayuki Kato, Keiichi Namba, Ignacio Fita, Makoto Miyata, David Aparicio

Nature communications Vol. 11 ( 1 ) page： 5188 - 5188 2020.10

Language：English Publishing type：Research paper (scientific journal)

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

DOI： 10.1038/s41467-020-18777-y

Macrolide-Resistant Bordetella pertussis, Vietnam, 2016-2017. Reviewed International journal

Kazunari Kamachi, Hong T Duong, Anh D Dang, T Hai, Do Do, Kentaro Koide, Nao Otsuka, Keigo Shibayama, Ha Thi Thu Hoang

Emerging infectious diseases Vol. 26 ( 10 ) page： 2511 - 2513 2020.10

Language：English Publishing type：Research paper (scientific journal)

Macrolide-resistant Bordetella pertussis emerged in Vietnam during 2016-2017. Direct analyses of swab samples from 10 patients with pertussis revealed a macrolide-resistant mutation, A2047G, in the 23S rRNA. We identified the MT104 genotype of macrolide-resistant B. pertussis (which is prevalent in mainland China) and its variants in these patients.

DOI： 10.3201/eid2610.201035

Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans. Reviewed International journal

Tsuyoshi Sekizuka, Chihiro Katsukawa, Makoto Kuroda, Keigo Shibayama, Ken Otsuji, Mitsumasa Saito, Akihiko Yamamoto, Masaaki Iwaki

Emerging infectious diseases Vol. 26 ( 10 ) page： 2457 - 2459 2020.10

Language：English Publishing type：Research paper (scientific journal)

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

DOI： 10.3201/eid2610.200086

National trend of blood-stream infection attributable deaths caused by Staphylococcus aureus and Escherichia coli in Japan. Reviewed International journal

Shinya Tsuzuki, Nobuaki Matsunaga, Koji Yahara, Yoshiaki Gu, Kayoko Hayakawa, Aki Hirabayashi, Toshiki Kajihara, Motoyuki Sugai, Keigo Shibayama, Norio Ohmagari

Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy Vol. 26 ( 4 ) page： 367 - 371 2020.4

Language：English Publishing type：Research paper (scientific journal)

There has been scarce evidence about deaths due to blood stream infection (BSI) in Japan so far. The main objective of this study is to understand the epidemiological trend of deaths caused by BSIs due to Staphylococcus aureus and Escherichia coli including Methicillin-resistant S. aureus (MRSA) and fluoroquinolone-resistant E. coli (FQREC) at national level. We annually estimated the number of BSI caused by S. aureus and E. coli between 2011 and 2017 across Japan using comprehensive data of bacterial culturing and drug susceptibilities collected in Japan Nosocomial Infection Surveillance (JANIS). The number of death was estimated by using BSI mortality obtained from previous studies in Japan. The number of BSI death attributable to S. aureus was estimated to 17,412 in 2011 and 17,157 in 2017, respectively, out of the whole population (126.8 million) in Japan. Among them, cases attributed to MRSA accounted for 5924 (34.0%) in 2011, and decreased to 4224 (24.6%) cases in 2017. On the other hand, the number of BSI death attributable to E. coli was estimated to 9044 in 2011 and increased to 14,016 in 2017. Among them, cases attributed to FQREC accounted for 2045 (22.6%) in 2011 and increased to 3915 (27.9%) cases in 2017. The number of BSI death attributable to MRSA has been decreasing and that attributable to FQREC has been increasing. This study provides the first annual estimate of disease burden of BSI caused by antimicrobial resistant (AMR) bacteria in Japan, and basis for formulating health policy to deal with AMR.

DOI： 10.1016/j.jiac.2019.10.017

Single-Tube Multiplex Polymerase Chain Reaction for the Detection of Genes Encoding Enterobacteriaceae Carbapenemase. Reviewed

Masanori Watahiki, Ryuji Kawahara, Masahiro Suzuki, Miyako Aoki, Kaoru Uchida, Yuko Matsumoto, Yuko Kumagai, Makiko Noda, Kanako Masuda, Chiemi Fukuda, Seiya Harada, Keiko Senba, Masato Suzuki, Mari Matsui, Satowa Suzuki, Keigo Shibayama, Hiroto Shinomiya

Japanese journal of infectious diseases Vol. 73 ( 2 ) page： 166 - 172 2020.3

Language：English Publishing type：Research paper (scientific journal) Publisher：National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee

A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.

DOI： 10.7883/yoken.JJID.2019.041

A prolonged multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales due to horizontal transmission of the IncN plasmid. Reviewed International journal

Takuya Yamagishi, Mari Matsui, Tsuyoshi Sekizuka, Hiroaki Ito, Munehisa Fukusumi, Tomoko Uehira, Miyuki Tsubokura, Yoshihiko Ogawa, Atsushi Miyamoto, Shoji Nakamori, Akio Tawa, Takahisa Yoshimura, Hideki Yoshida, Hidetetsu Hirokawa, Satowa Suzuki, Tamano Matsui, Keigo Shibayama, Makoto Kuroda, Kazunori Oishi

Scientific reports Vol. 10 ( 1 ) page： 4139 - 4139 2020.3

Language：English Publishing type：Research paper (scientific journal)

A multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales (IMP-6-CPE) occurred at an acute care hospital in Japan. This study was conducted to understand the mechanisms of IMP-6-CPE transmission by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and whole-genome sequencing (WGS), and identify risk factors for IMP-6-CPE acquisition in patients who underwent abdominal surgery. Between July 2013 and March 2014, 22 hospitalized patients infected or colonized with IMP-6-CPE (Escherichia coli [n = 8], Klebsiella oxytoca [n = 5], Enterobacter cloacae [n = 5], Klebsiella pneumoniae [n = 3] and Klebsiella aerogenes [n = 1]) were identified. There were diverse PFGE profiles and sequence types (STs) in most of the species except for K. oxytoca. All isolates of K. oxytoca belonged to ST29 with similar PFGE profiles, suggesting their clonal transmission. Plasmid analysis by WGS revealed that all 22 isolates but one shared a ca. 50-kb IncN plasmid backbone with blaIMP-6 suggesting interspecies gene transmission, and typing of plasmids explained epidemiological links among cases. A case-control study showed pancreatoduodenectomy, changing drains in fluoroscopy room, continuous peritoneal lavage and enteric fistula were associated with IMP-6-CPE acquisition among the patients. Plasmid analysis of isolates in an outbreak of IMP-6-CPE suggested interspecies gene transmission and helped to clarify hidden epidemiological links between cases.

DOI： 10.1038/s41598-020-60659-2

Complete Genome Sequence of Helicobacter suis Strain SNTW101c, Originally Isolated from a Patient with Nodular Gastritis. Reviewed International journal

Emiko Rimbara, Masato Suzuki, Hidenori Matsui, Masahiko Nakamura, Hirotaka Kobayashi, Shigetarou Mori, Keigo Shibayama

Microbiology resource announcements Vol. 9 ( 1 ) 2020.1

Language：English Publishing type：Research paper (scientific journal)

Helicobacter suis strain SNTW101c, which was originally obtained from a patient with nodular gastritis, has been maintained in mouse stomach because of difficulty culturing it in vitro Recently, we succeeded in culturing this strain in vitro Here, we report the complete genome sequence of H. suis strain SNTW101c.

DOI： 10.1128/MRA.01340-19

Periodic Genotype Shifts in Clinically Prevalent Mycoplasma pneumoniae Strains in Japan. Reviewed International journal

Tsuyoshi Kenri, Masato Suzuki, Tsuyoshi Sekizuka, Hitomi Ohya, Yoichiro Oda, Tsutomu Yamazaki, Hiroyuki Fujii, Toru Hashimoto, Hiroshi Nakajima, Chihiro Katsukawa, Makoto Kuroda, Keigo Shibayama

Frontiers in cellular and infection microbiology Vol. 10 page： 385 - 385 2020

Language：English Publishing type：Research paper (scientific journal)

Nationwide increases in Mycoplasma pneumoniae pneumonia cases in Japan were reported in 2011, 2012, 2015, and 2016. In this study, we isolated 554 M. pneumoniae strains in 4 areas in Japan (Kanagawa, Okayama, Osaka, and Saitama) between 2006 and 2019, and performed genotyping analysis. More than 80% of the strains isolated in 2011 and 2012 harbored type 1 p1 adhesin gene; however, strains harboring type 2 or its variant p1 gene increased in 2015 and 2016 and dominated after 2017. These findings suggested that a shift in the prevalent genotype of M. pneumoniae clinical strains occurred recently in Japan. More than 90% of the type 1 strains isolated after 2010 harbored macrolide-resistance mutations in their 23S rRNA gene, whereas most type 2 lineage strains had no such mutations. Consequently, the increase in type 2 lineage strains in Japan has reduced the macrolide resistance rate of clinical M. pneumoniae strains. During this analysis, we also identified M. pneumoniae strains carrying a novel variant type 1 p1 gene, and we classified it as type 1b. We then sequenced the genomes of 81 selected M. pneumoniae strains that we collected between 1976 and 2017 in Japan, and compared them with 156 M. pneumoniae genomes deposited in public databases to provide insights into the interpretation of M. pneumoniae genotyping methods, including p1 typing, multiple-locus variable-number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST), and typing by 8 single-nucleotide polymorphism markers (SNP-8). As expected, p1 typing, MLST, and SNP-8 results exhibited good correlation with whole-genome SNP analysis results in terms of phylogenetic relationships; however, MLVA typing results were less comparable to those of the other methods. MLVA may be useful for the discrimination of strains derived from a single outbreak within a limited area; however, is not reliable for classification of strains collected from distantly separated areas at different time points. This study showed the usefulness of genome-based comparison of M. pneumoniae for molecular epidemiology. Genome sequencing of more strains will improve our understanding of global propagation routes of this pathogen and evolutionary aspects of M. pneumoniae strains.

DOI： 10.3389/fcimb.2020.00385

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