Papers - SASAKI Takashi

Near-complete genome sequencing of the feline coronavirus serotype I strain FIPV-Aqua from a cat with feline infectious peritonitis in Japan.

Yoshikazu Tanaka, Eri Tanabe, Takashi Sasaki

Microbiology resource announcements   14 ( 6 ) e0035725  2025.06  [Refereed]  [International journal]

Authorship：   Last author

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Feline coronavirus causes the fatal disease feline infectious peritonitis (FIP), for which no effective vaccine is currently available. Here, we present the near-complete genome sequence of the Japanese strain FIPV-Aqua.

DOI PubMed

Emergence of community-associated methicillin-resistant Staphylococcus aureus ΨUSA300 among Japanese people with HIV, resulted from stepwise mutations in 2010s.

Koh Shinohara, Yuki Uehara, Katsuji Teruya, Takashi Sasaki, Tadashi Baba, Hidemasa Nakaminami, Pegah Kananizadeh, Yuh Morimoto, Yoshimi Kikuchi, Shinichi Oka

Scientific reports   13 ( 1 ) 8322 - 8322  2023.05  [Refereed]  [International journal]

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Although infection with the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 is extremely rare in Japan, the uniquely evolved clone ΨUSA300 has been reported in Japan. An outbreak of a distinct USA300 clone was recently reported in an HIV/AIDS referral hospital in Tokyo. The present study investigated the evolutionary origin and genetic diversity of USA300-related clones causing regional outbreaks among people living with HIV (PLWHIV) in Tokyo. MRSA isolates collected from PLWHIV in an HIV/AIDS referral center in Tokyo were subjected to whole-genome sequencing and their genetic features were compared with those of previously described USA300 MRSA genomes. Of the 28 MRSAs isolated in 2016-2019, 23 (82.1%) were identified as USA300, with 22 (95.6%) of the latter identified as ΨUSA300. Although the genomic structure of ΨUSA300 was identical to the structures of reference USA300 strains, one clade (cluster A) was found to have acquired 29 previously identified lineage-specific mutations in a stepwise manner. The estimated divergence dates of ΨUSA300 and Cluster A were 2009 and 2012, respectively. These findings suggested that the ΨUSA300 clone had spread among PLWHIVs in Tokyo in the early 2010s, with stepwise acquisition of lineage-specific nonsynonymous mutations.

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Complete Genome Sequencing of a Community-Associated Methicillin-Resistant Staphylococcus aureus ψUSA300 Strain JICS127, a Uniquely Evolved USA300 Lineage in Japan.

Koh Shinohara, Tadashi Baba, Takashi Sasaki, Katsuji Teruya, Yuki Uehara

Microbiology resource announcements   11 ( 9 ) e0071722  2022.09  [Refereed]  [International journal]

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A ψUSA300 clone of MRSA, a derivative of USA300, is uniquely found in Japan and has 12-bp deletion on ccrB2 in type IVa staphylococcal cassette chromosome mec element. We hereby present the complete genome of ψUSA300 strain JICS127.

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Genetic and phenotypic diversity of methicillin-resistant Staphylococcus aureus among Japanese inpatients in the early 1980s.

Hui Zuo, Yuki Uehara, Yujie Lu, Takashi Sasaki, Keiichi Hiramatsu

Scientific reports   11 ( 1 ) 5447 - 5447  2021.03  [Refereed]  [International journal]

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To trace the linkage between Japanese healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) strains in the early 1980s and the 2000s onward, we performed molecular characterizations using mainly whole-genome sequencing. Among the 194 S. aureus strains isolated, 20 mecA-positive MRSA (10.3%), 8 mecA-negative MRSA (4.1%) and 3 mecA-positive methicillin-susceptible S. aureus (MSSA) (1.5%) strains were identified. The most frequent sequence type (ST) was ST30 (n = 11), followed by ST5 (n = 8), ST81 (n = 4), and ST247 (n = 3). Rates of staphylococcal cassette chromosome mec (SCCmec) types I, II, and IV composed 65.2%, 13.0%, and 17.4% of isolates, respectively. Notably, 73.3% of SCCmec type I strains were susceptible to imipenem unlike SCCmec type II strains (0%). ST30-SCCmec I (n = 7) and ST5-SCCmec I (n = 5) predominated, whereas only two strains exhibited imipenem-resistance and were tst-positive ST5-SCCmec II, which is the current Japanese HA-MRSA genotype. All ST30 strains shared the common ancestor strain 55/2053, which caused the global pandemic of Panton-Valentine leukocidin-positive MSSA in Europe and the United States in the 1950s. Conspicuously more heterogeneous, the population of HA-MRSA clones observed in the 1980s, including the ST30-SCCmec I clone, has shifted to the current homogeneous population of imipenem-resistant ST5-SCCmec II clones, probably due to the introduction of new antimicrobials.

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Nicotine Oral Administration Attenuates DSS-Induced Colitis Through Upregulation of Indole in the Distal Colon and Rectum in Mice.

Akihito Nakajima, Tomoyoshi Shibuya, Takashi Sasaki, Yu Jie Lu, Dai Ishikawa, Keiichi Haga, Masahito Takahashi, Naoko Kaga, Taro Osada, Nobuhiro Sato, Akihito Nagahara

Frontiers in medicine   8   789037 - 789037  2021  [Refereed]  [International journal]

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Nicotine affects the gastrointestinal environment and modulates ulcerative colitis (UC). However, the associations among nicotine, gut metabolites, and UC are still largely unknown. We investigated whether orally administered nicotine affected gut metabolites and dextran sodium sulfate (DSS)-induced colitis. C57BL/6 male mice were orally administered nicotine solution in drinking water prior to inducing DSS-induced colitis. Short-chain fatty acids (SCFAs) and indole in gut contents and fecal samples were measured by GC-MS and hydroxylamine-based indole assays, respectively. Oral administration of nicotine increased indole concentration in feces, but, in contrast, SCFA values did not differ with nicotine administration. Indole levels were increased in the distal colon and rectum but not in the cecum and proximal colon. DSS-induced colitis was less severe clinically and histological changes were minimal in the rectum of orally nicotine-administered mice compared to mice drinking only water. 16S rRNA microbiome on the feces revealed an increasing in Clostridium and Porphyromonas in nicotine-administered mice. In conclusion, nicotine administration was associated with increased indole levels in the distal colon and rectum and attenuated DSS-induced colitis. Oral administration of nicotine may play a potential role in indole upregulation and prevention of UC.

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Corynebacterium Species of the Conjunctiva and Nose: Dominant Species and Species-Related Differences of Antibiotic Susceptibility Profiles.

Saichi Hoshi, Daisuke Todokoro, Takashi Sasaki

Cornea   39 ( 11 ) 1401 - 1406  2020.11  [Refereed]  [International journal]

Authorship：   Last author

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PURPOSE: Nondiphtherial Corynebacterium species are normal residents of human skin and mucosa, including the conjunctiva and nose, but can cause conjunctivitis and keratitis. Recently, resistance against various classes of antibiotics has been reported in Corynebacterium. The present study investigated the type of species and antibiotic susceptibilities of the conjunctival and nasal Corynebacterium species. METHODS: This study examined 183 strains of Corynebacterium species that were isolated from patients undergoing preoperative examinations for cataract surgery. Species were identified by RNA polymerase β-subunit-encoding gene (rpoB) sequencing. Antibiotic susceptibility tests were performed by the microdilution method according to the Clinical and Laboratory Standards Institute standard method M45. RESULTS: Corynebacterium macginleyi was the most predominant species (84%; 46 of 55) in the conjunctiva. The 2 major species in the nasal cavity were Corynebacterium accolens and Corynebacterium propinquum (44% and 31%, respectively), followed by Corynebacterium pseudodiphtheriticum (8%), Corynebacterium jeikeium (7%), and C. macginleyi (3%). In contrast to other nasal Corynebacterium species, only C. macginleyi showed a high susceptibility to macrolides. However, among nonconjunctival Corynebacterium species, C. propinquum, was unique in having a high resistance rate to levofloxacin (29%), comparable with that observed in C. macginleyi (36%). Penicillin G and tobramycin showed good susceptibility in almost all strains. CONCLUSIONS: Drug resistance against fluoroquinolones and macrolides was observed in Corynebacterium species, with the antibiotic susceptibility profiles correlating with differences of the species and niche. Nasal and conjunctival Corynebacterium profiles of drug resistance suggest habitat segregation strictly at the species level.

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An outbreak of USA300 MRSA among people with HIV in Japan.

Kazuhiko Ikeuchi, Eisuke Adachi, Takashi Sasaki, Masato Suzuki, Lay Ahyoung Lim, Makoto Saito, Michiko Koga, Takeya Tsutsumi, Yasutoshi Kido, Yuki Uehara, Hiroshi Yotsuyanagi

The Journal of infectious diseases   223 ( 4 ) 610 - 620  2020.10  [Refereed]  [International journal]

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BACKGROUND: USA300 produces Panton-Valentin leucocidin (PVL), and is known as a predominant community-associated methicillin-resistant Staphylococcus aureus (MRSA) strain in the United States, but it was extremely rare in Japan. We report here an outbreak of USA300 in people with HIV (PWH) in Tokyo, Japan. METHODS: We analyzed the cases of PVL-MRSA infection between 2010 and 2020 and screened for nasal colonization of PVL-MRSA in PWH who visited an HIV/AIDS referral hospital from December 2019 to March 2020. Whole-genome sequencing (WGS)-based single nucleotide polymorphism (SNP) analysis was performed for these isolates. RESULTS: During the study period, a total of 21 PVL-MRSA infections in 14 patients were identified after 2014. The carriage prevalence was 4.3% (12/277), and PVL-MRSA carriers were more likely to have sexually transmitted infections (STIs) within a year compared with patients who had neither the history of PVL-MRSA infection nor colonization (33.3% [4/12] vs 10.1% [26/258], P=0.03). SNP analysis showed that all 26 isolates were ST8-SCCmecⅣa-USA300. Twenty-four isolates were closely related (≤100 SNP differences) and had the nonsynonymous SNPs associated with carbohydrate metabolism and antimicrobial tolerance. CONCLUSIONS: An outbreak of USA300 has been occurring among PWH in Tokyo, and a history of STI was a risk of colonization.

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RNA Sequencing Identifies a Common Physiology in Vancomycin- and Ciprofloxacin-Tolerant Staphylococcus aureus Induced by ileS Mutations.

Madhuri Singh, Miki Matsuo, Takashi Sasaki, Tomomi Hishinuma, Norio Yamamoto, Yuh Morimoto, Teruo Kirikae, Keiichi Hiramatsu

Antimicrobial agents and chemotherapy   64 ( 10 )  2020.09  [Refereed]  [International journal]

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Little is known about the mechanisms by which ileS mutations induce vancomycin tolerance in Staphylococcus aureus This study showed that transcriptome profiles were similar in vancomycin-tolerant mutants and the IleRS-inhibitor-treated parent. Notably, ileS and relA, which induce a stringent response, were upregulated. The same mechanism was responsible for cross-tolerance to vancomycin and ciprofloxacin. These findings suggest that the accumulation of uncharged isoleucyl-tRNA following ileS mutations in S. aureus was responsible for drug tolerance.

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Epidemiology of methicillin-resistant Staphylococcus aureus in a Japanese neonatal intensive care unit.

Atsushi Tsujiwaki, Ken Hisata, Yudai Tohyama, Nobuaki Matsunaga, Yuki Uehara, Takashi Sasaki, Keiichi Hiramatsu, Toshiaki Shimizu

Pediatrics international : official journal of the Japan Pediatric Society   62 ( 8 ) 911 - 919  2020.08  [Refereed]  [International journal]

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BACKGROUND: There have been few reports on the genetic structure of the current population of methicillin-resistant Staphylococcus aureus (MRSA) from neonatal intensive care units (NICUs) in Japan. In the present study we conducted a molecular epidemiological analysis based on whole genome sequencing against MRSA strains in a Japanese NICU. METHODS: We performed genotyping by whole genome sequencing, polymerase chain reaction-based typing of Staphylococcal cassette chromosome mec (SCCmec) and polymerase chain reaction-based open-reading frame typing against 57 MRSA strains from fecal or nasal specimens from NICU patients in Juntendo University Shizuoka Hospital in 2013-2014. RESULTS: Forty-nine MRSA strains (86.0%) exhibited a clonal complex (CC) 1, and were divided into three sequence types (STs): ST2725 (n = 25), ST2764 (n = 21), and ST1 (n = 3). All CC1 MRSA strains had SCCmec IVa, and were resistant to new quinolones, which are limited in pediatric use, suggesting that these strains were derived from adult MRSA clones. Single nucleotide polymorphism differences of both ≤10 and >100 nucleotides were observed by pairwise, single nucleotide polymorphism analysis among ST2725 and ST2764 MRSA strains, respectively. Seven ST8 MRSA strains (12.2%) were isolated, and no strain exhibiting the Japanese hospital-associated MRSA genotype (ST5/SCCmec II) was isolated in this study. CONCLUSIONS: Our molecular epidemiological analysis suggested that ST2725 and ST2764 MRSA strains had genetic diversity that could not be explained only by a recent transmission event in the NICU. These MRSA clones might be disseminated in other Japanese hospital facilities as new endemic clones. Our results are expected to contribute to the improvement of infection control measures of MRSA in NICUs.

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A Soluble Fiber Diet Increases Bacteroides fragilis Group Abundance and Immunoglobulin A Production in the Gut.

Akihito Nakajima, Takashi Sasaki, Kikuji Itoh, Takashi Kitahara, Yoshinori Takema, Keiichi Hiramatsu, Dai Ishikawa, Tomoyoshi Shibuya, Osamu Kobayashi, Taro Osada, Sumio Watanabe, Akihito Nagahara

Applied and environmental microbiology   86 ( 13 )  2020.06  [Refereed]  [International journal]

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Immunoglobulin A (IgA) is essential for defense of the intestinal mucosa against harmful pathogens. Previous studies have shown that Bacteroidetes, the major phylum of gut microbiota together with Firmicutes, impact IgA production. However, the relative abundances of species of Bacteroidetes responsible for IgA production were not well understood. In the present study, we identified some specific Bacteroidetes species that were associated with gut IgA induction by hsp60-based profiling of species distribution among Bacteroidetes The levels of IgA and the expression of the gene encoding activation-induced cytidine deaminase (AID) in the large intestine lamina propria, which is crucial for class switch recombination from IgM to IgA, were increased in soluble high-fiber diet (sHFD)-fed mice. We found that Bacteroides acidifaciens was the most abundant Bacteroidetes species in both sHFD- and normal diet-fed mice. In addition, the gut IgA levels were associated with the relative abundance of Bacteroides fragilis group species such as Bacteroides faecis, Bacteroides caccae, and Bacteroides acidifaciens Conversely, the ratio of B. acidifaciens to other Bacteroidetes species was reduced in insoluble high-fiber diet fed- and no-fiber diet-fed mice. To investigate whether B. acidifaciens increases IgA production, we generated B. acidifaciens monoassociated mice and found increased gut IgA production and AID expression. Collectively, soluble dietary fiber increases the ratio of gut Bacteroides fragilis group, such as B. acidifaciens, and IgA production. This might improve gut immune function, thereby protecting against bowel pathogens and reducing the incidence of inflammatory bowel diseases.IMPORTANCE Immunoglobulin A (IgA) is essential for defense of the intestinal mucosa against harmful pathogens. Gut microbiota impact IgA production, but the specific species responsible for IgA production remain largely elusive. Previous studies have shown that IgA and Bacteroidetes, the major phyla of gut microbiota, were increased in soluble high-fiber diet-fed mice. We show here that the levels of IgA in the gut and the expression of activation-induced cytidine deaminase (AID) in the large intestine lamina propria, which is crucial for class switch recombination from IgM to IgA, were correlated with the abundance of Bacteroides fragilis group species such as Bacteroides faecis, Bacteroides caccae, and Bacteroides acidifaciensB. acidifaciens monoassociated mice increased gut IgA production and AID expression. Soluble dietary fiber may improve gut immune function, thereby protecting against bowel pathogens and reducing inflammatory bowel diseases.

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Genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib in a canine mast cell tumor cell line.

Sena Kurita, Ryo Miyamoto, Hiroyuki Tani, Masato Kobayashi, Takashi Sasaki, Kyoichi Tamura, Makoto Bonkobara

Journal of veterinary pharmacology and therapeutics   42 ( 6 ) 673 - 681  2019.11  [Refereed]  [International journal]

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One of the potential mechanisms underlying acquired resistance to toceranib in canine mast cell tumor (MCT) is the emergence of a secondary mutation in the KIT gene. Here, genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib were investigated in the toceranib-susceptible canine MCT cell line VI-MC, which carries a KIT-activating mutation resulting in a predicted p.(Asn508Ile) amino acid change in the receptor tyrosine kinase protein KIT. Two sublines were cloned from VI-MC and toceranib-resistant sublines then were established by continuous exposure to toceranib. The mutation status of KIT in parental VI-MC and its sublines was investigated using next-generation sequencing (NGS). Additionally, effects of secondary mutations on toceranib sensitivity in p.(Asn508Ile)-mutant KIT were examined. KIT secondary mutations, including those encoding p.(Asn679Lys)-, p.(Asp819Val)-, and p.(Asp819Gly)-mutant KIT, that confer toceranib insensitivity to p.(Asn508Ile)-mutant KIT emerged only in toceranib-resistant VI-MCs. These mutations were not detected by NGS in the parental VI-MC line or in the toceranib-naive cloned VI-MCs, although the parental line and sublines exhibited genetic heterogeneity in KIT that may have been caused by genetic evolution during clonal expansion. VI-MC clones with these secondary mutations in KIT appear to have arisen from subclones during treatment with toceranib rather than being pre-existing. However, further study using a higher resolution technique will be needed to confirm the developmental mechanism of KIT secondary mutation in canine MCT cells with acquired resistance to toceranib.

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A genetic variant of CYP2R1 identified in a cat with type 1B vitamin D-dependent rickets: a case report.

Takahiro Teshima, Sena Kurita, Takashi Sasaki, Hirotaka Matsumoto, Ayaka Niina, Daijiro Abe, Nobuo Kanno, Hidekazu Koyama

BMC veterinary research   15 ( 1 ) 62 - 62  2019.02  [Refereed]  [International journal]

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BACKGROUND: Vitamin D-dependent rickets is rare in animals and humans. Several types of this condition are associated with genetic variants related to vitamin D metabolism. This is the first report of type 1B vitamin D-dependent rickets in a cat. CASE PRESENTATION: Here, we describe the case of a 3-month-old female domestic short-haired cat previously fed on commercial kitten food that presented at our clinic with seizures, lethargy, and generalized pain. Serum and ionized calcium concentrations and 1,25-dihydroxycholecalciferol in this cat were low, and radiographs showed skeletal demineralization and abnormally wide growth plates on the long bones. Initially, simple vitamin D deficiency was suspected; however, the cat's profile, which included fed a well-balanced commercial diet, together with the findings of additional laboratory tests and the cat's unresponsiveness to various treatments, raised the suspicion of vitamin D-dependent rickets. Examination of the DNA sequences of CYP2R1 and CYP27B1 genes, which are genes linked with vitamin D metabolism, showed a CYP2R1 frameshift mutation in exon 5 (where T is deleted at position c.1386). This mutation alters the amino acid sequence from position 462, while the stop codon introduced at position 481 prematurely truncates the 501 amino acid full-length protein. With this knowledge, a new treatment regime based on a standard dose of calcitriol was started and this markedly improved the cat's condition. CONCLUSIONS: To the best of our knowledge, the present case is the first description of type 1B vitamin D-dependent rickets linked with a genetic variant of CYP2R1 in a cat.

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Genetic and Transcriptomic Analyses of Ciprofloxacin-Tolerant Staphylococcus aureus Isolated by the Replica Plating Tolerance Isolation System (REPTIS).

Miki Matsuo, Miyu Hiramatsu, Madhuri Singh, Takashi Sasaki, Tomomi Hishinuma, Norio Yamamoto, Yuh Morimoto, Teruo Kirikae, Keiichi Hiramatsu

Antimicrobial agents and chemotherapy   63 ( 2 ) e02019 - 18  2019.02  [Refereed]  [International journal]

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We developed a simple, efficient, and cost-effective method, named the replica plating tolerance isolation system (REPTIS), to detect the antibiotic tolerance potential of a bacterial strain. This method can also be used to quantify the antibiotic-tolerant subpopulation in a susceptible population. Using REPTIS, we isolated ciprofloxacin (CPFX)-tolerant mutants (mutants R2, R3, R5, and R6) carrying a total of 12 mutations in 12 different genes from methicillin-sensitive Staphylococcus aureus (MSSA) strain FDA209P. Each mutant carried multiple mutations, while few strains shared the same mutation. The R2 strain carried a nonsense mutation in the stress-mediating gene, relA Additionally, two strains carried the same point mutation in the leuS gene, encoding leucyl-tRNA synthetase. Furthermore, RNA sequencing of the R strains showed a common upregulation of relA Overall, transcriptome analysis showed downregulation of genes related to translation; carbohydrate, fat, and energy metabolism; nucleotide synthesis; and upregulation of amino acid biosynthesis and transportation genes in R2, R3, and R6, similar to the findings observed for the FDA209P strain treated with mupirocin (MUP0.03). However, R5 showed a unique transcription pattern that differed from that of MUP0.03. REPTIS is a unique and convenient method for quantifying the level of tolerance of a clinical isolate. Genomic and transcriptomic analyses of R strains demonstrated that CPFX tolerance in these S. aureus mutants occurs via at least two distinct mechanisms, one of which is similar to that which occurs with mupirocin treatment.

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Comparison of USA300 with non-USA300 methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit.

Takemi Murai, Kaoru Okazaki, Kazue Kinoshita, Yuki Uehara, Hui Zuo, Yujie Lu, Yuki Ono, Takashi Sasaki, Keiichi Hiramatsu, Yuho Horikoshi

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases   79   134 - 138  2019.02  [Refereed]  [International journal]

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OBJECTIVES: Reports of USA300 methicillin-resistant Staphylococcus aureus (MRSA) strain were still scarce in neonatal intensive care units (NICUs) and the relationship of USA300 MRSA to clinical infections is still controversial. The primary outcome was the incidence of MRSA infections caused by the USA300 and non-USA300 strains at a NICU in Japan. METHODS: This retrospective cohort study was conducted between November 2011 and October 2016 at Tokyo Metropolitan Children's Medical Center in Japan. All MRSA isolated after 48h of hospitalization were included for analysis by pulsed-field gel electrophoresis (PFGE) using the standard USA300 strain. Genes were tested for Panton-Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME). A whole genome sequence was performed for representative isolates of USA300. RESULTS: In total, 109 MRSA isolates were included for analysis. PFGE classified 34 and 75 isolates of USA300 and non-USA300 MRSA, respectively. Both PVL and ACME genes were detected in USA300 and non-USA300 strains at rate of 100% (34/34) and 5.3% (4/75), respectively (P<0.05). There was no statistically significant difference in the proportion of clinical diseases between USA- 300 and non-USA 300 strains. CONCLUSIONS: Infants with USA300 MRSA infection did not differ significantly from those with non-USA300 MRSA infection.

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Regional outbreak of community-associated methicillin-resistant Staphylococcus aureus ST834 in Japanese children.

Yuki Uehara, Takashi Sasaki, Tadashi Baba, Yujie Lu, Eri Imajo, Yuka Sato, Shigeru Tanno, Munehiro Furuichi, Miki Kawada, Keiichi Hiramatsu

BMC infectious diseases   19 ( 1 ) 35 - 35  2019.01  [Refereed]  [International journal]

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BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infection has recently become a challenging problem worldwide and in Japan. We experienced 10 pediatric patients infected with CA-MRSA and hospitalized from 2011 to 2014 in a tertiary care hospital in Saitama, Japan, and assessed the characteristic of the strains using a whole genome sequencing (WGS)-based approach. METHODS: CA-MRSA strains isolated from infected patients who required hospitalization for treatment were evaluated in this study. Antimicrobial susceptibility tests, molecular typing by PCR and pulse-field gel electrophoresis (PFGE) were performed to characterize MRSA strains. WGS was performed for detailed genetic analysis. RESULTS: A total of 582 MRSA strains (35.2%) were identified among 1625 S. aureus strains collected during the study period. Ten MRSA strains (1.7%) were defined as CA-MRSA clinically, and all were isolated from pediatric patients. All strains mainly caused purulent lymphadenitis, were susceptible to fluoroquinolone and tetracycline, exhibited sequence type (ST) 834 or its single-locus variants and contained staphylococcal cassette chromosome mec (SCCmec) type IVc. Phylogenic analysis by PFGE and WGS revealed close relatedness of all strains, with the number of single nucleotide polymorphisms ranging from 35 to 119 by WGS. Out of the ten strains, nine possessed the genomic island SaPISaitama2 containing tst, sec and sel genes. SaPISaitama2 comprises a mosaic of genomic islands SaPIm4 and SaPIm1 harbored by a hospital-associated MRSA strain Mu50. CONCLUSIONS: This study describes a regional outbreak of ST834-related CA-MRSA in children with a unique pathogenicity island in Japan. Pediatric patient tropism of this clone could be enhanced by susceptibility to fluoroquinolones and tetracyclines, which cannot be prescribed to children.

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Fecal Freezing Preservation Period Influences Colonization Ability for Fecal Microbiota Transplantation.

Takahashi M, Ishikawa D, Sasaki T, Lu YJ, Kuwahara-Arai K, Kamei M, Shibuya T, Osada T, Hiramatsu K, Nagahara A

J Appl Microbiol.   126 ( 3 ) 973 - 984  2019  [Refereed]

The Microbial Composition of Bacteroidetes Species in Ulcerative Colitis Is Effectively Improved by Combination Therapy With Fecal Microbiota Transplantation and Antibiotics.

Dai Ishikawa, Takashi Sasaki, Masahito Takahashi, Kyoko Kuwahara-Arai, Keiichi Haga, Shoko Ito, Koki Okahara, Akihito Nakajima, Tomoyoshi Shibuya, Taro Osada, Keiichi Hiramatsu, Sumio Watanabe, Akihito Nagahara

Inflammatory bowel diseases   24 ( 12 ) 2590 - 2598  2018.11  [Refereed]  [International journal]

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Background: We previously reported that fresh fecal microbiota transplantation (FMT) after triple-antibiotic therapy (amoxicillin, fosfomycin, and metronidazole [AFM]; A-FMT) synergistically contributed to the recovery of phylum Bacteroidetes composition associated with the endoscopic severity and treatment efficacy of ulcerative colitis (UC). Here, we performed further microbial analyses using a higher-resolution method to identify the key bacterial species in UC and determine whether viable Bacteroidetes species from donor feces were successfully colonized by A-FMT. Methods: The taxonomic composition of Bacteroidetes in 25 healthy donors and 27 UC patients at baseline was compared at the species level using a heat-shock protein (hsp) 60-based microbiome method. Microbiota alterations before and after treatment of UC patients were also analyzed in 24 cases (n = 17 A-FMT; n = 3 mono-AFM; n = 4 mono-FMT). Results: We found species-level dysbiosis within the phylum Bacteroidetes in UC samples, which was associated with reduced species diversity, resulting from hyperproliferation and hypoproliferation of particular species. Moreover, in responders treated with A-FMT, diversity was significantly recovered at 4 weeks after a fresh round of FMT, after which high degrees of similarity in Bacteroidetes species composition among recipients and donors were observed. Conclusions: A-FMT alleviated intestinal dysbiosis, which is caused by the loss of Bacteroidetes species diversity in patients with UC. Eradication of dysbiotic indigenous Bacteroidetes species by AFM pretreatment might promote the colonization of viable Bacteroidetes cells, thereby improving the intestinal microbiota dysbiosis induced by UC. Our findings serve as a basis for further investigations into the mechanisms of FMT.

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Development of a New Application for Comprehensive Viability Analysis Based on Microbiome Analysis by Next-Generation Sequencing: Insights into Staphylococcal Carriage in Human Nasal Cavities.

Yu Jie Lu, Takashi Sasaki, Kyoko Kuwahara-Arai, Yuki Uehara, Keiichi Hiramatsu

Applied and environmental microbiology   84 ( 11 ) e00517 - 18  2018.06  [Refereed]  [International journal]

Authorship：   Corresponding author

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The nasal carriage rate of Staphylococcus aureus in human is 25 to 30%, and S. aureus sporadically causes severe infections. However, the mechanisms underlying staphylococcal carriage remain largely unknown. In the present study, we constructed an rpoB-based microbiome method for staphylococcal species discrimination. Based on a microbiome scheme targeting viable cell DNA using propidium monoazide (PMA) dye (PMA microbiome method), we also developed a new method to allow the comprehensive viability analysis of any bacterial taxon. To clarify the ecological distribution of staphylococci in the nasal microbiota, we applied these methods in 46 nasal specimens from healthy adults. PMA microbiome results showed that Staphylococcaceae and Corynebacteriaceae were the most predominant viable taxa (average relative abundance: 0.435262 and 0.375195, respectively), and Staphylococcus epidermidis exhibited the highest viability in the nasal microbiota. Staphylococcus aureus detection rates from nasal specimens by rpoB-based conventional and PMA microbiome methods were 84.8% (39 of 46) and 69.5% (32 of 46), respectively, which substantially exceeded the values obtained by a culture method using identical specimens (36.9%). Our results suggest that Staphylococcaceae species, especially S. epidermidis, adapted most successfully to human nasal cavity. High detection of S. aureus DNA by microbiome methods suggests that almost all healthy adults are consistently exposed to S. aureus in everyday life. Furthermore, the large difference in S. aureus detection rates between culture and microbiome methods suggests that S. aureus cells frequently exist in a viable but nonculturable state in nasal cavities. Our method and findings will contribute to a better understanding of the mechanisms underlying carriage of indigenous bacteria.IMPORTANCE Metagenomic analyses, such as 16S rRNA microbiome methods, have provided new insights in various research fields. However, conventional 16S rRNA microbiome methods do not permit taxonomic analysis of only the viable bacteria in a sample and have poor resolving power below the genus level. Our new schemes allowed for viable cell-specific analysis and species discrimination, and nasal microbiome data using these methods provided some interesting findings regarding staphylococcal nasal carriage. According to our comprehensive viability analysis, the high viability of Staphylococcus species, especially Staphylococcus epidermidis, in human nasal carriage suggests that this taxon has adapted most successfully to human nasal tissue. Also, a higher detection rate of S. aureus DNA by microbiome methods (84.8%) than by a culture method (36.9%) suggests that almost all healthy adults are consistently exposed to Staphylococcus aureus in the medium and long term. Our findings will contribute to a better understanding of the mechanisms underlying the carriage of indigenous bacteria.

DOI PubMed

Risk factors of fecal colonization with extended-spectrum β-lactamase-producing Enterobacteriaceae in special nursing homes in Japan.

Kumi Yokoyama, Yuki Uehara, Takashi Sasaki, Keiichi Hiramatsu

Journal of general and family medicine   19 ( 3 ) 90 - 96  2018.05  [Refereed]  [Domestic journal]

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Objective: Japanese welfare facilities for the elderly are called as special nursing home (SNH), providing conventional-type with group care or unit-type with individual care. We investigated the risk factors of fecal colonization with extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) of elderly who required care at SNH in Japan. Methods: The feces discharged on diaper were obtained from the total of 100 residents with fecal incontinence in 9 SNHs located in Tokyo, Japan. The samples were cultured on ESBL selection agar, and ESBL-E were determined by the antimicrobial susceptibility test and genetic analysis. The status of the residents and the characteristics of facilities, especially about the incontinence care, were obtained by questionnaire methods. Statistical analysis was performed to determine the factors related to carriage of ESBL-E. Results: Extended-spectrum β-lactamase-producing Enterobacteriaceae was isolated from 53 of 100 SNH residents. Risk factors of colonization among the individual residents were not found. The prevalence of ESBL-E carriage was significantly higher in the 6 conventional-type facilities than in the 3 unit-type facilities (P = .015). The cart for diaper exchange was used in 5 of 6 conventional-type facilities in 9 SNHs, and their residents tended to show high of ESBL-E colonization rate. The residents living in unit-type facilities which do not use gloves for changing diaper tended to show high ESBL-E colonization rate than other 2 facilities using gloves. Conclusions: It is suggested that using the cart for changing diaper has relation to carry ESBL-E. In the facilities using cart, revision of their methods of excretion care will be needed.

DOI PubMed

Maternal High Fiber Diet during Pregnancy and Lactation Influences Regulatory T Cell Differentiation in Offspring in Mice

Akihito Nakajima, Naoko Kaga, Yumiko Nakanishi, Hiroshi Ohno, Junki Miyamoto, Ikuo Kimura, Shohei Hori, Takashi Sasaki, Keiichi Hiramatsu, Ko Okumura, Sachiko Miyake, Sonoko Habu, Sumio Watanabe

JOURNAL OF IMMUNOLOGY ( AMER ASSOC IMMUNOLOGISTS )  199 ( 10 ) 3516 - 3524  2017.11  [Refereed]

　View Summary

Short-chain fatty acids (SCFAs), the end products of dietary fiber, influence the immune system. Moreover, during pregnancy the maternal microbiome has a great impact on the development of the offspring's immune system. However, the exact mechanisms by which maternal SCFAs during pregnancy and lactation influence the immune system of offspring are not fully understood. We investigated the molecular mechanisms underlying regulatory T cell (T-reg) differentiation in offspring regulated by a maternal high fiber diet (HFD). Plasma levels of SCFAs in offspring from HFD-fed mice were higher than in those from no fiber diet-fed mice. Consequently, the offspring from HFD-fed mice had higher frequencies of thymic T-reg (tT(reg)) and peripheral T-regs. We found that the offspring of HFD-fed mice exhibited higher autoimmune regulator (Aire) expression, a transcription factor expressed in the thymic microenvironment, suggesting SCFAs promote tT(reg) differentiation through increased Aire expression. Notably, the receptor for butyrate, G protein-coupled receptor 41 (GPR41), is highly expressed in the thymic microenvironment and Aire expression is not increased by stimulation with butyrate in GPR41-deficient mice. Our studies highlight the significance of SCFAs produced by a maternal HFD for T-reg differentiation in the thymus of offspring. Given that Aire expression is associated with the induction of tT(regs), the maternal microbiome influences T-reg differentiation in the thymus of offspring through GPR41-mediated Aire expression.

DOI

Activity of tick antimicrobial peptide from Ixodes persulcatus (persulcatusin) against cell membranes of drug-resistant Staphylococcus aureus

Naruhide Miyoshi, Emiko Isogai, Keiichi Hiramatsu, Takashi Sasaki

JOURNAL OF ANTIBIOTICS   70 ( 2 ) 142 - 146  2017.02  [Refereed]

Authorship：   Last author  , Corresponding author

DOI

In Vitro Tolerance of Drug-Naive Staphylococcus aureus Strain FDA209P to Vancomycin

Madhuri Singh, Miki Matsuo, Takashi Sasaki, Yuh Morimoto, Tomomi Hishinuma, Keiichi Hiramatsu

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY ( AMER SOC MICROBIOLOGY )  61 ( 2 ) e01154 - 16  2017.02  [Refereed]

　View Summary

The mechanisms underlying bacterial tolerance to antibiotics are unclear. A possible adaptation strategy was explored by exposure of drug-naive methicillin-susceptible Staphylococcus aureus strain FDA209P to vancomycin in vitro. Strains surviving vancomycin treatment (vancomycin survivor strains), which appeared after 96 h of exposure, were slow-growing derivatives of the parent strain. Although the vancomycin MICs for the survivor strains were within the susceptible range, the cytokilling effects of vancomycin at 20-fold the MIC were significantly lower for the survivor strains than for the parent strain. Whole-genome sequencing demonstrated that ileS, encoding isoleucyl- tRNA synthetase (IleRS), was mutated in two of the three vancomycin survivor strains. The IleRS Y723H mutation is located close to the isoleucyl-tRNA contact site and potentially affects the affinity of IleRS binding to isoleucyl-tRNA, thereby inhibiting protein synthesis and leading to vancomycin tolerance. Introduction of the mutation encoding IleRS Y723H into FDA209P by allelic replacement successfully transferred the vancomycin tolerance phenotype. We have identified mutation of ileS to be one of the bona fide genetic events leading to the acquisition of vancomycin tolerance in S. aureus, potentially acting via inhibition of the function of IleRS.

DOI

Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B

Yoshikazu Tanaka, Yuka Sato, Takashi Sasaki

JOURNAL OF GENERAL VIROLOGY   98 ( 2 ) 190 - 200  2017.02  [Refereed]

Authorship：   Last author

DOI

Changes in Intestinal Microbiota Following Combination Therapy with Fecal Microbial Transplantation and Antibiotics for Ulcerative Colitis

Dai Ishikawa, Takashi Sasaki, Taro Osada, Kyoko Kuwahara-Arai, Keiichi Haga, Tomoyoshi Shibuya, Keiichi Hiramatsu, Sumio Watanabe

INFLAMMATORY BOWEL DISEASES ( LIPPINCOTT WILLIAMS & WILKINS )  23 ( 1 ) 116 - 125  2017.01  [Refereed]

　View Summary

Background: Fecal microbiota transplantation (FMT) is a potential therapeutic approach to restore normal intestinal microbiota in patients with ulcerative colitis (UC), which is associated with dysbiosis; however, treatment efficacy remains unclear. Hence, we studied the impact of antibiotic pretreatment with amoxicillin, fosfomycin, and metronidazole (AFM therapy) and FMT versus AFM alone. Methods: AFM therapy was administered to patients for 2 weeks until 2 days before FMT. Patients' spouses or relatives were selected as donor candidates. Donor fecal samples were collected on the day of administration and transferred into the patient's colon by colonoscopy within 6 hours. Microbiome analysis was performed by 16S rRNA next-generation sequencing. Results: Patients with mild-to-severe active UC (combination-therapy group, n = 21; AFM monotherapy group, n = 20) were included. Thirty-six patients completed this assessment (combination-therapy group, n = 17; AFM monotherapy group, n = 19). A higher clinical response was observed after combination therapy compared with AFM monotherapy at 4 weeks after treatment. After the 2-week AFM therapy, the Bacteroidetes composition was nearly abolished. The Bacteroidetes proportion recovered in clinical responders at 4 weeks after FMT was not observed in the AFM monotherapy group. Persistent antimicrobial-associated dysbiosis found in the AFM monotherapy group was reversed by FMT. The recovery rate of Bacteroidetes at 4 weeks after FMT correlated with endoscopic severity. Conclusions: FMT following antimicrobial bowel cleansing synergistically contributes to the recovery of the Bacteroidetes composition, which is associated with clinical response and UC severity. Thus, this therapeutic protocol may be useful for managing UC.

DOI

バンコマイシン投与中にMIC上昇を認めたMRSA臨床分離株の遺伝子変異

森 美紀, 上原 由紀, 平松 啓一, 菊池 賢, 佐々木 崇

日本化学療法学会雑誌 ( (公社)日本化学療法学会 )  64 ( Suppl.A ) 182 - 182  2016.05

Complete genome sequence of vancomycin-intermediate Staphylococcus aureus strain MI (HIP5827)

Tomomi Hishinuma, Yuki Katayama, Miki Matsuo, Takashi Sasaki, Keiichi Hiramatsu

Genome Announcements ( American Society for Microbiology )  4 ( 2 ) e00123 - 16  2016  [Refereed]  [International journal]

　View Summary

We report the complete genome sequence of vancomycin-intermediate Staphylococcus aureus (VISA) strain MI (HIP5827).

DOI PubMed

Apigenin as an anti-quinolone-resistance antibiotic

Yuh Morimoto, Tadashi Baba, Takashi Sasaki, Keiichi Hiramatsu

INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS ( ELSEVIER SCIENCE BV )  46 ( 6 ) 666 - 673  2015.12  [Refereed]

　View Summary

We previously reported the first 'reverse antibiotic' (RA), nybomycin (NYB), which showed a unique antimicrobial activity against Staphylococcus aureus strains. NYB specifically suppressed the growth of quinolone-resistant S. aureus strains but was not effective against quinolone-susceptible strains. Although NYB was first reported in 1955, little was known about its unique antimicrobial activity because it was before the synthesis of the first quinolone ('old quinolone'), nalidixic acid, in 1962. Following our re-discovery of NYB, we looked for other RAs among natural substances that act on quinolone-resistant bacteria. Commercially available flavones were screened against S. aureus, including quinolone-resistant strains, and their minimum inhibitory concentrations (MICs) were compared using the microbroth dilution method. Some of the flavones screened showed stronger antimicrobial activity against quinolone-resistant strains than against quinolone-susceptible ones. Amongst them, apigenin (API) was the most potent in its RA activity. DNA cleavage assay showed that API inhibited DNA gyrase harbouring the quinolone resistance mutation gyrA(Ser84Leu) but did not inhibit 'wild-type' DNA gyrase that is sensitive to levofloxacin. An API-susceptible S. aureus strain Mu50 was also selected using agar plates containing 20 mg/L API. Whole-genome sequencing of selected mutant strains was performed and frequent back-mutations (reverse mutations) were found among API-resistant strains derived from the API-susceptible S. aureus strains. Here we report that API represents another molecular class of natural antibiotic having RA activity against quinolone-resistant bacteria. (C) 2015 The Authors. Published by Elsevier B.V.

DOI

Molecular epidemiological study of feline coronavirus strains in Japan using RT-PCR targeting nsp14 gene

Yoshikazu Tanaka, Takashi Sasaki, Ryo Matsuda, Yosuke Uematsu, Tomohiro Yamaguchi

BMC VETERINARY RESEARCH   11 ( 57 )  2015.03  [Refereed]

Authorship：   Lead author

DOI

Treatment of a case of feline infectious peritonitis with cyclosporin A.

Tanaka Y, Sato Y, Takahashi D, Matsumoto H, Sasaki T

Vet Rec Case Rep.   3   e000134  2015  [Refereed]

Cellular peptidyl-prolyl cis/trans isomerase Pin1 facilitates replication of feline coronavirus.

Tanaka Y, Amano A, Morisaki M, Sato Y, Sasaki T

Antiviral Res.   7 ( 126 ) 1 - 7  2015  [Refereed]

Authorship：   Last author

Complete genome sequence of methicillin-resistant Staphylococcus schleiferi strain TSCC54 of canine origin

Takashi Sasaki, Sae Tsubakishita, Kyoko Kuwahara-Arai, Miki Matsuo, Yu Jie Lu, Yoshikazu Tanaka, Keiichi Hiramatsu

Genome Announcements ( American Society for Microbiology )  3 ( 5 ) e01268 - 15  2015  [Refereed]

Authorship：   Lead author  , Corresponding author

DOI

Complete Genome Sequence of a Drug-naive Classical Staphylococcus aureus Strain FDA209P.

Singh M, Sasaki T, Matsuo M, Morimoto Y, Aiba Y, Hiramatsu K

Genome Announc.   12 ( 3(6) ) e01343 - 15  2015  [Refereed]  [International journal]

Authorship：   Corresponding author

　View Summary

We report the complete genome sequence of the methicillin-sensitive Staphylococcus aureus (MSSA) strain FDA209P (ATCC 6538P and NCTC 7447).

DOI PubMed

Future Chemotherapy Preventing Emergence of Multi-Antibiotic Resistance.

Hiramatsu K, Sasaki T, Morimoto Y

Juntendo Medical Journal. ( The Juntendo Medical Society )  3 ( 3 ) 249 - 256  2015  [Refereed]

　View Summary

The authors and all of the faculty at the Department of Bacteriology (Dept. of Microbiology from April 2015) have studied methicillin-resistant Staphylococcus aureus (MRSA), a representative multi-drug-resistant pathogen prevalent globally. During this study, we have identified the extreme flexibility of this organism to survive antibiotic pressure. S. aureus is part of the normal flora of human beings, yet it possesses high pathogenic potential, being aptly called 'the department of toxins'. We are probably destined to continue our lives in association with this dangerous neighbor. For this reason, in 2008, KH launched a new project searching for novel antibiotics that are effective against MRSA. A newly found antibiotic called nybomycin has a curious property: it is effective against quinolone-resistant S. aureus strains (including most MRSA), but not against quinolone-susceptible ones. Moreover, the mutant strains derived from the parent S. aureus strain after treatment with nybomycin were cured of their quinolone resistance. Subsequently, we found an antibiotic with the same property in flavones, too. We designated these antibiotics with such unique properties reverse antibiotics (RA). By using RA and extant antibiotics in a well-controlled way, we should be able to establish sophisticated anti-microbial chemotherapy in the future.

DOI CiNii

Multi-drug-resistant Staphylococcus aureus and future chemotherapy

K. Hiramatsu, Y. Katayama, M. Matsuo, T. Sasaki, Y. Morimoto, A. Sekiguchi, T. Baba

JOURNAL OF INFECTION AND CHEMOTHERAPY ( SPRINGER JAPAN KK )  20 ( 9-10 ) 593 - 601  2014.09  [Refereed]

　View Summary

Staphylococcus (S.) aureus silently stays as our natural flora, and yet sometimes threatens our life as a tenacious pathogen. In addition to its ability to outwit our immune system, its multi-drug resistance phenotype makes it one of the most intractable pathogenic bacteria in the history of antibiotic chemotherapy. It conquered practically all the antibiotics that have been developed since 1940s. In 1961, the first MRSA was found among S. aureus clinical isolates. Then MRSA prevailed throughout the world as a multi-resistant hospital pathogen. In 1997, MRSA strain Mu50 with reduced susceptibility to vancomycin was isolated. Vancomycin-intermediate S. aureus (VISA), so named according to the CLSI criteria, was the product of adaptive mutation of S. aureus against vancomycin that had long been the last resort to MRSA infection. Here, we describe the genetic basis for the remarkable ability of S. aureus to acquire multi-antibiotic resistance, and propose a novel paradigm for future chemotherapy against the multiresistant pathogens. (C) 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

DOI

Suppression of Coronavirus Replication by Cyclophilin Inhibitors

Yoshikazu Tanaka, Yuka Sato, Takashi Sasaki

VIRUSES-BASEL   5 ( 5 ) 1250 - 1260  2013.05  [Refereed]

Authorship：   Last author

DOI

Genomic basis for methicillin resistance in Staphylococcus aureus

Keiichi Hiramatsu, Teruyo Ito, Sae Tsubakishita, Takashi Sasaki, Fumihiko Takeuchi, Yuh Morimoto, Yuki Katayama, Miki Matsuo, Kyoko Kuwahara-Arai, Tomomi Hishinuma, Tadashi Baba

Infection and Chemotherapy   45 ( 2 ) 117 - 136  2013  [Refereed]

　View Summary

Since the discovery of the first strain in 1961 in England, MRSA, the most notorious multidrug-resistant hospital pathogen, has spread all over the world. MRSA repeatedly turned down the challenges by number of chemotherapeutics, the fruits of modern organic chemistry. Now, we are in short of effective therapeutic agents against MRSA prevailing among immuno-compromised patients in the hospital. On top of this, we recently became aware of the rise of diverse clones of MRSA, some of which have increased pathogenic potential compared to the classical hospital-associated MRSA, and the others from veterinary sources. They increased rapidly in the community, and started menacing otherwise healthy individuals by causing unexpected acute infection. This review is intended to provide a whole picture of MRSA based on its genetic makeup as a versatile pathogen and our tenacious colonizer. © 2013 by The Korean Society of Infectious Diseases | Korean Society for Chemotherapy.

DOI

Characterization of Staphylococcus aureus isolates from faecal samples of the Straw-Coloured Fruit Bat (Eidolon helvum) in Obafemi Awolowo University (OAU), Nigeria

Babatunji Akobi, Oladipo Aboderin, Takashi Sasaki, Adebayo Shittu

BMC MICROBIOLOGY ( BIOMED CENTRAL LTD )  12 ( 12(1) ) 279  2012.11  [Refereed]

　View Summary

Background: Bats (Chiroptera) are one of the most diverse groups of mammals which carry out important ecological and agricultural functions that are beneficial to humans. However, they are increasingly recognized as natural vectors for a number of zoonotic pathogens and favourable hosts for zoonotic infections. Large populations of the Straw-Coloured Fruit Bat (Eidolon helvum) colonize the main campus of the Obafemi Awolowo University (OAU), Ile-Ife, Nigeria, but the public health implications of faecal contamination and pollution by these flying mammals is unknown. This study characterized S. aureus obtained from faecal samples of these migratory mammals with a view to determining the clonal types of the isolates, and to investigate the possibility of these flying animals as potential reservoir for zoonotic S. aureus infections. Results: One hundred and seven (107) S. aureus isolates were recovered from 560 faecal samples in eleven roosting sites from January 2008 to February 2010. A large proportion of the isolates were susceptible to antibiotics, and molecular characterization of 70 isolates showed that 65 (92.9%) were assigned in coagulase type VI, while accessory gene typing classified 69 isolates into the following: type I (12; 17.1%), type II (3; 4.3%), type III (1; 1.4%) and type IV (53; 75.7%). On the whole, the isolates were grouped in five (A-E) main genotypes. Of the ten representative isolates selected for multilocus sequence typing (MLST), nine isolates were assigned with new sequence types: ST1725, ST1726, ST1727, ST2463-ST2467 and ST2470. Phylogenetic analysis provided evidence that S. aureus isolates in group C were closely related with ST1822 and associated clones identified in African monkeys, and group D isolates with ST75, ST883 and ST1223. The two groups exhibited remarkable genetic diversity compared to the major S. aureus clade. Conclusions: Antibiotic resistance in faecal S. aureus isolates of E. helvum is low and multiple unique S. aureus lineages co-existed with E. helvum. The Straw-Coloured Fruit Bat in Ile-Ife, Nigeria is colonized predominantly by ST1725, ST1726, ST2463 and ST2470 with distinct genotypic characteristics that are rarely found in humans. This study has demonstrated on the possible existence of a reservoir of indigenous and anciently-divergent S. aureus clones among mammals in Africa.

DOI

Population Genetic Structures of Staphylococcus aureus Isolates from Cats and Dogs in Japan

Takashi Sasaki, Sae Tsubakishita, Yoshikazu Tanaka, Masayuki Ohtsuka, Isamu Hongo, Tsuneo Fukata, Hidenori Kabeya, Soichi Maruyama, Keiichi Hiramatsu

JOURNAL OF CLINICAL MICROBIOLOGY   50 ( 6 ) 2152 - 2155  2012.06  [Refereed]

Authorship：   Lead author  , Corresponding author

DOI

Suppression of feline coronavirus replication in vitro by cyclosporin A

Yoshikazu Tanaka, Yuka Sato, Shuichi Osawa, Mai Inoue, Satoka Tanaka, Takashi Sasaki

VETERINARY RESEARCH ( BIOMED CENTRAL LTD )  43 ( 43(1) ) 41  2012.04  [Refereed]

　View Summary

The feline infectious peritonitis virus (FIPV) is a member of the feline coronavirus family that causes FIP, which is incurable and fatal in cats. Cyclosporin A (CsA), an immunosuppressive agent that targets the nuclear factor pathway of activated T-cells (NF-AT) to bind cellular cyclophilins (CyP), dose-dependently inhibited FIPV replication in vitro. FK506 (an immunosuppressor of the pathway that binds cellular FK506-binding protein (FKBP) but not CyP) did not affect FIPV replication. Neither cell growth nor viability changed in the presence of either CsA or FK506, and these factors did not affect the NF-AT pathway in fcwf-4 cells. Therefore, CsA does not seem to exert inhibitory effects via the NF-AT pathway. In conclusion, CsA inhibited FIPV replication in vitro and further studies are needed to verify the practical value of CsA as an anti-FIPV treatment in vivo.

DOI

Rapid and Accurate Identification of Human-Associated Staphylococci by Use of Multiplex PCR

Shintaro Hirotaki, Takashi Sasaki, Kyoko Kuwahara-Arai, Keiichi Hiramatsu

JOURNAL OF CLINICAL MICROBIOLOGY ( AMER SOC MICROBIOLOGY )  49 ( 10 ) 3627 - 3631  2011.10  [Refereed]

　View Summary

Although staphylococci are identified by phenotypic analysis in many clinical laboratories, these results are often incorrect because of phenotypic variation. Genetic analysis is necessary for definitive species identification. In the present study, we developed a simple multiplex-PCR (M-PCR) for species identification of human-associated staphylococci, which were as follows: Staphylococcus aureus, S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. saprophyticus, and S. warneri. This method was designed on the basis of nucleotide sequences of the thermonuclease (nuc) genes that were universally conserved in staphylococci except the S. sciuri group and showed moderate sequence diversity. In order to validate this assay, 361 staphylococcal strains were studied, which had been identified at the species levels by sequence analysis of the hsp60 genes. In consequence, M-PCR demonstrated a sensitivity of 100% and a specificity of 100%. By virtue of simplicity and accuracy, this method will be useful in clinical research.

DOI

Origin and molecular evolution of the determinant of methicillin resistance in staphylococci (Antimicrobial Agents and Chemotherapy (2010) 54, 10 (4352?4359))

Sae Tsubakishita, Kyoko Kuwahara-Arai, Takashi Sasaki, Keiichi Hiramatsu

Antimicrobial Agents and Chemotherapy   55 ( 2 ) 946  2011.02  [Refereed]

DOI

健康な犬におけるメチシリン耐性Staphylococcus pseudintermedius（MRSP）の疫学調査

須藤哲長, 寺井陽子, 三枝早苗, 椿下早絵, 佐々木崇, 平松啓一

獣医臨床皮膚科 ( Japanese Society of Veterinary Dermatology )  17 ( 2 ) 79 - 83  2011  [Refereed]

　View Summary

In veterinary medicine, the use of broad-spectrum antibiotics has been increasing. The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs is now increasingly common all over the world, and has also become a serious issue in veterinary dermatology practice in Japan. In the present study, we surveyed nasal carriage of MRSP and methicillin-resistant S. aureus (MRSA) in 104 healthy dogs in Japan, and compared the results with those of 102 patient dogs under secondary veterinary medical care. Nasal carriage rates of MRSP in healthy and patient dogs were 4.8% and 21.6%, respectively, and that of patient dogs was a significantly higher value (p<0.001), suggesting that patient dogs under secondary veterinary medical care were under antibiotic selective pressure. The MRSP carriage rate in healthy dogs in Japan was similar to those reported overseas. MRSA nasal carriage rates in healthy and patient dogs were 0% and 1.96%, respectively, with no significant difference between the two (p=0.244). Under antibiotic selective pressures, MRSA carriage rate in dogs didn’t increase significantly, suggesting that dogs have low potential as a reservoir of MRSA.


DOI CiNii J-GLOBAL

Origin and Molecular Evolution of the Determinant of Methicillin Resistance in Staphylococci

Sae Tsubakishita, Kyoko Kuwahara-Arai, Takashi Sasaki, Keiichi Hiramatsu

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY ( AMER SOC MICROBIOLOGY )  54 ( 10 ) 4352 - 4359  2010.10  [Refereed]

　View Summary

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant pathogens around the world. MRSA is generated when methicillin-susceptible S. aureus (MSSA) exogenously acquires a methicillin resistance gene, mecA, carried by a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec), which is speculated to be transmissible across staphylococcal species. However, the origin/reservoir of the mecA gene has remained unclear. Finding the origin/reservoir of the mecA gene is important for understanding the evolution of MRSA. Moreover, it may contribute to more effective control measures for MRSA. Here we report on one of the animal-related Staphylococcus species, S. fleurettii, as the highly probable origin of the mecA gene. The mecA gene of S. fleurettii was found on the chromosome linked with the essential genes for the growth of staphylococci and was not associated with SCCmec. The mecA locus of the S. fleurettii chromosome has a sequence practically identical to that of the mecA-containing region (similar to 12 kbp long) of SCCmec. Furthermore, by analyzing the corresponding gene loci (over 20 kbp in size) of S. sciuri and S. vitulinus, which evolved from a common ancestor with that of S. fleurettii, the speciation-related mecA gene homologues were identified, indicating that mecA of S. fleurettii descended from its ancestor and was not recently acquired. It is speculated that SCCmec came into form by adopting the S. fleurettii mecA gene and its surrounding chromosomal region. Our finding suggests that SCCmec was generated in Staphylococcus cells living in animals by acquiring the intrinsic mecA region of S. fleurettii, which is a commensal bacterium of animals.

DOI

Multiplex-PCR Method for Species Identification of Coagulase-Positive Staphylococci

Takashi Sasaki, Sae Tsubakishita, Yoshikazu Tanaka, Arihito Sakusabe, Masayuki Ohtsuka, Shintaro Hirotaki, Tetsuji Kawakami, Tsuneo Fukata, Keiichi Hiramatsu

JOURNAL OF CLINICAL MICROBIOLOGY   48 ( 3 ) 765 - 769  2010.03  [Refereed]

Authorship：   Lead author  , Corresponding author

DOI

Reclassification of Canine Staphylococcal Strains Identified as Methicillin-resistant Staphylococcus aureus (MRSA) in a Clinical Laboratory

Kasai Tomoko, Saegusa Sanae, Sasaki Takashi

The Japanese Journal of Veterinary Dermatology ( Japanese Society of Veterinary Dermatology )  16 ( 3 ) 119 - 124  2010  [Refereed]

DOI DOI2 CiNii J-GLOBAL

Emergence of Quinolone-Resistant Bordetella pertussis in Japan

Masayuki Ohtsuka, Ken Kikuchi, Kenichiro Shimizu, Namiko Takahashi, Yuka Ono, Takashi Sasaki, Keiichi Hiramatsu

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY ( AMER SOC MICROBIOLOGY )  53 ( 7 ) 3147 - 3149  2009.07  [Refereed]

　View Summary

Six Bordetella pertussis strains isolated from children in Japan from 2004 to 2006 showed high-level resistance to nalidixic acid (NAL; MIC, > 256 mu g/ml) and decreased susceptibilities to fluoroquinolones. All of the NAL-resistant strains had the same D87G mutation in gyrA.

DOI

Genetic Diversity of Staphylocoagulase Genes (coa): Insight into the Evolution of Variable Chromosomal Virulence Factors in Staphylococcus aureus

Shinya Watanabe, Teruyo Ito, Takashi Sasaki, Shanshuang Li, Ikuo Uchiyama, Kozue Kishii, Ken Kikuchi, Robert Leo Skov, Keiichi Hiramatsu

PLOS ONE ( PUBLIC LIBRARY SCIENCE )  4 ( 5 ) e5714  2009.05  [Refereed]

　View Summary

Background: The production of staphylocoagulase (SC) causing the plasma coagulation is one of the important characteristics of Staphylococcus aureus. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood. Methodology/Principal Findings: We compared the nucleotide sequences of 105 SC genes (coa), 59 of which were determined in this study. D1 regions, which contain prothrombin-activating and -binding domains and are presumed to be the binding site of each type-specific antiserum, were classified into twelve clusters having more than 90% nucleotide identities, resulting to create two novel SC types, XI and XII, in addition to extant 10 types. Nine of the twelve SC types were further subdivided into subtypes based on the differences of the D2 or the central regions. The phylogenetical relations of the D1 regions did not correlate exactly with either one of agr types and multilocus sequence types (STs). In addition, genetic analysis showed that recombination events have occurred in and around coa. So far tested, STs of 126 S. aureus strains correspond to the combination of SC type and agr type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively. Conclusion: The data suggested that the evolution of coa was not monophyletic in the species. Chromosomal recombination had occurred at coa and agr loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome.

DOI

Smqnr, a new chromosome-carried quinolone resistance gene in Stenotrophomonas maltophilia

Kenichiro Shimizu, Ken Kikuchi, Takashi Sasaki, Namiko Takahashi, Masayuki Ohtsuka, Yuka Ono, Keiichi Hiramatsu

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY ( AMER SOC MICROBIOLOGY )  52 ( 10 ) 3823 - 3825  2008.10  [Refereed]

　View Summary

A new chromosome-carried quinolone resistance gene from Stenotrophomonas maltophilia, Smqnr, was characterized. The gene was present in type strain CCUG 5866 and was also detected in 24 clinical isolates and showed some allelic diversity. The expression of Smqnr in Escherichia coli decreased the susceptibilities of the E. coli isolates to several fluoroquinolones.

DOI

Is Histoplasma capsulatum a native inhabitant of Japan ?

Ken Kikuchi, Takashi Sugita, Koichi Makimura, Kensaku Urata, Takashi Someya, Takashi Sasaki, Katsuhiko Kamei, Masakazu Niimi, Keiichi Hiramatsu, Yoshimasa Uehara

MICROBIOLOGY AND IMMUNOLOGY ( BLACKWELL PUBLISHING )  52 ( 9 ) 455 - 459  2008.09  [Refereed]

　View Summary

Histoplasmosis is an infectious disease caused by inhaling spores of the fungal pathogen H. capsulatum and in Japan is considered an imported mycosis. However, some patients in Japan with histoplasmosis have no history of traveling overseas nor of risk of occupational exposure to Histoplasma. To investigate the possibility of native distribution of Histoplasma in Japan, 187 bat guano samples from 67 bat-inhabited caves in 17 prefectures were collected. These were examined for H. capsulatum by culture and Histoplasma-specific PCR in three independent laboratories. No H. capsulatum was detected by either method, therefore H. capsulatum is unlikely to be present in bat guano in Japanese caves.

DOI

多剤耐性緑膿菌に対するaztreonamとarbekacinによる併用療法

菊池 賢, 星 最智, 佐々木 崇, 堀 賢, 平松 啓一

感染症学雑誌 ( (一社)日本感染症学会 )  82 ( 5 ) 567 - 567  2008.09

Postantibiotic effects and bactericidal activities of Levofloxacin and gatifloxacin at concentrations simulating those of topical ophthalmic administration against fluoroquinolone-resistant, and fluoroquinolone-sensitive methicillin-resistant staphylococcus aureus strains

Saichi Hoshi, Ken Kikuchi, Takashi Sasaki, Chie Sotozono, Shigeru Kinoshita, Keiichi Hiramatsu

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY ( AMER SOC MICROBIOLOGY )  52 ( 8 ) 2970 - 2973  2008.08  [Refereed]

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The bactericidal activities and postantibiotic effects (PAEs) of levofloxacin and gatifloxacin at concentrations corresponding to those in antibiotic eye drops against methicillin-resistant Staphylococcus aureus strains were determined. Levofloxacin and gatifloxacin at concentrations simulating those in eye drops showed lower bactericidal activities and shorter PAEs against fluoroquinolone-resistant strains than against fluoroquinolone-sensitive strains.

DOI

多剤耐性緑膿菌に対するaztreonamとarbekacinによる併用療法

菊池 賢, 星 最智, 佐々木 崇, 堀 賢, 平松 啓一

感染症学雑誌 ( (一社)日本感染症学会 )  82 ( 臨増 ) 248 - 248  2008.03

Reclassification of phenotypically identified Staphylococcus intermedius strains

Takashi Sasaki, Ken Kikuchi, Yoshikazu Tanaka, Namiko Takahashi, Shinichi Kamata, Keiichi Hiramatsu

JOURNAL OF CLINICAL MICROBIOLOGY   45 ( 9 ) 2770 - 2778  2007.09  [Refereed]

Authorship：   Lead author

DOI

Methicillin-resistant Staphylococcus pseudintermedius in a veterinary teaching hospital

Takashi Sasaki, Ken Kikuchi, Yoshikazu Tanaka, Namiko Takahashi, Shinichi Kamata, Keiichi Hiramatsu

JOURNAL OF CLINICAL MICROBIOLOGY   45 ( 4 ) 1118 - 1125  2007.04  [Refereed]

Authorship：   Lead author

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