研究者詳細 - 佐々木　崇

論文 - 佐々木　崇

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

Yoshikazu Tanaka, Yuka Sato, Takashi Sasaki

JOURNAL OF GENERAL VIROLOGY ( MICROBIOLOGY SOC ) 98 ( 2 ) 190 - 200 2017年02月 [査読有り]

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cistransisomerase active sites of Cyps might be required for FCoV replication.

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年02月 [査読有り]

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

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 ( JAPAN ANTIBIOTICS RESEARCH ASSOC ) 70 ( 2 ) 142 - 146 2017年02月 [査読有り]

Persulcatusin (IP), which is an antimicrobial peptide found in Ixodes persulcatus midgut, is active against Gram-positive bacteria such as Staphylococcus aureus. Multidrug-resistant bacteria in particular methicillin-resistant S. aureus (MRSA), vancomycinintermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) are a worldwide clinical concern. In the present study, to explore the potential of IP as a new agent against multidrug-resistant S. aureus infections, we evaluated the antimicrobial activity of IP against multidrug-resistant S. aureus strains by MIC90, morphological observation with scanning electron microscope (SEM), and the calcein leakage assay of membrane integrity. Among the six antimicrobial peptides used in this study, IP exhibited the lowest MIC90 values for both vancomycin-susceptible and-resistant S. aureus strains. The IP MIC90 against a VISA strain was equivalent to vancomycin, while the MIC90 against VRSA was relatively low. SEM observations indicated that bacterial cells exposed to IP were crumpled and showed prominent structural changes. Moreover, IP influenced the cell membranes of both MRSA and VRSA in a mere 5 min, leading to leakage of the preloaded calcein. Although a VISA strain was resistant to the action of IP on cell membrane, the MIC90 of IP was lower than that of Nisin, suggesting that IP had another bactericidal mechanism in addition to cell membrane attack. Our results indicate that the synthetic tick antimicrobial peptide, IP exhibits strong antibacterial activity against multidrug-resistant S. aureus strains, including VRSA, via both cell membrane attack and another unknown mechanism. IP represents a promising candidate for a new anti-VRSA therapy.

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年01月 [査読有り]

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年05月

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年 [査読有り] [国際誌]

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年12月 [査読有り]

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 ( BIOMED CENTRAL LTD ) 11 ( 57 ) 2015年03月 [査読有り]

Background: Feline infectious peritonitis is a fatal disease of cats caused by infection with feline coronavirus (FCoV). For detecting or genotyping of FCoV, some RT-PCR plus nested PCR techniques have been reported previously. However, referring to the whole genome sequences (WGSs) registered at NCBI, there are no detection methods that can tolerate the genetic diversity among FCoV population. In addition, the quasispecies nature of FCoV, which consists of heterogeneous variants, has been also demonstrated; thus, a universal method for heteropopulations of FCoV variants in clinical specimens is desirable. Results: To develop an RT-PCR method for detection and genotyping of FCoV, we performed comparative genome analysis using WGSs of 32 FCoV, 7 CCoV and 5 TGEV strains obtained from NCBI. As the PCR target, we focused on the nsp14 coding region, which is highly conserved and phylogenetically informative, and developed a PCR method targeting nsp14 partial sequences. Among 103 ascites, 45 pleural effusion and 214 blood specimens from clinically ill cats, we could detect FCoV in 55 (53.4%), 14 (31.1%) and 19 (8.9%) specimens using the present method. Direct sequencing of PCR products and phylogenetic analysis allowed discrimination between type I-and II-FCoV serotypes. Our nsp14 amino acid sequence typing (nsp14 aa ST) showed that the FCoV clone with sequence type (ST) 42, which was the most predominant genotype of WGS strains, was prevalent in domestic cats in Japan. Conclusions: Our nsp14 PCR scheme will contribute to virus detection, epidemiology and ecology of FCoV strains.

DOI

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年 [査読有り]

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年 [査読有り]

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年 [査読有り] [国際誌]

DOI PubMed

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年 [査読有り]

Future Chemotherapy Preventing Emergence of Multi-Antibiotic Resistance.

Hiramatsu K, Sasaki T, Morimoto Y

Juntendo Medical Journal. ( 順天堂医学会 ) 3 ( 3 ) 249 - 256 2015年 [査読有り]

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年09月 [査読有り]

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 ( MDPI AG ) 5 ( 5 ) 1250 - 1260 2013年05月 [査読有り]

Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS), feline infectious peritonitis (FIP), mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA), could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

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年 [査読有り]

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年11月 [査読有り]

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 ( AMER SOC MICROBIOLOGY ) 50 ( 6 ) 2152 - 2155 2012年06月 [査読有り]

We determined the population genetic structures of feline and canine Staphylococcus aureus strains in Japan by multilocus sequence typing (MLST). Ecological analyses suggested that multiple feline-related S. aureus clones, including ST133, naturally occur as commensals and can cause endogenous infections in felines. In contrast, S. aureus populations do not likely include any clone that exhibits tropism in domestic dogs. Even if S. aureus infections occur in dogs, the pathologies are likely exogenous infections.

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年04月 [査読有り]

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年10月 [査読有り]

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

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