Researchers Database

sai ryusyu

    DepartmentofInfectionandImmunityDivisionofBacteriology Professor
Last Updated :2021/11/23

Researcher Information


  • (BLANK)


Research funding number

  • 50306932


J-Global ID

Research Interests

  • Bacteriophage   Bacteriology   infectious diseases   Microbiology   Virology   

Research Areas

  • Life sciences / Ecology and environmental science
  • Life sciences / Internal medicine - General
  • Life sciences / Virology
  • Life sciences / Bacteriology

Academic & Professional Experience

  • 2015/04 - Today  Jichi Medical Univ. Prof.
  • 2012/02 - 2015/03  Vice Director, RCAI, Kitasato University Institute
  • 2007/04 - 2012/01  Juntendo Univ. Associate professor
  • 2002/04 - 2007/03  Juntendo Univ. Lecturer
  • 1998/04 - 2002/03  Juntendo Univ. Asistant professor
  • 1997/07 - 1998/03  Juntendo Univ. Visiting researcher
  • 1995/07 - 1997/06  WHO Collaborating Center for Virus Reference and Research on HFRS (Hantavirus), Seoul, Korea. Postdoc
  • 1985/07 - 1994/06  The Fourth Millitary Medical UniversityUniversity affiliated 1th hospitalPhysician, Lecturer


  •        - 1994  第四医科大学 大学院 修士・博士(医学)
  •        - 1985  第四医科大学 医学部卒業 学士

Association Memberships

  • The Japan China Medical Association   THE JAPANESE SOCIETY FOR CLINICAL MICROBIOLOGY   JAPANESE SOCIETY FOR INFECTION PREVENTION AND CONTROL   American Society for Microbiology   日本感染症学会   日本化学療法学会   日本細菌学会   

Published Papers

  • Pourya Gholizadeh, Mohammad Aghazadeh, Reza Ghotaslou, Mohammad Ahangarzadeh Rezaee, Tahereh Pirzadeh, Longzhu Cui, Shinya Watanabe, Hadi Feizi, Hiva Kadkhoda, Hossein Samadi Kafil
    Annals of Clinical Microbiology and Antimicrobials 20 (1) 2021/12 
    AbstractClustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are one of the factors which can contribute to limiting the development and evolution of antibiotic resistance in bacteria. There are three genomic loci of CRISPR-Cas in Enterococcus faecalis. In this study, we aimed to assess correlation of the CRISPR-Cas system distribution with the acquisition of antibiotic resistance among E. faecalis isolates. A total of 151 isolates of E. faecalis were collected from urinary tract infections (UTI) and dental-root canal (DRC). All isolates were screened for phenotypic antibiotic resistance. In addition, antibiotic resistance genes and CRISPR loci were screened by using polymerase chain reaction. Genomic background of the isolates was identified by random amplified polymorphic DNA (RAPD)-PCR. The number of multidrug-resistant E. faecalis strains were higher in UTI isolates than in DRC isolates. RAPD-PCR confirmed that genomic background was diverse in UTI and DRC isolates used in this study. CRISPR loci were highly accumulated in gentamycin-, teicoplanin-, erythromycin-, and tetracycline-susceptible strains. In concordance with drug susceptibility, smaller number of CRISPR loci were identified in vanA, tetM, ermB, aac6’-aph(2”), aadE, and ant(6) positive strains. These data indicate a negative correlation between CRISPR-cas loci and antibiotic resistance, as well as, carriage of antibiotic resistant genes in both of UTI and DRC isolates.
  • Harumi Koibuchi, Yasutomo Fujii, Yusuke Sato’o, Takashi Mochizuki, Toshiyuki Yamada, Longzhu Cui, Nobuyuki Taniguchi
    Journal of Medical Ultrasonics 1346-4523 2021/08 
    Abstract Purpose We aimed to investigate whether low-intensity continuous and pulsed wave ultrasound (US) irradiation can inhibit the formation of Staphylococcus epidermidis biofilms, for potential application in the treatment of catheter-related bloodstream infections (CRBSI). Methods S. epidermidis biofilms that formed on the bottom surfaces of 6-well plates were irradiated on the bottom surface using the sound cell incubator system for different intervals of time. Results US irradiation with continuous waves for 24 h notably inhibited biofilm formation (p < 0.01), but the same US irradiation for 12 h had no remarkable effect. Further, double US irradiation with pulsed waves for 20 min inhibited biofilm formation by 33.6%, nearly two-fold more than single US irradiation, which reduced it by 17.9%. Conclusion US irradiation of a lower intensity (ISATA = 6–29 mW/cm2) than used in a previous study and lower than recommended by the Food and Drug Administration shows potential for preventing CRBSI caused by bacterial biofilms.
  • Aa Haeruman Azam, Xin-Ee Tan, Srivani Veeranarayanan, Kotaro Kiga, Longzhu Cui
    Antibiotics 10 (8) 999 - 999 2021/08 
    The bacteriophage (or phage for short) has been used as an antibacterial agent for over a century but was abandoned in most countries after the discovery and broad use of antibiotics. The worldwide emergence and high prevalence of antimicrobial-resistant (AMR) bacteria have led to a revival of interest in the long-forgotten antibacterial therapy with phages (phage therapy) as an alternative approach to combatting AMR bacteria. The rapid progress recently made in molecular biology and genetic engineering has accelerated the generation of phage-related products with superior therapeutic potentials against bacterial infection. Nowadays, phage-based technology has been developed for many purposes, including those beyond the framework of antibacterial treatment, such as to suppress viruses by phages, gene therapy, vaccine development, etc. Here, we highlighted the current progress in phage engineering technology and its application in modern medicine.
  • Katsuyuki Katahira, Shinya Watanabe, Kentaro Wakamatsu, Zenzo Nagasawa, Masayuki Kawasaki, Longzhu Cui
    Microbiology Resource Announcements 10 (27) 2021/07 
    We report the complete genome sequence of Mycobacterium heckeshornense strain JMUB5695, which was isolated from necrotizing granulomatous lesions in a lung cancer patient. The complete genome consists of a 4,865,109-bp chromosome with a GC content of 65.9% and contains no plasmids.
  • Ahmed Tabbabi, Shinya Watanabe, Daiki Mizushima, Abraham G. Caceres, Eduardo A Gomez, Daisuke S. Yamamoto, Longzhu Cui, Yoshihisa Hashiguchi, Hirotomo Kato
    Microorganisms 9 (1) 68 - 68 2020/12 
    Differences in the gut microbial content of Lutzomyia (Lu.) ayacuchensis, a primary vector of Andean-type cutaneous leishmaniasis in Ecuador and Peru, may influence the susceptibility of these sand flies to infection by Leishmania. As a first step toward addressing this hypothesis, a comparative analysis of bacterial and fungal compositions from Lu. ayacuchensis populations with differential susceptibilities to Leishmania was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing approaches were used to characterize the bacterial composition in wild-caught populations from the Andean areas of Ecuador and southern Peru at which the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and a population from the northern Peruvian Andes at which the transmission of Leishmania by Lu. ayacuchensis has not been reported. In the present study, 59 genera were identified, 21 of which were widely identified and comprised more than 95% of all bacteria. Of the 21 dominant bacterial genera identified in the sand flies collected, 10 genera had never been detected in field sand flies. The Ecuador and southern Peru populations each comprised individuals of particular genera, while overlap was clearly observed between microbes isolated from different sites, such as the number of soil organisms. Similarly, Corynebacterium and Micrococcus were slightly more dominant bacterial genera in the southern Peru population, while Ochrobactrum was the most frequently isolated from other populations. On the other hand, fungi were only found in the southern Peru population and dominated by the Papiliotrema genus. These results suggest that variation in the insect gut microbiota may be elucidated by the ecological diversity of sand flies in Peru and Ecuador, which may influence susceptibility to Leishmania infection. The present study provides key insights for understanding the role of the microbiota during the course of L. (L.) mexicana and L. (V.) peruviana infections in this important vector.
  • Kanate Thitiananpakorn, Yoshifumi Aiba, Xin-Ee Tan, Shinya Watanabe, Kotaro Kiga, Yusuke Sato’o, Tanit Boonsiri, Feng-Yu Li, Teppei Sasahara, Yusuke Taki, Aa Haeruman Azam, Yuancheng Zhang, Longzhu Cui
    Scientific Reports 10 (1) 16107 - 16107 2020/12 [Refereed]
    Abstract We first reported a phenomenon of cross-resistance to vancomycin (VCM) and daptomycin (DAP) in methicillin-resistant Staphylococcus aureus (MRSA) in 2006, but mechanisms underlying the cross-resistance remain incompletely understood. Here, we present a follow-up study aimed to investigate genetic determinants associated with the cross-resistance. Using 12 sets of paired DAP susceptible (DAPS) and DAP non-susceptible (DAPR) MRSA isolates from 12 patients who had DAP therapy, we (i) assessed susceptibility to DAP and VCM, (ii) compared whole-genome sequences, (iii) identified mutations associated with cross-resistance to DAP and VCM, and (iv) investigated the impact of altered gene expression and metabolic pathway relevant to the cross-resistance. We found that all 12 DAPR strains exhibiting cross-resistance to DAP and VCM carried mutations in mprF, while one DAPR strain with reduced susceptibility to only DAP carried a lacF mutation. On the other hand, among the 32 vancomycin-intermediate S. aureus (VISA) strains isolated from patients treated with VCM, five out of the 18 strains showing cross-resistance to DAP and VCM carried a mprF mutation, while 14 strains resistant to only VCM had no mprF mutation. Moreover, substitution of mprF in a DAPS strain with mutated mprF resulted in cross-resistance and vice versa. The elevated lysyl-phosphatidylglycerol (L-PG) production, increased positive bacterial surface charges and activated cell wall (CW) synthetic pathways were commonly found in both clinical isolates and laboratory-developed mutants that carry mprF mutations. We conclude that mprF mutation is responsible for the cross-resistance of MRSA to DAP and VCM, and treatment with DAP is more likely to select for mprF-mediated cross-resistance than is with VCM.
  • Tanit Boonsiri, Shinya Watanabe, Xin-Ee Tan, Kanate Thitiananpakorn, Ryu Narimatsu, Kosuke Sasaki, Remi Takenouchi, Yusuke Sato’o, Yoshifumi Aiba, Kotaro Kiga, Teppei Sasahara, Yusuke Taki, Feng-Yu Li, Yuancheng Zhang, Aa Haeruman Azam, Tomofumi Kawaguchi, Longzhu Cui
    Scientific Reports 10 (1) 16907 - 16907 2020/12 [Refereed]
    Abstract Staphylococcus aureus strains that are susceptible to the β-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire β-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0–256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of β-lactam resistance in OS-MRSA.
  • Teppei Sasahara, Ryusuke Ae, Akio Yoshimura, Koki Kosami, Kazumasa Sasaki, Yumiko Kimura, Dai Akine, Masanori Ogawa, Kenji Hamabata, Shuji Hatakeyama, Longzhu Cui
    BMC Geriatrics 20 (1) 481 - 481 2020/12 [Refereed]
    Abstract Background A high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization has been reported among residents in geriatric long-term care facilities (LTCFs). Some studies indicate that MRSA might be imported from hospitals into LTCFs via resident transfer; however, other studies report that high MRSA prevalence might be caused by cross-transmission inside LTCFs. We aimed to assess which factors have a large impact on the high MRSA prevalence among residents of geriatric LTCFs. Methods We conducted a cohort study among 260 residents of four geriatric LTCFs in Japan. Dividing participants into two cohorts, we separately analyzed (1) the association between prevalence of MRSA carriage and length of LTCF residence (Cohort 1: n = 204), and (2) proportion of residents identified as MRSA negative who were initially tested at admission but subsequently identified as positive in secondary testing performed at ≥2 months after their initial test (Cohort 2: n = 79). Results Among 204 residents in Cohort 1, 20 (9.8%) were identified as positive for MRSA. Compared with residents identified as MRSA negative, a larger proportion of MRSA-positive residents had shorter periods of residence from the initial admission (median length of residence: 5.5 vs. 2.8 months), although this difference was not statistically significant (p = 0.084). Among 79 residents in Cohort 2, 60 (75.9%) were identified as MRSA negative at the initial testing. Of these 60 residents, only one (1.7%) had subsequent positive conversion in secondary MRSA testing. In contrast, among 19 residents identified as MRSA positive in the initial testing, 10 (52.6%) were negative in secondary testing. Conclusions The prevalence of MRSA was lower among residents with longer periods of LTCF residence than among those with shorter periods. Furthermore, few residents were found to become MRSA carrier after their initial admission. These findings highlight that MRSA in LTCFs might be associated with resident transfer rather than spread via cross-transmission inside LTCFs.
  • ファージテクノロジーと現代医療
    氣駕 恒太朗, 崔 龍洙
    実験医学 38 (17) 207 - 213 2020/11 [Refereed]
  • Kotaro Kiga, Xin-Ee Tan, Rodrigo Ibarra-Chávez, Shinya Watanabe, Yoshifumi Aiba, Yusuke Sato'o, Feng-Yu Li, Teppei Sasahara, Bintao Cui, Moriyuki Kawauchi, Tanit Boonsiri, Kanate Thitiananpakorn, Yusuke Taki, Aa Haeruman Azam, Masato Suzuki, José R Penadés, Longzhu Cui
    Nature communications 11 (1) 2934 - 2934 2020/06 [Refereed][Not invited]
    The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.
  • Yoshifumi Aiba, Shinya Watanabe, Rieko Tsukahara, Naoka Umemoto, Kanate Thitiananpakorn, Tanit Boonsiri, Feng-Yu Li, Kotaro Kiga, Yusuke Sato’o, Xin-Ee Tan, Yusuke Taki, Aa Haeruman Azam, Yuancheng Zhang, Teppei Sasahara, Toshio Demitsu, Longzhu Cui
    Microbiology Resource Announcements 9 (23) 2020/06 [Refereed]
    The association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by Staphylococcus aureus remains controversial. Here, we report the complete genome sequence of the PVL-negative S. aureus strain JMUB1273, isolated from a patient with pervasive NSTI.
  • Shinya Watanabe, Bintao Cui, Kotaro Kiga, Yoshifumi Aiba, Xin Ee Tan, Yusuke Sato'o, Moriyuki Kawauchi, Tanit Boonsiri, Kanate Thitiananpakorn, Yusuke Taki, Fen Yu Li, Aa Haeruman Azam, Yumi Nakada, Teppei Sasahara, Longzhu Cui
    Frontiers in Microbiology 11 2020/02 
    In the original article, there was a mistake in Table 1 as published. “GC% of L. wadei JMUB3933, JMUB3934, JCM16777, Leptotrichia sp.-1 JMUB3936, L. shahii JCM16776, L. hofstadii JCM16775, L. trevisanii JMUB3870, JMUB4039, JMUB3935 and L. buccalis C-1013-b, Leptotrchia sp.-3 F0260, Leptotrichia sp. F0590, L. goodfellowi JCM16774 and Leptotrichia sp.-6W10393, and chromosome length of L. wadei JCM16777” were incorrect. The corrected Table 1 appears below. In the original article, there was an error. GC% of genome-sequenced strains was incorrect. A correction has been made to Results and Discussion, Comparative Analysis of Leptotrichia Genome, line 373-375: As shown in Table 1, the chromosome size of the genus Leptotrichia varies from 2,142,946 to 2,829,322 bp with GC contents of 29.5% to 31.7%. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.
  • Janyerkye Tulyeu, Hideki Kumagai, Eriko Jimbo, Shinya Watanabe, Koji Yokoyama, Longzhu Cui, Hitoshi Osaka, Makiko Mieno, Takanori Yamagata
    Microorganisms 7 (10) 463  2019/10 [Refereed][Not invited]
  • Tan XE, Neoh HM, Cui L, Hiramatsu K, Jamal R
    Canadian journal of microbiology 65 (8) 623 - 628 0008-4166 2019/08 [Refereed][Not invited]
  • Azam AH, Kadoi K, Miyanaga K, Usui M, Tamura Y, Cui L, Tanji Y
    Applied microbiology and biotechnology 103 (16) 6809 - 6823 0175-7598 2019/08 [Refereed][Not invited]
  • Dai Akine, Teppei Sasahara, Shinya Watanabe, Yohei Ishishita, Takashi Yamaguchi, Longzhu Cui, Yuji Morisawa
    IDCases 18 e00622 - e00622 2214-2509 2019/01 [Refereed]
    Klebsiella variicola, a member of K. pneumoniae phylogroup, can cause severe infectious diseases. We report a case of K. variicola meningitis after neurosurgery. The bacterium was isolated from blood and cerebrospinal fluid, and bacterial species identification was carried out by using both matrix-assisted laser-desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) and whole genome sequencing. Initially, the organism was misidentified as K. pneumoniae by VITEK®2; automated system in the clinical laboratory examination. The patient recovered with the combination of surgical drainage and antimicrobial treatment. To our knowledge, this is the first case report of post-surgical meningitis caused by K. variicola. As experienced in this case, the automated bacterial identification system popularly being used in the clinical laboratory might not be effective enough for bacterial species identification. The use of MALDI-TOF MS for microbial identification may be helpful to physicians for appropriate management of K. pneumoniae phylogroup infection.
  • Shinya Watanabe, Bintao Cui, Kotaro Kiga, Yoshifumi Aiba, Xin-Ee Tan, Yusuke Sato'o, Moriyuki Kawauchi, Tanit Boonsiri, Kanate Thitiananpakorn, Yusuke Taki, Fen-Yu Li, Aa Haeruman Azam, Yumi Nakada, Teppei Sasahara, Longzhu Cui
    Frontiers in microbiology 10 2838 - 2838 2019 [Refereed][Not invited]
    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.
  • Bintao Cui, Shinya Watanabe, Yusuke Sato'o, Fumiya Nihashi, Yoshifumi Aiba, Kotaro Kiga, Teppei Sasahara, Xin-Ee Tan, Moriyuki Kawauchi, Tanit Boonsiri, Kanate Thitiananpakorn, Yusuke Taki, Feng-Yu Li, Shiro Imokawa, Longzhu Cui
    Microbiology resource announcements 8 (4) 2019/01 [Refereed][Not invited]
    Severe community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) is relatively rare and is usually associated with rapid progression to death. Here, we report the complete genome sequence of the MRSA strain JMUB3031, which was isolated from a patient with fatal CAP.
  • Kanesaka I, Fujisaki S, Aiba Y, Watanabe S, Mikawa T, Katsuse A K, Takahashi H, Cui L, Kobayashi I
    Journal of Infection and Chemotherapy 25 (1) 1 - 5 2019/01 [Refereed][Not invited]
  • Shinya Watanabe, Yoshifumi Aiba, Xin-Ee Tan, Feng-Yu Li, Tanit Boonsiri, Kanate Thitiananpakorn, Bintao Cui, Yusuke Sato'o, Kotaro Kiga, Teppei Sasahara, Longzhu Cui
    BMC genomics 19 (1) 810 - 810 2018/11 [Refereed][Not invited]
    BACKGROUND: Staphylococcus caprae is an animal-associated bacterium regarded as part of goats' microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. RESULTS: We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. CONCLUSION: The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.
  • Ishida Y, Sakai E, Sato K, Sugiyama E, Mima K, Taneno A, Shimomura H, Cui L, Hirai Y
    Food Safety 6 (4) 151 - 155 2018/10 [Refereed][Not invited]
  • Yusuke Sato'o, Yoshifumi Aiba, Kotaro Kiga, Shinya Watanabe, Teppei Sasahara, Yasuhiko Hayakawa, Longzhu Cui
    Journal of microbiological methods 146 25 - 32 2018/03 [Refereed][Not invited]
    Electroporation is a common technique necessary for genomic manipulation of Staphylococci. However, because this technique has too low efficiency to be applied to some Staphylococcal species and strains, especially to coagulase-negative Staphylococcus (CNS) isolates, basic researches on these clinically important Staphylococci are limited. Here we report on the optimization of electroporation parameters and conditions as well as on the generation of a universal protocol that can be efficiently applicable to both CNS and Coagulase-positive Staphylococci (CPS). This protocol could generate transformants of clinical Staphylococcus epidermidis isolate, with an efficiency of up to 1400 CFU/μg of plasmid DNA. Transformants of 12 other clinically important Staphylococcal species, including CNS and CPS, were also generated with this protocol. To our knowledge, this is the first report on successful electroporation in nine these Staphylococcal species.
  • Xin-Ee Tan, Hui-min Neoh, Mee-Lee Looi, Siok Fong Chin, Longzhu Cui, Keiichi Hiramatsu, Salasawati Hussin, Rahman Jamal
    CANADIAN JOURNAL OF MICROBIOLOGY 63 (3) 260 - 264 0008-4166 2017/03 [Refereed][Not invited]
    Comparative proteomic profiling between 2 vancomycin-intermediate Staphylococcus aureus (VISA) strains, Mu50 Omega-vraSm and Mu50 Omega-vraSm-graRm, and vancomycin-susceptible S.aureus (VSSA) strain Mu50 Omega revealed upregulated levels of catabolic ornithine carbamoyltransferase (ArcB) of the arginine catabolism pathway in VISA strains. Subsequent analyses showed that the VISA strains have higher levels of cellular ATP and ammonia, which are by-products of arginine catabolism, and displayed thicker cell walls. We postulate that elevated cytoplasmic ammonia and ATP molecules, resulting from activated arginine catabolism upon acquisition of vraS and graR mutations, are important requirements facilitating cell wall biosynthesis, thereby contributing to thickened cell wall and consequently reduced vancomycin susceptibility in VISA strains.
  • Tan XE, Neoh HM, Looi ML, Tan TL, Hussin S, Cui L, Hiramatsu K, Jamal R
    Malaysian Journal of Microbiology 12 (6) 498 - 505 2016/05 [Refereed][Not invited]
  • Shinya Watanabe, Teppei Sasahara, Naoshi Arai, Kazumasa Sasaki, Yoshifumi Aiba, Yusuke Sato'o, Longzhu Cui
    Genome Announcements 4 (5) e01133-16  2169-8287 2016 [Refereed][Not invited]
    Acute phlegmonous gastritis is an uncommon endogenous bacterial gastritis presenting with a high mortality rate. Here, we report the complete genome sequence of an emm89 Streptococcus pyogenes strain, JMUB1235, which is the causative agent of acute phlegmonous gastritis.
  • Katsunori Masaki, Makoto Ishii, Masaki Anraku, Ho Namkoong, Ryo Miyakawa, Takeshi Nakajima, Koichi Fukunaga, Katsuhiko Naoki, Sadatomo Tasaka, Kenzo Soejima, Koichi Sayama, Kayoko Sugita, Satoshi Iwata, Longzhu Cui, Hideaki Hanaki, Naoki Hasegawa, Tomoko Betsuyaku
    The American journal of case reports 16 454 - 8 2015/07 [Refereed][Not invited]
    BACKGROUND: Increasing evidence has indicated that Staphylococcus aureus pneumonia complicated with influenza virus infection is often fatal. In these cases, disease severity is typically determined by susceptibility to antimicrobial agents and the presence of high-virulence factors that are produced by Staphylococcus aureus, such as Panton-Valentine leukocidin (PVL). CASE REPORT: We describe a rare case of fatal community-acquired pneumonia caused by methicillin-sensitive Staphylococcus aureus (MSSA), which did not secrete major high-virulence factors and coexisted with influenza type B infection. The 32-year-old previously healthy male patient presented with dyspnea, high fever, and cough. His roommate had been diagnosed with influenza B virus infection 3 days earlier. Gram-positive clusters of cocci were detected in the patient's sputum; therefore, he was diagnosed with severe pneumonia and septic shock, and was admitted to the intensive care unit. Despite intensive antibiotic and antiviral treatment, he died of multiple organ failure 5 days after admission. His blood culture from the admission was positive for MSSA, and further analysis revealed that the strain was negative for major high-virulence factors, including PVL and enterotoxins, although influenza B virus RNA was detected by PCR. CONCLUSIONS: Physicians should pay special attention to patients with pneumonia following influenza and Staphylococcus aureus infection, as it may be fatal, even if the Staphylococcus aureus strain is PVL-negative and sensitive to antimicrobial agents.
  • Nihonyanagi S, Sunakawa K, Cui L, Masaki T, Wada T, Hoshiyama T, Nakamura M, Takayama Y, Kanoh Y, Ogawa A, Shichiri M, Hanaki H
    Clinical case reports 3 (2) 76 - 80 2015/02 [Refereed][Not invited]
    We report here a very rare case of primary meningococcal arthritis of the knee joint without clinical features associated with meningococcemia, meningitis, or meningococcal complications. The patient suffered from diabetes mellitus and had experienced two episodes of joint trauma. Intravenous infusion of ampicillin/sulbactam for 18 consecutive days was successful.
  • Michie Saito, Yuki Katayama, Tomomi Hishinuma, Akira Iwamoto, Yoshifumi Aiba, Kyoko Kuwahara-Arai, Longzhu Cui, Miki Matsuo, Nanae Aritaka, Keiichi Hiramatsua
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 58 (9) 5024 - 5035 0066-4804 2014/09 [Refereed][Not invited]
    Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (>= 1 x 10(-6)). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated "slow VISA" (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to similar to 24 mg/liter when determined after 72 to similar to 144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase beta-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB(R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.
  • Hideaki Hanaki, Longzhu Cui, Yurika Ikeda-Dantsuji, Taiji Nakae, Junichi Honda, Katsunori Yanagihara, Yoshio Takesue, Tetsuya Matsumoto, Keisuke Sunakawa, Mitsuo Kaku, Kazunori Tomono, Kunihiko Fukuchi, Shinya Kusachi, Hiroshige Mikamo, Tohru Takata, Yoshihito Otsuka, Osanori Nagura, Shigeki Fujitani, Yosuke Aoki, Yoshio Yamaguchi, Kazuhiro Tateda, Junichi Kadota, Shigeru Kohno, Yoshihito Niki
    JOURNAL OF INFECTION AND CHEMOTHERAPY 20 (9-10) 527 - 534 1341-321X 2014/09 [Refereed][Not invited]
    We conducted an antibiotic susceptibility survey of 830 blood-borne methicillin resistant Staphylococcus aureus collected from nationwide hospitals in Japan over a three-year period from January 2008 through May 2011. Antibiotic susceptibility was judged according to the criteria recommended by the Clinical Laboratory Standard Institute. Over 99% of the MRSA showed to be susceptible to teicoplanin, linezolid, sulfamethoxazole/trimethoprim and vancomycin, and over 97% of them were susceptible to daptomycin, arbekacin and rifampin. The majority of the MRSA strains showed resistant to minocycline, meropenem, imipenem, clindamycin, ciprofloxacin, cefoxitin, and oxacillin in the rates of 56.6, 72.9, 73.7, 78.7, 89.0, 99.5, and 99.9%, respectively. Among the MRSA strains, 72 showed reduced susceptibility to vancomycin, including 8 strains (0.96%) of vancomycin-intermediate S. aureus (VISA), 54 (6.51%) of heterogeneous vancomycin-intermediate S. aureus (hVISA), and 55 (5.63%) of beta-lactam antibiotics-induced vancomycin resistant S. aureus (BIVR). Unexpectedly, among the 54 hVISA and 55 BIVR, 45 isolates (83.3% and 81.8%, respectively) showed both hVISA and BIVR phenotypes. A new trend of vancomycin resistance found in this study was that VISA strains were still prevalent among the bacteremic specimens. The high rates of the hVISA/BIVR two-phenotypic vancomycin resistance, and the prevalence of VISA in the bloodborne MRSA call attention in the MRSA epidemiology in Japan. (C) 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
  • 'Slow VISA' (sVISA), a novel phenotype of vancomycin resistance, obtained in vitro from hVISA strain Mu3.
    Michie Saito, Yuki Katayama, Tomomi Hishinuma, Akira Iwamoto, Yoshifumi Aiba, Kyoko Kuwahara-Arai, Longzhu Cui, Miki Matsuo, Nanae Aritaka, Keiichi Hiramatsu, Michie Saito, Yuki Katayama contribute equally to this study
    Antimicrobial Agents and Chemotherapy 58 (9) 5024 - 5035 2014 [Refereed][Not invited]
  • Miki Matsuo, Longzhu Cui, Jeeyoung Kim, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 57 (12) 5843 - 5853 0066-4804 2013/12 [Refereed][Not invited]
    Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10(-6) or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the beta subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.
  • Yoshifumi Aiba, Yuki Katayama, Tomomi Hishinuma, Hiroko Murakami-Kuroda, Longzhu Cui, Keiichi Hiramatsu
    Antimicrobial Agents and Chemotherapy 57 (10) 4861 - 4871 0066-4804 2013/10 [Refereed][Not invited]
    Three types of phenotypic expression of β-lactam resistance have been reported in methicillin-resistant Staphylococcus aureus (MRSA): heterogeneous, homogeneous, and Eagle-type resistance. Heterogeneous-to-homogeneous conversion of β-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) in addition to the expression of the mecA gene. Eagle-type resistance is a unique phenotype of chr occurring in pre-MRSA strain N315 whose mecA gene expression is strongly repressed by an intact mecI gene. We here report that certain mutations of the rpoB gene, encoding the RNA polymerase β subunit, belong to chr. We studied homogeneous MRSA (homo-MRSA) strain N315ΔIP-H5 (abbreviated as ΔIP-H5), which was obtained from hetero-MRSA strain N315ΔIP by selection with 8 mg/liter imipenem. Whole-genome sequencing of ΔIP-H5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino acid replacement of Asn by Ile at position 967 of RpoB. The effect of the rpoB(N967I) mutation was confirmed by constructing a revertant H5 rpoB(I967N) strain as well as an N315-derived mutant, N315 rpoB(N967I). H5 rpoB(I967N) regained the hetero-resistance phenotype, and the N315 rpoB(N967I) strain showed an Eagle-type phenotype similar to that of the typical Eagle-type MRSA strain N315h4. Furthermore, subsequent whole-genome sequencing revealed that N315h4 also had a missense mutation of rpoB(R644H). Introduction of the rpoB(N967I) mutation was accompanied by decreased autolysis, prolonged doubling time, and tolerance to bactericidal concentrations of methicillin. We consider that rpoB mutations are the major cause for heterogeneous-to-homogeneous phenotypic conversion of β-lactam resistance in MRSA strain N315 and its derived strains. Copyright © 2013, American Society for Microbiology. All Rights Reserved.
  • Hidehito Matsui, Juri Kimura, Masato Higashide, Yoshio Takeuchi, Kuniyuki Okue, Longzhu Cui, Taiji Nakae, Keisuke Sunakawa, Hideaki Hanaki
    Clinical and Vaccine Immunology 20 (9) 1381 - 1387 1556-6811 2013/09 [Refereed][Not invited]
    Group B Streptococcus (GBS Streptococcus agalactiae) is a leading cause of serious neonatal infections. The Centers for Disease Control and Prevention recommends GBS screening for all pregnant women during the 35th to 37th weeks of gestation. Although GBSscreening has been performed mainly by the culture-based method, it takes several days to obtain a reliable result. In this study, we developed a rapid immunochromatographic test (ICT) for the detection of GBS-specific surface immunogenic protein in 15 min using an overnight enrichment culture. The ICT was prepared using two anti-Sip monoclonal antibodies. This ICT was able to detect recombinant Sip levels of 0.5 ng/ml, or about 10 6 CFU/ml of GBS cells, in tests with 9 GBS strains of different serotypes. The cross-reactivity test using 26 species of microorganism showed no detectable false-positive result. Reactivity of the ICT with 229 GBS strains showed one false-negative result that was attributable to the production of truncated Sip. Among 260 enrichment cultures of vaginal swabs, 17 produced red to orange pigments in Granada medium, and they were all GBS and Sip positive. Among 219 pigment-negative cultures, 12 were GBS positive and 10 were Sip positive. Two Sip-negative cultures contained GBS cells below the limit of detection by the ICT. Among 207 GBS-negative cultures, only one was Sip positive, which was attributable to GBS cell debris. Thus, the sensitivity and specificity of the ICT appeared to be 93.1% and 99.6%, respectively. The newly developed ICT is readily applicable to clinical use in the detection of GBS. Copyright © 2013, American Society for Microbiology.
  • Jun Yamakawa, Mayumi Aminaka, Katsuko Okuzumi, Hiroyoshi Kobayashi, Yuki Katayama, Shigemi Kondo, Ayako Nakamura, Toyoko Oguri, Satoshi Hori, Longzhu Cui, Teruyo Ito, Jingxun Jin, Hisashi Kurosawa, Kazuo Kaneko, Keiichi Hiramatsu
    JOURNAL OF INFECTION AND CHEMOTHERAPY 18 (3) 406 - 409 1341-321X 2012/06 [Refereed][Not invited]
    Vancomycin-intermediate Staphylococcus aureus (VISA) and its precursor, heterogeneous VISA (hVISA), are increasingly the cause of vancomycin treatment failure. Prolonged glycopeptide treatment causes the emergence of these pathogens. However, we recently reported that hVISA can be generated by methicillin-resistant S. aureus (MRSA) exposure to imipenem (Katayama et al., Antimicrob Agents Chemother. 53:3190-6). We report here a retrospective prevalence study of VISA and hVISA on 750 MRSA clinical strains isolated from 31 Japanese national university hospitals in 1990, the year before the introduction of injectable vancomycin into clinical use in Japan in 1991. No VISA strain was identified, but population analysis identified 38 hVISA strains (5.1%) from 19 hospitals. We also determined the nucleotide sequences of vraSR, walRK, clpP, and rpoB genes whose mutations are known to be associated with vancomycin resistance. When compared with vancomycin-susceptible MRSA strain N315, six of the 38 hVISA strains possessed nonsynonymous mutations in vraSR, seven in walRK, and two in rpoB genes, Thirteen of 38 (34.2%) hVISA strains possessed at least one of these mutations. Results were consistent with our hypothesis that hVISA was present in Japanese hospitals before the clinical introduction of vancomycin.
  • Longzhu Cui, Hui-min Neoh, Akira Iwamoto, Keiichi Hiramatsu
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 (25) E1647 - E1656 0027-8424 2012/06 [Refereed][Not invited]
    Genome inversions are ubiquitous in organisms ranging from prokaryotes to eukaryotes. Typical examples can be identified by comparing the genomes of two or more closely related organisms, where genome inversion footprints are clearly visible. Although the evolutionary implications of this phenomenon are huge, little is known about the function and biological meaning of this process. Here, we report our findings on a bacterium that generates a reversible, large-scale inversion of its chromosome (about half of its total genome) at high frequencies of up to once every four generations. This inversion switches on or off bacterial phenotypes, including colony morphology, antibiotic susceptibility, hemolytic activity, and expression of dozens of genes. Quantitative measurements and mathematical analyses indicate that this reversible switching is stochastic but self-organized so as to maintain two forms of stable cell populations (i.e., small colony variant, normal colony variant) as a bet-hedging strategy. Thus, this heritable and reversible genome fluctuation seems to govern the bacterial life cycle; it has a profound impact on the course and outcomes of bacterial infections.
  • Akira Komoto, Longzhu Cui, Noriko Ebata, Yukiko Watanabe, Miki Matsuo, Yuki Katayama, Piyama Petcharoen, Keiichi Hiramatsu
    Juntendo Medical Journal 58 498 - 505 2012 [Refereed][Not invited]
  • Miki Matsuo, Tomomi Hishinuma, Yuki Katayama, Longzhu Cui, Maria Kapi, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 (9) 4188 - 4195 0066-4804 2011/09 [Refereed][Not invited]
    The clinical vancomycin-intermediate Staphylococcus aureus (VISA) strain Mu50 carries two mutations in the vraSR and graRS two-component regulatory systems (TCRSs), namely, vraS(I5N) and graR(N197S) (hereinafter designated graR*). The clinical heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3 shares with Mu50 the mutation in vraS that encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR*, carrying the mutated two-component response regulator graR*, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy of graR* into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR* thus obtained remained hVISA (MIC <= 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50 rpoB gene, encoding the RNA polymerase beta subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR* with rifampin gave rise to rpoB mutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR* were found to possess rpoB mutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneous rpoB mutants with various degrees of increased vancomycin resistance.
  • Mitsutaka Shoji, Longzhu Cui, Risa Iizuka, Akira Komoto, Hui-min Neoh, Yukiko Watanabe, Tomomi Hishinuma, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 (8) 3870 - 3881 0066-4804 2011/08 [Refereed][Not invited]
    Vancomycin-intermediate Staphylococcus aureus (VISA) is generated from vancomycin-susceptible Staphylococcus aureus by multiple spontaneous mutations. We previously reported that sequential acquisition of mutations in the two-component regulatory systems vraSR and graRS was responsible for the VISA phenotype of strain Mu50. Here we report on the identification of a novel set of regulator mutations, a deletion mutation in two-component regulatory system walRK (synonyms, vicRK and yycFG), and a truncating mutation in a proteolytic regulatory gene, clpP, responsible for the raised vancomycin resistance in a laboratory-derived VISA strain, LR5P1-V3. The contributory effect of the two mutations to vancomycin resistance was confirmed by introducing the walK and clpP mutations into the vancomycin-susceptible parent strain N315LR5P1 by a gene replacement procedure. The vancomycin MIC of N315LR5P1 was raised from 1 to 2 mg/liter by the introduction of the walK or clpP mutation, but it was raised to 4 mg/liter by the introduction of both the walK and clpP mutations. The vancomycin MIC value of the double mutant was equivalent to that of strain LR5P1-V3. Like VISA clinical strains, LR5P1-V3 and the double mutant strain LR5P1walK*clpP* exhibited a thickened cell wall, slow growth, and decreased autolytic activity. Transcriptional profiles of the mutants with gene replacements demonstrated that introduction of both the walK and clpP mutations could alter expression of dozens or hundreds of genes, including those involved in cell envelope and cellular processes, intermediary metabolism, and information pathway. A mutation prevalence study performed on 39 worldwide clinical VISA strains showed that 61.5, 7.7, 10.3, and 20.5% of VISA strains harbored mutations in walRK, clpP, graRS, and vraSR, respectively. The mutation of walRK was most frequently carried by VISA strains. Together, these results suggested that the mutations of walK and clpP identified in LR5P1-V3 constitute a new combination of genetic events causing vancomycin resistance in Staphylococcus aureus.
  • Yukiko Watanabe, Longzhu Cui, Yuki Katayama, Kishii Kozue, Keiichi Hiramatsu
    JOURNAL OF CLINICAL MICROBIOLOGY 49 (7) 2680 - 2684 0095-1137 2011/07 [Refereed][Not invited]
    Of 38 vancomycin-intermediate Staphylococcus aureus (VISA) clinical strains, 27 (71%) possessed a mutation(s) in rpoB encoding the beta-subunit of RNA polymerase. Furthermore, 95.6% of the rifampin-resistant mutants obtained from 9 methicillin-resistant S. aureus (MRSA) clinical isolates showed decreased vancomycin susceptibilities. These data indicate the involvement of an rpoB mutation in VISA phenotype expression.
  • Longzhu Cui, Taisuke Isii, Minoru Fukuda, Tomonori Ochiai, Hui-min Neoh, Ilana Lopes Baratella da Cunha Camargo, Yukiko Watanabe, Mitsutaka Shoji, Tomomi Hishinuma, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 54 (12) 5222 - 5233 0066-4804 2010/12 [Refereed][Not invited]
    We have previously reported the establishment of a Staphylococcus aureus laboratory strain, 10*3d1, having reduced susceptibility to daptomycin and heterogeneous vancomycin-intermediate S. aureus (VISA) phenotype. The strain was generated in vitro by serial daptomycin selection (Camargo, I. L., H. M. Neoh, L. Cui, and K. Hiramatsu, Antimicrob. Agents Chemother. 52: 4289-4299, 2008). Here we explored the genetic mechanism of resistance in the strain by whole-genome sequencing and by producing gene-replaced strains. By genome comparison between 10*3d1 and its parent methicillin-resistant Staphylococcus aureus (MRSA) strain N315 Delta IP, we identified five nonsynonymous single nucleotide polymorphisms (SNPs). One of the five mutations was found in the rpoB gene encoding the RNA polymerase beta subunit. The mutation at nucleotide position 1862 substituted the 621st alanine by glutamic acid. The replacement of the intact rpoB with the mutated rpoB, designated rpoB(A621E), conferred N315 Delta IP with the phenotypes of reduced susceptibility to daptomycin and hetero-VISA. The rpoB(A621E)-mediated resistance conversion was accompanied by a thickened cell wall and reduction of the cell surface negative charge. Being consistent with these phenotypic changes, microarray data showed that the expression of the dlt operon, which increases the cell surface positive charge, was enhanced in the rpoB(A621E) mutant. Other remarkable findings of microarray analysis of the rpoB(A621E) mutant included repression of metabolic pathways of purine, pyrimidine, arginine, the urea cycle, and the lac operon, enhancement of the biosynthetic pathway of vitamin B2, K1, and K2, and cell wall metabolism. Finally, mutations identified in rplV and rplC, encoding 50S ribosomal proteins L22 and L3, respectively, were found to be associated with the slow growth, but not with the phenotype of decreased susceptibility to vancomycin and daptomycin, of 10*3d1.
  • Ito T, Kuwahara K, Hisata K, Okuma K, Cui L, Hiramatsu K
    Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 84 (5 Suppl 1) 23 - 33 0387-5911 2010/09 [Refereed][Not invited]
  • Yuki Katayama, Hiroko Murakami-Kuroda, Longzhu Cui, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 53 (8) 3190 - 3196 0066-4804 2009/08 [Refereed][Not invited]
    Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates are considered to have emerged from VAN-susceptible S. aureus (VSSA) by spontaneous mutation during VAN exposure. We previously reported that laboratory mutant H14, obtained from VSSA strain Delta IP by exposure to imipenem (IPM), showed overexpression of the vraSR two-component system and a typical hVISA phenotype. In the present study, to elucidate the mechanism of VSSA conversion to hVISA, we further characterized strain H14 by determining its whole-genome sequence, morphology, cell wall synthetic activity, and gene expression. Genome sequencing revealed that H14 harbored a mutated vraS (designated vraS(H14)) that caused an amino acid substitution (S(329)-> L). This mutation is different from the VraS mutation (N(5)-> I) identified in representative clinical hVISA strain Mu3. However, H14 exhibited a phenotype similar to that of Mu3, including heterogeneous resistance to VAN, enhanced cell wall synthetic activity, and vraSR overexpression. Replacement of the vraS gene of Delta IP with the mutated vraS(H14) gene confirmed that the S(329)-> L substitution was responsible for both the upregulation of vraSR and conversion to the hVISA phenotype. This conversion was also achieved by using the vraS gene of Mu3, which carries a mutation (N(5)-> I), but not with the native vraS gene of strain N315. Finally, we carried out a study to analyze the appearance of hVISA from VSSA by exposure of Delta IP to selective concentrations of VAN and beta-lactam antibiotics. A total of 8 and 5 hVISA isolates were detected among 50 isolates selected with VAN and IPM, respectively. Among the 13 hVISA mutants, mutation in vraSR was detected only in mutant strain H14, suggesting that additional mutational mechanisms can be responsible for evolution to the hVISA phenotype. We conclude that exposure not only to VAN but also to beta-lactams may select for reduced glycopeptide susceptibility in S. aureus.
  • Longzhu Cui, Hui-min Neoh, Mitsutaka Shoji, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 53 (3) 1231 - 1234 0066-4804 2009/03 [Refereed][Not invited]
    We describe here the genetic analysis of a vancomycin-susceptible Staphylococcus aureus (VSSA) strain, Mu50 Omega, a strain related to vancomycin-intermediate S. aureus (VISA) strain Mu50. Using a combination of Mu50 Omega whole-genome sequencing and genome engineering, we observed a stepwise evolution of vancomycin resistance from VSSA to VISA after the mutated vraS and graR genes of Mu50 were engineered into Mu50 Omega.
  • Ilana Lopes Baratella da Cunha Camargo, Hui-Min Neoh, Longzhu Cui, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 52 (12) 4289 - 4299 0066-4804 2008/12 [Refereed][Not invited]
    In order to better understand the mechanism of daptomycin resistance, we generated a daptomycin-nonsusceptible derivative strain, strain 10*3d1 (MIC = 3.0 mu g/ml), by in vitro exposure of methicillin-resistant Staphylococcus aureus strain N315 Delta IP (MIC = 0.5 mu g/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 10*3d1-10 (MIC = 1.0 mu g/ml), by passaging 10*3d1 in drug-free medium for 10 days. The resultant triple-isogenic strains were analyzed for their phenotypes and gene expression by microarray analysis. No significant differences in the membrane fluidities of 10*3d1 and 10*3d1-10 compared to the membrane fluidity of N315 Delta IP were observed. Resistant strain 10*3d1 had the highest membrane potential, followed by strains 10*3d1-10 and N315 Delta IP. The vancomycin and teicoplanin MICs also increased. Teichoic acid genes (tagA, tagG), mprF encoding lysyl-phosphatidylglycerol, and cls encoding cardiolipin synthase were downregulated in 10*3d1 and 10*3d1-10. The vraF and vraG genes, which encode ATP binding cassette transporter proteins, were upregulated in 10*3d1. The vraSR two-component regulatory system was upregulated, and electron microscopy revealed that the cell wall of 10*3d1 was significantly thicker than that of the parental strain. Taken together, daptomycin exposure selected a daptomycin-nonsusceptible strain with a phenotype similar to that of heterogeneous vancomycin-intermediate S. aureus and a transcription profile that partially overlapped that of heterogeneous vancomycin-intermediate S. aureus.
  • Yukiko Watanabe, Hui-min Neoh, Longzhu Cui, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 52 (11) 4207 - 4208 0066-4804 2008/11 [Refereed][Not invited]
  • Tengku Zetty Maztura Tengku Jamaluddin, Kyoko Kuwahara-Arai, Ken Hisata, Masahiko Terasawa, Longzhu Cui, Tadashi Baba, Chie Sotozono, Shigeru Kinoshita, Teruyo Ito, Keiichi Hiramatsu
    JOURNAL OF CLINICAL MICROBIOLOGY 46 (11) 3778 - 3783 0095-1137 2008/11 [Refereed][Not invited]
    For the past few years, we have been observing the dissemination of methicillin-resistant staphylococci in the community. From 2001 to 2003, an evaluation of nasal samples from 1,285 children in five day-care centers and two kindergartens in three districts in Japan revealed that methicillin-resistant coagulase-negative staphylococci (MRC-NS) have been widely disseminated in the Japanese community. Their prevalence is much greater than community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Forty-nine children (3.81%) were colonized with MRSA, whereas 390 children (30.35%) were colonized with MRC-NS. These MRC-NS strains predominantly harbored a pair of cassette chromosome recombinase types A2 and B2 (ccrAB2). Of these, 40.8% harbored type IVa staphylococcal cassette chromosome mec (SCCmec) elements, a distinct/characteristic type of SCCmec in pandemic clones of CA-MRSA. Interestingly, there was also a high frequency of nontypeable strains which possessed atypical structures compared to previous SCCmec types. Among the MRC-NS, the majority of strains (63.59%) were methicillin-resistant Staphylococcus epidermidis (MRSE). Their genotypes, as judged from pulsed-field gel electrophoresis (PFGE), were highly diverse. They were so diverse that there was no sign of an immediate transmission of any MRSE clone among children in the same institutions. In a previous report, we expounded that a few CA-MRSA clones with distinct SCCmec types were disseminated among children in the same institutions. Au contraire, with the case of CA-MRSE, there was no single genotype of CA-MRSE disseminated among children even in the same institution or class.
  • Junnosuke Otsuka, Yasumitsu Kondoh, Tomoyuki Amemiya, Akio Kitamura, Teruyo Ito, Satoshi Baba, Longzhu Cui, Keiichi Hiramatsu, Tomoko Tashiro, Hideo Tashiro
    MOLECULAR AND CELLULAR PROBES 22 (1) 1 - 13 0890-8508 2008/02 [Refereed][Not invited]
    We have developed a microarray-based assay for the genotyping of Staphylococcus aureus strains. A DNA microarray consisting of 221 genes with 390 oligonucleotide probes was designed to identify characteristic genes or gene alleles of S. aureus. The 221 genes were chosen on the basis of the following criteria: (i) genes used as control for the microarray system, (ii) virulence genes, (iii) resistance genes and their regulators, and (iv) genes constituting genomic islands, e. g., SCCmec. The microarray system was established by determining the method to prepare targets by random-primer labeling with chromosomal DNA and the conditions for hybridization. We verified the system by using DNAs of seven strains, the genome of which has been fully sequenced. Furthermore, the presence of 32 genes and the types of SCCmec elements and coagulase genes carried by another 27 strains were examined and compared with the results of PCR. As a result, the presence or absence of 182 genes out of the 221 genes was verified. Our data showed the usefulness of the oligonucleotide microarray based assay in identifying important marker sets, such as toxin genes, resistance genes, SCCmec elements, and coagulase genes, for the molecular epidemiology of S. aureus. (C) 2007 Elsevier Ltd. All rights reserved.
  • Hui-Min Neoh, Longzhu Cui, Harumi Yuzawa, Fumihiko Takeuchi, Miki Matsuo, Keiichi Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 52 (1) 45 - 53 0066-4804 2008/01 [Refereed][Not invited]
    Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR, designated graR*, but not intact graR, designated graRn, could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR* expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR* increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.
  • Hui-Min Neoh, Satoshi Hori, Mitsutaka Komatsu, Toyoko Oguri, Fumihiko Takeuchi, Longzhu Cui, Keiichi Hiramatsu
    Annals of Clinical Microbiology and Antimicrobials 6 13  1476-0711 2007/10 [Refereed][Not invited]
    Background: The aim of this study was to determine whether clinical outcome of patients with methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia was correlated with vancomycin susceptibility of the corresponding strains. Methods: A retrospective study on MRSA bacteraemia was performed at a teaching hospital between January 1998 and October 2005 by linking vancomycin susceptibility profiles of patients' isolates with hospitalization data. Results: A total of 20 out of 209 MRSA bacteraemia patients were treated with vancomycin for at least 5 days with adequate trough levels, and fulfilled the study's inclusion and exclusion criteria. Twenty-two S. aureus isolates from these patients' blood cultures were identified as MRSA, including two hetero-VISA from separate patients and two VISA with vancomycin MIC of 4 mg/L from one patient. Between patients who showed 'good' vancomycin response and patients who did not, there was a significant difference (p < 0.01) in their corresponding MRSAs' vancomycin susceptibility expressed by 'area under curve' (AUC) of population analysis. Significant correlations were found between AUC and initial vancomycin therapeutic response parameters of 'days till a febrile' (r = 0.828, p < 0.01) and 'days till CRP ≤ 30% of maximum' (r = 0.627, p < 0.01) Conclusion: Our study results cautionhealthcare personnel that early consideration should be given to cases with a poor vancomycin treatment response that could signify the involvement of MRSA with reduced susceptibility to vancomycin. © 2007 Neoh et al licensee BioMed Central Ltd.
  • Neoh Hui-min, 堀 賢, 小松 充孝, 小栗 豊子, 竹内 史比古, 崔 龍洙, 平松 啓一
    順天堂医学 順天堂医学会 53 (2) 243 - 250 0022-6769 2007/06 
    バンコマイシンにおける感受性の低下と治療初期効果の鈍化について、1998年から2005年に当院で発生したメチシリン耐性黄色ブドウ球菌(MRSA)による血流感染症を後方視的(retrospective)に研究し、新たに相関を認めた。バンコマイシンヘテロ耐性株に対するバンコマイシンの初期治療効果の指標のうち、"解熱に要した期間"および"CRP(C-reactive protein)低下が最高値の30%以下に要した期間"では、それぞれ相関係数がr=0.828(p<0.01)、およびr=0.627(p<0.01)であり、バンコマイシンの初期治療効果の鈍化に有意な影響を与えていた。このことから、バンコマイシンによる治療の初期治療にもかかわらず解熱に要した期間やCRPの低下する期間が遷延している場合には、早期にバンコマイシンヘテロ耐性株の存在を疑う必要があることが示唆された。(著者抄録)
  • Hiroko Kuroda, Makoto Kuroda, Longzhu Cui, Keiichi Hiramatsu
    FEMS MICROBIOLOGY LETTERS 268 (1) 98 - 105 0378-1097 2007/03 [Refereed][Not invited]
    SaeRS is a two-component system that has been characterized as a positive regulatory system for the expression of several virulence factors, including coagulase, alpha-, beta- and gamma-haemolysins, nuclease, and fibronectin-binding proteins in Staphylococcus aureus. Previously, the SaeRS system was found to be induced at the transcriptional level by beta-lactam. Here, we found that subinhibitory concentrations of beta-lactam induce haemolytic activity in the S. aureus N315 strain but not in the saeRS null mutant KSA. Comparison of the transcriptional profile of the N315 and KSA strains by microarray analysis reveals that the SaeRS system modulates the regulation of coagulase (coa), alpha-, beta- and gamma-haemolysins (hla, hlb and hlg), nuclease (SA0746), fibrinogen-binding proteins (emp, efb, SA1000 and SA1004), fibronectin-binding protein B (fnbB), and 13 other genes. Further, the use of cefoxitin as a signal inducer reveals that the SaeRS system appears to modulate 22 additional genes as a secondary regulon, including the staphylococcal accessory regulators SarA and SarT and the Clp protease ATPase subunits ClpB and ClpL. These observations suggest that beta-lactam is able to induce the SaeRS system, which acts as a crucial signal transduction system for S. aureus pathogenicity rather than antimicrobial resistance.
  • Faris G. Bakri, Nisreen Abu Al-Hommos, Asem Shehabi, Randa G. Naffa, Longzhu Cui, Keiich Hiramatsu
    SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES 39 (5) 457 - 460 0036-5548 2007 [Refereed][Not invited]
    A 49-y-old male with erythrodermic psoriasis developed persistent bacteraemia for 3 months due to methicillin-resistant Staphylococcus aureus despite antimicrobial therapy. The skin was the likely focus. Three consecutive isolates from the blood and 1 from the nose were identical and had vancomycin MIC of 4 mg/l.
  • LZ Cui, E Tominaga, HM Neoh, K Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 50 (3) 1079 - 1082 0066-4804 2006/03 [Refereed][Not invited]
    We present here findings of a strong positive correlation between reduced daptomycin susceptibility and vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). This correlation is related to cell wall thickening, suggesting that, similar to the case with vancomycin resistance in VISA, the physical barrier of a thickened cell wall may contribute to daptomycin resistance in S. aureus.
  • LZ Cui, A Iwamoto, JQ Lian, HM Neoh, T Maruyama, Y Horikawa, K Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 50 (2) 428 - 438 0066-4804 2006/02 [Refereed][Not invited]
    As an aggressive pathogen, Staphylococcus aureus poses a significant public health threat and is becoming increasingly resistant to currently available antibiotics, including vancomycin, the drug of last resort for gram-positive bacterial infections. S. aureus with intermediate levels of resistance to vancomycin (vancomycin-intermediate S. aureus [VISA]) was first identified in 1996. The resistance mechanism of VISA, however, has not yet been clarified. We have previously shown that cell wall thickening is a common feature of VISA, and we have proposed that a thickened cell wall is a phenotypic determinant for vancomycin resistance in VISA (L. Cui, X. Ma, K. Sato, et al., J. Clin. Microbiol. 41:5-14, 2003). Here we show the occurrence of an anomalous diffusion of vancomycin through the VISA cell wall, which is caused by clogging of the cell wall with vancomycin itself. A series of experiments demonstrates that the thickened cell wall of VISA could protect ongoing peptidoglycan biosynthesis in the cytoplasmic membrane from vancomycin inhibition, allowing the cells to continue producing nascent cell wall peptidoglycan and thus making the cells resistant to vancomycin. We conclude that the cooperative effect of the clogging and cell wall thickening enables VISA to prevent vancomycin from reaching its true target in the cytoplasmic membrane, exhibiting a new class of antibiotic resistance in gram-positive pathogens.
  • F Takeuchi, S Watanabe, T Baba, H Yuzawa, T Ito, Y Morimoto, M Kuroda, LZ Cui, M Takahashi, A Ankai, S Baba, S Fukui, JC Lee, K Hiramatsu
    JOURNAL OF BACTERIOLOGY 187 (21) 7292 - 7308 0021-9193 2005/11 [Refereed][Not invited]
    Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S. haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S. haemolyticus, Staphylococcus aureus, and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the "oriC environ," likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S. haemolyticus, S. aureus, and S. epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S. haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.
  • LZ Cui, JQ Lian, HM Neoh, E Reyes, K Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 49 (8) 3404 - 3413 0066-4804 2005/08 [Refereed][Not invited]
    Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to similar to 128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.
  • K Hisata, K Kuwahara-Arai, M Yamanoto, T Ito, Y Nakatomi, LZ Cui, T Baba, M Terasawa, C Sotozono, S Kinoshita, Y Yamashiro, K Hiramatsu
    JOURNAL OF CLINICAL MICROBIOLOGY 43 (7) 3364 - 3372 0095-1137 2005/07 [Refereed][Not invited]
    Methicillin-resistant Staphylococcus aureus (MRSA), regarded as a tenacious pathogen in the hospital, has recently become increasingly prevalent as a community pathogen. We evaluated the prevalence and characteristics of methicillin-resistant staphylococci in the Japanese community by testing nasal samples of 818 children of five day care centers and two kindergartens in three districts. We found that methicillin-resistant staphylococci are already prevalent among healthy children. Among 818 children, 35 children (4.3%) carried MRSA and 231 children (28.2%) carried methicillin-resistant coagulase-negative staphylococci (MRC-NS). The types of staphylococcal cassette chromosome mec (SCCmec) found among 44 MRSA isolates were as follows: type IIa, 11 isolates; type IIb, 19 isolates; and type IV, 14 isolates. The type IIb SCCmec element was a new SCCmec element found in this study. Eleven (25%) strains which belonged to clonal complex 5 (CC5) carried type IIa SCCmec, and they produced type 2 coagulase and toxic shock syndrome toxin 1. They were indistinguishable from health care-associated MRSA (H-MRSA) strains in Japan, represented by strain N315. On the other hand, 33 (75%) strains, most of which belonged to CC78 or CC91, carried small SCCmec elements, such as type IIb or type IV, and they produced type 1 or type 3 coagulase and exfoliative toxin. The data indicated that MRSA clones distinct from H-MRSA have disseminated in healthy children. The fact that MRC-NS strains were prevalent in the community suggested that they might serve as a reservoir for the SCCmec element carried by MRSA strains disseminated in the community.
  • K Hiramatsu, LZ Cui, K Kuwahara-Arai
    LANCET 364 (9434) 565 - 566 0140-6736 2004/08 [Refereed][Not invited]
  • Ito T, Kuwahara K, Hisata K, Okuma K, Cui L, Hiramatsu K
    Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 78 (6) 459 - 469 0387-5911 2004/06 [Refereed][Not invited]
  • A Isnansetyo, LZ Cui, K Hiramatsu, Y Kamei
    INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS 22 (5) 545 - 547 0924-8579 2003/11 [Refereed][Not invited]
    Antibacterial activity of 2,4-diacetylphloroglucinol (DAPG) was evaluated against 23 vancomycin-resistant Staphylococcus aureus (VRSA) strains isolated from several Asian and European countries, Brazil, South Africa and USA, and against vancomycin-resistant Enterococcus spp (VRE) genotypes A, B and C. DAPG was active against a wide range of VRSA isolates as well as vancomycin hetero-resistant S. aureus (h-VRSA) at MIC 4 mg/l. This substance also had moderate activity against both VRE-A and -B at MIC 8 mg/l, but not against VRE-C at up to 16 mg/l. The activity of DAPG did not directly correlate with levels of vancomycin resistance in VRSA and VRE. These results suggest that DAPG might be useful in developing new antibiotics against VRSA. (C) 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
  • R Yoshida, K Kuwahara-Arai, T Baba, LZ Cui, JF Richardson, K Hiramatsu
    JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY 51 (2) 247 - 255 0305-7453 2003/02 [Refereed][Not invited]
    Staphylococcus aureus clinical isolate 61/5896 exhibited methicillin resistance (MIC 64 mg/L), but lacked mecA, which encodes penicillin-binding protein 2'. The strain was isolated in England in 1961, and exhibited unstable heterogeneous methicillin resistance. When cultivated in drug-free medium, the methicillin resistance of 61/5896 increased after three daily passages, then decreased and was completely lost after 12 days' passage. Electron microscopy revealed that strain 61/5896 had a thicker and rougher cell wall than its methicillin-susceptible derivatives. It produced about three times more penicillin-binding protein 2 (PBP2) than methicillin-susceptible derivatives. The strain was characteristically a non-producer of autolytic enzyme, though the phenotype, which was lost easily, was not directly correlated with methicillin resistance.
  • L Cui, XX Ma, K Sato, K Okuma, FC Tenover, EM Mamizuka, CG Gemmell, MN Kim, MC Ploy, N El Solh, Ferraz, V, K Hiramatsu
    JOURNAL OF CLINICAL MICROBIOLOGY 41 (1) 5 - 14 0095-1137 2003/01 [Refereed][Not invited]
    We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and, K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 X 10(-6) to 1.64 X 10(-3). The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin (r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.
  • T Baba, F Takeuchi, M Kuroda, H Yuzawa, K Aoki, A Oguchi, Y Nagai, N Iwama, K Asano, T Naimi, H Kuroda, L Cui, K Yamamoto, K Hiramatsu
    LANCET 359 (9320) 1819 - 1827 0140-6736 2002/05 [Refereed][Not invited]
    Background A new type of meticillin-resistant Staphylococcus aureus (MRSA), designated community-acquired MRSA, is becoming increasingly noticeable in the community, some strains of which cause fatal infections in otherwise healthy individuals. By contrast with hospital-acquired MRSA, community-acquired MRSA is more susceptible to non beta-lactam antibiotics. We investigated the high virulence potential of certain strains of this bacterium. Methods We ascertained the whole genome sequence of MW2, a strain of community-acquired MRSA, by shotgun cloning and sequencing, MW2 caused fatal septicaemia and septic arthritis in a 16-month-old girl in North Dakota, USA, in 1998. The genome of this strain was compared with those of hospital-acquired MRSA strains, including N315 and Mu50. Findings Meticillin resistance gene (mecA) in MW2 was carried by a novel allelic form (type IVa) of staphylococcal cassette chromosome mec (SCCmec), by contrast with type II in N315 and Mu50. Type IVa SCCmec did not carry any of the multiple antibiotic resistance genes reported in type II SCCmec. By contrast, 19 additional virulence genes were recorded in the MW2 genome. All but two of these virulence genes were noted in four of the seven genomic islands of MW2. Interpretation MW2 carried a range of virulence and resistance genes that was distinct from those displayed on the chromosomes of extant S aureus strains. Most genes were carried by specific allelic forms of genomic islands in the MW2 chromosome. The combination of allelic forms of genomic islands is the genetic basis that determines the pathogenicity of medically important phenotypes of S aureus, including those of community-acquired MRSA strains.
  • GA Oliveira, AM Dell'Aquila, RL Masiero, CE Levy, MS Gomes, L Cui, K Hiramatsu, EM Mamizuka
    INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY 22 (7) 443 - 448 0899-823X 2001/07 [Refereed][Not invited]
    OBJECTIVE: To evaluate the possible presence of vancomycin-resistant Staphylococcus aureus (VRSA) in a Brazilian hospital. DESIGN: Epidemiological and laboratory investigation of nosocomial VRSA. METHODS: 140 methicillin-resistant S aureus strains isolated between November 1998 and October 1999 were screened for susceptibility to vancomycin. The screening was carried out by using brain-heart infusion agar (BHIA) supplemented with 4, 6, and 8 mug/mL of vancomycin. The minimum inhibitory concentration (MIC) determination was carried out as standardized by the National Committee for Clinical Laboratory Standards using the broth macrodilution, agar-plate dilution, and E-test methods. PATIENTS: Hospitalized patients exposed to vancomycin. RESULTS: 5 of the 140 isolates had a vancomycin MIC of 8 mug/mL by broth macrodilution, agar plate dilution, and E-test methods. Four VRSA strains were isolated from patients in a burn unit who had been treated with vancomycin for more than 30 days, and one from an orthopedic unit patient who had received vancomycin treatment for 7 days. Pulsed-field gel electrophoresis characterized four of the VRSA strains as belonging to the Brazilian endemic clone. All five strains were negative for vanA, vanB, and vanC genes by polymerase chain reaction. Transmission electron microscopy of the five strains revealed significantly thickened cell walls. One patient died due to infection caused by the VRSA strain. CONCLUSIONS: This is the first report of isolation of VRSA in Brazil and the first report of isolation of multiple VRSA strains from one facility over a relatively short period of time. This alerts us to the possibility that VRSA may be capable of nosocomial transfer if adequate hospital infection control measures are not taken (Infect Control Hosp Epidemiol 2001;22:443-448).
  • N Aritaka, H Hanaki, LZ Cui, K Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 45 (4) 1292 - 1294 0066-4804 2001/04 [Refereed][Not invited]
    We tested the combined activity of vancomycin and seven beta -lactam antibiotics against Staphylococcus aureus clinical strain Mu3, which displays heterogeneous resistance to vancomycin, When combined with vancomycin, four of the seven tested beta -Lactams exhibited an additive effect at or near their MICs, while all showed an antagonistic effect at lower, sub-MIC levels. This study implicated the unpredictable nature of combination therapy of beta -lactams and vancomycin against S. aureus with reduced susceptibility to vancomycin.
  • M Kuroda, T Ohta, Uchiyama, I, T Baba, H Yuzawa, Kobayashi, I, LZ Cui, A Oguchi, K Aoki, Y Nagai, JQ Lian, T Ito, M Kanamori, H Matsumaru, A Maruyama, H Murakami, A Hosoyama, Y Mizutani-Ui, NK Takahashi, T Sawano, R Inoue, C Kaito, K Sekimizu, H Hirakawa, S Kuhara, S Goto, J Yabuzaki, M Kanehisa, A Yamashita, K Oshima, K Furuya, C Yoshino, T Shiba, M Hattori, N Ogasawara, H Hayashi, K Hiramatsu
    LANCET 357 (9264) 1225 - 1240 0140-6736 2001/04 [Refereed][Not invited]
    Background Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. Methods Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. Findings The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. in the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. Interpretation The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.
  • LZ Cui, H Murakami, K Kuwahara-Arai, H Hanaki, K Hiramatsu
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 44 (9) 2276 - 2285 0066-4804 2000/09 [Refereed][Not invited]
    Staphylococcus aureus Mu50, which has reduced susceptibility to vancomycin, has a remarkably thickened cell wall with an increased proportion of glutamine nonamidated muropeptides. In addition, Mu50 had enhanced glutamine synthetase and L-glutamine D-fructose-6-phosphate aminotransferase activities, which are involved in the cell-wall peptidoglycan synthesis pathway. Furthermore, significantly increased levels of incorporation of C-14-labeled D-glucose into the cell wall was observed in Mu50. Unlike a femC mutant S, aureus strain, increased levels of production of nonamidated muropeptides in Mu50 was not caused by lower levels of glutamine synthetase activity but was considered to be due to the glutamine depletion caused by increased glucose utilization by the cell to biosynthesize increased amounts of peptidoglycan. After the cells were allowed to synthesize cell wall in the absence or presence of glucose and glutamine, cells with different cell-wall thicknesses and with cell walls with different levels of cross-linking were prepared, and susceptibility testing of these cells demonstrated a strong correlation between the cell-wall thickness and the degree of vancomycin resistance. Affinity trapping of vancomycin molecules by the cell wall and clogging of the outer layers of peptidoglycan by bound vancomycin molecules were considered to be the mechanism of vancomycin resistance of Mu50, The reduced cross-linking and the increased affinity of binding to vancomycin of the Mu50 cell mall presumably caused by the increased proportion of nonamidated muropeptides may also contribute to the resistance to some extent.
  • LZ Cui, H Kasegawa, Y Murakami, H Hanaki, K Hiramatsu
    SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES 31 (2) 208 - 209 0036-5548 1999 [Refereed][Not invited]
    We report on a rare fatal case of postoperative toxic shock syndrome caused by infection with a highly virulent methicillin-resistant Staphylococcus aureus strain, designated Sak-1, which was found to be characteristic in its increased production of toxic shock syndrome toxin 1 in human whole blood (about 30-fold more than produced in Tod Hewitt broth). The strain also produced a high level of toxic shock syndrome toxin 1 in the circulating blood of mice experimentally infected with the strain.
  • Daisuke Kato, Hideaki Hanaki, Longzhu Cui, Toyoko Oguri, Keiichi Hiramatsu
    Japanese Journal of Antibiotics 51 (12) 735 - 744 0368-2781 1998/12 [Refereed][Not invited]
    Two hundred and thirteen clinical strains of coagulase-negative staphylococci isolated in Japan between 1980 and 1997 were analyzed for glycopeptide susceptibility by determing MIC using both Mueller-Hinton agar (MHA) and Brain Heart Infusion agar (BHIA) plates. Of 37 Staphylococcus epidermidis strains isolated between 1980 and 1981, all were susceptible to vancomycin and teicoplanin on both MHA and BHIA. However, of 122 isolates of Staphylococcus epidermidis isolated between 1994 and 1997, 1 (0.8%) was intermediate to vancomycin on MHA and 39 (32%) were intermediate on BHIA, while 3 (2.5%) and 27 (22.1%) were intermediate or resistant to teicoplanin on MHA and BHIA, respectively. It was demonstrated that the susceptibilities of the strains in 1990s to vancomycin and teicoplanin were significantly decreased compared with those in 1980s. Population analysis was performed with six strains each of Staphylococcus epidermidis and Staphylococcus haemolyticus (three with vancomycin MIC≤8 μg/ml and three with vancomycin MIC≤4 μg/ml using BHIA). The population curves of the Staphylococcus epidermidis strains showed a homogeneous pattern of susceptibility. Whereas, those for two Staphylococcus haemolyticus strains (vancomycin MIC = 8 μg/ml using BHIA) showed a typical heterogeneous pattern. Vancomycin-resistant mutants (MIC> 32 μg/ml) were obtained with a high frequency of 10-4- -5 from the strains by one-step selection with 16 μg/ml of vancomycin.

Books etc

  • Please see above site


  • 国内外ファージ研究の状況と推進すべき研究開発戦略に関する提言
    崔 龍洙, 氣駕 恒太朗, 渡邊真弥  JST-CRDS(研究開発の俯瞰報告書)  2019/03  [Not refereed][Not invited]
  • 溶連菌感染症の歴史
    渡邊真弥, 崔龍洙  小児科  59-  (11)  1511  -1518  2018/10  [Refereed][Invited]
  • M. Matsuo, L. Cui, J. Kim, K. Hiramatsu  INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS  42-  S54  -S54  2013/06  [Not refereed][Not invited]
  • Staphylococcus aureusにおいてrpoB変異はリネゾリド感受性を改善する(The rpoB mutation improves linezolid susceptibility in Staphylococcus aureus)
    江端 望, 香本 晃良, 崔 龍洙, 渡辺 由紀子, 松尾 美記, 片山 由紀, 平松 啓一  日本細菌学雑誌  67-  (1)  134  -134  2012/02  [Not refereed][Not invited]
  • Staphylococcus aureusにおいてrpoB遺伝子変異はβラクタム系抗生物質耐性に関与する(A mutation of rpoB gene contributes to β-lactam antibiotic resistance in Staphylococcus aureus)
    相羽 由詞, 片山 由紀, 菱沼 知美, 崔 龍洙, 平松 啓一  日本細菌学雑誌  67-  (1)  138  -138  2012/02  [Not refereed][Not invited]
  • Imipenemによるヘテロバンコマイシン耐性黄色ブドウ球菌(Heterogeneous Vancomycin-Intermediate S.aureus;hVISA)の選択
    片山 由紀, 黒田 博子, 崔 龍洙, 平松 啓一  日本化学療法学会雑誌  57-  (Suppl.A)  200  -200  2009/04  [Not refereed][Not invited]
  • MRSA感染症におけるLinezolidの位置づけ 基礎的検討からの提言
    内山 倫宏, 桑原 京子, 片山 由紀, 崔 龍洙, 馬場 理, 平松 啓一  日本化学療法学会雑誌  55-  (Suppl.A)  145  -145  2007/04  [Not refereed][Not invited]
  • K Hiramatsu, L Cui, M Kuroda, T Ito  TRENDS IN MICROBIOLOGY  9-  (10)  486  -493  2001/10  [Not refereed][Not invited]
    Significant advances have been made in recent years in our understanding of how methicillin resistance is acquired by Staphylococcus aureus. Integration of a staphylococcal cassette chromosome mec(SCCmec) element into the chromosome converts drug-sensitive S. aureus into the notorious hospital pathogen methicilin-resistant S. aureus(MRSA), which is resistant to practically all beta -lactam antibiotics. SCCmec is a novel class of mobile genetic element that is composed of the mec gene complex encoding methicillin resistance and the ccr gene complex that encodes recombinases responsible for its mobility. These elements also carry various resistance genes for non-beta -lactam antibiotics. After acquiring an SCCmec element, MRSA undergoes several mutational events and evolves into the most difficult-to-treat pathogen in hospitals, against which all extant antibiotics including vancomycin are ineffective. Recent epidemiological data imply that MRSA has embarked on another evolutionary path as a community pathogen, as at least one novel SCCmec element seems to have been successful in converting S. aureus strains from the normal human flora into MRSA.
  • Please see above site
    [Not refereed][Not invited]

Industrial Property Rights

  • 細菌毒素検出培地
  • 微生物の生理性検出方法
  • 高度薬剤耐性遺伝子

Awards & Honors

  • 2014/06 北里柴三郎記念賞
    受賞者: 崔 龍洙
  • 2010/06 日本化学療法学会学術奨励賞
  • その他:
    受賞者: 崔 龍洙

Research Grants & Projects

  • Development of novel genetic testing, genotyping and therapeutic methods for implementing effective countermeasure against drug-resistant bacteria
    国立研究開発法人 日本医療研究開発機構 (AMED):新興・再興感染症に対する革新的医薬品等開発推進研究事業
    Date (from‐to) : 2020/04 -2022/03 
    Author : 崔龍洙, 氣駕恒太朗, 渡邊真弥, 相羽由詞, 佐藤祐介, 笹原 鉄平, 永沢 善三, タンシンイー
  • 長期滞在型高齢者福祉施設における効率的な感染症対策プログラムの開発
    Date (from‐to) : 2018/08 -2021/03 
    Author : 笹原 鉄平, 崔 龍洙
  • 薬剤耐性菌に対する新規追尾型抗菌治療法の開発
    Date (from‐to) : 2017/08 -2020/03 
    Author : 崔龍洙, 氣駕恒太朗, 渡邊真弥, 相羽由詞, 佐藤祐介, 笹原 鉄平
  • グリコペプチド耐性ブドウ球菌におけるグリコペプチド耐性メカニズムの解明
    Date (from‐to) : 2017/07 -2019/03 
    Author : 崔 龍洙
  • ファージを用いた新規抗菌療法の開発
    Date (from‐to) : 2017/06 -2019/03 
    Author : 崔 龍洙
  • Infectious diseases
  • Bacterial genomics
  • Drug resistance in Staphylococus aureus

Committee Membership

  • 2018/05 - Today   Scientific Reports; Editorial Board Member   Scientific Reports

Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.