Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Jpn. J. Infect. Dis., 54, 17-22, 2001 Original Article Molecular and EpidemiologlCal Study of the First Outbreak of vanB Type Vancomycin-Resistant Enterococcusfaecalis in Japan Kozue Oana*, Yoshiyuki Kawakami, Makoto Ohnishil , Masayo lshikawa2, Masako Hirota2, MinoruTozuka2, Kenichi Atarashi3, Kousuke Baba3, Kyoko Fujiki3, Mitsuo Okazaki4, Takayuki Honda5 and Tetsuya HayashiI Department ofMedical Technology, School ofAllied Medical Sciences, Shinshu University, 2central Clinical Laboratories, Shinshu UniversiO; Hospital and 5Department of Laboratory Medicine, Shinshu UniversiO, School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, lDepartmen i of Microbiology, Miyazaki Medical College, Kiyotake 5200, Miyazaki 889-1 692, 3Department of lnfection Control Committee, Hokushin General Hospital, Nishi 1-5-63, Nakano 383-0021 and 4Gene Research Center and Department ofApplied Biology, FaculoJ of Textile Science and Technology, Shinshu UniversiO!, Tokita 3-1511, Ueda 38618567, Japan (Received October 24, 2000. Accepted March 1 , 2001) SUMMARY: In July, 1 999, an outbreak of vancomycin-resistant EnteTlDCOCCuSfaecalis (VREF) with the vanB genotype occurred for the first time in Japan at Hokushin General Hospital, Nakano City, Nagano Prefecture. Four VREF strains were isolated from the clinical specimens of four Inpatients, and 16 VREF strains were isolated by the screenlng Of asymptomatic ca汀iers and by suⅣeillance of the hospital environment. All or the isolates possessed vanB genes. In a pulsed-field gel electrophoresis analysts, 1 9 out of 20 VREF isolates exhibited the indistinguishable restriction endonuclease digestion pattems of the chromosomal DNA. Additional investigation by Southem hybridization using the vanB probe implied that the vanB gene of these 19 isolates was encoded on a 1 10-kb plasmid. These findings indicate that the outbreak was prlnCIPally caused by a slngle clone. The restriction endonuclease digestion pattems of the remainlng Slngle isolate was different from those of the other isolates. The vα〝β gene was encoded on the chromosome. MArERIALS AND METHODS INTRODUCTION Particularly in Europeand the United States, vancomycln- Isolation, identirICation, and screenlng Of VRE: Hokushin resistant enterococci (VRE) have emerged as a significant General Hospital is a 640-bed hospital with four.Wards (Wards cause of nosocomial infections and colonizations since their A-D). Four VREF (designated H 1-H4) Were lSOlated from first description in 1988 (I). In Japan, a vanB genotype the clinical specimens of fわur aged inpatients (76 to 87 years Entey10COCCuS gallinarum was first isolated in 1 996 (2). Since old) on Wards A and B during a 3-week period in July 1999. then, Only two sporadic cases ofVRE infection, both of which Eight VREF strains were isolated by the screenlng Of asymp- were due to E・faecium (3,4), had been rep.orted in Japan・ tomatic camiers (P-numbered isolates),and eight were isolated One case was an 81-year-Old female inpatlent With acute by the surveillance of the hospital environment on Wards A pyelonephritis caused by a vanA gen.otype VRE (3), and the and a (E-numbered isolates). other was a 27-year-old female inpatient With postl0Perative Identification ofE. faecalis was made by Vitek GPI Cards abdominal lymphocyst infection by a vanB genotype VRE (bioMerieux Vitek systems, Inc., Hazelwood, Mo., USA) and the MicroScan WalkAway system (Dade Intemational, West (4). However, in July of 1999, an outbreak ofnosocomial infectionsof vancomycin-resistant Ejaecalis (VREF) Occurred Sacramento, Calif・, USA)・ To screen the asymptomatic at Hokushin General Hospital, Nakano City, Nagano Prefec- camerS, 8 1 9 rectal swabs and/or sputa were collected from ture (5). ln this report, we describe the results ofmolecular 759 inpatients and 57 inpatients'family members. Rectal swabs and epidemiological studies of the 20 VREF strains isolated were collected from the 969 hospital personnel as well. The from the outbreak, which was the first VREF outbreak in rectal swabs were collected uslng Sterilized cotton swabs Japan. The results indicate that a slngle clone with the vα〝β moistenedwith sterilized saline.払ch rectal swab or one loopfull genotype prlnClpally caused the outbreak. Our data also 0feach sputum was spread onto the Enterococcosel agar plates suggested that the vanB gene of the clone was encoded on a supplemented with 8 LLg/ml of vancomycin (Nippon Becton 1 1 0-kb plasmid. Dickinson, Tbkyo). For the surveillance of VRE in the hospital environment, *Corresponding author: Tel: +8 1 -263-37-2387, Fax: +8 1 -263-37- specimens were collectedfrom I ,054 hospital environmental 2370, E-mail: [email protected] sites on Wards A and a by swabbing an area orabout 100 17 cm2 With sterilized cotton swabs moistened with sterilized cycle, 94oC for 30 see, 58oC for 30 see, and 720C for I min saline. SpecimensWere subjected to the isolation and identi- for the next 40 cycles, and then 72oC for 7 min for the last fication procedures described above. After disinfecting the cycle・ PCR products were analyzed on 2% agarose gel prepared with 0.5 X Tris-borate-EDTA (TBE) buffer ( 1 X TBE buffer: ward environment, the bacteriological investlgation of the 840 sites of Wards A and B was carried out by the same procedure 89 mM TriS, 89 mM boric acid, and 2.5 mM EDTA lpH8.0]) containing ethidium bromide (0.5 LLg/ml). E.faecalis strain in order to ascertain the elimination of VRE contamination. Antimicrobial susfePtit'ility testing: Resistance levels of each VRE isolate agalnSt Various antimicrobial agents except RVl , which was provided by Dr. Y. Arakawa (National hstitute of Infectious Diseases, Tokyo), was used as a vanB-positive teicoplanin were detemined both by Vitek GPS Cards and control strain. the MicroScan WalkAway system according to the instruc- Pulsed-rleld gel electrophoresis (PFGE): Preparation of tions provided with each system. Both systems yielded the intact genomic DNAs and restriction endonuclease-digestion same susceptibility results. Since we only used the standard of the DNAs were perf♭med as described previously (7), protocols provided by the manufactures, the exact minimum except that achromopeptidase (Wako Junyaku, Tokyo) was inhibitory concentrations (MICs) were not determined. In this Included inthe lysis solution at afinal concentration of4 mg/ml. All the restriction endonucleases used were purchased from Takara Shuzo (Shiga). Electrophoresis was performed on 1 % agarose gel prepared with 0.5 X TBE buffer uslng a CHEF report, the values obtained from the analyses using the two systems were described as the MICs. Susceptibilities to teicoplanin were determined by the disk di肌sion method MAPPER system (Bio-Rad Laborator!es・ Hercules, Calif・, using Sensidisks (Nippon Becton Dickinson). Detection of vanA, vanB, and vanC genes by polymerase chain reaction (PCR): Oligonucleotide primers for the USA) according to the manufacture's Instruction. Running conditions were described in the figure legend of Fig. 2. amplification of vanA, vanB, and vanCl genes were prepared SoutherTI hybridizatiOn analysis: DNAs were transferred as previously described (6). Bacterial isolates to be tested from PFGE gels to nylon membranes (Hybond N';Amershamm were grown at 350C ovemight on sheep blood agar plates (Nippon Becton Dickinson). Colonies were suspended in 300 Pharmacia Biotech, Buckinghamshire, UK) using a VacuGene blotter apparatus(Amershamm Pharmacia Biotech). The vanB 〝l of distilled water and were boiled f♭r 10 min. Boiled probe, a 635 bp fragment of the vanB coding region, Was samples were chilled quickly on ice and were subjected to prepared by PCR, and was purified from an agarose geluslng centrifugation at 30,000 G for 5 min, and the supematants were used as templates for PCR. PCR reaction mixtures of a Gene clean II Kit (Bio 101, Carlsbad, Calif., USA). Probe 25 1Ll final volume contained 5 LEI of the sample, 1 LEI of uslng an ECLTM Direct Nucleic Acid Labeling and Detection each primer solution (25 LLM), 2.5 LEI of dNTP mixture (2 mM each), 0.15 LEI of Taq Gold Polymerase (50 units/LEI, System (Amershamm Phamacia Biotech). labeling, hybridization, and signal detection were ca汀ied out Roche MolecularBiochemicals, Mamieim, Germany), 2.5 LLI RESULTS of 1 0 X PCR buffer supplemented with Taq Gold Polymerase, and 12.85 Lil ofdistilled water. PCR was performed on a DNA thermal cycler (Gene Amp 9600; Applied Biosystems, Foster Isolation ofVRE: During a 31Week period in July 1999, four inpatients in Wards A or B yielded VREF (Hl-H4 in Table 1). Each isolate was originated &om the sputum of an City, Calif., USA) with cycles of95oC for 12 min for the first Table 1 ・ 20 vanB genotype VRE isolates and their antimicrobial susceptibilities isolate Date of ハ Drug Ward Origin Numbc, isolation Source VCM B Inpatient H 1 9, July Sputum A Inpati en I H2 19,July Urine A 27,July Urine Inpatient H3 B Inpati en t H4 31,July Rectalswab B Inpatient P3 5 I , Aug. Rectal swab a Inpatient P3 8 I , Aug. Rectal swab B Inpatient P40 I , Aug. Rectal swab A Inpatient P93 1 , Aug. Rectal swab B 1 , Åug. Rectal swab Inpatient P473 A Inpatient P48 8 1 A Inpatient P 1 I 30 1 , Åug. Rectal swab , Aug. Rectal ≧ ≧ ≧ ≧ー ≧ ≧ ≧ ≧ ≧ ≧ 1 , Åug. Rectal swab 4, B Envi ron men I E20 7 4, Aug. bed side table LLg/ml GM ′b ∠U ′b 上U AV 2′○ b 0∠U′b ′0 ∠U b′ 0 ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ 1 1 1 一1 - 1 )* AMK. ′0 b6′0 ∠U′b KV′b 上U 0/b ′ b ′ 0 b ′ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ 2 2 2 0 2 2 lZ 2 2 1 l 1 - 1 - 1 ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ 64 64 糾 64 64 64 ≧ ≧ ≧ ≦ ≧ ≧ ≧ ≧ ≧ ≧ 史U0O 80 01 0 8CXU 史 80 0 0 moor B Environment E208 4, Åug. over table a Environment E2 1 3 4, a Environment E222 4, Åug. bed side table noor a Environment E225 4, Åug. bed pan A Environment E398 4, B Environment E5 1 8 4, Aug. slipper Aug. ward (MIC, ABPC swab B Environment E20 1 Aug. ward SSSSSSS levels PCG 5 A Inpatient P8065 Aug. ward 32 32 32 32632 32 32 32 32 32 resistance TEIC moor *Al1 the values were the ones determined by the Vitek GPS Cards and the MicroScan WalkAway system uslng the standard protocolsthatthe manufactures provided. VCM: vancomycin, TEIC: teicoplanin, PCG: benzylpenicillin, ABPC: ampicillin, GM: gentamicin, AMK: amikacin, LVFX: levonoxacin, S: susceptible. 18 M P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 N 635b p Fig. L Detection orvd〃β genes in 20 VRE isolates. PCR products were analyzed on 2% agarose gel. Lanes: M: molecular weight marker, P: vanB-positive strain (E.faecalis RVl), N: negative control, 1: Hl, 2: H2, 3: H3, 4: H4, 5: P35, 6: P38, 7: P40,8: P93,9: P473, 10: P488, ll: P1130, 12: P8065, 13: E201, 14: E207, 15: E208, 16: E213, 17: E222, 18:E225, 19: E398,20: E518. 85lyear-Old female with bronchitis (Hl ),from the urine of a 76-year-old female with cerebral infarction (H2), from the urine of an 87-year-old female with cerebral infarction (H3), contrast, P93 exhibited completely different PFGE pattems in the case of the NotI, SmaI, and ApaI-digestion (Fig. 2A, and from the rectal swab of a 76-year-old female suffering from gallbladder cancer (H4). From the sputum of the 85- Southern hybridization analysis using the vanB probe: ln the Southem hybridization analysts Of the NotI-digests of year101d female and the urine of the 76-year-Old female, genomic DNAs, the 1 9 isolates exhibited the same hybridiza- methicillin-resistant Staphylococcus aureus (MRSA) and tion pattem as that of the vanB probe, namely, a weak signal 2B, and data not shown, respectively). Pseudomonas aeruginosa were also isolated, respectively. of叩prOXimately 1 10 kb in size was obseⅣed (Fig. 2A). Their MRSA was also isolated from the stool of the 76-year-Old SmaI- and ApaI-digests also gave 1 10-kb signals (Fig. 2B, female and the sputum and stool of the 87-year-Old female. and data not shown, respectively). In contrast, P93 exhibited strongly suggested an outbreak due to VRE, 819 rectal swabs completely different hybridization pattems: a >400-kb signal in the NotI-digest and a 200-kb signal in the SmaI-digest (Fig. and/or sputafrom all of the 759 inpatients and 57 inpatients' 2A and 2B, respectively). Interestingly, Vα〃β-hybridization Since the frequent isolation ofVRE within a short period family members who visited the hospital were examined for slgnals of the 19 isolates were the same size fわr all three the colonization ofVRE. Rectal swabs丘om all 969 hospital restriction enzymes, and the strengths of the slgnals were personnel were also examined. By this screenlng, VRE were slgnificantly weaker than both those of the control vanB- isolated丘.om the rectal swabs of 8 inpatients (P-numbered positive strain and the P93. In addition, the 〃ofⅠ-digests or isolates in Table 1): 4 from inpatients in Ward A and 4from Inpatients in Ward B. No VRE was isolated from the hospital the 19 isolates exhibited no visible DNA band at the positions personnel and inpatients'family members. In addition, We the vanB genes of the 19 isolates were located on Ilo-kb examined 1 ,054 environment sites in Wards A and B for VRE contamination. VRE were isolatedfrom 8 sites in Wards A and plasmids which had no restriction sites for the three enzymes. B (E-numbered isolates in Table 1). No VRE was recovered plasmid DNAs which were linearized by mechanical or non- of the 1 10-kb signals. These血ta suggest the possibility that Furthemore, the data suggest that only a small portion or from the hospital environment after disinfection. specific cleavage gave weak 1 10-kb signals. Strong slgnals Characteristics of the 20 VRE isolates: With one excep- were actually observed at the positions ofplugs, which were tion (an isolate designated P93), susceptibilities to various most likely from the intact, circular plasmid DNAs that did antibiotics were the same fわr all the VRE isolates. As shown not migrate on the PFGE gels (Fig. 2A and 2B). Furthermore, in Table 1 , 1 9 isolates exhibited a high degree of resistance to Southem hybridization analysis of undigested genomic DNAs vancomycin (MICs were 32 〝g/ml or higher) as well as to of血e 19 isolates also gave weak 1 10-kb signals, whereas the benzylpenicillin, gentamlCln, amikacin, and levofloxacin, but undigested DNAsfrom the control strain and isolate P93 gave were susceptible to aminobenzylpenicillin. In contrast, isolate no signal (Fig. 2C). These results also suggest that Vα〃β genes P93 exhibited a lower MIC value to vancomycin (16 LLg/ml), and was susceptible to benzylpenicillin and levofloxacin. The of the 19 isolates are located on 1 10-kb plasmids. Although our repeated efforts to isolate plasmid DNA from these 19 susceptibilityto aminobenzylpenicillin was also different from that of the other 19 isolates (MIC - 0.5 jig/ml). the Southem hybridization analyses uslng Other restriction VRE were for unknown reasons unsuccessful, the results of All of the VRE isolates, including P93, revealed the typical enzymes were consistent with the fbllowlng notion. That is, Ⅵ血B phenotype, i.e., resistant to vancomycin but susceptible Sam- and EcoRI-digested genomic DNAsfrom the 19 isolates to teicoplanin (8). These isolates possessed the vanB gene, as gave 76-kb and 7.8-kb signals, respectively, and the signals demonstrated by PCRusing vanB-specific primers (Fig. 1 ). No PCR products were detected when either the vanA- or vanC-specific prlmerS Were used. at the positions ofplugs disappeared (Fig. 2D and 2E). DISCUSSION In the PFGE analysis Of the restriction endonuclease diges- tion pattems of the genomic DNAs, all of the isolates except P93 showed almost the same NotI or SmaI-digestion pattem An outbreak of VREF occurred at Hokushin General Hospital, Nakano City, Nagano Prefecture, in July, 1 999 (5). (Fig. 2A and 2B, respectively). Apal-digestion pattems of the All of the 20 VRE isolates possessed vanB genes.Antimicrobial 19 isolates were also indistinguishable (data not shown), susceptibility testing and PFGE analys?s revealed that 1 9 out indicatlng that they were derived from a slngle clone. In of the 20 isolates were clonal, indicatlng that this outbreak 19 (a) (b) (A) MIAsIS2P1 2 34 5 67 891011121314151617181920MZMIMIAsIS2P1 2 34 567 891011121314151617181920M2Ml (B) MIASISZP1 2 34 5 67 891011121314151617181920M2MIMIASISZ P1 2 34 5 67891011121314151617181920M2Ml Fig. 2. PFGE analyses and Southem hybridization analyses using a vanB probe・ Genomic DNAswere digestedwith NotI (A), SmaI (B), none (C), SalI (D), or EcoRI (E), and were subjected to PFGE analyses (a); then, Southernhybridization analyses were conducted usingthe vanB probe (b). Lanes: Ml : lambda phage DNA ladder standard (Bio-Rad Laboratories), M2: low-molecular-weight ladder standard (Bio-Rad Laboratories), M3: lambda StyI digest (Nippon Gene), P: a vanB- positive control E.faecalis strain (RVl), A: a vancomycin-susceptible E・faecalis strain (ATCC 19433), Sl and S2: vanc0mycin-susceptible E.faecalis strains (clinical isolates in Shinshu UniversityHospital), I : Hl, 2: H2, 3: H3, 4: H4, 5: P35, 6: P38,7:P40,8: P93,9: P473, 10:P488, ll:Pl130, 12:P8065, 13: E201, 14: E207, 15:E208, 16:E213, 17:E222, 18: E225, 19: E398, 20: E5 18. Running conditions for PFGE (pulse time andrurLning time) were as follows: (A) and (B): 0・47 scc-17・33 sec fわr 20 h 18 min (10-200 kb range fわr the CHEF MAPPER apparatus), (C): 0.22 sec-26・29 sec f♭r 15 h 16 min (5-300 kb), (D): 2.98 see-8.53 see for26h 56 min (201100 kb), (E) 0・ll see-0・36 see for20h 30 min (5125 kb)・ was prlnClpally caused by a slngle clone・ The route through the vanB gene is encoded on a 1 10-kb plasmid, thoughWe could which this clone was brought in the hospital remains unknown, not isolate the plasmid DNA by the conventional procedure・ but transmission of the clone within the hospital may have The vanB gertes are usually encoded on the chromosome (9- occurred due to the contaminated environment, since血e clone 1 1) and occaslOnally on plasmids (12- 17). Plasmids encoding was isolated from various sites in the hospital. the vanB genes were reported to be 60-kb in size by Boyce et al. The results of Southem hybridization analyses implied that (12) and Rice et al. (17), and were larger than 50 kb or 60 MDa 20 Fig. 2. 1Continued. Another important result was the isolation of VRE, which NotI, SmaI, or ApaI-digested genomic DNAs, which we employed in this study, is the most effective and accurate method at present. In order to detemine the level of contamination of VRE in hospital facilities and to organize the most approprlate cooperation system to the VRE infection, the was designated as P93, from an asymptomatic carrier・ This systematic screenlng Of asymptomatic carriers and the isolate also possessed a一昭〃β gene, but showed PFGE pa仕ems surveillance of high-risk environments is recommended・ (approximately 90 kb), as reported by Woodfond et all ( 14, 1 6)・ Molecular analysts Of the plasmid will provide important infbmation to aid an understanding of the molecular mechanism of vanB gene transmission among enterococcal strains・ different from the other 1 9 isolates, indicatlng that the isolate was of different ongin. Susceptibilities of this isolate to various ACKNOWLEDGMENTS antibiotics were indeed different from those of the other We thank Dr. Y. Arakawa of the National Institute of Infectious Diseases, Tokyo, for providing E. faecalis strain RVl. isolates. It isunlikely that the vanB gene ofP93 was transferred from the predominant clones because the vanB gene of P93 is not located on a plasmid but on the chromosome・ It is instead more likely that the P93 was isolated by chance during the systematic screenlng We Performed. This finding Implies REFERENCES that VRE may already have been distributed widely throughout the communlty and hospital facilities in Japan・ ln this regard, 1. Wood丘)rd, N. (1 998): Glycopeptide-resistant enterococci: it is important to mention our recent finding that VRE with a a decade of experience. ∫. Med. Microbiol・, 47, 849-862・ ⅥnA or ⅥlnB phenotype had colonized in the gastrointestinal 2. lshii, Y., 0lm0, A., Kashitani, S., lwata, M. and Yamaguchi, tracts of a small but slgnificant portion of healthy schoolchil- K. (1996): Identification of VanB-type vancomycin dren in the community (18). Healthy individuals whose resistance in EnterDCOCCuS gallinarum from Japan・ J・ gastrointestinal tracts have been colonized by VRE could be reservoirs of VRE in the community. Thus, all communities Infect. Chemother., 2, 1021105. 3. Fujita, N., Tanimoto, K. and Ike, Y. (1998): First report of the isolation of high-level vancomycln resistant Entero- and hospltal facilities in Japan appear to be presented with certainrisks ofencountenng VRE infTection. Fortunately, VRE Coccusfaecium from a patient in Japan. Antimicrob・ infection has thus far been rare in Japan. More attention should Agents Chemother・, 42, 21 50・ 4. Imafuku, Y., Yoshida, H., Sato, T., Yamada, H., Sato, A. and Hiramatsu, K. (1999): A case oflymphocystinfection caused by vanB type VRE. J. Jpn. Assoc. Infect・ Dis・, be paid to the emergence ofVRE, and the appropnate use of glycopeptides should be emphasized. lt is also important to establish rapid and accurate methods of epidemiologlCal investlgation of VRE. In this regard, the PFGE analysts Of 73, 473-476 (in Japanese)・ 21 Potter-Bynoe, G., Sherman, C. B., Romulo, R. L C., 5. Abrashi, K., Baba, K., Fujiki, K., Tdkamoto, T., Nishimura, Fortna, S. and Medeiros, A. A. (1994): Outbreak of H. and Honda, T. (2000): hvestigation ofthe丘rst outbreak multidrug-resistant En ten?coc?usfaecium with transferable vanB class vancomycln reSIStanCe. J. Clin. Microbio1., of vancomycin-resistant Enterococcus (VRE) in Japan. J. Jpn. Soc. lntem. Med., 89, Suppl. 272 (in Japanese)・ 6. Dutka-Malen, S., Evers, S. and Courvalin, P. (1995): 32, 1148-1153. Detection of glycopeptide resistance genotypes and 13. Woodfbrd, N., Jones, B.し., Baccus, Z., Ludlam, H. A. identification to the species level of clinically relevant and Brown, D. F. J. (1995): Linkage ofvancomycin and enterococci by PCR. ∫. Clin. Microbio1., 33, 24-27. high-level gentamicin resistance genes on the same 7. Furihata, K., Sato, K. and Matsumoto, H. (1995): plasmid in a clinical isolate ofEnterococcusfaecalis・ J・ Construction of a combined Notl/Smal physical and Antimicrob. Chemother., 35, 1 79- 1 84. genetic map of Moraxella (Branhamella) catarrhalis strain ATCC25238. Microbiol. Immuno1., 39, 745-75 1. 8. Arthur, M. and Courvalin, P. (1993): Genetics and mecha- 14. Woodford, N., Momison, D., Johnson, A. P., Bateman, A. nisms ofglycopeptide resistanceinenterococci.Antimicrob. in enterococci. Microb. Drug Resist., 1, 235-240・ 15. Chadwick, P. R., Oppenheim, B. A., Fox, A., Woodford, N., Morgenstern, G. R. and Scarffe, J. H・ (1996): Epi- C., HastlngS, ∫. G. M., Elliott, T. S. ∫. and Cookson, B・ ( 1 995) : Plasmid-mediated vα〝β glycopeptide resistance Agents Chemother., 37, 1563-1571. 9. Quintiliani, R. and Courvalin, P. (1994): Conjugal transfer of the vancomycln resistance deteminant vα〃β between demiology of an outbreak due to glycopeptide-resistant enterococci involves the movement of large genetic Enterococcus faecium on a leukaemia unit. J. Hosp. elements from chromosome to chromosome. FEMS Microbiol. Lett., 1 19, 359-364. 16. Woodfbrd, N., Chadwick, P. 氏., Morrison, D. and Infect., 34, 171-182. 10. quintiliani, 良. and Courvalin, P. (1996): Characteriza- Cookson, B. D. ( 1 997): Strains or glycopeptide-resistant tion ofTn1547, a composite transposonflanked by the Enterococcusfaecium canalter their van genotypes during an outbreak. J. Clin. Microbio1., 35, 2966-2968. 17. Rice, L B., Carias, L L., Donskey, C. L and Rudin, S. IS16 and IS256-like elements, that confers vancomycln resistance in Enterococcusfaecalis BM428 1. Gene, 1 72, D. (1 998): Transferable, plasmid-mediated VanB-type 1-8. ll. Carias, L L., Rudin, S. D.,Donskey, C. J. andRice, L B. glycopeptide resistance in Enterococcus faecium・ AntimlCrOb. Agents Chemother., 42, 963-964. (1 998): Genetic linkage and cotransfer of a novel, vanB- 18. Kawakami, Y., Yamaguchi, M., Oana, K., Hama, N., containing transposon (Tn5382) and a low-affinity Takahashi, Y., Akahane, T., Okimura, Y., YanaglSaWa, E. penicillin-binding prptein 5 gene in a clinical vancomycln- resistant Enterococcusfaecium isolate. J. Bacteri01., 1 80, and Katsuyama, T. (1999): Incidence of vancomycin- 4426-4434. resistant enterococci in stool specimens from healthy 12. 占oyce, ∫. M., Opal, S. M., Chow, ∫. W., Zervos, M. ∫., Japanese schoolchildren. Med. S°i. Res., 27, 61-62. 22