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 and4Gene 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 vanBgenotype 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 wereisolated by the screenlng Of asymptomatic ca汀iers and by suⅣeillance of the hospital environment. All or theisolates possessed vanB genes. In a pulsed‑field gel electrophoresis analysts, 1 9 out of 20 VREF isolates exhibitedthe indistinguishable restriction endonuclease digestion pattems of the chromosomal DNA. Additional investiga‑
tion 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 otherisolates. The vα〝β gene was encoded on the chromosome.

MArERIALS AND METHODSINTRODUCTION
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 fromfirst 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). Sinceold) 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 acuteby the surveillance of the hospital environment on Wards A

pyelonephritis caused by a vanA gen.otype VRE (3), and theand 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) andthe 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 asymptomaticat Hokushin General Hospital, Nakano City, Nagano Prefec‑

camerS, 8 1 9 rectal swabs and/or sputa were collected fromture (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 inrectal swabs were collected uslng Sterilized cotton swabs

Japan. The results indicate that a slngle clone with the vα〝βmoistenedwith 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 platessuggested 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: koana@gipac.shinshu‑u.acjp

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 thecycle･ PCR products were analyzed on 2% agarose gel preparedwith 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 ofeach VRE isolate agalnSt Various antimicrobial agents except

RVl , which was provided by Dr. Y. Arakawa (National hstituteof Infectious Diseases, Tokyo), was used as a vanB‑positive

teicoplanin were detemined both by Vitek GPS Cards andcontrol strain.

the MicroScan WalkAway system according to the instruc‑

Pulsed‑rleld gel electrophoresis (PFGE): Preparation oftions provided with each system. Both systems yielded theintact genomic DNAs and restriction endonuclease‑digestion

same susceptibility results. Since we only used the standardof 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 thisIncluded inthe lysis solution at afinal concentration of4 mg/ml.
All the restriction endonucleases used were purchased fromTakara 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 twosystems were described as the MICs. Susceptibilities toteicoplanin 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 theUSA) 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 preparedSoutherTI hybridizatiOn analysis: DNAs were transferred

as previously described (6). Bacterial isolates to be tested

from PFGE gels to nylon membranes (Hybond N';Amershammwere grown at 350C ovemight on sheep blood agar plates(Nippon Becton Dickinson). Colonies were suspended in 300

Pharmacia Biotech, Buckinghamshire, UK) using a VacuGeneblotter apparatus(Amershamm Pharmacia Biotech). The vanB〝l of distilled water and were boiled f♭r 10 min. Boiledprobe, a 635 bp fragment of the vanB coding region, Was

samples were chilled quickly on ice and were subjected toprepared 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 ofa 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 (2mM 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 LLIRESULTSof 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 anCity, 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
B

InpatientH

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.

RectalswabaInpatient

P3

8

I

,

Aug.

Rectalswab

B

Inpatient

P40

I

,

Aug.

Rectalswab

A Inpatient P93

1

,

Aug.

Rectalswab

B

1 , Åug.

Rectal swab

A Inpatient P48 8

1

RectalA Inpatient P 1 I 30

1 , Åug.

Inpatient

P473

,

Aug.

A Inpatient P8065

1 , Åug.

B Environment E20 1

4,

B Envi ron men I E20 7

4, Aug.

Aug.

B Environment E208

4, Åug.

a Environment E2 1 3

4,a Environment E222

4, Åug.

Aug.

TEIC

≧ ≧ ≧ ≧ー ≧ ≧ ≧ ≧ ≧ ≧ 32 32 32 32632 32 32 32 32 32

SSSSSSSlevels

PCG

(MIC,

ABPC

LLg/mlGM′b∠U ′b上U AV2′○ b 0∠U′b ′0∠U b′ 0

≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ 1 1 1 一1 ‑ 1′0 b6′0 ∠U′b KV′b上U 0/b′ b′ 0 b′

≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ 1 l1 ‑ 1 ‑ 1

2 2 2 0 2 2 lZ 2 2

)*

AMK.
≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ ≧ 64 64 糾 64 64 64≧ ≧ ≧ ≦ ≧ ≧ ≧ ≧ ≧ ≧ 史U0O80 01 08CXU史80 0 0

5

swab

Rectal swab
Rectal swab

ward

moor

bed side table
over table

ward

noor

bed side tablea Environment E225

4, Åug.

bed pan

A Environment E398

4,

ward

B Environment E5 1 8

4, Aug.

Aug.

resistance

VCM

moor

slipper*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 pFig. L Detection orvd〃β genes in 20 VRE isolates. PCR products were analyzed on 2% agarose gel. Lanes: M: molecularweight 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 a76‑year‑old female with cerebral infarction (H2), from theurine 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 sufferingfrom 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 ofyear101d 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) andtion 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). TheirMRSA 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

Since the frequent isolation ofVRE within a short period
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

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 werepersonnel 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‑numberedpositive 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 hospitalthe 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 andplasmids 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 orfrom 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 weretion (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 toSouthem hybridization analysis of undigested genomic DNAsvancomycin (MICs were 32 〝g/ml or higher) as well as toof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. Theof 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 fromthat 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 typicalenzymes 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, asgave 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‑ orvanC‑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. Inof 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: vanc0‑
mycin‑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 (5‑300kb), (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 throughthe 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 contami‑
nation of VRE in hospital facilities and to organize the mostapproprlate cooperation system to the VRE infection, thewas designated as P93, from an asymptomatic carrier･ Thissystematic screenlng Of asymptomatic carriers and theisolate 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 mecha‑
nism 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 otherWe 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 transferredfrom 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

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22