Jpn. J. Infect. Dis., 53, 2000

Laboratory and Epidemiology Communications

Molecular Epidemiology of a Methicillin‑Resistant
Staphylococcus aureus Infection
Aki Kaneko, Asayuki Iwail, Katsutoshi Saruta, Tomoko Fujino, Akio Nakamura,
Yoshinori Hamadal and Teruo Kirikae*

In ternational Medical Center ofJapan,
Toyama 1‑21‑1, Shinjuku‑ku, Too)0 162‑8655 andJNational Sanatorium Kagawa‑Children i Hospital,

Zentsuuji 2603, Zentsuuji, Kagawa 76518507
Communicated by Hiroshi Ybshikura(Accepted December 25, 2000)

Nosocomial infection caused by methicillin‑resistant

serotyping (Denka Seiken).

Asshown in Fig. 1, six different PFGE pattems ofSmaI

Staphylococcus aureus (MRSA) is a seriouP problem in

DNA digests were observed. Four BglI, four BstXI and six

immunocompromised hosts such as cancer patlentS ( 1 ).
In a ward fわr chemotherapy ln a Chil血en's hospital withCopI PFGE pattems were detected (Fig･ 2).As for ribosomal

40 beds, a patient with leukemia contracted acute dematitis.

DNA spacer reglOnS, bands with 220 bp, 270 bp and 300 bp

MRSA (No.516) was isolated from the affected part of thewere detected. Isolate No. 391 had all three bands, and isolate
Nos. 518 and 519 had 270 and 300 bp bands (Fig. 3). Fourbody. The patient was repeatedly ln and out or this andanother hospital･ MRSA had not been isolatedfrom patientsin the wards in the previous 2 years. However, nine MRSA

different sized plasmids of 2 kb, 2.5 kb and 4.3 kb werepatients with ca汀lage in other wards in the same hospital,detected (Fig. 4). Isolate Nos. 389 and 391 had no plasmid.
Isolate Nos. 388, 390, 392, 393, 516 and 517 had aplasmid
sized 2 kb. Isolate Nos. 518 and 519 had plasmids sized 2.5

before and during the time the patient was in the hospital. All

kb and 4.3 kb. Sensitivityto antibiotics is shown in Table I;

isolates (Nos. 388‑393 and 517‑519) had been obtained fromthe isolates were subjected to chromosomal DNAtyping byusing pulsed‑field gel electrophoresis (PFGE) (CHEF
MapperTM: Bio‑Rad Laboratories, Hercules, Calif., USA),typlng by length polymorphism of ribosomal DNA spacerM12345678910M MW(kb)A) BglI

regions by a polymerase chain reaction (PCR) assay (2),plasmid DNA typing by uslng agarOSe gel electrophoresis,

antibiotic resistance (WalkAwayTM, Dade Behring, Deerfield,Ill., USA), enterotoxin serotyping (SET‑RPLA: Denka SeikenCo., Tokyo), toxic shock syndrome toxin‑1 (TSSTJl)
production (TST‑RPLA: Denka Seiken), and coagulase

B) Bst XI

Sma I

M12345678910MMW(kb)C) CpoI3'ム一2一l l 爪フ S;9 14294 4 73 ,m2421 ,41 4 ,7胡Fig. 1. Pulsed‑field gel electrophoresis ofSmaI‑digested genomic DNA

from MRSA isolates.
Lane 1‑6: No.388‑393 and Lane 7‑10: No. 516‑519, respectively.
Fig. 2. Pulsed‑field gel electrophoresis of genomic DNA. Genomic
DNA was digested by BglI (A), BstXI (B) or CpoI (C). Lane 1:
No. 388, Lane 2: No. 390, Lane 3: No. 389, Lane 4: No. 392, Lane 5:

M: low range PFG Marker.
*Corresponding author: Fax: +8 1 ‑3‑3202‑7364, E‑mail: tkirikae

No. 517,Lane6:No. 518, Lane 7: No. 519, Lanes: No. 391, Lane9:No. 393, Lane 10: No. 516. M: low range PFG Marker.

@ri.imcj. go.jp242Jpn. J. Infect. Dis., 53, 2000

SA V‑VIM 1 23456789 10MM 123456789 10M.4.3Kb. 2.5Kb
I 2.OKb

Fig･ 3･ Detection of S･ aureus‑SpeciflC ribosomal DNA spacer reg10nSFig･ 4. Agarose gel electrophoresis ofplasmid DNA.
Lane 1‑6: No･ 388‑393 and Lane 7‑10: No. 516‑519, respectively.
M: 1 Kb DNA Ladder Marker.

by PCR･ DNA coding for S･ aureus‑specific ribosomal DNA spacer

reg10nS WaSamPlified by PCR using a pair of oligonucleotide prlmerS
detecting S. aureus 16S ribosomal DNA and 23S ribosomal DNA
regions (ref. 2, SAV and SAVI). Lane 1‑6: No. 3881393 andLane 7‑

10: No･ 5161519, respectively. M: loo bp DNA Ladder Marker.

Table 1 ･Antibiotic pattem classified by antibiotic pattern of 24 antibiotics agalnSt MRSA
Antibiotics Antibiotics listed

PIPC

FOM

CLDM

EMGMMrNOAMK

S良氏R良I S S I I▲

S l▲ S T⊥ S

ABKRFPST

VC M

SSSSS

SSSSS

SSSSSpattem in fbotnote#abcde

氏R良氏氏

良sRRR

R良sRR良氏sR良

良良良I良

SSSSS

"Listed antibiotics are ampicillin, clarithromycln, Cefditoren, cefazolin, cefpodoxime, cefpirome, cefotiam, cefotaxime, cefozopran,nomoxef, imipenem, oxacillin, benzyl‑penicillin･ PIPC: piperacillin, FOM: fosfomycin, CLDM: clindamycin, EM: erythromycin, GM:gentamicin, MINO: minocycline, AMK:amikacin, ABK: arbekacin, RFP: rifampicin, ST: sulfamethoxazole‑trimethoprim, VCM:vancomycin, R: resistant, S: susceptible, I: intermediate

Table 2. Phenotypic and genotypic characterization of the S. aureus isolates
PFGE

PCR

SmaI BglI BstXI Cpol v&vIJuly

388389t emb el r

Sら PA

sputumAntibiotic PlasmidEnterotoxin TSST‑1pattem pattem･￠ β ￠ hO β LAJ

DCDFCadddCddACEEadbb

J .1 月 J 〟 A

1l 1 2 1 1abaCda

121211l 1 3 tJaaea

1 l つJ つJCCCICC+++ l++

11 1IHmH 11++++1111

NICU nasalcavity

390

391Asputum

Btrachea

3 92

NICU skin393

Bnasal/LU 7 0 0ノskinc D IC 良

5 5 5 5 = = 日〓 =cavity.月 ｣ β βα β X 〜んI I I1 1

trach eaNUcarity
nasal cavity

there were five different pattems. Isolate No. 391 produced

In this study, PFGE analysts Clearly showed that MRSA

none ofenterotoxintypes A to D. Isolate Nos. 388‑390, 392,

No･ 516 isolated in the child chemotherapy ward was not

393 and 5 17 produced enterotoxin type C. Isolate Nos. 5 16,

introduced from other wards in the hospital (Table 2). The518 and 5 19 produced enterotoxin types li and C. Isolate No.

combination of PFGE with other genotypings and phenotypings

391 produced coagulase type III but no TSST‑ I ; other isolates

appears to be useful for epidemiologlCal analysts.

produced TSST‑I and coagulasetype II (Table 2).
The data are summarized in Table 2. Isolate No. 5 16 fromthe child chemotherapyunit (ward C) was different from otherisolates with respect to various markers. Isolate No. 388fromward A was closely related to No. 390 from the same ward.
Isolate No.5 18 from Neonatal Intensive Care Unit (NICU)

REFERENCES1. Baltimore, R. S. (1998): Neonatal nosocomial infections.
Semin. Perinato1., 22, 25‑32.

resembled No. 5 19 from ward B. Two MRSA isolates from2･ Samta, K., Matsunaga, T, Kono, M., Hoshina, S., Ikawa,S･, Sakai, 0. and Machida, K. (1997): Rapid identifica‑NICU, No￡. 389 and 392, Were almost identical to each other,tion and typing Staphylococcus aureus by nested PCR

but isolate No. 5 18 which was isolatedfrom the same ward

amplifiedribosomal DNA spacer reglOn. FEMS Microbiol.

4 months later was distantly related to the two isolates.

Lett., 146, 271‑278.

243