Investigation of nosocomial infection caused by arbekacin-resistant, methicillin-resistant Staphylococcus aureus

ELSEVIER

BACTERIOLOGY

Investigation of Nosocomial Infection Caused by Arbekacin-Resistant, Methicillin-Resistant Staphylococcus aureus Yuka Obayashi, Jiro Fujita, Satoshi Ichiyama, Satoko Hojo, Kiyoshi Negayama, Chie Takashima, Hiroshi Miyawaki, Tadao Tanabe, Yasufumi Yamaji, Koichi Kawanishi, and Jiro Takahara

An outbreak of coagulase VU-producing, arbekacin (ABK)resistant, methicillin-resistant Staphylococcus aureus (MRSA) occurred between September 1994 and December 1995, involving five different wards. Twenty-one patients developed skin, wound, drainage, OY respiratory tract colonization with coagulase VII-producing, (ABK)-resistant MRSA. Phenotypic characteristics (production of enterotoxin and TSST-1, antimicrobial susceptibility) and molecular-typing procedure (plasmid DNA profile, pulsed-field gel electrophoresis [PFGEI and arbitrarily primed polymerase chain reaction LAP-PCRI of chromosomal DNA) in isolated strains were

compared. Plasmid analysis identified four different profiles and 19 of 22 strains recovered had identical patterns. PFGE of chromosomal DNA identified three different subtypes and 18 (81.8%) isolates shared the same subtype. AP-PCR also demonstrated that most strains had the same phenotypic characteristics. Although traditional epidemiological methods; for example, coagulase typing, plays a central role in hospital infection control, combination of plasmid DNA profile, AP-PCR, and PFGE may prove to be a particularly informative means of 0 1997 Elsevier tracking the nosocomial spread of MRSA. Science Inc.

INTRODUCTION

bial susceptibility and production of extracellular enzymes, retain their central role in characterization of strains. Recently, combination of phenotypic methods and molecular procedures has proved effective in distinguishing MRSA strains. Previously, we and other investigators suggested that DNA fingerprinting by arbitrarily primed polymerase chain reaction (Al’-PCR) was useful in identifying nosocomial infection caused by MRSA (Van Belkum et al. 1993; Van Belkum et al. 1995; Hojo et al. 1995) It is known that arbekacin (ABK) is effective against methicillin-resistant StaphyZococc~~s aureus (MRSA) resistant to aminoglycoside, such as gentamicin and tobramycin (Inoue et al. 1994). Recently, we experienced a nosocomial outbreak of arbekacin-

Efficient measures for the prevention of nosocomial MRSA infection depend on the ability to differentiate individual strains of MRSA accurately and to track the nosocomial spread of microbial strains. Traditional methods for strain typing, such as antimicroFrom the First Department of Internal Medicine (YO, JF, SH, HM, YY, JT), Department of Clinical Laboratory (KN, CT, KK), Kagawa Medical University, Miki-cho, Kita-gun, Kagawa, the Nagaoya University, School of Medicine (SI), Nagoya, and the University of Occupational and Environmental Health (TT), Kitakyushu, Japan. Address reprinf requests to Dr. Jiro Fujita, First Department of Internal Medicine, Kagawa Medical University, Kagawa, 761-07, Japan.

DIAGN MICROBIOL INFECT DIS 1997;28:53-59 0 1997 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0732~8893/97/$17.00 PI1 SO732-8893(97)00005-9

Y. Obayashi

54

resistant MRSA in patients in our medical university hospital. We attempted traditional methods and molecular procedures to classify the strains. Thus, in this present study, three molecular procedures were attempted to classify MRSA strains. In addition, the efficacy of AP-PCR was compared with both PFGE and plasmid analysis.

MATERIALS Bacterial

AND METHODS

Strains

From September 1994 to December 1995, 21 coagulase VII-producing MRSA were collected in Kagawa University Hospital (613 beds). One MRSA strain that had coagulase type II was also evaluated as a control. Identification of MRSA was performed by plating each isolate on agar containing 2% sodium chloride and 4 pg/ml of oxacillin. The strains of MRSA used in the present study are shown in Table 1. The organism was isolated from pharyngeal swabs, sputum samples, urine, wound gauze, skin exudate, and pus from the drainage tube. Eleven of these patients were on the sixth floor East (nine of them admitted to the Department of Dermatology, the remainder admitted to the Department of Dental Surgery, or to the Department of Surgery). Eight of these patients were admitted to the third floor East or West (the Department of Surgery). KOl is the first strain that was isolated on the third floor. We became aware of the patient’s visiting dermatologist 3 days

TABLE Strain KO K02 K03 K04 K05 K06 K07 K08 K09 KO KO KO KO KO KO KO KO KO KO KO KO KO

1

10 11 12 13 14 15 16 17 18 19 20 21 22

et al.

after admission. It was suggested this strain was transmitted to this patient through the dermatologist’s hands when the patient received a skin biopsy. Four strains were isolated from the gauze of patients who were admitted to the Department of Surgery (third floor). One strain (KO 1) was isolated from pus from the drainage tube from a patient who suffered from mediastinitis after mitral valve replacement, but the infection was treated successfully (third floor). Two strains (KO 7, KO 15) were from urine. Three strains (KO 11, KO 14, KO 16) were from sputum specimen. A blood culture was negative in all cases. None of these patients died of MRSA infection. One MRSA strain with coagulase type II (KO 4) was obtained from a pharyngeal swab from a nurse. The dates of isolation of these MRSA strains in the five different wards are shown in Figure 1.

Typing Methods Coagulase Typing Coagulase typing of the strains was performed using specific antisera against eight different Staphylococcus aweus coagulase types (Denka Seiken, CAT #310801, Tokyo, Japan). Detection of Extracellular Products Production of enterotoxin was determined by the reversed passive latex agglutination kit for the detection of staphylococcal enterotoxin A, B, C, and D (Denka Seiken, SET-RPLA, CAT #340702, Tokyo, Ja-

1 Origin of MRSA in This Study Age and Sex 65 60 72 23 40 92 77 62 75 82 67 51 28 66 69 70 48 64 64 85 27 82

M F F F M M M M M M M F M M M M F M M F M M

Source Drainage Gauze Gauze Pharyngeal swab Gauze Skin exudate Urine Skin exudate Skin exudate Skin exudate Sputum Skin exudate Nasal discharge Sputum Urine Sputum Skin exudate Skin exudate Gauze Skin exudate Eye Pus

Underlying

Disease

Mitral stenosis Menetrier’s disease Lung cancer Rectal cancer Diabetes mellitus Lung cancer Paget’s Disease Ulcer of the leg Paget’s disease Esophageal cancer Pemphigus Ileus Diabetes mellitus Inguinal herniation Head and neck cancer Decubitus Toxic epidermal necrosis Pyothorax tuberculosis Bullous pemphigoid Retinal rupture Acute parotitis

Floor

Month Isolated

3rd west 3rd west 3rd east Nurse 3rd east 6th east 3rd east 6th east 6th east 6th east 3rd east 6th east 3rd east 6th east 6th east 6th east 6th east 6th east 3rd east 6th east 5th west Outpatient

Sept. 1994 Nov. 1994 Dec. 1994 Jan. 1995 Jan. 1995 Feb. 1995 Feb. 1995 Feb. 1995 Feb. 1995 Apr. 1995 May 1995 June 1995 June 1995 Nov. 1994 Aug. 1995 Aug. 1995 Aug. 1995 Nov. 1995 Nov. 1995 Nov. 1995 Dec. 1995 Dec. 1995

Nosocomial

Infection

1994ocr NOV

The

6th

floor

of ABK-Resistant

DEC

1995lAN FEB

55

MRSA

MAR

MAY

Am

JUNE

AUG

JLILY

"CT

SEP

DEC

NOV

,996 JAN

east K06

t

;

tt

QPe KO8

!t

t

t

t

oPe K09

t

KOIOIt QPe

t\t

t

t

t

i

oPe

KO 12

tt K014

t

tit

t

t

t Koi5* KO16

\

it

it

oPe

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oPe

KO 17 L *Pe

t KO 18

t

K020

Ope

tt

3rd floor east

The K03

tt

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KO 5 7128 I oPe

t

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t

KO II

"r

tt

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KO 13 1 /

tt

t

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The

3rd

floor

t

t ttt

west

KOl itttt oPe jKO2

/ +-I QPe

OPe

Outpatient The

5th

K022 t

clinic floor

west K04

Nurse 1994 OCT FIGURE

1

t NOV

DEC

1995 JAN

tt FEB

MAR

t Am

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\

I

oPe

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JUNE

JULY

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SEP

ocx

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19% IAN

The dates of isolation of MESA strains in the five different wards; arrow indicates dates of isolations.

56

pan). Production of the toxic shock syndrome toxin 1 (TSST-1) was determined by the reversed passive latex agglutination kit for the detection of staphylococcal TSST-1 (Denka Seiken, TSST-RPLA, CAT #340801, Tokyo, Japan). Susceptibility of Chemotherapeutic Agexts Isolates of MRSA were tested for their susceptibility to 15 antibiotics by determining the MIC. The activity of each antimicrobial agent was determined by measuring the MIC of each agent with the MIC 2000 Plus System (Dynatech Laboratories, Inc., USA). The antibiotics evaluated in this study were ampicillin (ABPC), piperacillin (PIPC), cefazolin (CEZ), cefotiam (CTM), cefmetazole (CMZ), erythromycin (EM), amikacin (AMK), minocycline (MINO), levofloxacin (LVFX), imipenem (IPM), flomoxef (FMOX), oxacillin (MPIP), arbekacin (ABK), vancomycin (VCM), and gentamicin (GM). Plasmid DNA Analysis (Tanabe and Udo 1995) To obtain plasmid DNA, an overnight culture was harvested, washed with TE buffer (25 mM Tris, 25 mM EDTA, pH 8.0), resuspended in 200 ~1 of TE containing lysozyme (1 mg/ml, L9772, Sigma Chemical Co., St. Louis, MO, USA), lysostaphin (50 pg/ml, L4402, Sigma), and RNase A (20 pg/ml, R5503, Sigma), then incubated for 30 min at 37°C. To this, 0.2 ml of 0.2 M NaOH with 1% (w/v) sodium dodecyl sulfate was added, and this mixture was allowed to stand on ice for 10 min, then 0.15 ml of 3 M potassium-5 M acetate was added, and the mixture was cooled for another 10 min on ice. The mixture was centrifuged at 12,000 g for 5 min, and the supernatant containing plasmid DNA was extracted once with buffer-saturated phenol mixture. The DNA was electrophoresed through a 1.2% agarose gel, stained with ethidium bromide, and photographed. Lambda/Hind III DNA was used as the size standard. Arbitrarily Primed Polymerase Chain Reaction For AI’-PCR (Van Belkum et al. 1993; Van Belkum et al. 1995; Hojo et al. 1995), total MRSA DNA was prepared using InstaGene Purification Matrix (BioRad Laboratories, Inc., CAT #732-6030, Richmond, CA, USA). Bacterial DNA was investigated by APPCR using the PCR KZ primer (CCCATGTGTACGCGTGTGGG) and Ml3 reverse primer (GGAAACAGCTATGACCATG). PCR was performed with a buffer of pH 8.5 and MgCl, 3.5 mmol/l. To optimize the conditions of AP-PCR, the DNA thermal cycler was programmed for 2 cycles of 92°C for 5 min (denaturation), 40°C 5 min (lowstringency annealing), 72°C 5 min (extension), 40 cycles of 92°C 1 min (denaturation), 60°C 1 min (high-stringency annealing), and 72°C 2 min (extension). The amplification products were resolved in loading buffer, electrophoresed through 10/20% gra-

Y. Obayashi

dient polyacrylamide ethidium bromide.

gels,

and

stained

et al.

with

a

Pulsed-Field Gel Electroyhoresis DNA fingerprinting by PFGE was performed according to a previously reported method (Ichiyama et al. 1991). An overnight culture was harvested with saline-EDTA solution (0.15 M NaCl, 10 mM EDTA [pH S.O]), and resuspended in Pett IV solution (1 M NaCl, 10 mM EDTA [pH 8.01). The suspension was mixed with an equal volume of 1.2% low-meltingtemperature agarose (Wako Pure Chemical Co., Osaka, Japan) and allowed to solidify in loo-p1 mold. The block was incubated overnight at 37°C in a lysis solution (1 M NaCl, 0.1 M EDTA [pH 8.01, 0.5% [w/v] Brij 58, 0.2% [w/v] deoxycholate, 0.5% [w/v] Sarkosyl) supplemented with lysozyme (1 mg/ml, Wako Pure Chemical Co.) and acromopeptidase (4 mg/ml, Wako Pure Chemical Co.). The block was incubated overnight at 50°C in ES solution (0.2 M EDTA [pH 8.01, 1% [w/v] Sarkosyl) supplemented with proteinase K (0.1 mg/ml, Wako Pure Chemical Co.) and was then treated with 1 mM phenylmethylsulfonyl fluoride (Sigma) in TE buffer (10 mM TrisHCL [pH 8.01, 1 mM EDTA [pH 8.01) for 4 h and washed four times with TE buffer. Thinly sliced sections of block (about 10 pm) were digested with 10 U of Sma I (Takara Shuzo Co., Kyoto, Japan) for 18 h and then electrophoresed through a 0.9% agarose gel in TBE buffer (0.1 M Tris, 0.1 M boric acid, 2 mM EDTA [pH 8.01) at 20°C by using the contourclamped homogeneous electric field (CHEF) system (Pulsaphor Plus; Pharmacia LKB Biotechnology, Uppsala, Sweden). The conditions for electrophoresis were 200 V for 15 h, with pulse times ranging from 10 to 100 s. Thereafter, the gels were stained with ethidium bromide, washed with distilled water, and photographed. Lambda DNA concatemers (BioRad Laboratories, Inc.) was used as the size standard. Interpretiae Criteria In AP-PCR and PFGE, isolates are determined to be genetically indistinguishable if their gel electrophoresis patterns have the same number of bands, and the corresponding bands are the same apparent size. Isolates are determined to be phenotypically indistinguishable if the patterns of the production of extracellular toxins as well as the patterns of the production of extracellular enzymes are the same.

RESULTS The results of phenotypic and genotypic characteristics of the strains are shown in Table 2. No strain produced enterotoxin or TSST-1. All strains except KO 4 (which has a coagulase type II) were classified as having coagulase type VII. Plasmid screening

Nosocomial

Infection

of ABK-Resistant

TABLE

2

Strain

Coagulase

Plasmids

KO 1 K02 K03 K04 K05 K06 K07 K08 K09 KO 10 KO 11 KO 12 KO 13 KO 14 KO 15 KO 16 KO 17 KO 18 KO 19 KO 20 KO 21 KO 22

VII VII VII II VII VII VII VII VII VII VII VII VII VII VII VII VII VII VII VII VII VII

PCS1 PCS1 pcs2, pCLP pcs1, pcs2, pCL1 PCS1 PCS1 PCS1 PCS1 PCS1 PCS1 PCS1 PCS1 PCS1 PCS1 PCS1 PCS1 pcs1, pcs3, pcS4 PCS1 PCS1 PCS1 PCS1

MRSA

Summary of Results of Phenotypic Genotypic Characteristics

pCS1”

57

and

PFGE

Al’-PCR

A A A B A A A A A A A A A A A A A’ A A A’ A’ A

I I I II I I I I I I I I I I I I I I I I I I

a PCS shows plasmids that have molecular weights smaller than the chromosomal DNA. b pCL shows plasmid that has a molecular the chromosomal DNA.

weight larger than

demonstrated four different patterns (Figure 2). However, 19 of 21 (86.3%) MRSAs shared the same plasmid (pCS1). One MRSA (K04) did not have pCS1. PFGE using Sma I (Figure 3) displayed two closely related profiles (PFGE type A and A’). These types were different from KO 4 (MRSA, this was typed to be B). Most of the isolates showing PFGE type A pattern preceded those isolates showing PFGE type A’. This fact suggested that minor chromosomal DNA rearrangement occurred during the outbreak. By AI’-PCR (Figure 4), KO 1 through KO 3, and KO 5 through KO 22 had identical DNA fingerprinting patterns (Type I). KO 4 (MRSA, which was obtained from the nurse) had a unique pattern (Type II). The result of susceptibility of chemotherapeutic agents showed that MRSA strains tested were highly resistant to ampicillin, piperacillin, cefazolin, cefotiam, cefmetazole, erythromycin, amikacin, minocycline, levofloxacin, imipenem, flomoxef, oxacillin, arbekacin, and gentamicin.

DISCUSSION In the present report, we demonstrated a nosocomial outbreak of ABK-resistant and coagulase VII-

producing MRSA strains mainly in the dermatology ward (the east sixth floor). We believe that this strain is an epidemic, because it was not present in other units, not present in the hospital before the study period, and also disappeared after the intervention. In Japan, ABK is frequently used to treat MRSA (Inoue et al. 1994). ABK-resistant MRSA strains and coagulase VII-producing MRSA had been recently reported. It was reported that 25 among 387 MRSA strains collected in Japan are resistant to ABK, in addition, 89% of isolates with type VII coagulase are ABK-resistant, while 2.5% of those with type II coagulase, 6.3% of those with type III coagulase, and no isolate with type IV coagulase are ABK-resistant (Inoue et al.). It was also reported that among 116 MRSA strains, 48 (41.3%) produce coagulase VII; in addition, coagulase VII-producing MRSA strains are more sensitive to clidamycin and more resistant to minocycline than are other MRSA strains (Kono and Arakawa 1995). The mechanism of the ABK-resistance should be considered. ABK is not modified by the AGsmodifying enzyme, ANT (4’)-1. It was reported that, although ABK is modified by the enzyme AAC-6/ APH-2”, which can inactivate aminoglycosides, the modification rate is low (Kondo et al. 1993). It was also reported that ABK-resistant mutants are isolated from gentamicin-resistant MRSA at a frequency of 1O-4 to 10P5 when the selection is made with MIC of ABK (Suzuki et al. 1994). In addition, isolated ABK-

1234 pCL1 Chromosome pcs4 pcs3 pcs2 PCS1

FIGURE 2 Gel electrophoresis patterns for the plasmid of selected MRSA isolates: lane 1: KO-1; lane 2: KO-4; lane 3: KO-5; lane 4: KO-18. pCL represents a plasmid with a molecular weight larger than the chromosomal DNA. PCS represents plasmids with molecular weights smaller than the chromosomal DNA.

Y. Obayashi

58

M

12

3

4

5

6

I7

8

et al.

Ml23456789

9101112M

bp 1000

700 500 400 300 200

FIGURE 3 Gel electrophoresis patterns for the PFGE products digested with restriction endonuclease Stna 1 of selected MRSA isolates: lane 1: KO-1; lane 2: KO-2; lane 3: KO-3; lane 4: KO-4; lane 5: KO-5; lane 6: KO-6; lane 7: KO-7; lane 8: KO-8; lane 9: KO-9; lane 10: KO-10; lane 11: KO-11; lane 12: KO-12. Lambda/Hind III DNA was used as the size standard (M).

resistant pMS91 plasmid APH-2”,

mutant that possess

from

a laboratory

strain

a gentamicin-resistant

that encodes a modifying shows the increase of

MS353/ gene on a

enzyme AAC-6/ aminoglycosides-

modifying enzymes, which is responsible for ABKresistance of MRSA (Suzuki et al.). In the present study, we could not evaluate the mechanism of ABKresistance, it was suggested that plasmid pCS1 might carry the ABK-resistant genes. In our present study, a nosocomial outbreak was experienced mainly in the dermatology ward. A nosocomial outbreak of MRSA in patients who had severe psoriasis or skin ulcers (Venezia et al. 1992) has been reported. In this report, it was suggested that oatmeal baths in a common bathtub may be the cause of nosocomial transmission (Venezia et al.). In addition, the outbreak of mupirocin-resistant MRSA on a dermatology ward in patients with exfoliating dermatologic disorders (Layton et al. 1993) was also reported. In this report, it was suggested that changing of blood pressure cuffs between patients and more stringent cleaning of communal areas is important to prevent nosocomial transmission (Layton et al.). In our present study, although it was impossible to culture nosocomial MRSA strain from the environment, the hands of physician and nurses, as well as a common bathtub might be candidates.

FIGURE 4 Gel electrophoresis patterns for the AP-PCR products of selected MRSA isolates: lane 1: KO-1; lane 2: KO-2; lane 3: KO-3; lane 4: KO-4; lane 5: KO-5; lane 6: KO-6; lane 7: KO-7; lane 8: KO-8; lane 9: KO-9. M represent a size marker.

To prevent nosocomial infection caused by MRSA, it is essential to differentiate individual strains of MRSA using reliable typing methods. In our study, coagulase, antibiogram, PFGE, and AP-PCR classified 21 isolates as being of the same type. Thus, the study showed that these typing techniques adequately grouped these epidemiologically related isolates. However, to evaluate and compare different typing methods for the epidemiologic characterization of MRSA strains, we should have included more controls. Therefore, our study had limitations to evaluate correctly the ability of these techniques to differentiate this epidemic strain from similar but epidemiologically unrelated strains. The clinical utility of DNA fingerprinting by PFGE in classifying isolates of MRSA has been confirmed (Ichiyama et al. 1991; Layton et al. 1993; Sabria-Lea1 et al. 1994; Leonard et al. 1995; Hartstein et al. 1995). This method has been frequently used to differentiate the MRSA strains. Plasmid analysis has been also frequently used to investigate the MRSA strains. However, this method has some limitations when MRSA does not have a plasmid. AP-PCR has also been recently used to differentiate MRSA strains (Van Belkum et al. 1993; Van Belkum et al. 1995; Hojo et al. 1995). Using these strains, in the present study, we compared the efficacy of DNA fingerprinting by the recently introduced AP-PCR technique for an epidemiological investigation of MRSA. This approach was compared with conventional phenotypic systems, such as biochemical profiles and compared

Nosocomial

Infection

of ABK-Resistant

MRSA

with PFGE and plasmid analysis. The result of APPCR correlated well with that of macrorestriction endonuclease analysis of genomic DNA using Sma I and PFGE. Of the three methods, AP-PCR provides quicker results with minimal technical complexity. In the present study, for arbitrarily primed PCR, bacterial DNA was extracted using a commercial kit, also conferring the advantages of simplicity and rapidity.

59

However, it was also noted that Al’-PCR typing had problems with respect to reproducibility and standardization. In conclusion, we experienced the nosocomial outbreak of ABK-resistant, coagulase VII-producing MRSA strains. Through this experience, we found that AP-PCR and PFGE to be very similar in effectiveness for distinguishing MRSA.

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gel electrophoresis in characterizing strains of methicillin resistant Staphylococcus aureus from a recent hospital outbreak. J Clin Microbial 33:2723-2727. Sabria-Lea1 M, Morthland VH, Pedro-Botet ML, Sopena N, Gimenez-Perez M, Branchini ML, Pfaller MA (1994) Molecular epidemiology for local outbreaks of methicillin resistant Staphylococcus aureus (MRSA). The need for several methods. Eur J Epidemiol 10:325-330. Suzuki T, Fujita K, Nagamachi Y (1994) Emergence of arbekacin-resistant strains among methicillin-resistant Staphylococcus aureus. Japan J Antibiot 47:634-639. Tanabe T, Udou T (1995) Molecular epidemiologic analysis of plasmids in clinical isolates of methicillin resistant Staphylococcus aureus. Environ Inject 10:18-22. Van Belkum A, Kluytmans J, Van Leeuwen W, Bax R, Quint W, Peters E, Fluit A, Vandenbroucke-Grauls C, Van den Brule A, Koeleman H, Melchers W, Meis J, Elaichouni A, Vaneechoutte M, Moonens F, Maes N, Struelens M, Tenover F, Verbrugh H (1995) Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains. J Clin Microbial 33:15371547. Van Belkum A, Bax R, Peerbooms I’, Goessens WH, van Leeuwen N, Quint WG (1993) Comparison of phage typing and DNA fingerprinting by polymerase chain reaction for discrimination of methicillin-resistant Staphylococcus aureus strains. J Clin Microbial 31:79&803. Venezia RA, Harris V, Miller C, Peck H, San Antonio M (1992) Investigation of an outbreak of methicillinresistant Staphylococcus aureus in patients with skin disease using DNA restriction patterns. Infect Control Hosp Epidemiol 13:472476.