Comparison of phenotypic methods and DNA hybridization for detection of methicillin resistant Staphylococcus aureus

0095-1137/94/$04.00+0

Copyright (C 1994, AmericanSociety for Microbiology

Comparison of Phenotypic Methods and DNA Hybridization for

Detection of Methicillin-Resistant

Staphylococcus

aureus

PASCALE

RICHARD,'*

MICHEL

MEYRAN,2

ESTELLE

CARPENTIER,'

ANDRE

THABAUT,2

AND HENRIB. DRUGEON1

Laboratoire deBacteriologie B, Hopital G.R. Laennec, 44035 Nantes

Cedex,'

and Laboratoire deBiologie Medicale,

Hopitald'Instruction des Arm&es Begin, 94160Saint-Mande2France

Received22September 1993/Returned formodification 12November1993/Accepted 24November 1993

One hundred

thirty-eight

Staphylococcusaureusisolatesfrompatientswith severestaphylococcalinfections were collected in 15 French hospitals.Detectionof the mecgenewasperformed bydot blothybridizationwith a specific DNA probe. Dot blot results were used to characterizethe isolates as methicillin susceptible (77 isolates) or resistant (61 isolates). The isolates were screened for methicillin resistance by an agar spread method on Mueller-Hintonplates containingoxacillin (2and 10,ug/ml) and were incubated at37°C,with 108

CFU as the inoculum. MICs of oxacillin and methicillin were determined by the agar dilution method on

Mueller-Hinton plateswithoutNaCl, by using105 CFU per spot, after24 and48hof incubationat30 or37°C.

ModeratelyelevatedMICswerefound for 20 isolates (14.5%).The mecgenewasdetectedin six (30%)ofthe

isolates expressinga low levelof resistance tomethicillin and/or oxacillin.Asdeterminedbycomparison with probehybridization results,thespread plate methodwith oxacillin at 2,ug/mlwas moresensitive(sensitivity,

100%) and specific (specificity, 100%o) than agardilution with either methicillin oroxacillin in identifying methicillin resistanceorsusceptibility. Determinations of methicillinandoxacillinMICsbytheagardilution

methodhad aspecificity of 99to100%dependingontheconditions ofincubation,butthesensitivitywasbelow

85%whateverthe durationortemperature of incubation.

The presence of a low-affinity penicillin-binding protein (PBP 2a) encoded by themecgeneis the main factor respon-sible for methicillin resistance instaphylococci(2, 14).Themec

gene and its product (PBP 2a) have never been found in

methicillin-susceptible Staphylococcus aureus,while theyhave

been detected in almost all methicillin-resistant S. aureus

isolates(MRSA) examinedsofar(1,2, 7, 11, 14,18, 21, 24,25).

Despite the invariable presence of these resistance

determi-nants in MRSA, phenotypic expression of methicillin resis-tance is strain specific andis oftenheterogeneous (4, 12, 13).

Thus, routine susceptibility tests may fail to identify MRSA,

particularly strains expressing a very low level of resistance. Although incubation at temperaturesbelow 37°C, prolonged incubation, use of media containing NaCl, and use of large inocula have been proposed for improving the detection of MRSA, there isnouniversal agreementonthechoice of either

an optimal procedure orMIC breakpoints for the

identifica-tion of methicillin resistance(4,5,8, 12,17, 19,23).Detection of the mec gene by DNA hybridization or PCR has been proved useful foridentification of MRSA (1, 7, 11, 14, 18, 21, 24, 25).Although these methods require specialized staff and

equipmentand maynotbeappliedin everyclinical

microbiol-ogylaboratory, they provideauseful tool for the evaluation of

procedures based onphenotypic expression of resistance. S. aureus isolates with a borderline level of resistance to methicillinoroxacillin have beenreported from time to time (7, 15, 16, 22). Three mechanisms of resistance have been

associated with this phenotype: mec-encoded resistance (ex-pression class 1), overproduction of penicillinase, and modifi-cations of normal PBPs (7, 15, 16, 22). Since there is no evidence thattheefficacyofmethicillinoroxacillin isimpaired

*Correspondingauthor. Mailing address: Laboratoire de

Bacteri-ologie A, Institut de Biologie des H6pitaux de Nantes, 9 Quai Moncousu,44035 NantesCedex, France. Phone: 40 08 39 58. Fax: 40 08 41 14.

for isolates with overproduction of penicillinase, it seems

importantto differentiate these isolates from those with

mec-related resistance (3). However, the frequencyof the border-line resistance phenotype and the relative frequency of the

corresponding resistance determinants havenotbeen studied

extensively.

The purposes of this study were to compare phenotypic

methods foridentificationof MRSA with detection of themec

gene byDNAhybridizationand toevaluate the frequency of

mec-encoded borderline resistance among French S. aureus

isolates.

(Part ofthis workwas presented at the 32nd Interscience

Conference on Antimicrobial Agents and Chemotherapy,

Anaheim, Calif., 1992.)

MATERIALS ANDMETHODS

Isolates. In1990, themicrobiologists from 15 French

hospi-tals (in Bordeaux,Brest, Clermont-Ferrand, Dijon, Grenoble,

Lille,Lyon,Montpellier, Nantes, Nice, Paris-Begin,Paris-Saint

Antoine, Poitiers, andStrasbourg)wererequested tosend all

isolates of S. aureus collected from patientswith systemic or

deep-seated infectionsto the Groupe d'Etude des Infections

S6veres

a Staphylocoques. A total of 138 isolates were

col-lected: 118 fromblood,8from skinorwoundabscesses,7from intravenouscatheters, and 5from othersources.

Spreadplatemethod.Allisolateswerescreened for oxacillin

resistance by using a modification of the spread plate test

describedby Archer and Pennell (1). A 100-,ulportion ofan

overnightMueller-Hinton broth (Difco Laboratories) culture

(approximately 108 CFU) was spread on Mueller-Hinton

plates (Difco Laboratories) containing 5% NaCl and oxacillin

atconcentrations of 2 and 10p,g/ml.Anygrowthafter2days of incubation at 37°C was considered indicative of resistance.

MIC determinations. MICsofoxacillin and methicillinwere

determinedby agardilutionon Mueller-Hinton plates (Difco

613

on May 15, 2020 by guest

http://jcm.asm.org/

TABLE 1. Detection ofmethicillin resistanceby DNAhybridizationand routinemicrobiologicsusceptibilitytests in 138 S. aureusisolatesa

No.of No.of isolates with indicated MIC

(jig/ml)

under incubationconditions shown isolates with

spreadplate Oxacillin Methicillin

Hybridization result result

300C24h 30°C 48h 370C24h 37°C 48h 300C24 h 300C 48h 37°C 24 h 37°C48 h

OSb ORc

c2 .4 c2 .4 c2 .4 s2 .4 c8 .16 '8 .16 c8 .16 s8 .16

mecpositive(n =61) 0 61 26 35 23 38 16 45 14 47 10 51 10 51 12 49 12 49

mecnegative(n =77) 77 0 76 1 76 1 77 0 77 0 77 0 77 0 77 0 77 0

Total 77 61 102 36 100 39 93 45 91 47 87 51 87 51 89 49 89 49

aDetection ofthe mecgenewasperformed by dot-blot hybridization withaspecific DNA probe. The isolates werescreenedforoxacillin resistance byanagar spread platemethodonoxacillin(2and 10,ug/ml)-containing Mueller-Hinton plates incubatedat370C, with108CFU astheinoculum. MICsofoxacillinandmethicillin were determined by the agar dilution methodonMueller-Hinton plates without NaCl, using105CFUper spot, after24and48hofincubationat30or37°C.

bOS, oxacillin susceptible, i.e.,isolatesnotgrowingonthe plates with 2

p.g

of oxacillin per ml. cOR,oxacillinresistant,i.e., isolates growingontheplateswith2 ,ug ofoxacillin perml.

Laboratories) without NaCl, after24and 48 h ofincubationat

30 or 37°C, by using

105

CFU per spot.

Susceptibility

and

resistance breakpointswerec2 and

.4

,ug/mlfor oxacillin and

.8

and

.16

,ug/mlfor methicillin(19, 20).MICsofoxacillin in the presence of clavulanic acid were also determined by the

agardilutionmethodafterincubationat30°C,by using afixed

concentration of clavulanate (4

jig/mI).

,-Lactamase assays were performed with nitrocefin disks (Cefinase; bioMerieux

SA).

Dot blot hybridization. The mec-specific DNA probe was obtained from JohnKornblum,Public Health Research Insti-tute, New York, N.Y. The origin of this probe has been

described previously (22). Forhybridization experiments, the

probewasradiolabeled with

32P

byusingarandom-priming kit

(Amersham FranceSA, LesUllis, France). Whole-celllysates

were prepared from 1 ml of an overnight broth culture. Bacterial cellswere pelleted by centrifugation andthen

resus-pendedandincubatedat37°Cfor 30 min inthefollowing lysis

buffer: 25 mMTrishydrochloride (pH 8.0), 10mMEDTA, 16

jig

oflysostaphinperml(Sigma Chemical Co.,St.Louis,Mo.)

and 8

jig

of muramidase per ml (Sigma). The resulting

protoplastswerelysedwith sodiumdodecylsulfate(0.1%)and

20 ,ugofproteinaseK perml

(Sigma)

for 1 hat37°C. Samples

(25

RI

each)oflysateswereloadedintothe wells ofadot blot

apparatus (Schleicher & Schuell, Inc., Keene, N.H.) and

transferred to a nitrocellulose membrane. Hybridization and

washingswerecarriedoutbystandard methods(13).

Autora-diographswere exposedfor 4 hat -70°C.

Interlaboratoryagreement.Susceptibility testingand

detec-tionof themecgenewere

performed

attwo separate

labora-tories,and resultswerereportedto an independent

investiga-tor for

comparison.

For strains which showed

discrepancies

betweenhybridization results andsusceptibility testingresults

andfor all strains withalow levelofresistance,the

hybridiza-tion resultswereverified.Atransportation medium(bacterial

strains storage medium; Sanofi

Diagnostics

Pasteur,

Marnes-La-Coquette,France)wasinoculatedfrom the culture

used

for

DNApreparation andsent tothe second

laboratory

for MIC

determinations in duplicate. The MIC determination results

were validated if the differences between all determinations

werewithin onelog 2 dilution. Forsixisolates, the

discrepan-cies between thehybridizationandMIC resultswereattributed

to a contamination of the storage culture used for the first determination.Forthe other 26isolatesthatwereretested,the

hybridization andsusceptibility testing resultswere

reproduc-ible.

RESULTS

Comparisonoftheprobe and spread plate results (Table1).

Theplates were read after 24 and 48 h of incubation because the first reading was difficult for some isolates (13 isolates)

giving pinpoint colonies. The reading was easier after 48 h of

incubation, but the interpretation remained unchanged.

Amongthe 138 staphylococcal isolates examined, 77 isolates

(55.8%) did not grow on the plates containing the lowest concentration of oxacillin (2 p.g/ml) and were classified as

oxacillin susceptible, while 59 isolates (42.7%) grew on the

plates containing the highest concentration of oxacillin (10

,ig/ml)

and were considered oxacillin resistant. Two isolates

(1.5%) grew onlyontheplates containing oxacillin at 2 ,ug/ml. Of the 61 isolatesgrowingontheplates containing oxacillinat

2,ug/ml, 100% gaveapositive signal with the mec probe, while 100% of the 77oxacillin-susceptible isolates showed no detect-able hybridization. Thus, compared with the hybridization procedure, the spread plate test with oxacillin at 2 ,ug/ml hada

sensitivity and a specificity of 100%; with oxacillin at 10,ug/ml,

thesensitivity and the specificity of the spread plate testwere

96.7 and97.4%, respectively.

Comparison of the probe and MIC results (Table 1). The MICs of methicillin were .8 jig/ml for all mec-negative isolates, and theMICs of oxacillin were .2 ,ug/ml for 76 of the 77 mec-negative isolates. Under all test conditions, oxacillin MICs for 7 (11.5%) of the 61 mec-positive isolates were.2 jig/ml and methicillin MICs for 9 (14.75%)were .8 jig/ml. However, MICs of both oxacillin and methicillin wereeither inferior or equal to the National Committee for Clinical

LaboratoryStandards(NCCLS) breakpointsforsusceptibility

foronly fourmec-positive isolates (6.5%). With the NCCLS

breakpoints,MIC determinationswerehighly specific

(speci-ficity, 99 to 100% under allconditions)but lackedsensitivity. After 24 h ofincubation, theagar dilution had sensitivities of

57% (30°C) and 73% (37°C)for oxacillin and sensitivitiesof

83% (30°C) and 80% (37°C) formethicillin. When the

incu-bationlasted 48h,the resultsobtained with methicillin didnot

change but the sensitivities for oxacillin increased to 62%

(30°C) and 77% (37°C). Changing the MIC breakpointsdid notresult in sensitivity andspecificityboth greater than 90%.

Low-level resistance. Twenty isolates (14.5%) expressed a

loworborderline level of resistancetomethicillinoroxacillin

(MICsof

methicillin,

4 to 16

,ig/ml;

MICs of

oxacillin,

1 to4

,ug/ml) under any of the test conditions. Six of the 61

mec-positive isolates (9.8%) fell in this category. Four of them

(6.5%) would have been consideredsusceptibletoboth

on May 15, 2020 by guest

http://jcm.asm.org/

TABLE 2. Characteristics of borderlineMRSA

Oxacillin spread MIC(s)(,ug/ml) under indicated incubation conditions

Strain" Presence of plateresults" Oxacillin Methicillin

penlicillinase

2pLg/ml 10 ,ug/ml 30(C,24 h 30°C, 48 h' 37°C, 24 h 37°C, 48 h 30°C, 24 h 30°C,48 h 37°C,24 h 37°C,48h

mecnegative

82 + - - 0.25 0.25, 0.125 0.5 0.5 4 4 4 4

84 + - - 0.5 2,2 0.125 0.125 1 1 1 1

103 + - - 0.5 2, 2 0.125 0.125 1 1 1 1

125 + - - 0.25 0.25, 0.25 0.25 0.25 4 4 2 4

128 + - - 0.5 1, 1 0.25 0.25 2 2 1 1

143 + - - 0.5 0.5,0.5 1 1 2 2 2 2

145 + - - 0.125 0.125, 0.25 1 1 1 1 1 1

154 + - - 0.5 0.5, 0.5 0.25 0.5 4 4 2 2

167 + - - 0.25 0.25,0.125 0.5 0.5 2 4 4 4

168 + - - 0.125 0.25,0.25 0.25 0.5 2 2 4 4

169 + - - 2 2,2 0.125 0.125 2 2 1 2

171 - - - 4 4,4 0.25 0.25 2 2 1 1

179 - - - 1 0.5,0.5 0.5 0.5 4 8 4 4

200 + - - I 1, 1 1 2 2 2 2 2

mecpositive

61 + + + 2 2, 2 2 2 0.25 0.5 4 4

66 + + - 1 2,0.5 0.125 0.125 1 2 2 2

68 + + - 0.5 0.5, 1 0.5 1 2 2 2 1

97 + + + 0.25 0.5, 0.5 0.5 0.5 16 32 16 16

112 + + + 0.25 0.5,0.5 4 4 16 16 16 16

183 + + + 0.25 0.25, 0.5 0.25 0.25 4 4 4 4

"iiec negative anditIecpositive,nohybridization and hybridization with the mec-specific probe, respectively.

hAfter 48 h of incubation at

37"C.

+,growth; -,absence of growth.

First and second values were obtained without and with clavulanic acid (4 ,ug/ml),respectively.

cillin and oxacillin

according

totheirMICs. With the

exception

of two

isolates,

all

mec-positive

borderline resistant isolates

grewon

plates containing

oxacillinatthe

highest

concentration

(10

,ug/ml)

in the

spread

plate

test

(Table 2).

Of the 77

mec-negative

isolates,

14

(18.2%)

had aborderline resistance

tooxacillin

(9

of

77,

i.e.,

11.6%) and/or

methicillin

(6

of

77,

i.e.,

7.8%),

butnonegrewonthe

plates containing

2

p.g

ofoxacillin

perml

(Table 2).

Clavulanicacid

(4 ,ug/ml)

didnot

significantly

reduce the MICs of oxacillin for these borderline-resistant

isolates,

andtwoofthemwere

penicillinase negative (Table 2).

TheMICs of

penicillin

Gfor these two

penicillinase-negative

strainswere0.05

.g/ml.

DISCUSSION

Because the choice of antimicrobial

therapy

and isolation

procedures

for

patients

with

staphylococcal

infectionsdepends

on the

susceptibility

of the isolates to

methicillin,

there is a

need for reliable methods for detection of methicillin

resis-tance.

However,

because ofthe

heterogeneous expression

of

resistance,

somemethicillin-resistant isolatesmaybe

misiden-tifiedasmethicillin

susceptible

by

the conventional methodsof in vitro

testing.

Various

procedures

have been

proposed

for

improving

the detection of methicillin

resistance,

but there is

no standardized

procedure

for the detection of methicillin resistance that hasbeen

accepted

worldwide

(4,

6, 12, 19,20).

Foragardilution and brothmicrodilution

testing,

the NCCLS

recommends the addition of2% NaCl, incubationat 35°Cfor

24 h and the use of either methicillin or oxacillin, although

oxacillin is

preferred

becauseof itsstabilityand the

reproduc-ibility

of itstestresults(19,

20).

Inaddition,forconfirmation of

resistance,

the NCCLS recommends an agar screen method

using

6

pLg

of oxacillin per ml in Mueller-Hinton plates

containing

4% NaCl andincubatedfor 24 hat35°C.InFrance,

theComite Francais del'Antibiogrammesuggestsusing oxacil-lin instead of methiciloxacil-lin and either adding 5% NaCl to the medium and incubatingthe plates at 37°C for 18 h or using Mueller-Hinton medium and incubatingtheplatesat30°Cfor 18 h (5). In addition, there isno universal agreement on the

MIC breakpoints for defining methicillin resistance. Isolates

for which the MICs of oxacillin are higher than 2 ,ug/ml are

usuallyclassified asMRSA,while isolates for which the MICs

of methicillin arehigher than 2, 4,or 8 ,ug/mlareconsidered resistant(5, 8, 17, 19,20). These problems about the choice of

an optimal method for the detection of methicillin resistance

aredue to the absence ofareference procedure. Because of

the mec gene's invariable presence in MRSA, its detection

provides an accurate method for identification of MRSA,

independent of environmental conditions that may affect the

phenotypic expressionofresistance (1, 7, 11, 18, 21,24, 25).

As determinedbycomparison withDNAhybridization, the

spread plate method and agar dilution were accurate in

identifying methicillin-susceptibleS. aureus. The agar dilution

testhad apoorsensitivity for the detection of MRSAdespite

the use ofan increased inoculum (105 CFU) comparedwith the NCCLS recommendations (104 CFU). This is probably relatedtothe absence of NaCl inthetestmedium,since other authors obtained similar results using Mueller-Hinton agar without NaCl and noted a substantial improvement of

sensi-tivityin the presence ofNaCl(10, 11). AccordingtoHuang et

al., the best agreementbetween the NCCLS reference broth

testandagardilutionwasachieved when 2% NaClwasadded

to the medium, although this cannot be achieved without

sacrificing

specificity (10). For oxacillin, more mec-positive

isolatesweredetectedafter incubationat37thanat30°C; this

is unlikely to be a temperature effect since, as previously

reported, incubation at 30 instead of 37°C improved the

sensitivityof the agar dilution testfor methicillin (11, 12).

on May 15, 2020 by guest

http://jcm.asm.org/

The spread plate method was the most sensitive phenotypic method for the detection of MRSA, probably because it uses both a high salt content and a large inoculum. Gerberding et al. have previously noted that agar screening with oxacillin at 6 ,ug/ml lackedspecificity when a larger inoculum(107CFU) was used,especially when NaCl was added to the medium (9). In our study, the specificity of the spread plate test was better, despite the use of lower concentrations of oxacillin (2 ju.g/ml)

and of a greater inoculum(108 CFU), probably because we did notselect for troublesome isolates, while most strains tested by Gerberdingetal. expressedborderline resistance (9).

We found a borderline resistance to methicillin and/or oxacillin in 20 of 138 French S. aureus isolates (14.5%) from

patients with severe infections. mec-encoded resistance

ac-counted for this phenotype in 30% (6 of 20) of the isolates. Of

77mec-negative isolates,14(18%) had a borderline resistance

to oxacillin and/or methicillin, a quite high frequency that might be due in part to the large inoculum (105CFU)used for agar dilution. However, using an inoculum of 103 CFU, de Lencastre etal. found MICs of methicillin of -4pug/mlfor 5% (11 of 215) of recent methicillin-susceptible S. aureus isolates, compared with 8% (6 of 77) (P = 0.4) in our study (7). The spread plate test with oxacillin at 2 p.g/ml was useful in identifying the strains with mec-encoded resistance among isolates with ambiguous MICs. Four mec-negative isolates yielding oxacillin MICs of -2

[Lg/ml

with an inoculum of 105

CFU did not grow on the agar screen plates with 2 pug of oxacillin per mldespite the useofalargerinoculum; thiswas

possiblyrelatedtotemperature,since the MICs ofoxacillinat

37°Cwere .0.25 ,ug/ml for all these isolates. Two of the 14

mec-negative isolates with aborderline level of resistance had

no detectable r-lactamase activity. Althoughwe did not look for the biochemical basis of resistance, it is likely that the borderline resistance phenotypewasalsoindependent of pen-icillinase production in the 12 other mec-negative isolates, since the addition of clavulanic acid didnotsignificantlyreduce the MICsofoxacillin. Thesefindingsare consistent with those of de Lencastreet al.,who havesuggested that, in additionto

the presence ofapenicillinase, many contemporaryisolates of S.aureushavealow-level intrinsic resistancetomethicillin that

ispossiblyrelatedtoaltered PBPs(7, 22).

Since the spreadplate method used in thisstudyidentified MRSA withasensitivityandaspecificityof

100%,

this method should be usedroutinelyfordetection of methicillin resistance. Determinations of methicillin and oxacillin MICs by agar dilution at 30 and 37°C, after 24 and 48 h of incubation on

Mueller-HintonplateswithoutNaCl,hadapoorsensitivityand gave ambiguous results for 14.5% of the isolates. Compared

with MIC determinations, the spread plate method with

ox-acillinat2 ,Lg/mlwasmuchmoreefficient for identification of mec-encoded resistance in isolates expressing a low level of resistance. Detection of the mec gene should be used as the procedure of referencewhenroutinesusceptibilitytesting gives

ambiguous results.

ACKNOWLEDGMENTS

We are grateful toJohn Kornblum for providingthe DNAprobe used in the presentstudy,to ourcolleaguesfrom theGrouped'Etude des Infections Severes a Staphylocoques for supplying the isolates included in thestudy,andtoHerveRichet forsuggestionsandhelpful

discussion.

REFERENCES

1. Archer, G. L., and E. Pennell. 1990. Detection of methicillin resistance instaphylococci by using a DNA probe. Antimicrob. Agents Chemother. 34:1720-1724.

2. Beck,W.D., B.Berger-Bachi, and F. H. Kayser. 1986.Additional

DNAinmethicillin-resistantStaplhylococcusaureus andmolecular cloningofnwec-specificDNA. J. Bacteriol. 165:373-378. 3. Chambers, H. F., G. Archer, andM.Matsuhashi. 1989.Low-level

methicillinresistance in strains ofStaphylococcusaureus. Antimi-crob.AgentsChemother. 33:424-428.

4. Chambers, H.F.,andC.J.Hackbarth. 1987. Effect ofNaCl and nafcillin on penicillin-binding protein 2a and heterogeneous ex-pressionof methicillin resistance in Staphylococcusalreus. Anti-microb. Agents Chemother. 31:1982-1988.

5. Comite de l'Antibiogrammede la SocieteFrancaise de Microbi-ologie. 1992. Communique 1992du Comitede l'Antibiogramme de laSociet FrancaisedeMicrobiologie.Pathol. Biol. 40:741-748. 6. Coudron,P.E., D. L. Jones, H. P. Dalton, and G. L.Archer.1986. Evaluationoflaboratory tests for detection of methicillin-resistant Stalphylococcus aurcus and Staphylococcus epidermidis. J. Clin. Microbiol. 24:764-769.

7. deLencastre, H.,A. M.SaFigueiredo, C. Urban, J. Rahal, andA. Tomasz. 1991. Multiplemechanisms ofmethicillinresistance and improvedmethods fordetection in clinical isolates of

Staphylococ-cus aureus. Antimicrob. Agents Chemother. 35:632-639. 8. French, G. L., J. Ling, Y. W. Hui, and H. K. T. Oo. 1987.

Determination of methicillin-resistance in Staphylococcus aureus

by agar dilution and disc diffusion methods. J. Antimicrob. Che-mother. 20:599-608.

9. Gerberding, J. L., C. Miick,H. H.Liu, andH. F.Chambers. 1991. Comparisonofconventionalsusceptibilitytestswithdirect detec-tion ofpenicillin-bindingproteini 2a in borderline

oxacillin-resis-tant strains ofStaphylococcus aureus. Antimicrob. Agents Che-mother.35:2574-2579.

10. Huang, M. B.,T. E.Gay, C. N. Baker, S. N.Banerjee, and F. C. Tenover. 1993. Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-baseddilution methods. J. Clin. Microbiol. 31:2683-2688. 11. Ligozzi, M.,G. M. Rossolini,E. A.Tonin, andR. Fontana. 1991.

Nonradioactive DNAprobefordetection of gene for methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Che-mother. 35:575-578.

12. Madiraju,M. V. V. S.,D.P.Brunner,and B.J.Wilkinson. 1987.

Effects of temperature,NaCl,andmethicillinonpenicillin-binding proteins, growth,peptidoglycansynthesis,andautolysisin methi-cillin-resistant Staphylococcus aureus. Antimicrob. Agents Che-mother. 31:1727-1733.

13. Maniatis,T. E., F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

14. Matsuhashi, M., M. D. Song, F. Ishino, M. Wachi, M. Doi, M. Inoue,K.Ubukata,N.Yamashita,and M. Konno.1986. Molecular cloning of the gene of a penicillin-binding protein supposed to

cause high resistance to ,B-lactam antibiotics in Staphylococcus

aureus.J. Bacteriol. 167:975-980.

15. McDougal,L.K.,andC.Thornsberry. 1986. The roleof

3-lacta-mase in staphylococcal resistance to penicillinase-resistant peni-cillinisandcephalosporins. J.Clin. Microbiol. 23:832-839. 16. Montanari,M.P.,E.Tonin,F.Biavasco,andP.E.Varaldo. 1990.

Further characterization of borderline methicillin-resistant

Staph-ylococcusaureusandanalysisofpenicillin-bindingproteins. Anti-microb.AgentsChemother. 34:911-913.

17. Mouton, R. P., S. L. T. Mulders, J. De Knijff,andJ. Hermans.

1989. Comparison oftest systems forrecognition ofmethicillin resistance inStaphyloco-ccusaureus.Eur.J.Clin. Microbiol. Infect. Dis. 8:968-973.

18. Murakami, K.,W. Minamide, K.Wada, E. Nakamura, H.

Ter-aoka, andS. Watanabe. 1991. Identification of

methicillin-resis-tantstrains ofstaphylococcibypolymerasechain reaction.J.Clin. Microbiol. 29:2240-2244.

19. National Committee for Clinical Laboratory Standards. 1990. Methods for dilution antimicrobialsusceptibilitytestsforbacteria that grow aerobically, 2nd ed. Approved standard M7-A2. Na-tional Committee for Clinical Laboratory Standards, Villanova,

Pa.

20. National Committee for Clinical Laboratory Standards. 1992. Performance standards for antimicrobial susceptibility testing.

on May 15, 2020 by guest

http://jcm.asm.org/

M1(0-S4. National Committee for Clinical Laboratory Standards, Villanova,Pa.

21. Tokue,Y.,S. Shoji, K.Satoh,A.Watanabe, andM.Motomiya.1992. Comparison ofa polymerase chain reaction assay and a

conven-tional microbiologic method for detection ofmethicillin-resistant Staphylococcusaureus.Antimicrob. Agents Chemother. 36:6-9.

22. Tomasz, A., H. B. Drugeon, H. M. de Lencastre, D. Jabes, L.

McDougall, and J. Bille. 1989. New mechanism for methicillin resistance in Staphylococcus aureus:clinical isolates that lack the

PBP 2ageneandcontain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrob. Agents Che-mother. 33:1869-1874.

23. Tomasz, A., S. Nachman, and H. Leaf. 1991. Stable classes of phenotypic expression in methicillin-resistant clinical isolatesof staphylococci. Antimicrob. Agents Chemother. 35:124-129. 24. Ubukata, K., S.Nakagami,A.Nitta,A.Yamane,S.Kawakami,M.

Sugiura, andM. Konno. 1992.Rapid detectionofthe mecAgene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products.J. Clin. Microbiol.

30:1728-1733.

25. Unal,S., J. Hoskins, J. E. Flokowitsch, C. Y. E. Wu, D. A. Preston, andP. L. Skatrud. 1992. Detection ofmethicillin-resistant staph-ylococci by using the polymerase chain reaction. J. Clin. Microbiol. 30:1685-1691.

on May 15, 2020 by guest

http://jcm.asm.org/