0095-1137/94/$04.00+0
Copyright© 1994,American SocietyforMicrobiology
Rapid Detection of
Methicillin-Resistant
Staphylococcus
aureus
Strains
Not
Identified by Slide
Agglutination
Tests
P.
KUUSELA,l*
P.HILDtN,l
K. SAVOLAINEN,1 M. VUENTO,20. LYYTIKAINEN,3 ANDJ.VUOPIO-VARKILA4Departmentof BacteriologyandImmunology, University of
Helsinki,4
andDepartmentsofInfectionEpidemiology3
andSpecial BacterialPathogens,'
NationalPublic Health Institute, Helsinki,andDepartmentof Biology, University ofJyvaskyla,
Jyvaskyli,2
FinlandReceived 22June 1993/Returned for modification 10August 1993/Accepted 15 October1993
Seventy-nine
methicillin-resistantStaphylococcusaureus(MRSA) strains,isolatedduring1980to1990,were classifiedasMRSAAggl- (14 strains) and MRSA Aggl+ (65strains)strainsonthe basis oftestresultsin slide agglutination assays designed to detect fibrinogen-binding protein (clumping factor) and protein A on the staphylococcal surface. Sodiumdodecyl
sulfate-polyacrylamide
gel electrophoresisanalysis
revealed thatlysostaphin
digests of MRSAAggl- strains containedahigh-molecular-weight protein whichwas notdetected indigestsof MRSA Aggl+ strains. Immunization of rabbits with anMRSAAggl strain producedanantiserum which agglutinated all MRSA Aggl strains and also 64 of 65 MRSA Aggl+ strains. Only 1 of 68coagulase-negative staphylococcishowedagglutinationin this assay. The anti-MRSA Aggl antiserum reacted
mainly
witha230-kDa staphylococcal surfaceprotein but also witha 175-kDaprotein,probably
formedbyproteolysis
of the former andafewslightly
smallerproteins.These couldnotbeimmunologically
detected inlysostaphindigests ofMRSAAggl+strains.Purified antibodiesreacting with the 230-kDaprotein agglutinated
all MRSAAggl- strains, indicatingthat the protein is locatedonthesurfaces ofstaphylococci. The results suggestatentative role for the 230-kDaproteinoritsfragmentsasanovel targettodevelopmoreefficientrapid identification methods for S. aureus, includingMRSA.
Identification of Staphylococcus aureus, an important
human pathogen, is based on typical morphology, positive
coagulation reaction, production ofthermostable nuclease,
and utilization ofvarious sugars as a carbohydrate source
(14). These methods are laborious and time-consuming,
requiring incubation for several hours before the reaction
resultcanbe recorded. Toovercomethesedrawbacks, slide
agglutination tests employing particles coated either with
fibrinogen or with fibrinogen and immunoglobulin G have
been developed forrapid detection of proteinAand/or the
fibrinogen-binding protein(clumping factor) associatedwith
the surfaceof S. aureus,respectively.Innumerous
compar-ative studies, these tests have shownhigh sensitivities and
specificitiesforS. aureus (1, 2, 4, 6, 8, 27). Afew reports,
however, indicate that 1 to 25% ofmethicillin-resistant S.
aureus (MRSA) strains are not detected by these assays
(MRSAAggl- strains) (4, 17, 21, 22, 26).
In this article we describe the identification of a
high-molecular-weight protein present in lysostaphin digests of
MRSA Aggl- strains. A similar type of protein was also
foundinMRSA strainsidentified by slide agglutinationtests
(MRSA Aggl+),
albeit in much lower concentrations. Wefurther demonstrate that a direct bacterial agglutination
assayemploying antiserum against an MRSA Aggl- strain
detects bothtypesof MRSA strains withhigh sensitivityand
specificity.
* Correspondingauthor. Mailingaddress: Department of Bacte-riology and Immunology, University of Helsinki, P.O. Box 21, 00014Helsinki, Finland.Phone:358-0-43461. Fax: 358-0-434 6382. Electronic mail address: [email protected].
MATERIALS AND METHODS
Bacterial strains. A total of 79 methicillin-resistant S. aureusstrainswerecollectedduringtheperiod from1980to 1990 at the Department ofBacteriology and Immunology,
UniversityofHelsinki, Helsinki,Finland. Thestrainswere
isolated fromclinical samplesobtained in 12different
hospi-tals or outpatient health centers in the southern part of
Finland. Seventy-eightof the isolates wererecoveredfrom
differentpatients; from one patient, both an MRSA
Aggl-strainandanMRSAAggl+ strainwere isolatedat a1-week
interval. In order to minimize thepossibility ofdealingwith
thesamebacterial strain in differentpatients,aperiod ofat
least 3 months wasrequiredbetween theisolation datesfor
samples originating fromthe samehospital. Also, 20
methi-cillin-susceptible S. aureus (MSSA) strains per year were
collectedascontrols for slide
agglutination
tests.Thestrainswere stored in milk-glycerol at -70°C and cultivated for
experiments on sheep blood agar plates for 20 to 24 h at
37°C. All the strains were coagulase, DNase, and urease
producers and formed acid from maltose and trehalose.
MRSAAggl- strains were additionally confirmed by
API-Staph (BioMerieux S.A.)asS. aureusstrains. ATCCstrains
(9144, 12600, 25923, and29213 [S. aureus]; 27840 [S.
capi-tis]; 35538 [S. caprae];29974[S.cohnii]; 12228and14990[S.
epidermidis];
35539 [S.gallinarum];
29970 [S.haemolyti-cus];
29885 [S. hominis]; 11249[S. hyicus]; 29663 [S.inter-medius];
43809 [S.lugdunensis];
15305 [S. saprophyticus]; 43808 [S.schleiferiJ;
29060 [S.sciuri];
27851 [S. simulans]; 27836 [S.warneri];
and 29971 [S. xylosus]) and neonatalsepticemiaS. epidermidis strainscollectedduringa
nation-wide surveillance of bacteremic diseases in children since 1985 (7) were obtained from the collection of the National Public HealthInstitute, Helsinki, Finland. The strains were storedat-70°Cin10% skim milk until use.
Antimicrobial
susceptibility.
Antimicrobial susceptibility143
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144 KUUSELA ET AL.
was determined with Neo-Sensitabs disks (A/S Rosco) and
Mueller-Hinton IImedium(BBL, Becton Dickinson Micro-biology Systems). Methicillin resistancewasidentified with
1-,ug oxacillin disks onMueller-Hinton II agarplates
incu-batedat30°C. Oxacillin MICsweredeterminedby the plate dilution method on Mueller-Hinton II agarplates with 4%
NaCl and incubation at37°C. Strains for which the MIC of oxacillin was >4
p,g/ml
were regarded as methicillinresis-tant.
Phagetyping.Phage typingwasperformed with the
inter-national phage set (5) in the Staphylococcus Reference LaboratoryattheNational Public Health Institute.
Agglutination tests. For all agglutination experiments, strainswerecultivatedonsheep bloodagarplates overnight
at37°C. The slide agglutinationtestswereperformed
accord-ing to the instructions of the manufacturers. Staphyslide-Test (BioMerieux) is a hemagglutination test employing fibrinogen-coated (test reagent) and uncoated (control
re-agent) sheep erythrocytes todetect the clumping factor on
theS. aureus surface. Staphaurex (Wellcome Diagnostics)
and ANI S. aureus TEST (Ani Biotech OY, Helsinki,
Finland) arelatexagglutination tests in whichparticles are
coated withfibrinogen and immunoglobulin Gto detect the surface-associated clumping factor and protein A,
respec-tively.Latexparticlesareeithersuspended (Staphaurex) or
dried reagent dotsonacard(ANIS.aureusTEST). All three
tests are sensitive and specific for S. aureus (20). Direct
bacterial agglutination testswere performed bymixingtwo tothreecolonies ofstaphylococci with absorbed and diluted (1:7) anti-MRSA Aggl- antiserum or with concentrated
purified anti-230-kDa-protein antibodieson acoverslip.
Ag-glutination was recorded after 10 to 30 s. Serum from nonimmunized rabbitswas usedas acontrol.
Antiserumagainst MRSAAggl- strains. Antiserum against
a representative MRSA Aggl- strain was produced by
immunizing rabbits twice subcutaneouslyat2-week intervals with 109 heat-killed bacteria mixed in Freund's complete adjuvant. Ten days after the last booster, blood was
col-lected andserumwasisolated. The antiserumwasabsorbed
twice with intact S. epidermidis ATCC 12228 (2 x
109
bacteria per ml of antiserum for 2 h at 4°C) grown in Todd-Hewittbroth. For a few experiments, the antibodies againstthe 230-kDaproteinwereisolatedfrom antiserumby adsorbing the antibodies to nitrocellulose membranes
con-taining the protein band. After washings with phosphate-buffered saline (PBS), the antibodieswere eluted by incu-batingthe membranes for 10 minin1.0 M acetate,pH 2.0, and then the eluatewas neutralized and concentrated.
Analysisoflysostaphin digests. Forlysostaphin digestion, staphylococciweregrownin Todd-Hewitt brothovernightat 37°C, collected by centrifugation, and washed twice with PBS. Finally, the bacterialdensitywas adjustedto
approx-imately 2 x 1010 bacteria per ml. Digestion was
accom-plished by incubating0.5 ml of bacterialsuspensionfor 2 hat 37°Cwith10p,gof recombinantlysostaphin (Applied Micro-biology, Inc.,NewYork, N.Y.)and 4pgeach of RNase and DNase(Sigma)inthepresenceof0.5 mM phenylmethylsul-fonyl fluoride(Sigma) and ethylmaleimide (Sigma). Unbro-ken bacterial cellswereremovedby centrifugation, and the supernatantswereincubated for15minat80°Ctoinhibit the
enzymes.Finally, proteinconcentrations in thedigestswere
determinedasdescribedpreviously (19).
Lysostaphin digests were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (16) withslabscontaining 8% acrylamide.Thegelswerestained
with Coomassie blue for protein or, when needed,
subse-TABLE 1. Characteristics of MRSAAgg-strains
Susceptibilitycategory'
Strain No. of
type isolates" Erythro- Clinda- Tobra- Phage type"
mycin mycin mycin
a 4 R R R 81/42E/47/54/75/84/85
b 1 S S R 81/42E/47/54/75/84/85
c 2 S S S 81/42E/47/54/75/84/85
d 2 R R R 85
e 5 R S R 85
aTotalof 14 isolates were used.
IAllstrains were susceptible to vancomycin,rifampin, fusidic acid,
netilm-icin, tetracycline, andco-trimoxazole.S, susceptible; R, resistant.
CRead at 100 times the routine test dilution. Thefollowing phages were used: 3A, 3C, 6, 29, 42E, 47, 52, 52A, 53, 54, 55, 71, 75, 77, 79, 80, 81, 83A, 84,85, 94, 95, and96.
quently transferred electrophoretically to nitrocellulose
membranes(24). Membraneswerepretreated for1hat room
temperaturewith PBScontaining 5% (wt/vol) defatted milk
powder and 1%
(vol/vol)
Triton X-100 and then washedtwice with TEN-Tween buffer (0.05 M Tris-HCl
[pH
7.5],0.025MEDTA, 0.15 M NaCl,0.5%
[vol/vol]
Tween20). Themembraneswerefirst probed withapredetermined dilution
ofanti-MRSA Aggl- antiserum or control serum and then
with horseradishperoxidase-conjugated F(ab')2 fragments of
sheep antibodies to rabbit immunoglobulinG
(Jackson
Im-munoResearch); all probes were diluted in TEN-Tween
buffer.Finally, themembranes werewashed four times with
TEN-Tween buffer and once with PBS. The bands were
visualized by incubating the membranes in50mlof50 mM
acetatebuffer, pH 5.0, containing 3-amino-9-ethylcarbazole
(10
mg),
N,N'-dimethylformamide(2.5
ml), and 30%hydro-genperoxide (30 ,ul).
Statistics.Statisticalcomparison between MICs for MRSA
Aggl+ and MRSA Aggl- strains was done by Student's t
test.
RESULTS
Characterization of MRSAAggl- strains. A total of79
MRSAstrains, isolated during 1980to1990,wereincluded in
the study. The number of isolates varied between2and 12
eachyear. Eleven ofthese strainsshowednoagglutination
reaction with three commercial slide agglutination assays
designed for detection of S. aureus. Three MRSA strains
displayed variable agglutination results in repeated
testings
with differentassays.These strainswere,however, recorded
as MRSAAggl- strains in the analysis. The
proportion
ofMRSA Aggl- strainsamong all MRSA strainswas 17.7%.
There was no statistical difference between the MICs of
oxacillin for the MRSA Aggl+ group
(median,
128tLg/ml;
range, 4 to512
p,g/ml)
and that for the MRSAAggl- group(median, 128 ,uglml; range, 64 to 256
,ug/ml) (data
notshown).
All 220 MSSA strains collectedduring
the sameperiodwere
correctly
identifiedwith these assays.Susceptibility
toantibiotics andphagetyping.
Thesuscep-tibilities ofMRSAAggl- strains tovarious antibiotics are
shown inTable1. Tostudy whether theisolates
represented
individual strains, the antibiotic
susceptibility
patterns andphagetypes of the strainswere determined
(Table 1).
TheMRSAAggl- strainswereshowntorepresentfive different
strain types
(a through e) consisting
of twophage
types.Neither of the
phage
types was common among MRSAAggl+ strains collected
during
thesameinterval.J.CLIN. MICROBIOL.
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RAPID DETECTION OF MRSA 145 A
1 2 3 4 5 6 7 8 9 10 11 12 13 14
200
116-
97-A
wis!w v; . *O 4w.i
42 -1
SZ
~~t~W'*
t.2.,,
8# |.W S.,,-k
30-B
200
116-
97-
66-:j .,
42-.... :4,, 30-
ft-- . .
W . -aw
:>iv..
UIf;
*^*,.,i.,.
**Xs.NNo,-eaX4-
_
=;=4-
b Z 4 x.
.__
..._ _
_,-at; j: \,,. ;|. +:.
FIG. 1. SDS-PAGE analysis of
lysostaphin
digests (48p.g
of protein per slot) of 14 MRSA Agg- (A) and 14 representative MRSAAggl+(B)strains. Thearrow indicates the 230-kDaprotein not detectedin the digests of MRSAAggl+ strains. Migration of molecular weight markers is shown on the left (weights are in thousands).SDS-PAGEanalysis oflysostaphin digests. When cellwall
lysostaphin digestsofvariousMRSA strainswere analyzed
by SDS-PAGE,acleardifferencewas seenbetween
digests
ofMRSAAggl- strains and those of MRSA
Aggl+
strains.Digests of11MRSAAggl- strains containeda
protein
withamolecularweightof230,000 which couldnotbevisualized
by protein staining in digests of the MRSA Aggl+ strains
(Fig. 1A,lanes 1through 11, and 1B, lanes1through
14).
Inthedigestof one MRSAAggl-strain, the respective protein
bandmigrated slightlyfaster, corresponding to an
approxi-mate molecular weight of 190,000 (Fig. 1A, lane 12). In
digests of two MRSAAggl- strains, norespective protein
band could be seen in thisregionof thegel(Fig. 1A,lanes 13 and 14).
Immunoblotting. In order to study the expression of the 230-kDa protein, rabbits were immunized with an MRSA Aggl- strain harboring the protein. In immunoblotting of lysostaphin digests of MRSA Aggl- strains, anti-MRSA Aggl- antibodies absorbed with S. epidennidis visualized
principally the 230-kDa protein and additionally a 175-kDa
protein (Fig. 2A). These were not detected in immunoblots of digests from MRSA Aggl+ strains. In MRSA Aggl-digests, the antiserum also detected two smaller proteins
withapproximate molecular weights of 110,000 and 80,000
(Fig. 2A, lanes 1 through 11). In the digest of one MRSA Aggl- strain which gave alternatingresultsin slide aggluti-nation assays, theantiserum stained two polypeptideswith approximate molecular weights of 190,000 and 97,000(Fig. 2A, lane 12). Similarly, in digests oftwo other alternating MRSA Aggl- strains, the antiserum detected mainly a 175-kDa polypeptide (Fig. 2A, lanes 13 and 14). These
polypeptides were not detected in digests ofMRSA
Aggl+
strains. With high concentrations of MRSA Aggl+ digests,
the antiserum detectedtwoproteins with approximate
mo-lecularweightsof120,000to 125,000 and 100,000to105,000
which, however, stained much less intensively (data not
shown). Nonimmunized rabbit serum did not stain any of
these proteins (Fig. 2A, lower panel, lanes 1 through
14).
One polypeptide was visualized with control serum in
di-gests of MRSAAggl-andMRSAAggl+ strains (Fig.2Aand
B, respectively). In MRSA Aggl- digests, the molecular
weightseemed to be constantincontrast totheonefound in
MRSA Aggl+ digests, which varied slightly. Purified
anti-bodies to the 230-kDa protein stained not only the
corre-sponding 230-kDaband but also the 175-kDa
protein,
indi-cating that the onewith a lower molecular
weight
is mostprobably generated from thelargerone by
proteolytic
deg-radation(datanot shown).
Direct bacterial agglutination assay. In direct bacterial
agglutination assays, rabbit antiserum obtainedby
immuni-zation with an MRSAAggl- strain and absorbed with S.
epidermidis detected all 14 MRSAAggl- strains
(Table
2).
The antiserum also detected 64of65 MRSAAggl+ strains
and 20 of 32 MSSA strains.
Interestingly,
noneofthe 52 S.epidennidis strains, which included both
methicillin-resis-tantandmethicillin-susceptible strains, and only1strain
(an
S. hominis isolate)of 16 othercoagulase-negative
staphylo-cocci gave a positive result in direct agglutination assay
A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 200_ - ;
97
-66
-gggggggp---'up
44
-30
-200
-97
-66
-44 -30
-B
1 2 3 4 5 6 7 8 9 1011 12 13 14
- I*e
isX,4 "l -,,'"!
MlS
m man
FIG. 2. Immunoblottinganalysis of lysostaphin digests (5.2to 6.0pgofprotein perslot) of14 MRSAAggl- (A) and14representative MRSAAggl+ (B)strains with absorbedanti-MRSA Aggl- antiserum (upperpanels) and normal rabbitserum as acontrol(lowerpanels). Migrationof molecularweight markers isshown onthe left(weightsareinthousands).Fordetails,seeMaterialsandMethods.
VOL. 32,1994
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146 KUUSELA ET AL.
TABLE 2. Direct bacterialagglutination with antiserumagainst MRSAAggl- strains
No. of strains
Organism(s) Showing agglutinationwitha:
Total Anti-MRSA Anti-230-kDa
Aggl- protein NRS
MSSA 32 20 NT 0
MRSA Aggl- 14 14 14 0
MRSAAggl+ 65 64 ob 0
S. epidennidisc 52 0 NT 0
Coagulase-negative 16 id NT 0
staphylococci
a NRS, normal rabbit serum; NT,nottested.
b14 of65 strains were tested.
c42methicillin-resistant and 10methicillin-susceptible strains.
dS.hominis.
(Table 2). Purified antibodies tothe 230-kDaprotein agglu-tinated all 14 MRSAAggl-strains, indicating that 230- and 175-kDa proteins are exposed on thebacterial surface. Of
the 64 MRSAAggl+ strains, 14 weretested. Noneofthem
agglutinated with the samepurified antibodies(Table 2).
DISCUSSION
The present results confirm earlier findings that among
clinical MRSA isolates there are strains which cannot be identified by slide agglutination assays designed to detect fibrinogen-binding protein(clumping factor)orproteinAon
the staphylococcal surface. In our study, their frequency was17.7%ofMRSAisolates,whichisgreaterthan hasbeen reportedearlier(4, 17, 21, 22,26).Ruaneetal.(22)described failureratesof 17 and25% forStaphaurex and Staphyslide-Test, respectively, among 73 MRSA strains obtained from
three different hospitals in California but also claimed that
thehighpercentageoffalse-negativeresultsmight have been
duetoastrain endemictothatarea. Thesamepossibility is
not completely excluded in the present investigation, al-though five different clones based on phage types and antibiograms could be found amongMRSAAggl- isolates.
The MRSAAggl- strainsdo not representa subpopulation
with especially high or, alternatively, only moderate
resis-tancetowardsoxacillin, as theMICsofoxacillin for MRSA
Aggl- strains and those for MRSA Aggl+ strains do not showanystatistically significantdifference.
The failure of slide agglutination tests to detect MRSA Aggl- strains may suggest that protein A and clumping factorarepoorlyavailableonthe bacterial surface. We have
shownthatthesestrains harbor anextrasurface protein with
anapproximate molecularweight of230,000 whichwasnot detected in digests of MRSA Aggl+ ones. This raised the
possibility that antibodies against MRSA Aggl- strains would provideatoolfordetecting MRSAstrains negative in
slide agglutination assays.
Indirectbacterialagglutination assays, anti-MRSA
Aggl-antiserum detected all the MRSAAggl- strains and also 64 of 65 MRSA Aggl+ strains. The results thus indicate that MRSAAggl+ strainsalsocontain theantigenicstructureson
their surfaces. Thetestalsoshowed ahigh specificity, since
onlyone S. hominisstrain of 68 coagulase-negative
staphy-lococciwaspositive in thisassay.
Immunoblotting analysis showed that the immunological reactivity of the antiserumwas mainlydirected toward the
230-kDa protein and additionallytoa175-kDaprotein.When
largeramountsofproteinwereloadedontothegel, smaller
proteinscould be detected in digestsofbothMRSA
Aggl-and MRSA
Aggl+
strains. Together with the finding that isolated anti-230-kDa-protein antibodies also stained the175-kDaprotein in immunoblotting experiments,the present
results favor theidea that the 230-kDa protein may exist in
different molecular formsonboth MRSAAggl- and MRSA
Aggl+
strains, although in much smaller quantities on thelatterones. This is also in agreement with the finding that
agglutinationof MRSAAggl+ strains by theantiserum was
much weaker than that of MRSA Aggl- strains. There is,
however, a possibility that the smallerpolypeptides detected in digests of both MRSA groups are notrelated toeither the
230- or 175-kDa protein but represent another
antigen-antibody system. This has to be studied in more detail by
using antibodies raised againstthepurified230-kDaprotein.
The 48-kDa pentaglycine cross-linking protein and the
74-kDa modified penicillin-binding protein (PBP-2'), prod-ucts of two methicillin resistance genes, femA and mec,
respectively, appear to have molecularweightsconsiderably
lower than 230,000 (3, 25). Therefore, the 230-kDa protein
identified inthepresentstudy does not seem to berelatedto
these factors and represents a previously uncharacterized
protein. It is also expressed on the bacterium, since purified
antibodies to the 230-kDa proteincaused theagglutinationof
all MRSAAggl- strains. Interestingly,twocommercialslide
agglutination assays have been introduced recently which
take advantage of specific bindingofmonoclonal antibodies
either to the surface protein(s) (Slidex; BioMerieux) or to
type 5 and 8capsularpolysaccharides (Pastorex Staph-Plus;
Sanofi Diagnostics Pasteur) ofS. aureus. In a few
compar-ative studies, these assays also efficiently detected MRSA
strainswhich remained negativein slide agglutination tests,
strains similar to those used in our study (9, 10, 13). At
present, it is not known how the staphylococcal surface protein(s) detected by the Slidex testrelates to the 230- and 175-kDaproteins described in thisarticle.Guzman et al. (11) alsodescribed an enzyme-linked immunosorbent assay em-ploying antigenic differences of excreted staphylococcal
glucosaminidase todifferentiate variousstaphylococcal
spe-cies. This test was also able toidentifyMRSA strains which did not produce protein A or staphylocoagulase orboth.
Studies in progress in our laboratory indicate that the availability ofbinding proteins for ligands such as fibronec-tin, laminin, and collagens (12, 15, 18, 23), as well as the availability ofprotein A and clumping factor, is reducedon the surface of the MRSA Aggl- strains included in this study. However, in immunoblots of lysostaphin digests of MRSAAggl- strains, thecontrolrabbitserum also detected a polypeptide with a molecular weight corresponding roughly to the molecular weight of protein A, although it could not be detected on the staphylococcal surface. This can be explained by the possibility that protein A and/or other binding proteins are prevented by steric hindrance
from interacting with their counterparts. It remains to be
seenwhetherthe230-kDa protein has this type ofinhibitory
role. Itwill also be interestingto see whether agglutination assays which use antibodies to the 230-kDa protein or its synthetic peptides will provide a more applicable assay system to identify S. aureus strains which have surface
proteinswith altered compositions.
ACKNOWLEDGMENTS
SirpaKuisma isacknowledged for excellent technical assistance. Aino Takala is kindlythanked forproviding the neonatalsepticemia S. epidermidis strains.
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