Rapid detection of methicillin-resistant

Staphylococcus aureus strains not identified

by slide agglutination tests.
P Kuusela, P Hildn, K Savolainen, M Vuento, O Lyytikinen and
J Vuopio-Varkila
J. Clin. Microbiol. 1994, 32(1):143.

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JOURNAL OF CLNICAL MICROBIOLOGY, Jan. 1994, p. 143-147 Vol. 32, No. 1
0095-1137/94/$04.00+0
Copyright 1994, American Society for Microbiology

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,2 0. LYYTIKAINEN,3
AND J. VUOPIO-VARKILA4
Department of Bacteriology and Immunology, University of Helsinki,4 and Departments of Infection
Epidemiology3 and Special Bacterial Pathogens,' National Public Health Institute, Helsinki,
and Department of Biology, University ofJyvaskyla, Jyvaskyli,2 Finland
Received 22 June 1993/Returned for modification 10 August 1993/Accepted 15 October 1993

Downloaded from http://jcm.asm.org/ on April 19, 2014 by PENN STATE UNIV

Seventy-nine methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated during 1980 to 1990, were
classified as MRSA Aggl- (14 strains) and MRSA Aggl+ (65 strains) strains on the basis of test results in slide
agglutination assays designed to detect fibrinogen-binding protein (clumping factor) and protein A on the
staphylococcal surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that
lysostaphin digests of MRSA Aggl- strains contained a high-molecular-weight protein which was not detected
in digests of MRSA Aggl+ strains. Immunization of rabbits with an MRSAAggl strain produced an antiserum
which agglutinated all MRSA Aggl strains and also 64 of 65 MRSA Aggl+ strains. Only 1 of 68
coagulase-negative staphylococci showed agglutination in this assay. The anti-MRSA Aggl antiserum reacted
mainly with a 230-kDa staphylococcal surface protein but also with a 175-kDa protein, probably formed by
proteolysis of the former and a few slightly smaller proteins. These could not be immunologically detected in
lysostaphin digests of MRSA Aggl+ strains. Purified antibodies reacting with the 230-kDa protein agglutinated
all MRSA Aggl- strains, indicating that the protein is located on the surfaces of staphylococci. The results
suggest a tentative role for the 230-kDa protein or its fragments as a novel target to develop more efficient rapid
identification methods for S. aureus, including MRSA.

Identification of Staphylococcus aureus, an important MATERIALS AND METHODS

human pathogen, is based on typical morphology, positive
coagulation reaction, production of thermostable nuclease,
and utilization of various sugars as a carbohydrate source
(14). These methods are laborious and time-consuming,
requiring incubation for several hours before the reaction
result can be recorded. To overcome these drawbacks, slide
agglutination tests employing particles coated either with
fibrinogen or with fibrinogen and immunoglobulin G have
been developed for rapid detection of protein A and/or the
fibrinogen-binding protein (clumping factor) associated with
the surface of S. aureus, respectively. In numerous compar-
ative studies, these tests have shown high sensitivities and
specificities for S. aureus (1, 2, 4, 6, 8, 27). A few reports,
however, indicate that 1 to 25% of methicillin-resistant S.
aureus (MRSA) strains are not detected by these assays
(MRSA Aggl- 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
found in MRSA strains identified by slide agglutination tests
(MRSA Aggl+), albeit in much lower concentrations. We
further demonstrate that a direct bacterial agglutination
assay employing antiserum against an MRSA Aggl- strain
detects both types of MRSA strains with high sensitivity and
specificity.
*
Corresponding author. Mailing address: Department of Bacte-
riology and Immunology, University of Helsinki, P.O. Box 21,
00014 Helsinki, Finland. Phone: 358-0-434 61. Fax: 358-0-434 6382.
Electronic mail address: pkuusela@cc.helsinki.fi.
143
Bacterial strains. A total of 79 methicillin-resistant S.
aureus strains were collected during the period from 1980 to
1990 at the Department of Bacteriology and Immunology,
University of Helsinki, Helsinki, Finland. The strains were
isolated from clinical samples obtained in 12 different hospi-
tals or outpatient health centers in the southern part of
Finland. Seventy-eight of the isolates were recovered from
different patients; from one patient, both an MRSA Aggl-
strain and an MRSA Aggl+ strain were isolated at a 1-week
interval. In order to minimize the possibility of dealing with
the same bacterial strain in different patients, a period of at
least 3 months was required between the isolation dates for
samples originating from the same hospital. Also, 20 methi-
cillin-susceptible S. aureus (MSSA) strains per year were
collected as controls for slide agglutination tests. The strains
were stored in milk-glycerol at -70C and cultivated for
experiments on sheep blood agar plates for 20 to 24 h at
37C. All the strains were coagulase, DNase, and urease
producers and formed acid from maltose and trehalose.
MRSA Aggl- strains were additionally confirmed by API-
Staph (BioMerieux S.A.) as S. aureus strains. ATCC strains
(9144, 12600, 25923, and 29213 [S. aureus]; 27840 [S. capi-
tis]; 35538 [S. caprae]; 29974 [S. cohnii]; 12228 and 14990 [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 neonatal
septicemia S. epidermidis strains collected during a nation-
wide surveillance of bacteremic diseases in children since
1985 (7) were obtained from the collection of the National
Public Health Institute, Helsinki, Finland. The strains were
stored at -70C in 10% skim milk until use.
Antimicrobial susceptibility. Antimicrobial susceptibility
144 KUUSELA ET AL.
was determined with Neo-Sensitabs disks (A/S Rosco) and
Mueller-Hinton II medium (BBL, Becton Dickinson Micro-
biology Systems). Methicillin resistance was identified with
1-,ug oxacillin disks on Mueller-Hinton II agar plates incu-
bated at 30C. Oxacillin MICs were determined by the plate
dilution method on Mueller-Hinton II agar plates with 4%
NaCl and incubation at 37C. Strains for which the MIC of
oxacillin was >4 p,g/ml were regarded as methicillin resis-
tant.
Phage typing. Phage typing was performed with the inter-
national phage set (5) in the Staphylococcus Reference
Laboratory at the National Public Health Institute.
Agglutination tests. For all agglutination experiments,
strains were cultivated on sheep blood agar plates overnight
at 37C. The slide agglutination tests were performed 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 to detect the clumping factor on
the S. aureus surface. Staphaurex (Wellcome Diagnostics)
and ANI S. aureus TEST (Ani Biotech OY, Helsinki,
Finland) are latex agglutination tests in which particles are
coated with fibrinogen and immunoglobulin G to detect the
surface-associated clumping factor and protein A, respec-
tively. Latex particles are either suspended (Staphaurex) or
dried reagent dots on a card (ANI S. aureus TEST). All three
tests are sensitive and specific for S. aureus (20). Direct
bacterial agglutination tests were performed by mixing two
to three colonies of staphylococci with absorbed and diluted
(1:7) anti-MRSA Aggl- antiserum or with concentrated
purified anti-230-kDa-protein antibodies on a coverslip. Ag-
glutination was recorded after 10 to 30 s. Serum from
nonimmunized rabbits was used as a control.
Antiserum against MRSA Aggl- strains. Antiserum against
a representative MRSA Aggl- strain was produced by
immunizing rabbits twice subcutaneously at 2-week intervals
with 109 heat-killed bacteria mixed in Freund's complete
adjuvant. Ten days after the last booster, blood was col-
lected and serum was isolated. The antiserum was absorbed
twice with intact S. epidermidis ATCC 12228 (2 x 109
bacteria per ml of antiserum for 2 h at 4C) grown in
Todd-Hewitt broth. For a few experiments, the antibodies
against the 230-kDa protein were isolated from antiserum by
adsorbing the antibodies to nitrocellulose membranes con-
taining the protein band. After washings with phosphate-
buffered saline (PBS), the antibodies were eluted by incu-
bating the membranes for 10 min in 1.0 M acetate, pH 2.0,
and then the eluate was neutralized and concentrated.
Analysis of lysostaphin digests. For lysostaphin digestion,
staphylococci were grown in Todd-Hewitt broth overnight at
37C, collected by centrifugation, and washed twice with
PBS. Finally, the bacterial density was adjusted to approx-
imately 2 x 1010 bacteria per ml. Digestion was accom-
plished by incubating 0.5 ml of bacterial suspension for 2 h at
37C with 10 p,g of recombinant lysostaphin (Applied Micro-
biology, Inc., New York, N.Y.) and 4 pg each of RNase and
DNase (Sigma) in the presence of 0.5 mM phenylmethylsul-
fonyl fluoride (Sigma) and ethylmaleimide (Sigma). Unbro-
ken bacterial cells were removed by centrifugation, and the
supernatants were incubated for 15 min at 80C to inhibit the
enzymes. Finally, protein concentrations in the digests were
determined as described previously (19).
Lysostaphin digests were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (16)
with slabs containing 8% acrylamide. The gels were stained
with Coomassie blue for protein or, when needed, subse-
J. CLIN. MICROBIOL.
TABLE 1. Characteristics of MRSA Agg- strains
Susceptibility category'
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
aTotal of 14 isolates were used.
I All strains were susceptible to vancomycin, rifampin, fusidic acid, netilm-
icin, tetracycline, and co-trimoxazole. S, susceptible; R, resistant.
C Read at 100 times the routine test dilution. The following phages were
Downloaded from http://jcm.asm.org/ on April 19, 2014 by PENN STATE UNIV
used: 3A, 3C, 6, 29, 42E, 47, 52, 52A, 53, 54, 55, 71, 75, 77, 79, 80, 81, 83A,
84, 85, 94, 95, and 96.
quently transferred electrophoretically to nitrocellulose
membranes (24). Membranes were pretreated for 1 h at room
temperature with PBS containing 5% (wt/vol) defatted milk
powder and 1% (vol/vol) Triton X-100 and then washed
twice with TEN-Tween buffer (0.05 M Tris-HCl [pH 7.5],
0.025 M EDTA, 0.15 M NaCl, 0.5% [vol/vol] Tween 20). The
membranes were first probed with a predetermined dilution
of anti-MRSA Aggl- antiserum or control serum and then
with horseradish peroxidase-conjugated F(ab')2 fragments of
sheep antibodies to rabbit immunoglobulin G (Jackson Im-
munoResearch); all probes were diluted in TEN-Tween
buffer. Finally, the membranes were washed four times with
TEN-Tween buffer and once with PBS. The bands were
visualized by incubating the membranes in 50 ml of 50 mM
acetate buffer, pH 5.0, containing 3-amino-9-ethylcarbazole
(10 mg), N,N'-dimethylformamide (2.5 ml), and 30% hydro-
gen peroxide (30 ,ul).
Statistics. Statistical comparison between MICs for MRSA
Aggl+ and MRSA Aggl- strains was done by Student's t
test.
RESULTS
Characterization of MRSA Aggl- strains. A total of 79
MRSA strains, isolated during 1980 to 1990, were included in
the study. The number of isolates varied between 2 and 12
each year. Eleven of these strains showed no agglutination
reaction with three commercial slide agglutination assays
designed for detection of S. aureus. Three MRSA strains
displayed variable agglutination results in repeated testings
with different assays. These strains were, however, recorded
as MRSA Aggl- strains in the analysis. The proportion of
MRSA Aggl- strains among all MRSA strains was 17.7%.
There was no statistical difference between the MICs of
oxacillin for the MRSA Aggl+ group (median, 128 tLg/ml;
range, 4 to 512 p,g/ml) and that for the MRSA Aggl- group
(median, 128 ,uglml; range, 64 to 256 ,ug/ml) (data not
shown). All 220 MSSA strains collected during the same
period were correctly identified with these assays.
Susceptibility to antibiotics and phage typing. The suscep-
tibilities of MRSA Aggl- strains to various antibiotics are
shown in Table 1. To study whether the isolates represented
individual strains, the antibiotic susceptibility patterns and
phage types of the strains were determined (Table 1). The
MRSA Aggl- strains were shown to represent five different
strain types (a through e) consisting of two phage types.
Neither of the phage types was common among MRSA
Aggl+ strains collected during the same interval.
VOL. 32, 1994

200
116-
97-

42
30-
-1

B
1 2

*_=X;s.No,-e4aX_
SZ~*:^>tWi',v*.t.2 ,
3

Aw
wi
s! v;
4 5 6 7 8 9 10 11

. *O
8#
.W
12 13

4w
|
.i

S.,, -k
14
RAPID DETECTION OF MRSA

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
with approximate molecular weights of 110,000 and 80,000
(Fig. 2A, lanes 1 through 11). In the digest of one MRSA
145

Aggl- strain which gave alternating results in slide aggluti-

nation assays, the antiserum stained two polypeptides with
approximate molecular weights of 190,000 and 97,000 (Fig.

Downloaded from http://jcm.asm.org/ on April 19, 2014 by PENN STATE UNIV

200 2A, lane 12). Similarly, in digests of two other alternating
116- MRSA Aggl- strains, the antiserum detected mainly a
97-
W . -aw - . 175-kDa polypeptide (Fig. 2A, lanes 13 and 14). These
66- polypeptides were not detected in digests of MRSA Aggl+
:j .,
UIf; b Z 4 x . strains. With high concentrations of MRSA Aggl+ digests,
42 - .... :4,, the antiserum detected two proteins with approximate mo-
30 - _
.
_ _, -
...
lecular weights of 120,000 to 125,000 and 100,000 to 105,000
ft- at; j: \,,. ;|. +:. which, however, stained much less intensively (data not
FIG. 1. SDS-PAGE analysis of lysostaphin digests (48 p.g of shown). Nonimmunized rabbit serum did not stain any of
protein per slot) of 14 MRSA Agg- (A) and 14 representative these proteins (Fig. 2A, lower panel, lanes 1 through 14).
MRSA Aggl+ (B) strains. The arrow indicates the 230-kDa protein One polypeptide was visualized with control serum in di-
not detected in the digests of MRSA Aggl+ strains. Migration of gests of MRSA Aggl- and MRSA Aggl+ strains (Fig. 2A and
molecular weight markers is shown on the left (weights are in B, respectively). In MRSA Aggl- digests, the molecular
thousands). weight seemed to be constant in contrast to the one found in
MRSA Aggl+ digests, which varied slightly. Purified anti-
bodies to the 230-kDa protein stained not only the corre-
SDS-PAGE analysis of lysostaphin digests. When cell wall sponding 230-kDa band but also the 175-kDa protein, indi-
lysostaphin digests of various MRSA strains were analyzed cating that the one with a lower molecular weight is most
by SDS-PAGE, a clear difference was seen between digests probably generated from the larger one by proteolytic deg-
of MRSA Aggl- strains and those of MRSA Aggl+ strains. radation (data not shown).
Digests of 11 MRSA Aggl- strains contained a protein with Direct bacterial agglutination assay. In direct bacterial
a molecular weight of 230,000 which could not be visualized agglutination assays, rabbit antiserum obtained by immuni-
by protein staining in digests of the MRSA Aggl+ strains zation with an MRSA Aggl- strain and absorbed with S.
(Fig. 1A, lanes 1 through 11, and 1B, lanes 1 through 14). In epidermidis detected all 14 MRSA Aggl- strains (Table 2).
the digest of one MRSA Aggl- strain, the respective protein The antiserum also detected 64 of 65 MRSA Aggl+ strains
band migrated slightly faster, corresponding to an approxi- and 20 of 32 MSSA strains. Interestingly, none of the 52 S.
mate molecular weight of 190,000 (Fig. 1A, lane 12). In epidennidis strains, which included both methicillin-resis-
digests of two MRSA Aggl- strains, no respective protein tant and methicillin-susceptible strains, and only 1 strain (an
band could be seen in this region of the gel (Fig. 1A, lanes 13 S. hominis isolate) of 16 other coagulase-negative staphylo-
and 14). cocci gave a positive result in direct agglutination assay

A B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14

200_ - ;
97 -
66 -gggggggp---'up is X,4
-
"l
I*e
-,,'"!
44 -
30 -

200 -
97 -
66 - MlS

44 -
m man
30 -
FIG. 2. Immunoblotting analysis of lysostaphin digests (5.2 to 6.0 pg of protein per slot) of 14 MRSA Aggl- (A) and 14 representative
MRSA Aggl+ (B) strains with absorbed anti-MRSA Aggl- antiserum (upper panels) and normal rabbit serum as a control (lower panels).
Migration of molecular weight markers is shown on the left (weights are in thousands). For details, see Materials and Methods.
146 KUUSELA ET AL.
TABLE 2. Direct bacterial agglutination with antiserum against
MRSA Aggl- strains
No. of strains
Organism(s) Showing agglutination witha:
Total Anti-MRSA Anti-230-kDa
Aggl- protein
MSSA 32 20 NT
MRSA Aggl- 14 14 14
MRSA Aggl+ 65 64 ob
S. epidennidisc 52 0 NT
Coagulase-negative 16 id NT
staphylococci
a NRS, normal rabbit serum; NT, not tested.
b 14 of 65 strains
were tested.
c 42 methicillin-resistant and 10 methicillin-susceptible strains.
d S. hominis.
(Table 2). Purified antibodies to the 230-kDa protein agglu-
tinated all 14 MRSA Aggl- strains, indicating that 230- and
175-kDa proteins are exposed on the bacterial surface. Of
the 64 MRSA Aggl+ strains, 14 were tested. None of them
agglutinated with the same purified 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) or protein A on
the staphylococcal surface. In our study, their frequency
was 17.7% of MRSA isolates, which is greater than has been
reported earlier (4, 17, 21, 22, 26). Ruane et al. (22) described
failure rates of 17 and 25% for Staphaurex and Staphyslide-
Test, respectively, among 73 MRSA strains obtained from
three different hospitals in California but also claimed that
the high percentage of false-negative results might have been
due to a strain endemic to that area. The same possibility is
not completely excluded in the present investigation, al-
though five different clones based on phage types and
antibiograms could be found among MRSA Aggl- isolates.
The MRSA Aggl- strains do not represent a subpopulation
with especially high or, alternatively, only moderate resis-
tance towards oxacillin, as the MICs of oxacillin for MRSA
Aggl- strains and those for MRSA Aggl+ strains do not
show any statistically significant difference.
The failure of slide agglutination tests to detect MRSA
Aggl- strains may suggest that protein A and clumping
factor are poorly available on the bacterial surface. We have
shown that these strains harbor an extra surface protein with
an approximate molecular weight of 230,000 which was not
detected in digests of MRSA Aggl+ ones. This raised the
possibility that antibodies against MRSA Aggl- strains
would provide a tool for detecting MRSA strains negative in
slide agglutination assays.
In direct bacterial agglutination assays, anti-MRSA Aggl-
antiserum detected all the MRSA Aggl- strains and also 64
of 65 MRSA Aggl+ strains. The results thus indicate that
MRSA Aggl+ strains also contain the antigenic structures on
their surfaces. The test also showed a high specificity, since
only one S. hominis strain of 68 coagulase-negative staphy-
lococci was positive in this assay.
Immunoblotting analysis showed that the immunological
reactivity of the antiserum was mainly directed toward the
230-kDa protein and additionally to a 175-kDa protein. When
J. CLIN. MICROBIOL.
larger amounts of protein were loaded onto the gel, smaller
proteins could be detected in digests of both MRSA Aggl-
and MRSA Aggl+ strains. Together with the finding that
isolated anti-230-kDa-protein antibodies also stained the
175-kDa protein in immunoblotting experiments, the present
results favor the idea that the 230-kDa protein may exist in
NRS different molecular forms on both MRSA Aggl- and MRSA
0
Aggl+ strains, although in much smaller quantities on the
0
latter ones. This is also in agreement with the finding that
0 agglutination of MRSA Aggl+ strains by the antiserum was
0 much weaker than that of MRSA Aggl- strains. There is,
0 however, a possibility that the smaller polypeptides detected
in digests of both MRSA groups are not related to either the
230- or 175-kDa protein but represent another antigen-
Downloaded from http://jcm.asm.org/ on April 19, 2014 by PENN STATE UNIV
antibody system. This has to be studied in more detail by
using antibodies raised against the purified 230-kDa protein.
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 molecular weights considerably
lower than 230,000 (3, 25). Therefore, the 230-kDa protein
identified in the present study does not seem to be related to
these factors and represents a previously uncharacterized
protein. It is also expressed on the bacterium, since purified
antibodies to the 230-kDa protein caused the agglutination of
all MRSA Aggl- strains. Interestingly, two commercial slide
agglutination assays have been introduced recently which
take advantage of specific binding of monoclonal antibodies
either to the surface protein(s) (Slidex; BioMerieux) or to
type 5 and 8 capsular polysaccharides (Pastorex Staph-Plus;
Sanofi Diagnostics Pasteur) of S. aureus. In a few compar-
ative studies, these assays also efficiently detected MRSA
strains which remained negative in 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 test relates to the 230- and
175-kDa proteins described in this article. Guzman et al. (11)
also described an enzyme-linked immunosorbent assay em-
ploying antigenic differences of excreted staphylococcal
glucosaminidase to differentiate various staphylococcal spe-
cies. This test was also able to identify MRSA strains which
did not produce protein A or staphylocoagulase or both.
Studies in progress in our laboratory indicate that the
availability of binding proteins for ligands such as fibronec-
tin, laminin, and collagens (12, 15, 18, 23), as well as the
availability of protein A and clumping factor, is reduced on
the surface of the MRSA Aggl- strains included in this
study. However, in immunoblots of lysostaphin digests of
MRSA Aggl- strains, the control rabbit serum 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
seen whether the 230-kDa protein has this type of inhibitory
role. It will also be interesting to 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
proteins with altered compositions.
ACKNOWLEDGMENTS
Sirpa Kuisma is acknowledged for excellent technical assistance.
Aino Takala is kindly thanked for providing the neonatal septicemia
S. epidermidis strains.
VOL. 32, 1994
The work was supported by the Paulo Foundation.
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