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Journal of Clinical Microbiology, April 1999, p. 1190-1192, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid Identification of Staphylococcus
aureus by Using Fluorescent Staphylocoagulase Assays
M. G.
Holliday,*
M.
Ford,
J. D.
Perry, and
F. K.
Gould
Department of Microbiology, Freeman Hospital,
Newcastle upon Tyne, NE7 7DN, United Kingdom
Received 13 August 1998/Returned for modification 13 October
1998/Accepted 21 December 1998
 |
ABSTRACT |
Two rapid (1-h) assays for the detection of Staphylococcus
aureus staphylocoagulase were developed by using the fluorogenic thrombin substrates
N-t-boc-Val-Pro-Arg-7-amido-4-methylcoumarin (VPA) and
N-t-boc-
-benzyl-Asp-Pro-Arg-7-amido-4-methylocoumarin (BB). The assays were compared to the tube coagulase test and latex
agglutination (LA) (Sanofi Diagnostics Pasteur, Guildford, Surrey,
United Kingdom) by using 406 clinical isolates of staphylococci, and
they produced positive and negative predictive values of 99.2 and
99.1% for LA, 98.9 and 92.7% for VPA, and 98.9 and 99.1% for BB.
Fluorescent assays used colonies from solid media, thereby eliminating
the need for broth cultures, and were performed in microtiter trays,
thus making them suitable for large-scale screening.
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TEXT |
Staphylococcus aureus is
one of the most frequently encountered pathogens in clinical samples
(7, 10), while methicillin-resistant S. aureus (MRSA) is an important nosocomial pathogen (9,
15). A rapid, reliable test to identify these organisms is
of considerable relevance (10, 15). The detection of
staphylocoagulase by the tube coagulase test (1, 4,
16) is the "gold standard" for identification of
S. aureus from human sources (3, 7, 13, 16);
however, variations in the quality of the plasma (3, 12, 16)
and problems in the interpretation of the test (1, 3, 4, 11-13,
16) can lead to misidentification (1, 12).
Tube coagulase tests are incubated for 24 h (1, 3, 12, 13,
16) to ensure detection of weak coagulase producers, thus
increasing the risk of false-negative results due to proteolysis (4, 11) or false-positive results due to the activation of prothrombin by proteases (4, 11) or metalloproteases
(4). Latex agglutination (LA) tests which detect
S. aureus clumping factor, protein A, and capsular
polysaccharides (7, 10) may give false-positive results with
other organisms (11a) and staphylococci (10) and
may have low sensitivity with MRSA (7, 10).
Chromogenic and fluorogenic staphylocoagulase assays have been
developed and used to study purified staphylocoagulase (5, 6) and broth supernatants (3, 4, 11). This report
details two new fluorescent assays for staphylocoagulase using colonies on solid media and compares these to the classical tube coagulase test
and an improved LA test using clinical isolates of staphylococci.
Bacterial strains.
A total of 406 clinical isolates of
staphylococci were tested. Isolates were grown and maintained on
Columbia horse blood agar. All isolates were identified as
catalase-positive, gram-positive cocci and by the tube coagulase test.
Tube coagulase test.
Strains were incubated for 18 h in
10 ml of brain heart infusion (BHI) broth (Lab M, Bury, United Kingdom)
at 37°C. A 200-µl volume of this broth was added to 500 µl of
Bacto Coagulase Plasma (Difco Laboratories, East Molesey, United
Kingdom) in a sterile plastic universal container and mixed. Tests were
incubated at 37°C and examined at 4 and 24 h. Coagulation was
graded as 1+ to 4+ (12). S. aureus (NCTC
6571) was included as a positive control and Staphylococcus
epidermidis (NCTC 11047) was included as a negative control with
each run.
LA.
The Pastorex Staph Plus test (Sanofi Diagnostics
Pasteur, Guildford, United Kingdom), which detects clumping
factor, staphylococcal protein A, and capsular polysaccharides was used
according to the manufacturer's instructions.
Fluorescent staphylocoagulase assays.
Isolates were
subcultured onto Columbia blood agar overnight before testing. Tests
were performed in round-bottomed microtiter trays. A 25-µl volume of
BHI broth containing 0.2% bovine albumin/fraction V (Sigma Ltd.,
Poole, United Kingdom) and 25 µl of prothrombin (factor V)
diluted at 1 mg/ml in distilled water (Sigma) were pipetted into each
well. Four distinct colonies or a 2-mm streak through confluent growth
of the organism tested was emulsified in the BHI-prothrombin solution
to a turbidity greater than a no. 10 MacFarland standard.
The plates were incubated at 37°C for 40 min before 100 µl of N-t-boc-Val-Pro-Arg-7-amido-4-methylcoumarin
(VPA) (Sigma) (0.5 mg/ml in 0.05 M Tris, pH 8.0) was added, the
solution was mixed, and the plates were incubated for a further 20 min.
The degree of fluorescence was determined by using a Labtech Biolite F1
fluorescent microtiter plate reader (Labtech Ltd., Litchfield, United
Kingdom) with filters set at 365 and 440 nm and sensitivity at 28. Readings above 8,000 were taken as positive. Positive, negative, and
reagent controls were included with each batch, with S. aureus NCTC 6571 as the positive control and S. epidermidis NCTC 11047 as the negative control. The same technique
was repeated with
N-t-boc--benzyl-Asp-Pro-Arg-7-amido-4-methylcoumarin (BB)
(Sigma) (0.5 mg/ml in 0.05 M Tris, pH 8.0).
Of the 406 clinical isolates tested, 297 were positive by the tube
coagulase test and were identified as
S. aureus. Of
these,
295 (99.3%) were LA positive while 294 (98.9%) were positive
by
the two fluorescent assays (Table
1).
Two strains identified
as
S. aureus by the API STAPH
system (Biomerieux, Basingstoke,
United Kingdom) and by the tube
coagulase test were reproducibly
LA negative. Forty MRSA strains were
positive with all three assays.
Two
S. aureus strains gave negative results with both
VPA and BB fluorescent assays. One
S. aureus strain was
VPA negative
and BB positive, and one strain was VPA positive and
BB negative
(Table
1). Of the 109 coagulase-negative strains, one
was repeatedly
LA positive and was identified by the API
STAPH system as
S. epidermidis.
Eight strains (7%)
were VPA positive, while one strain (0.9%)
(different from the
previous eight) was BB positive (Table
2).
The positive and negative predictive values of the Pastorex LA test,
the VPA fluorescent assay, and the BB fluorescent assay
were 99.3 and
99.1%, 98.9 and 92.7%, and 98.9 and 99.1%,
respectively.
Tube coagulase tests are widely used to detect staphylocoagulase
production for identification of
S. aureus (
1,
3,
4,
7,
16). Problems have been encountered with this test due
to
variations in the type of plasma used, incubation time, and
degree of
clotting achieved, and definitive results may be unavailable
until up
to 24 h after initial isolation (
3,
12,
13,
16).
Direct detection of the staphylocoagulase-prothrombin
complex could offer a rapid, reliable assay for identification of
S. aureus (
4), and specific and sensitive
chromogenic assays for
staphylocoagulase have been described
(
3,
4). The more sensitive
fluorogenic substrate VPA has
been used to study staphylocoagulase
kinetics and proved to be rapid,
accurate, and simple to use (
5,
8). Previous chromogenic and
fluorogenic assays have been based
on detection of staphylocoagulase in
broth culture fluids or in
semipurified form. These assays are not
suitable for use in routine
laboratories, where most organisms are
grown on solid media. Staphylocoagulase
is present and exposed on the
cell surface (
2) and is released
into culture fluids through
proteolysis (
5). False-positive
reactions observed with
culture fluid staphylocoagulase assays
may be due to direct activation
of prothrombin by trypsin or papain-like
proteases (
4).
Proteolytic release of staphylocoagulase may
result in degradation of
its COOH terminus, producing heterogeneity
(
5,
6), and may
explain discrepancies observed between tube
coagulase and direct
staphylocoagulase tests using culture fluids.
The use of colonies grown
on solid media as described in this
study may remove these problems
while dispensing with the need
to grow strains in broth culture first.
A linear relationship
exists between reaction rates and
staphylocoagulase production
(
4), with optimal production
depending on supply of growth
factors from a rich medium
(
7). This study shows that overnight
growth of
S. aureus on Columbia blood agar plates produces sufficient
staphylocoagulase for detection in 1 h. Fluorescence values ranged
from 9,100 to 76,000, with the majority between 30,000 and 60,000.
The
positive control, negative control, and reagent blank gave
average
readings of 30,000, 3,000, and 2,000,
respectively.
Extending assay incubation time beyond 1 h slightly
increased fluorescence values with
S. aureus strains
but significantly
increased the number of false-positive reactions
observed (data
not shown). Between 0% (
8) and 20%
(
5) of coagulase-negative
staphylococci (CNS) can produce
positive reactions with chromogenic
assays. Pseudocoagulase
activity in CNS has been described (
3,
14) and may
contribute to this false-positive reaction
rate.
In this study, 7.3 and 0.9% of the CNS were positive by the VPA and BB
assays, respectively (Table
2). Protease inhibitors
such as hirudin and
aprotinin can prevent false-positive reactions
in clotting assays
(
4) but failed to prevent false-positive
reactions with
fluorogenic substrates in this study (data not
shown), suggesting that
these may be due to mechanisms other than
proteolytic activation of
prothrombin.
Fluorescent staphylocoagulase assays appear to be simple, rapid, and
specific tests for identification of
S. aureus colonies
grown on solid media. The BB assay is as sensitive as the VPA
assay but
is more specific. Our results suggest that VPA or BB
assays are valid
alternatives to LA or the tube coagulase test
for the identification of
S. aureus or could provide rapid and
reliable
confirmation of LA tests in the laboratory. Fluorogenic
assays are well
suited to large-scale screening and can provide
quantitative values
with standardized
inocula.
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ACKNOWLEDGMENTS |
This work was supported by a research grant from the Freeman
Hospital Clinical Research Fund.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Freeman Hospital, High Heaton, Newcastle upon Tyne, NE7 7DN, United Kingdom. Phone: (0191) 2231248. Fax: (0191) 2231224. E-mail: malcolm.holliday{at}tfh.nuth.northy.nhs.uk.
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Journal of Clinical Microbiology, April 1999, p. 1190-1192, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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