JOURNAL OF CLINICAL MICROBIOLOGY, July 2001, p.

25812583
0095-1137/01/$04.000 DOI: 10.1128/JCM.39.7.25812583.2001
Copyright 2001, American Society for Microbiology. All Rights Reserved.

Vol. 39, No. 7

Performance of the Chromogenic Medium CHROMagar Staph

Aureus and the Staphychrom Coagulase Test in the Detection
and Identification of Staphylococcus aureus
in Clinical Specimens
ANNE CARRICAJO,1 AXEL TRENY,2 NATHALIE FONSALE,1 MICHELE BES,3
MARIE ELISABETH REVERDY,3 YVES GILLE,2 GERALD AUBERT,1
2
AND ANNE MARIE FREYDIERE *
pital de lAntiquaille, F-69321 Lyon cedex 5,2
Ho
pital de Bellevue, F-42055 Saint Etienne,1 Ho
and Ho
pital Edouard Herriot, F-69437 Lyon cedex 03,3 France
Received 12 March 2001/Returned for modification 13 April 2001/Accepted 7 May 2001

CHROMagar Staph aureus (CSAM) (CHROMagar Microbiology, Paris, France) is a new chromogenic medium designed to enable detection of colonies of Staphylococcus aureus by their pink color. A total of 775 specimens were cultured in parallel on CHROMagar Staph aureus and conventional media. Among the 267 S. aureus strains recovered on at least one medium, 263 were isolated on CSAM medium (sensitivity, 98.5%), and
245 (sensitivity, 91.8%) were isolated on conventional media. The specificity of presumptive identification of S. aureus on the basis of pink colony color on CSAM medium was 97% (493 of 508). This specificity increased to 100%
when coagulase detection with the Staphychrom coagulase test was added and to 98.8% when S. aureus surface
components were detected by agglutination in the Pastorex Staph Plus test. Susceptibility testing of 67 S. aureus
strains, performed in parallel on pink CSAM colonies and on colonies grown on blood agar, gave similar results. Thus, rapid and accurate recognition and identification of S. aureus isolates were achieved with CSAM
as the primary isolation medium, followed by the staphylocoagulase Staphychrom test. Antimicrobial susceptibility testing (disk-diffusion method or ATB STAPH System) can be performed directly on pink CSAM colonies.
staphylococcal species are also coagulase positive (e.g., Staphylococcus intermedius) even if they are rarely detected in human specimens (9, 14). The Staphychrom coagulase test is a
fluorogenic staphylocoagulase test based on human prothrombin and protease inhibitors and specifically detects S. aureus
coagulase (8; A. Treny, M. Bes, N. Fonsale, A. Carricajo, M. E.
Reverdy, and A. M. Freydiere, Abstr. 11th Eur. Congr. Clin.
Microbiol. Infect. Dis., abstr. P1512, p. 326327, 2001).
We assessed the performance of CSAM by culturing 775
clinical samples; all pink colonies were then submitted to the
Staphychrom coagulase test. Results were compared with
those obtained with conventional culture media, the Pastorex
Staph Plus agglutination kit, and the conventional coagulase
tube test.
(This work was presented in part at the 100th General Meeting of the American Society for Microbiology, Los Angeles,
Calif., 21 to 25 May 2000.)

Staphylococcus aureus causes severe suppurative infections

associated with high morbidity and mortality. Its isolation from
a patient with an infectious syndrome usually leads to specific
antibiotic treatment. S. aureus can be missed when the clinical
specimen contains a mixed flora. This is especially the case
when other staphylococcal species with an identical colony
appearance are present or when swarming colonies of Proteus
or Pseudomonas cover those of S. aureus. Misidentification of
S. aureus in a clinical sample can have serious clinical repercussions.
S. aureus colonies grown on a chromogenic medium, such
as CHROMagar Staph aureus (CHROMagar Microbiology,
Paris, France) (CSAM) are pink, unlike colonies of other
Staphylococcus species. CSAM has been reported to yield a
higher detection rate for S. aureus in plurimicrobial samples.
The reported sensitivity of the CSAM method is 95.5%, compared to 81.9% with conventional methods (Gaillot et al. [6]).
Pink colonies grown on CSAM can be rapidly confirmed to be
S. aureus by using agglutination kits, such as Pastorex Staph
Plus, which simultaneously detects the clumping factor, protein
A, and capsular antigens. The specificity of this test is only
75.5%, however (13), as species like Staphylococcus schleiferi
and Staphylococcus lugdunensis, which are sometimes involved
in human diseases, may also give positive reactions (2, 4, 7,
12). S. aureus is also identified by coagulase testing, but other

MATERIALS AND METHODS

Clinical specimens. From August 1999 to June 2000 we tested 775 clinical
specimens, comprising 431 wound samples, 6 urine samples, 4 stool samples, 98
blood culture supernatants, 5 bronchoalveolar lavage samples, 3 sputum samples,
198 tracheal aspirates, 5 drainage fluid samples, and 65 nasal specimens. Four
hundred sixty-eight specimens were studied at Antiquaille hospital (Lyon,
France), and 307 were studied at Bellevue hospital (Saint Etienne, France), using
the same protocol. Nonfluid specimens were suspended in physiological (0.85%)
saline, and 0.01 ml of this suspension was streaked on the different plates.
Media. Specimens were streaked on CSAM agar plates (CHROMagar Microbiology), Columbia agar plates supplemented with 5% horse blood (bioMerieux,
Marcy-lEtoile, France), and chocolate agar plates (bioMerieux). All the plates
were randomly inoculated at the same time and examined after 24 and 48 h of
incubation at 37C. CSAM plates and conventional agar plates were read independently by different technicians.

* Corresponding author. Present address: Laboratoire de microbiologie, Ho

pital Debrousse, 29 rue Soeur Bouvier, 69322 Lyon cedex
05, France. Phone: 33 4 72 38 58 16. Fax: 33 4 72 38 55 35. E-mail: am
[email protected].
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2582

CARRICAJO ET AL.

J. CLIN. MICROBIOL.

TABLE 1. Number of S. aureus isolates recovered from 775 clinical

specimens and number of false negatives by using
CSAM and a standard method
Method

No. of isolates identified

as S. aureus

No. of false
negatives

Sensitivity
(%)

Standard methoda
CSAMb
Any media

245
263
267c

22
4

91.8
98.5
100

a
Suggestive colony morphology and Gram staining, positive catalase test, and
positive latex agglutination.
b
Mauve colony and suggestive Gram staining.
c
Total number of S. aureus isolates recovered by at least one method.

Identification of S. aureus. All pink colonies grown on CSAM agar were Gram
stained and agglutinated with the Pastorex Staph Plus kit; coagulase production
was detected using the Staphychrom test according to the manufacturers recommendations. Colonies grown on conventional agar were suspected to be
staphylococcal on the basis of their morphology, Gram staining, and catalase
positivity. Catalase-positive colonies and gram-positive cocci were agglutinated
with the Pastorex Staph Plus kit, and coagulase production was detected with
EDTA-rabbit plasma (Difco Laboratories, Detroit, Mich.). When the result of
the latex agglutination test differed from that of the coagulase test, identification
was performed with the API ID32 STAPH gallery (bioMerieux) or the Accuprobe test (bioMerieux), which detects S. aureus-specific rRNA sequences.
Susceptibility testing. Antimicrobial susceptibility testing was performed by
picking colonies directly from CSAM and conventional media. The disk-diffusion
method on Mueller-Hinton agar (bioMerieux) was used at Antiquaille hospital
according to the recommendations of the Comite Francais de lAntibiogramme
(3). The methicillin susceptibility test was performed with a 5-g oxacillin disk,
a 108-CFU/ml inoculum, and incubation at 30C for 24 h. The automated ATB
STAPH System (bioMerieux) was used at Bellevue hospital, according to the
manufacturers recommendations. Briefly, a 0.5 McFarland emulsion of isolated colonies in sterile saline was added to 7 ml of ATB medium (MuellerHinton broth supplemented with 5% NaCl). The final inoculum was transferred
into an oxacillin (2 g/ml) well and incubated for 24 h at 35C. The antibiotics
tested were penicillin G, oxacillin, erythromycin, lincomycin, pristinamycin, tetracycline, kanamycin, tobramycin, gentamicin, rifampin, fusidic acid, fosfomycin,
pefloxacin, cotrimoxazole, vancomycin, and teicoplanin.

RESULTS
Detection of S. aureus. A total of 267 S. aureus strains (25%
methicillin-resistant strains) were isolated from CSAM and/or
conventional media (Table 1). Two hundred sixty-three isolates grew on CSAM: 242 (92%) grew after 24 h and 21 (8%)
after 48 h. Two hundred forty-three isolates grew on conventional media. Four isolates were not detected on CSAM and 22
were not detected on conventional media (13 isolates were
masked or inhibited by a gram-negative species, 7 were mixed
with several other coagulase-negative staphylococci, and 2 isolates corresponded to samples containing too few colonies).
The sensitivities of CSAM and conventional media for growing
S. aureus were 98.5 and 91.8%, respectively.
Fifteen pink colonies that grew on CSAM were subsequently
identified as species other than S. aureus. None of them produced coagulase in the Staphychrom coagulase test, while
six agglutinated in the Pastorex Staph Plus test and were identified as Staphylococcus simulans, Staphylococcus intermedius,
S. schleiferi, or Staphylococcus warneri by using the API STAPH
gallery (Table 2). The isolates which were neither coagulase
positive nor agglutination positive belonged to other coagulase-negative Staphylococcus species or were micrococci (Table 2). Five of these 15 strains were detected after 48 h of
incubation. In addition, 55 Corynebacterium spp., 3 Candida

albicans isolates, and 1 unidentified gram-negative bacillus

developed a pink aspect on CSAM medium. Except for one
C. albicans isolate and one Corynebacterium isolate, which were
detected after 24 h of incubation, all the other strains took 48 h
to grow. For gram-positive cocci, the specificity of CSAM for
the presumptive identification of S. aureus was 97%. This specificity increased to 98% with the Pastorex Staph Plus test, and
to 100% with the Staphychrom coagulase test (Table 2).
Susceptibility testing was performed on 67 S. aureus isolates
(47 by the disk-diffusion method and 20 with the ATB STAPH
system); 11 of these 67 S. aureus strains (16%) were methicillin-resistant strains. Full agreement was obtained between pink
colonies on CSAM media and corresponding colonies on conventional media, for all the antibiotics tested.
DISCUSSION
The use of CSAM medium for the detection of S. aureus,
followed by Staphychrom coagulase testing, yielded an overall
sensitivity of 98% and a specificity of 100%. The four S. aureus
isolates which were not detected on CSAM but grew on conventional media corresponded to samples containing only one
to five colonies per plate, and the lack of growth on CSAM
might have been a consequence of random seeding. CSAM
medium permitted the detection of 22 S. aureus isolates that
were not detected on conventional media, notably in plurimicrobial samples. The good visibility of the pink colonies on
CSAM facilitates the recognition of potential S. aureus isolates
and thus increases the detection rate.
These results confirm data reported by Gaillot et al. (6), who
isolated 310 S. aureus strains on CSAM from among 2,000
clinical samples; they obtained a sensitivity of 95.5% for
CSAM versus 81.9% for a conventional method. CSAM has
also been used for the detection of S. aureus nasal carriage:
Laudat et al. (P. Laudat, A. Gendre, and C. Chillou, Abstr.
20th Interdiscip. Meet. Anti-Infect. Chemother., Soc. Fr. Microbiol. Section Agents Antimicrob. Soc. Pathol. Infect., abstr.
343/P2, 2000) detected 26 S. aureus carriers with CSAM and
TABLE 2. Number and nature of false-positive S. aureus strains
identified using CSAM alone, CSAM plus the Pastorex Staph
Plus test, and CSAM plus the Staphychrom coagulase test
No. of strains with false-positive
S. aureus identification using a:
Strain(s)

Micrococcus spp.
Staphylococcus epidermidis
Staphylococcus simulans
Staphylococcus intermedius
Staphylococcus warneri
Staphylococcus schleiferi
Other coagulase-negative
Staphylococcus spp.
Total
a

CSAMb

CSAMb
Pastorex Staph
Plus test

CSAMb
Staphychrom
coagulase test

2
3
3c
1
1
1
4

0
0
3c
1
1
1
0

0
0
0
0
0
0
0

15

Specificities were the following: for CSAM, 97%; for CSAM Pastorex
Staph Plus test, 98.8%; for CSAM Staphychrom coagulase test, 100%.
b
Mauve colony and suggestive Gram staining, but not an S. aureus strain.
c
These three strains were isolated from different specimens from the same
patient.

VOL. 37, 2001

CSAM AND STAPHYCHROM COAGULASE TEST

only 22 with blood agar plates. Higher-than-normal detection

rates have also been described with several other chromogenic
media designed for urinary tract pathogens, C. albicans, and
salmonellae (1, 5, 10, 11).
The rapid Staphychrom coagulase test was more specific
than the latex-agglutination test Pastorex Staph Plus for S. aureus identification, since the six non-S. aureus strains that
yielded both pink colonies and a positive latex agglutination
test were correctly identified as belonging to other staphylococcal species. Thus, the use of CSAM combined with the
rapid Staphychrom coagulase test for confirmation of S. aureus identification seems to be an optimal strategy, since it
avoids the frequently recommended combination of two tests
for accurate identification of S. aureus (an agglutination test
and the tube coagulase test) (13; Treny et al., 11th ECCMID).
Although CSAM medium is more expensive than conventional medium, it may be cost effective, since it eliminates the
need for numerous catalase and latex agglutination tests for
non-S. aureus strains grown on conventional media (A. M.
Freydiere, M. Be`s, C. Roure, and A. Carricajo, Abstr. 100th
Gen. Meet. Am. Soc. Microbiol., abstr. C230, p. 183, 2000). On
CSAM, only Staphylococcus strains yielding pink colonies require further testing, thereby reducing handling and reagent
costs. The Staphychrom coagulase test is slightly more fastidious and less rapid (2 h) than the Pastorex Staph Plus test.
Finally, susceptibility testing performed directly on pink
CSAM colonies yields results that are in perfect agreement
with those obtained with colonies grown on conventional media.
ACKNOWLEDGMENTS
We thank Alain Rambach and CHROMagar Microbiology for the
gift of CHROMagar Staph aureus medium and International Microbio
for the gift of the Staphychrom reagent. We are also grateful to the
laboratory technicians, to J. Etienne for his helpful advice and his
encouragement, and to D. Young for his valuable help in editing the
English version of this paper.

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