Previous Article | Next Article 
Journal of Clinical Microbiology, June 2000, p. 2378-2380, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
New Chromogenic Identification and Detection of
Staphylococcus aureus and Methicillin-Resistant
S. aureus
John
Merlino,1,2,3,*
Marcel
Leroi,1
Ross
Bradbury,1
Duncan
Veal,2 and
Colin
Harbour3
Department of Microbiology and Infectious
Diseases, Concord Repatriation General Hospital, Concord
2139,1 Department of Biological
Sciences, Macquarie University, Concord 2109,2
and Department of Infectious Diseases, Faculty of Medicine,
University of Sydney, 2006 Sydney,3 New South
Wales, Australia
Received 13 September 1999/Returned for modification 13 November
1999/Accepted 15 February 2000
 |
ABSTRACT |
This paper describes a new chromogenic plate medium, CHROMagar
Staph aureus (CHROMagar, Paris, France), for the identification of
Staphylococcus aureus on the basis of colony pigmentation. The abilities of CHROMagar Staph aureus, thermostable nuclease (DNase),
and mannitol salt agar (MSA) to identify S. aureus isolates (n = 114) and discriminate between S. aureus and coagulase-negative staphylococci (CoNS;
n = 22) were compared. CHROMagar Staph aureus proved
to be more sensitive and specific than DNase and MSA, allowing a
reliable, simple, and rapid method for the identification of S. aureus isolates. All CoNS encountered in this study with the exception of S. chromogenes could be easily differentiated
from S. aureus on this medium. The supplementation with 4 µg of oxacillin or methicillin per ml allowed simple identification
of methicillin resistance in hospital-acquired S. aureus
strains which show multiple-drug resistance profiles.
Community-acquired methicillin-resistant S. aureus strains
showing non-multi-drug resistance profiles require further evaluation
on this new chromogenic medium. Methicillin or oxacillin resistance of
all S. aureus isolates was confirmed by the detection of
penicillin-binding protein 2a, encoded by the mecA gene,
using the latex slide agglutination MRSA-Screen test (PBP 2' Test,
DR900M; Oxoid).
| |
TEXT |
Staphylococcus
aureus is one of the most frequently identified pathogens in
clinical laboratories, while methicillin-resistant S. aureus
(MRSA) is an important nosocomial pathogen (6). Considerable effort has been expended by numerous investigators (7-9) in
the development of reliable media for differentiation of S. aureus from coagulase-negative staphylococci (CoNS). S. aureus has most frequently been associated with the coagulation of
plasma, the fermentation of mannitol (mannitol salt agar [MSA]), the
production of thermostable nuclease (DNase), egg yolk lipase hydrolysis
(lipovitellin-salt-mannitol agar [LSM]), and the production of
natural pigment (6).
Reliable and rapid methods to identify these organisms are crucial in
any clinical laboratory. The tube coagulase test is regarded as the
gold standard; however, variations in levels in plasma and incubation
times and problems in interpretation can lead to misidentification. An
alternative approach is to incorporate chromogenic substrates into a
suitable isolation medium. Detection of the activities of specific
bacterial enzymes, indicated by color change, negates the need for
time-consuming and costly biochemical identification (10).
The present study examines the use of a new chromogenic plate medium,
CHROMagar Staph aureus, for the identification of S. aureus
and detection of MRSA. The criteria for medium evaluation included
colony growth reaction, color reproducibility for the identification of
S. aureus, and ease of color detection of MRSA when the
medium was supplemented with methicillin or oxacillin. The study was
divided into two parts. Part 1 compared the accuracy of reactions of
S. aureus on CHROMagar Staph aureus, DNase, and MSA with the
accuracy of reactions of CoNS. Part 2 compared the detection of MRSA on
the new chromogenic medium when it was supplemented with 4 µg of
methicillin or oxacillin per ml to detection on a standard
Iso-Sensitest agar dilution screening plate containing the same
concentration of methicillin. Methicillin or oxacillin resistance was
confirmed by the detection of penicillin-binding protein 2a (PBP 2a)
expressed by the mecA gene using the latex slide
agglutination MRSA-Screen test (PBP 2' Test, DR900M; Oxoid) (11,
12).
(This paper was presented in part at the IXth International Congress of
Bacteriology and Applied Microbiology Meeting, Sydney, Australia.)
Bacterial cultures.
One hundred twenty-six significant
staphylococcal isolates were examined at random upon isolation from
clinical samples received for examination in the Department of
Microbiology and Infectious Diseases at Concord Repatriation General
Hospital, Concord, Australia. These included S. aureus (104 isolates), S. epidermidis (7 isolates), S. capitis (5 isolates), S. haemolyticus (3 isolates),
S. warneri (2 isolates), S. hominis (2 isolates),
S. cohnii (1 isolate), and S. simulans (1 isolate). These isolates represented clinical samples from
sterile (blood, tissue, bone, eye) or nonsterile (sputum, superficial
wound, urine) sites. In addition, 10 non-multi-drug-resistant community-acquired MRSA isolates were supplied from the
bacteriophage-typing laboratory at Royal Prince Alfred Hospital,
Sydney, Australia.
Culture media.
CHROMagar Staph aureus was provided by the
CHROMagar Company, Paris, France, and imported to Australia by DUTEC
Diagnostics (a division of DUTEC Pty. Ltd., Sydney, Australia). The
medium contained agar (15 g/liter), peptones (40 g/liter), NaCl (25 g/liter), and a proprietary chromogenic mix (3.5 g/liter). The medium
was prepared as instructed by the manufacturer by avoiding heating at
over 100°C. Methicillin or oxacillin (4 µg/ml) was added when the
agar was cooled at 48°C. Each plate contained 20 ml of agar medium
dispensed into 90-mm-diameter petri dishes. Horse blood (5%) agar
(Oxoid-Columbia base-CM331; Oxoid, Victoria, Australia), MSA
plates (Oxoid-CM85), and DNase (Oxoid-CM321) were prepared according to
the manufacturer's instructions. S. aureus ATCC 25923 was
used to monitor batch variability. Horse blood agar was used for
organism viability testing.
Identification and susceptibility methods.
Gram stain
reactions, morphology, reactions to catalase (3% [wt/vol] hydrogen
peroxide, BDH-UN2014; Kilsyth, Victoria, Australia), mannitol salt
fermentation, reactions to slide and tube coagulase (lyophilized rabbit
plasma; bioMerieux, Marcy l'Etoile, France), and DNase activity were
used for identification. API20 Staph galleries (bioMerieux) and
STAPH-ZYM kits (Rosco, Taastrup, Denmark) were used for confirmatory
species identification. On CHROMagar Staph aureus any growth appearing
pink to mauve (Fig. 1) was interpreted as
positive for the presence of S. aureus. Susceptibility of
isolates was confirmed by agar plate dilution using a multipoint
inoculation system (10). Alternatively, a disk agar
diffusion method (National Committee for Clinical Laboratory Standards)
using methicillin (5 µg) disks (OXOID) was also used (8).
E-test strips (Australian Laboratory Services, Pty. Ltd., Sydney,
Australia) confirmed the MICs for certain isolates (data not shown).
S. aureus ATCC 25923 was used to monitor antibiotic potency
for quality control. Testing was performed using a multipoint
inoculating technique (MAST systems) as described previously
(9). Briefly, each pin transferred 104 to
105 CFU of inoculum per ml to the surface of the culture
medium. Moisture on the surfaces of agar plates was removed by air
drying (lid kept ajar) at 35°C for 1 h prior to inoculation. All
plates with inoculated medium were incubated at 30°C and at 35 to
37°C in ambient air. CHROMagar plates were incubated in the dark as instructed by the manufacturer. All S. aureus strains
showing methicillin and oxacillin resistance were confirmed by the
MRSA-Screen latex agglutination test. The test was performed in
accordance with the manufacturer's instructions and has been described
in previous studies (11, 12). Beta-lactamase enzyme testing
was performed with cefinase disks (BBL, Cockeysville, Md.) as
instructed by the manufacturer. For statistical analysis, CHROMagar
Staph aureus agar results and those obtained with other conventional media were compared for sensitivity and specificity of the media.
View larger version (68K):
[in this window]
[in a new window]
|
FIG. 1.
Comparisons of different agar media showing various
reactions of 18 selected Staphylococcus isolates and one
CoNS isolate mixed with enterococci. (A) CHROMagar Staph aureus with no
antibiotic supplementation showing pink-to-red reactions for all
S. aureus isolates. Row 4 spot 1 shows a blue color reaction
for CoNS mixed with enterococci. Row 5 spots 2 and 3 show a white color
for CoNS. (B) MSA showing yellow positive reactions and pink-to-red
negative reactions of isolates. (C) CHROMagar Staph aureus supplemented
with oxacillin showing the oxacillin (methicillin) resistance of
isolates. The color of MRSA isolates is pink to red. (D) DNase agar
showing clearing around positive isolates.
|
|
CHROMagar Staph aureus accuracy in detecting S. aureus.
Of 114 S. aureus isolates tested on CHROMagar Staph aureus,
all grew and were identified chromogenically as S. aureus by
a pink-to-mauve color change after 18 to 24 h of incubation. In contrast, DNase and MSA were successful in identifying S. aureus in 112 of 114 of isolates (98%). Of 22 CoNS tested on
CHROMagar Staph aureus in this study, 21 isolates produced
colorless or cream-colored colonies. One CoNS (S. chromogenes) produced a pale-pink pigment. This could not be
easily differentiated from the pigment produced by S. aureus
colonies. Mannitol was fermented in 8 of 22 CoNS isolates (5 S. capitis isolates, 1 S. simulans isolate, 1 S. chromogenes isolate, and 1 S. cohnii isolate) on MSA
(36.5%), while 1 CoNS isolate (S. capitis) produced a
positive reaction on DNase. Hence, in comparison to DNase and MSA,
CHROMagar Staph aureus gave superior sensitivity and specificity for
identification of S. aureus (Table
1).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Comparison of the levels of accuracy of reactions of
S. aureus isolates on CHROMagar Staph aureus, DNase, and MSA
and of coagulase testing of CoNS after 18 to 24 h of incubation
|
|
CHROMagar Staph aureus accuracy in detecting MRSA.
Following
addition of methicillin and oxacillin (4 µg/ml) to the chromogenic
medium, there was inhibition of all methicillin-sensitive strains, with
full correlation to results from methicillin- and oxacillin-supplemented standard agar (Iso-Sensitest) dilution testing
(Table 2). Multi-drug-resistant MRSA
strains were reliably detected on the medium (100%) with similar color
changes, and all were positive for PBP 2a. However,
non-multi-drug-resistant community-acquired MRSA grew inconsistently on
the chromogenic medium. Only 4 of 12 (30%) such isolates grew on the
supplemented CHROMagar (Table 2). Different incubation temperatures
(30, 35, and 37°C) did not affect this result (data not shown). All
12 strains were PBP 2a positive by the rapid MRSA-Screen latex test. The MICs (measured by E-test) for these isolates were not significantly different from those for the other MRSA tested. All
methicillin-resistant CoNS grew as colorless or cream-colored colonies.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Detection of MRSA on CHROMagar Staph aureus when it was
supplemented with 4 µg of methicillin per ml and at antibiotic
susceptibility breakpointsa
|
|
In conclusion, CHROMagar Staph aureus achieved a higher
sensitivity and specificity than those of two commonly used media,
DNase and MSA, in identifying
S. aureus isolates. The new
chromogenic
medium facilitated clear distinction between
coagulase-positive
and -negative staphylococci, permitting a reliable,
simple, and
rapid method of identification of
S. aureus in
this study. Results
are encouraging. Further evaluation is needed with
other species
of CoNS not encountered or described in this study. The
supplementation
with oxacillin or methicillin allows nosocomial
multi-drug-resistant
MRSA to be detected, which was not the case with
non-multi-drug-resistant
community-acquired MRSA.
Non-multi-drug-resistant community-acquired
MRSA are of increasing
clinical significance and represent a growing
proportion of
community-acquired
S. aureus infections from outpatient
clinics. They have been isolated from soft tissue, abscess, skin,
blood, bone, eye, genital, and respiratory infections (
1,
4).
These do not represent nosocomial isolates which have
spread in
the community from hospitals, since most have susceptibility
and
phage-typing patterns (data not shown) distinct from those of
multi-drug-resistant hospital MRSA isolates. Cases of
community-acquired
non-multi-drug-resistant MRSA have been reported
in Chicago, Ill.,
Australia, and New Zealand (
1,
5,
11). The cause of these
organisms' growth anomaly on the test
chromogenic medium remains
unclear but may reflect active
cotransportation of methicillin
intracellularly with the chromogenic
moiety. In the presence of
methicillin or oxacillin, the
chromogenically linked substrates
may affect the cell membrane
potential during permeation, leading
to nonspecific membrane
disorganization or induced cell death.
Research is currently being
undertaken to define these phenomena
in these community-acquired
non-multi-drug-resistant MRSA strains.
Further evaluation of this new
chromogenic medium with direct
clinical specimens is needed before this
medium can be used for
routine direct screening for
MRSA.
| |
ACKNOWLEDGMENTS |
We thank Alison Vickery for supplying some strains for this
study and Matthias Dorsch, Paul Attfield, Thusitha Gunasekera, and
Andrew Boyd for discussions. The skilled support of Mohammad Siddique
for medium preparation and the technical assistance of the Department
of Microbiology and Infectious Diseases at Concord Repatriation General
Hospital are recognized. We also thank the CHROMagar, Oxoid, DUTEC
Diagnostics, and Rosco companies for supplying materials for this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Infectious Diseases, Concord Repatriation General
Hospital, Hospital Rd., Concord 2139, New South Wales, Australia.
Phone: (612) 9767 6658. Fax: (612) 9767 7868. E-mail:
john{at}micr.crg.cs.nsw.gov.au.
| |
REFERENCES |
| 1.
|
Collignon, P.
1999.
Increased incidence of methicillin-resistant strains of Staphylococcus aureus in the community.
J. Infect. Dis.
179:1592[CrossRef][Medline]. (Letter.)
|
| 2.
|
Collignon, P.,
I. Gosbell,
A. Vickery,
G. Nimmo,
T. Stylianopoulos, and T. Gottleib on Behalf of the Australian Group on Antimicrobial Resistance.
1998.
Community-acquired methicillin-resistant Staphylococcus aureus in Australia.
Lancet
352:145-146[CrossRef][Medline]. (Letter.)
|
| 3.
|
Herold, B. C.,
L. C. Immergluck,
M. C. Maranan,
D. S. Lauderdale,
R. E. Gaskin,
S. Boyle-Vavra,
C. D. Leitch, and R. S. Daum.
1998.
Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk.
JAMA
279:593-598[Abstract/Free Full Text].
|
| 4.
|
Kloos, W. E., and J. H. Jorgensen.
1985.
Staphylococci, p. 143-153.
In
E. H. Lennette, A. Balows, W. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C.
|
| 5.
|
Lally, R. T.,
M. N. Ederer, and B. F. Woolfrey.
1985.
Evaluation of mannitol salt agar with oxacillin as a screening medium for methicillin-resistant Staphylococcus aureus.
J. Clin. Microbiol.
22:501-504[Abstract/Free Full Text].
|
| 6.
|
Merlino, J.,
R. Gill, and G. J. Robertson.
1996.
Application of lipovitellin-salt-mannitol agar for screening, isolation, and presumptive identification of Staphylococcus aureus in a teaching hospital.
J. Clin. Microbiol.
34:3012-3015[Abstract].
|
| 7.
|
Merlino, J.,
S. Siarakas,
G. J. Robertson,
G. R. Funnell,
T. Gottlieb, and R. Bradbury.
1996.
Evaluation of CHROMagar Orientation for differentiation and presumptive identification of gram-negative bacilli and Enterococcus species.
J. Clin. Microbiol.
34:1788-1793[Abstract].
|
| 8.
|
National Committee for Clinical Laboratory Standards.
1993.
Performance standards for antimicrobial disk susceptibility tests, 5th ed.
Approved standard M2-A5. National Committee for Clinical Laboratory Standards, Villanova, Pa.
|
| 9.
|
O'Brien, F. G.,
J. W. Pearman,
M. Gracey,
T. V. Reley, and W. B. Grubb.
1999.
Community strain of methicillin-resistant Staphylococcus aureus involved in a hospital outbreak.
J. Clin. Microbiol.
37:2858-2862[Abstract/Free Full Text].
|
| 10.
|
Reuther, J. W. A.
1968.
A simplified system for the identification of staphylococci by multipoint inoculation of test media.
J. Med. Microbiol.
22:179-182[Abstract/Free Full Text].
|
| 11.
|
Van Griethuysen, A.,
M. Pouw,
N. Van Leeuwen,
M. Heck,
P. Willemse,
A. Buiting, and J. Kluytmans.
1999.
Rapid slide latex agglutination test for detection of methicillin resistance in Staphylococcus aureus.
J. Clin. Microbiol.
37:2789-2792[Abstract/Free Full Text].
|
| 12.
|
van Leeuwen, W. B.,
C. van Pelt,
A. Luijendijk,
H. A. Verbrugh, and W. H. F. Goessens.
1999.
Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-Screen latex agglutination test.
J. Clin. Microbiol.
37:3029-3030[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, June 2000, p. 2378-2380, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Bocher, S., Smyth, R., Kahlmeter, G., Kerremans, J., Vos, M. C., Skov, R.
(2008). Evaluation of Four Selective Agars and Two Enrichment Broths in Screening for Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol.
46: 3136-3138
[Abstract]
[Full Text]
-
van Loo, I. H. M., van Dijk, S., Verbakel-Schelle, I., Buiting, A. G. M.
(2007). Evaluation of a chromogenic agar (MRSASelect) for the detection of meticillin-resistant Staphylococcus aureus with clinical samples in The Netherlands. J Med Microbiol
56: 491-494
[Abstract]
[Full Text]
-
Diederen, B. M. W., van Leest, M.-L., van Duijn, I., Willemse, P., van Keulen, P. H. J., Kluytmans, J. A. J. W.
(2006). Performance of MRSA ID, a New Chromogenic Medium for Detection of Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol.
44: 586-588
[Abstract]
[Full Text]
-
Hedin, G., Fang, H.
(2005). Evaluation of Two New Chromogenic Media, CHROMagar MRSA and S. aureus ID, for Identifying Staphylococcus aureus and Screening Methicillin-Resistant S. aureus. J. Clin. Microbiol.
43: 4242-4244
[Abstract]
[Full Text]
-
Diederen, B., van Duijn, I., van Belkum, A., Willemse, P., van Keulen, P., Kluytmans, J.
(2005). Performance of CHROMagar MRSA Medium for Detection of Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol.
43: 1925-1927
[Abstract]
[Full Text]
-
Kipp, F., Kahl, B. C., Becker, K., Baron, E. J., Proctor, R. A., Peters, G., von Eiff, C.
(2005). Evaluation of Two Chromogenic Agar Media for Recovery and Identification of Staphylococcus aureus Small-Colony Variants. J. Clin. Microbiol.
43: 1956-1959
[Abstract]
[Full Text]
-
Perry, J. D., Davies, A., Butterworth, L. A., Hopley, A. L. J., Nicholson, A., Gould, F. K.
(2004). Development and Evaluation of a Chromogenic Agar Medium for Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol.
42: 4519-4523
[Abstract]
[Full Text]
-
Flayhart, D., Lema, C., Borek, A., Carroll, K. C.
(2004). Comparison of the BBL CHROMagar Staph aureus Agar Medium to Conventional Media for Detection of Staphylococcus aureus in Respiratory Samples. J. Clin. Microbiol.
42: 3566-3569
[Abstract]
[Full Text]
-
Perry, J. D., Rennison, C., Butterworth, L. A., Hopley, A. L. J., Gould, F. K.
(2003). Evaluation of S. aureus ID, a New Chromogenic Agar Medium for Detection of Staphylococcus aureus. J. Clin. Microbiol.
41: 5695-5698
[Abstract]
[Full Text]
-
Blanc, D. S., Wenger, A., Bille, J.
(2003). Evaluation of a Novel Medium for Screening Specimens from Hospitalized Patients To Detect Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol.
41: 3499-3502
[Abstract]
[Full Text]
-
Kluytmans, J., Van Griethuysen, A., Willemse, P., Van Keulen, P.
(2002). Performance of CHROMagar Selective Medium and Oxacillin Resistance Screening Agar Base for Identifying Staphylococcus aureus and Detecting Methicillin Resistance. J. Clin. Microbiol.
40: 2480-2482
[Abstract]
[Full Text]
-
Merlino, J., Watson, J., Rose, B., Beard-Pegler, M., Gottlieb, T., Bradbury, R., Harbour, C.
(2002). Detection and expression of methicillin/oxacillin resistance in multidrug-resistant and non-multidrug-resistant Staphylococcus aureus in Central Sydney, Australia. J Antimicrob Chemother
49: 793-801
[Abstract]
[Full Text]
-
Simor, A. E., Goodfellow, J., Louie, L., Louie, M.
(2001). Evaluation of a New Medium, Oxacillin Resistance Screening Agar Base, for the Detection of Methicillin-Resistant Staphylococcus aureus from Clinical Specimens. J. Clin. Microbiol.
39: 3422-3422
[Full Text]
Top
Abstract
Text
