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Journal of Clinical Microbiology, February 2000, p. 752-754, Vol. 38, No. 2
London Health Sciences
Centre1 and Department of Microbiology
and Immunology, The University of Western
Ontario,2 London, Ontario, Canada
Received 7 July 1999/Returned for modification 26 August
1999/Accepted 12 November 1999
The National Committee for Clinical Laboratory
Standards has recently changed the oxacillin breakpoint from
Coagulase-negative staphylococci
(CoNS) are a major cause of bacteremia in hospitalized patients
(6, 11). Many CoNS isolates are resistant to penicillin and
oxacillin by virtue of beta-lactamase and PBP 2a production,
respectively (5). It is therefore common practice to use
vancomycin as an initial therapy for such infections. Due to the
emergence of vancomycin-resistant enterococci (17), the
Hospital Infection Control Practices Advisory Committee has recommended
curtailing the use of vancomycin (7). Hence, it is important
for clinical laboratories to distinguish between oxacillin-susceptible and oxacillin-resistant CoNS. Recently, it
was shown that the oxacillin breakpoint of The CoNS strains analyzed in our study were clinically
significant isolates collected from patients at three teaching
hospitals in our city or were obtained from other researchers. Isolates were identified by conventional biochemical tests as recommended in the
Manual of Clinical Microbiology (9) and by other
investigators (2), susceptibility to desferrioxamine
(10), and cellular fatty acid profile analysis
(15). The biochemical tests included acid production from
carbohydrates (arabinose, cellobiose, fructose, galactose, glucose,
lactose, maltose, mannitol, mannose, melezitose, raffinose, salicin,
sorbitol, sucrose, trehalose, xylitol, and xylose); hydrolysis of
arginine, esculin, and urea; decarboxylation of ornithine; detection of
beta-glucosidase, coagulase, DNase, gelatinase, phosphatase,
pyroglutamate aminopeptidase, oxidase, and Tween 80 lipase; acetoin
production; resistance to furazolidone and novobiocin; and nitrate
reduction. Isolates were kept frozen at The MIC of oxacillin (0.125 to 4.0 mg/liter) was determined by using
agar dilution methods according to NCCLS guidelines. Briefly,
for each organism, colonies isolated from overnight growth were
selected to prepare direct suspensions in tryptic soy broth. The
suspensions were adjusted to a 0.5 McFarland standard by using a
MicroScan Turbidity Meter (Dade International Inc., Sacramento, Calif.). The 0.5 McFarland suspensions were diluted 1:10. Growth control Mueller-Hinton agar (Oxoid Inc., Nepean, Ontario, Canada) and oxacillin salt (2%) Mueller-Hinton agar plates were
inoculated with the final suspensions by using a replicator delivering
approximately 104 CFU in each spot. Plates were incubated
at 35°C and were read after 24 h of incubation in ambient air.
Staphylococcus aureus ATCC 29213, S. aureus ATCC
43300, and S. aureus ATCC 33591 were included in each
run as control organisms. The MIC was recorded as the
lowest concentration of oxacillin that completely inhibited growth. Oxacillin powder was obtained from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada).
To detect the mecA gene by PCR, growth from blood agar
plates was suspended in sterile water, and the turbidity was adjusted to a 0.5 McFarland standard. To 1 ml of this suspension, an equal volume of Chelex 100 was added, followed by boiling for 7 min. After
centrifugation at 18,000 × g for 15 min, 1 µl of the
supernatant was used in 25 µl of amplification mixture. The
amplification mixture contained a standard amount of PCR buffer; 3.5 mM
MgCl2; 18.75 pM dUTP; 6.25 pM each dATP, dCTP, and dGTP; 5 pM each primer; and 1.25 U of Taq (Life Technologies,
Burlington, Ontario, Canada) and 0.125 U of heat-labile uracil DNA
glycosylase (Boehringer Mannheim Biochemicals). The primers used to
detect the nuc and mecA genes have been
previously described and amplify 310- and 270-bp fragments,
respectively (3, 16). Amplification was carried out by use
of a programmable GeneAmp PCR system 9600 (Perkin-Elmer Cetus) with a
10-min cycle at 94°C followed by 30 cycles of denaturation at 94°C
for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 8 min.
Amplified products were detected by electrophoresis through a 2%
agarose gel containing ethidium bromide (0.5 mg/liter), and bands were
observed under UV light. Each run included S. aureus ATCC
25923, S. epidermidis ATCC 12228, and a local
epidemiological strain of methicillin-resistant S. aureus
(MRSA) known as the Ontario strain as controls.
A total of 493 CoNS isolates belonging to 11 species were tested.
The number of organisms of each species tested and the oxacillin MICs
for strains with and without mecA are shown in Table
1. mecA-positive strains were
not found among S. capitis, S. lugdunensis, S. schleiferi, S. simulans, and S. xylosus. Among other species, the percentage of
mecA-positive strains varied considerably and ranged from
9.0% for S. saprophyticus to 83.3% for S. haemolyticus.
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Correlation of Oxacillin MIC with mecA
Gene Carriage in Coagulase-Negative Staphylococci
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
4 mg/liter to 0.5 mg/liter to detect methicillin-resistant
coagulase-negative staphylococci (CoNS) because the previous
breakpoint lacked sensitivity. To determine the correlation between the
new oxacillin breakpoint and the presence of the
mecA gene, 493 CoNS of 11 species were tested. The presence
of the mecA gene was determined by PCR, and oxacillin susceptibility was determined by the agar dilution method with Mueller-Hinton agar containing 2% NaCl and oxacillin (0.125 to 4.0 mg/liter). The new breakpoint correctly classified all CoNS
strains with mecA as methicillin resistant and strains of Staphylococcus epidermidis, S. haemolyticus, and S. hominis
without mecA as methicillin susceptible. The breakpoint of
0.5 mg/liter was not specific for S. cohnii, S. lugdunensis, S. saprophyticus, S. warneri, and S. xylosus, in that it categorized 70 of
74 strains of these species without
mecA (94.6%) as methicillin resistant. The results
of this study indicate that the new oxacillin breakpoint accurately identifies strains of CoNS with mecA
but is not specific for strains of certain species of CoNS without
mecA.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
4 mg/liter
recommended by the National Committee for Clinical Laboratory
Standards (NCCLS) lacked sensitivity and was unable to
classify many mecA-positive CoNS as oxacillin resistant.
Marshall and coworkers found that lowering the oxacillin breakpoint to
define resistance significantly improved the accuracy of the
susceptibility tests (11). Accordingly, the NCCLS
has redefined the breakpoints for oxacillin susceptibility for CoNS, so that organisms for which the MIC is 0.5 mg/liter are
considered resistant and those for which the MIC is 
0.25 mg/liter are
considered susceptible (13). In this report, we examined the
validity of these new breakpoints.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C and subcultured twice
before being tested.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Correlation between the oxacillin MIC and the
presence of the mecA gene
Based on the new breakpoints and the presence of the
mecA gene, the organisms could be classified into the
following four categories. Category I included S. epidermidis, S. haemolyticus, and S. hominis. More than half of the isolates in this category were
mecA positive. The percentage of mecA-positive
strains was highest for S. haemolyticus (83.3%), followed
by S. epidermidis (61.9%) and S. hominis
(51.8%). The MIC for all the isolates with mecA was
0.5 mg/liter, and that for isolates without mecA was 0.25 mg/liter. Category II included S. cohnii,
S. saprophyticus, and S. warneri.
These organisms had mecA-positive strains, but the
percentage of such strains was much lower than that for the previous
category, ranging from 9 to 28.5%. The oxacillin MIC for strains with
mecA was consistently 0.5 mg/liter, but the MIC for 91.3%
(42 of 46) of strains without mecA was similar. Category III
included S. lugdunensis and S. xylosus.
All strains of these species lacked the mecA gene;
nevertheless, the new breakpoints classified these strains as
resistant to oxacillin. Category IV included S. capitis, S. schleiferi, and S. simulans.
These organisms were similar to those of category III in that all
lacked mecA. However, they differed from the former in that
they were correctly categorized as oxacillin sensitive by the new
susceptibility breakpoints.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Detection of methicillin resistance in staphylococci is complex,
mainly because the resistance is often heterogenous and only 1 of
104 to 108 cells express the resistance trait
(5). Standard susceptibility testing is less accurate in
determining methicillin resistance in mecA-positive CoNS
than in MRSA (18). The oxacillin MICs for
mecA-positive CoNS have been shown to be lower than those for mecA-positive S. aureus (11), and
this finding led the NCCLS to establish new oxacillin MIC
breakpoints for CoNS. The correlation between the presence of the
mecA gene and the oxacillin MIC was determined using strains
of S. epidermidis, S. haemolyticus, S. hominis, and less common CoNS species; however, about half of the
isolates of CoNS in that study (11) were not identified to
the species level. In the present study, the proportions of mecA-positive strains differed significantly among various
species of CoNS, and the new oxacillin MIC breakpoints were less
accurate when applied to S. cohnii, S. saprophyticus, S. warneri, S. lugdunensis, and S. xylosus. For all strains with mecA and 70 of 74 strains without mecA (94.6%) of these species, the
oxacillin MICs were 0.5 mg/liter.
S. epidermidis, S. haemolyticus, and S. hominis are the most commonly isolated species in bacteremias due
to CoNS and account for approximately 95% of such bacteremias
(11, 12). The new interpretative guidelines correctly
classify strains of these species with and without mecA as
oxacillin resistant and oxacillin susceptible, respectively. Many
clinical laboratories do not routinely identify the species of clinical
isolates of CoNS; therefore, the oxacillin breakpoint of 0.5
mg/liter will correctly identify all CoNS strains with mecA
but may also report a few strains without mecA, especially
of species of categories II and III, as falsely methicillin resistant.
However, in practice, because the vast majority of clinically
significant CoNS are S. epidermidis, followed by S. haemolyticus and S. hominis, errors will be
relatively few. Therefore, the use of the new recommendations to
identify methicillin-resistant CoNS can be justified and appears to be practical.
Occasionally, CoNS other than S. epidermidis, S. haemolyticus, and S. hominis cause serious infections
(8, 14), and in certain infections, such as shunt-associated
meningitis and endocarditis, beta-lactam drugs are the preferred
therapy. Susceptibility tests will define all CoNS for which the
oxacillin MIC is 0.5 mg/liter as methicillin resistant, regardless of
the species and the status of the mecA gene. As a result,
some patients may unnecessarily be denied the benefit of relatively
nontoxic and useful beta-lactam antibiotics. In order to accommodate
such cases, an alternative approach to detect methicillin resistance
can be adopted. Instead of relying on phenotypic susceptibility tests
for the detection of methicillin resistance, tests that detect the
mecA gene or PBP 2a can be used. PCR and DNA probes have
been successfully used to detect the mecA gene (1, 16,
18). Archer and Pennell have demonstrated that the detection of
the mecA gene is more sensitive and specific than oxacillin
susceptibility testing in identifying methicillin-resistant CoNS
(1). Recently, a latex agglutination test that detects PBP
2a has been marketed (4), although to date it has only been
shown to be sensitive for MRSA. If it is demonstrated to be effective
at detecting PBP 2a in CoNS, it will have several advantages. It is
faster than PCR, DNA probing, and susceptibility testing, is
technically simple, and can be easily performed in small
laboratories. With this approach, organisms positive for the
mecA gene or PBP 2a can be reported as methicillin resistant. Strains lacking these markers can be tested for
their susceptibility to oxacillin, and breakpoints recommended for
S. aureus can be applied, as the new guidelines for CoNS
were drawn to increase the accuracy of oxacillin susceptibility tests
and not for pharmacokinetic reasons. Before such recommendations are accepted, our findings must be confirmed by other studies with a larger
number of CoNS isolates.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Clinical Microbiology and Infection Control, Westminster Tower, London Health Sciences Centre, Box 5010, London, Ontario, Canada N6A 4G5. Phone: (519) 685-8149. Fax: (519) 685-8203. E-mail: Zafar.hussain{at}lhsc.on.ca.
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REFERENCES |
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Fung-Tomc, J. C., Minassian, B., Kolek, B., Huczko, E., Aleksunes, L., Stickle, T., Washo, T., Gradelski, E., Valera, L., Bonner, D. P.
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