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Journal of Clinical Microbiology, June 1999, p. 2047-2050, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Disk with High Oxacillin Content Discriminates between
Methicillin-Resistant and Borderline Methicillin-Susceptible
Staphylococcus aureus Strains in Disk Diffusion Assays
Using a Low Salt Concentration
Ann Cathrine
Petersson,1,2,*
Carl
Kamme,1,2 and
Håkan
Miörner2
Clinical Microbiology Laboratory, University
Hospital,1 and Department of Infectious
Diseases and Medical Microbiology, University of
Lund,2 Lund, Sweden
Received 29 September 1998/Returned for modification 11 December
1998/Accepted 25 February 1999
 |
ABSTRACT |
A separation between mecA+ strains of
Staphylococcus aureus and strains lacking mecA
was achieved by the disk diffusion assay and the agar dilution method,
utilizing disks containing 5 µg of oxacillin and inocula of
approximately 5 × 105 CFU/spot, respectively,
provided that agar with 0 to 0.5% NaCl and incubation at 30°C were
employed. The 5-µg oxacillin disks clearly discriminated between
borderline methicillin-susceptible and mecA+
strains. The oxacillin MICs were more affected by the inoculum density
and salt concentration than were the methicillin MICs, and oxacillin
MICs of 4 to 16 µg/ml were obtained for strains lacking
mecA. Significantly higher levels of 
-lactamase
production and reduced oxacillin susceptibilities were recorded for
strains lacking mecA, in particular strains of phage group
V, when agar with 
2% NaCl was used than when agar with 0 to 0.5%
NaCl was employed. The results indicate that the borderline
methicillin-susceptible phenotype is a salt-dependent in vitro
phenomenon of questionable clinical relevance.
| |
TEXT |
Strains of Staphylococcus
aureus with reduced susceptibility to penicillinase-resistant
penicillins are categorized as follows: (i) methicillin-resistant
S. aureus (MRSA), which produce the low-affinity penicillin
binding protein (PBP) 2a, encoded by the mecA gene
(25); (ii) strains with modified PBPs (29) due to altered penicillin binding capacity as a result of point mutations or
due to hyperproduction of PBPs (8, 10); and (iii) borderline methicillin-susceptible S. aureus (BSSA), also referred to
as borderline oxacillin-resistant S. aureus, generally
considered to be due to hyperproduction of type A -lactamase by
strains of phage group V, the 94-96 complex, that harbor the pBW15
plasmid (17, 18).
A routine method for the detection of strains with reduced
susceptibility should be quick and easy to perform with good precision and accuracy. In order to improve identification of MRSA strains, several parameters, such as the preparation and size of the inocula, incubation time and temperature, and medium type, brand, and
composition have been evaluated (2, 6, 9, 16, 28). The disk
diffusion method is often recommended, but an international consensus
has not been reached. The use of disks containing 1 µg of oxacillin for identification of oxacillin-resistant strains and of
amoxicillin-clavulanic acid disks for discrimination between MRSA and
BSSA strains is recommended by the National Committee for Clinical
Laboratory Standards and the Swedish Reference Group for Antibiotics
(20, 26), while the use of disks with 5 µg of oxacillin is
recommended in France (5). Additionally, a consensus has not
been reached with respect to the incubation temperature and the
composition of the media. Reports have shown that neither the disk
diffusion method nor MIC determination is able to identify
heterogeneous or salt-intolerant mecA+ strains
as MRSA or to discriminate between BSSA and MRSA strains (7, 9,
11, 14, 19).
The aim of the present study was to evaluate the impact of NaCl on (i)
oxacillin and methicillin MICs; (ii) the ability of disks with 5 µg
of oxacillin, versus those with amoxicillin-clavulanic acid, to
discriminate between MRSA and BSSA strains; and (iii) the production
and release of -lactamase in the disk diffusion method.
(This study was presented in part at the 8th International Symposium on
Staphylococci and Staphylococcal Infections, Aix-les-Bains, France, 23 to 26 June 1996.)
Organisms and test conditions.
In the present study, 107 S. aureus strains identified by established methods
(13) were used. The presence of the mecA gene was
determined by PCR (22) with a Gene Amp PCR System 2400 (Perkin-Elmer). S. aureus ATCC 29213 and ATCC 25923 and one
clinical mecA+ strain of S. haemolyticus (D38) were used as reference strains. Fifty-six
strains were mecA+ and categorized as MRSA.
Twenty of the strains lacking mecA were categorized as BSSA
(methicillin MIC 4 µg/ml [19]) by using agar with
2% NaCl (17) and incubating at 30°C (23).
-Lactamase was produced by 49 mecA+ strains
and by 49 strains lacking mecA (21). The strains
were characterized by phage typing (3). Inocula from
overnight cultures on blood agar plates were suspended in 50 mM
phosphate buffer (pH 7.0) to match the turbidity of a McFarland 0.5 standard. Cation-supplemented Mueller-Hinton agar (BBL Microbiology
System, Cockeysville, Md.) with 0, 0.5, or 2% (wt/vol) NaCl was used
throughout the study. Agar with 3 or 5% NaCl was included in a
semiquantitative -lactamase test, and agar with 5% NaCl was used
for MIC determinations. Statistical analyses included Fisher's exact
test, Student's t test, and McNemar's test (1).
Susceptibility of and discrimination between MRSA, BSSA, and
methicillin-sensitive S. aureus (MSSA).
Oxacillin
(Sigma Chemical Co., St. Louis, Mo.) and methicillin (Astra,
Södertälje, Sweden) MICs were determined for 36 mecA+ strains and 51 strains lacking
mecA by the agar dilution method. Incubation at 30°C for
24 h was employed (23). Significantly higher MICs
(P < 0.001) were obtained for the strains lacking mecA, in particular the BSSA strains, and for the highly
heterogeneous MRSA strains when agar with 2 or 5% NaCl was used than
when agar with 0 or 0.5% NaCl was employed (Table
1). However, a separation between
mecA+ strains and those lacking mecA
was not achieved, and the oxacillin MICs obtained for MRSA strains that
showed high MICs at 0 to 0.5% NaCl were one to four twofold dilution
steps lower when agar with 2 or 5% NaCl was used (P < 0.001). Similar results have been reported elsewhere (9,
19), and it has been proposed that NaCl concentrations above
2.5% not be used due to the salt intolerance of some clones of MRSA
(11). Our results support this view, but we would advocate the use of NaCl at a concentration no higher than 0.5% for MIC determinations.
A separation between mecA+ strains and those
lacking mecA was achieved when a larger inoculum
(approximately 5 × 105 CFU/spot) was used instead of
the recommended 5 × 104 CFU/spot (20)
(Table 1). The oxacillin MICs were more affected by the inoculum size
than were the methicillin MICs, in particular for the heterogeneous
MRSA. A few BSSA strains showed oxacillin MICs of 4 to 16 µg/ml.
Inoculum dependence of oxacillin MICs and reduced specificity upon agar
screening have also been reported by others (7). These
results may be explained by the lower degree of stability of oxacillin
to -lactamases than that of methicillin (17, 24). In the
present study, the methicillin MICs classified all
mecA+ strains as resistant (16 µg/ml) and
all strains lacking mecA as susceptible (
8 µg/ml)
(20, 26) when the larger inoculum was used. Classification
of the strains lacking mecA as BSSA or MSSA varied under
different growth conditions.
Incubation at 30 and 35°C for 24 h was employed in the disk
diffusion method. Transmitted light was used for inspection of
zones
for minute or single colonies and for recording of zone
diameters. The
strains lacking
mecA (BSSA and MSSA) could not
be clearly
separated from
mecA+ strains when disks with 1 µg of oxacillin or with 20 µg of amoxicillin
plus 10 µg of
clavulanic acid (AB Biodisk, Solna, Sweden) were
used (Table
2). After incubation at 35°C, 33 and
61% of the strains
lacking
mecA showed no zone of
inhibition on agar with 0 and 2%
NaCl, respectively (data not shown).
Poor performances by these
disks have been reported (
9,
14),
and we earlier recommended
the use of disks containing 5 µg of
oxacillin in combination with
PDM agar (Biodisk) for
S. aureus and novobiocin-resistant coagulase-negative
staphylococci
(
22). However, the disks containing 1 µg of oxacillin
proved to be the most suitable for identification of
methicillin-resistant,
novobiocin-sensitive, coagulase-negative
staphylococci (
22).
Disks with a high content of oxacillin
have been recommended previously
(
5,
16,
27), although
evaluations have produced less favorable
results (
2). The
poor performance in these studies could be
due to the fact that
species-specific breakpoints (
22) were
not applied; i.e.,
S. aureus and coagulase-negative strains were
not separated
(
2,
16), and detection of the
mecA gene was
not
feasible at that time. In the present study, a clear separation
between
the
mecA+ strains and the strains lacking
mecA was obtained with the disks
containing 5 µg of
oxacillin when agar with 0 to 0.5% NaCl and
incubation at 30°C were
employed (Table
2). The zone histograms
for the strains lacking
mecA showed a shift toward smaller zones,
indicating
enhanced growth at 35°C compared to 30°C. The opposite
pattern was
observed for the
mecA+ strains, yielding a low
degree of discrimination between strains
with and without the
mecA gene. These results were in accordance
with those
reported by French and coworkers, who advocated incubation
at 30°C
(
6).
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TABLE 2.
Distribution of zone diameters obtained with strains
lacking mecA and with mecA+ strains
under different conditions, using disks containing 5 µg of
oxacillin or 20 µg of amoxicillin with 10 µg of
clavulanic acida
|
|
The influence of salt concentration on -lactamase
production.
The BSSA phenotype has been correlated with extensive
production of type A -lactamase among strains of phage group V, the 94-96 complex (17, 18). Based on the 1974 results of Kim and Chipley, who showed that the release of staphylococcal -lactamase increases in an environment containing 5 to 10% NaCl (12),
a semiquantitative method designed to reflect the milieu in the disk
diffusion method was applied to ascertain whether the salt concentration could affect the production and release of -lactamase during susceptibility testing. Duplicate 1-µl drops (approximately 105 CFU/spot) were inoculated onto agar supplemented with
0.5 µg of methicillin/ml as a -lactamase inducer (1.0 µg/ml was
used for strains with methicillin MICs of 4 µg/ml). After 24 h
at 30°C, the agar plates were flooded with nitrocefin (BBL
Microbiology Systems; 200 µg/ml). After 40 min ± 15 s at
room temperature, the zone diameters of hydrolyzed nitrocefin
surrounding the colony spots were recorded with an accuracy of 0.1 mm.
The total intra- and interassay variation, 0.6532 mm, was calculated
from results obtained by testing six strains on five different
occasions with two or four tests of each strain per agar plate
(1).
At 0.5% NaCl, all strains produced similar amounts of
-lactamase;
there was no deviating strain or group of strains. These
results were
used as reference values. At 2 to 5% NaCl, a correlation
between the
salt concentration and the
-lactamase production
was evident (Fig.
1). Four patterns were identified,
corresponding
to zones obtained for four reference strains: type A
-lactamase,
NCTC 9789; type B, 22260; type C, Sal77; and type D,
FAR10 (D.
Kernodle, Nashville, Tenn.). The type A pattern, produced by
27
MRSA, 13 BSSA, and 14 MSSA strains, was characterized by a large
zone (
12.0 mm) when agar with
2% NaCl was used. Strains with
the
type B pattern (11 MRSA, 4 BSSA, and 6 MSSA) showed unchanged
or
reduced zone sizes at the higher salt concentrations compared
to 0.5%
NaCl. Strains with the type C pattern (seven MRSA, two
BSSA, and five
MSSA) showed zone sizes intermediate between those
of the type A and
type B patterns. The type D pattern (four MRSA
and five MSSA) differed
from the other three patterns in that
the zone size of the hydrolyzed
nitrocefin showed small changes
between 0.5, 2, and 3% NaCl but
increased substantially at 5%.
The type A, B, and C patterns differed
significantly (
P < 0.001)
at all salt concentrations,
whereas the type D occasionally differed
from that of type A or B. The
increased
-lactamase production
by the type A strains was reflected
in significantly higher MICs
and smaller zones than those of other
strains at high salt concentrations
but not at 0 to 0.5% NaCl. The
type A
-lactamase was mainly produced
by strains of phage group V,
which was in accordance with results
published by McMurray and
coworkers (
18).
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FIG. 1.
Ranges of changes in -lactamase production by 31 strains of S. aureus lacking mecA grown in
the presence of 2, 3, and 5% NaCl versus production in medium with
0.5% NaCl. Based on changes in -lactamase production, the strains
were classified as type A ( ), type B
(-...-), type C
(.....), and type D
(---) -lactamase producers.
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|
In conclusion, the agar dilution method performed according to
recommendations provides only an approximation of the degree
of
susceptibility and does not reveal MRSA strains. An inoculum
of
5 × 10
5 CFU/spot has to be used for screening for
MRSA, and a method
of verifying the presence or absence of the
mecA gene in strains
that grow at oxacillin concentrations
of
4 µg/ml is necessary.
The amoxicillin-clavulanic acid disk
should be replaced by the
disk containing 5 µg of oxacillin, and agar
with a low salt concentration
and incubation at 30°C should be
employed for the identification
of MRSA by the disk diffusion method.
Since the BSSA phenotype
is related to the salt concentration, we
propose that it does
not constitute a true therapeutic problem but
rather is a laboratory
phenomenon; this is supported by the fact that
BSSA infections
can be treated with the same drugs as are used for
treatment of
MSSA infections (
4,
15).
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FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Microbiology Laboratory, Sölvegatan 23, S-223 62 Lund, Sweden.
Phone: 46 46 173250. Fax: 46 46 189117. E-mail:
Ann-Cathrine.Petersson{at}skane.se.
| |
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Journal of Clinical Microbiology, June 1999, p. 2047-2050, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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