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Journal of Clinical Microbiology, January 1998, p. 273-274, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evaluation of Screening and Commercial Methods for
Detection of Methicillin Resistance in Coagulase-Negative
Staphylococci
Zafar
Hussain,1,2,*
Luba
Stoakes,1
Robert
Lannigan,1,2
Susan
Longo,1 and
Barbara
Nancekivell1
Department of Microbiology, London Health
Sciences Centre,1 and
Department of
Microbiology and Immunology, University of Western
Ontario,2 London, Ontario, Canada
Received 26 August 1997/Accepted 22 September 1997
 |
ABSTRACT |
The National Committee for Clinical Laboratory Standards recommends
48 h of incubation by the oxacillin salt agar screen (OSAS) method
for the detection of methicillin-resistant coagulase-negative staphylococci (CoNS). An earlier identification of methicillin resistance is desirable. The time to detection of the mecA
gene by PCR was compared with the times to detection by OSAS, by the oxacillin disk diffusion (ODD) method, and with MicroScan Gram Positive
Combo type 6 panels (MicroScan Inc. Sacramento, Calif.) and Vitek
GPS-SA cards (bioMérieux Vitek Inc., Hazelwood, Mo.). The
combination of the Vitek card and the ODD method detected 92 of 99 methicillin-resistant strains of CoNS at 24 h; however, 6 mecA-positive strains were phenotypically methicillin
susceptible. We conclude that most methicillin-resistant CoNS can be
detected and the results can be reported after overnight incubation by a combination of methods.
| |
TEXT |
The recent data from National
Nosocomial Infection Surveillance indicate the increasing importance of
gram-positive organisms (13). The increase in methicillin
resistance has accompanied the increase in the prevalence of
staphylococci as nosocomial pathogens. In North America, the proportion
of strains of Staphylococcus aureus resistant to methicillin
rose from 2.4% in 1975 to 29% in 1991 (2), and more than
50% of isolates of coagulase-negative staphylococci (CoNS) are
resistant to beta-lactams (13). The use of vancomycin has
drastically increased (17) because it is the drug of choice
for the treatment of methicillin-resistant staphylococci. The emergence
of vancomycin-resistant Enterococcus sp. (VRE) has further
compounded the problem (3, 9). To check the spread of VRE,
the Hospital Infection Control Advisory Committee has recommended that
the use of vancomycin be curtailed (5). Early identification
of methicillin resistance in staphylococci could help to control the
unnecessary use of vancomycin. Thus, a rapid and reliable method for
the detection of methicillin resistance is needed.
Methicillin-resistance S. aureus (MRSA) can be reliably detected after 24 h of incubation; however, for CoNS 48 h is
often required before resistance can be ruled out (11).
We compared the time to mecA detection by PCR with the times
to detection by the National Committee for Clinical Laboratory Standards (NCCLS) oxacillin salt agar screen (OSAS) method, by the
oxacillin disk diffusion (ODD) method, and with MicroScan Gram Positive
Combo type 6 panels (DADE MicroScan Inc. Sacramento, Calif.) and Vitek
GPS-SA cards (bioMérieux Vitek Inc., Hazelwood, Mo.). The
objective of the study was to determine which method or combination of
methods could accurately and most rapidly predict methicillin
resistance in CoNS.
One hundred eighty-one strains of CoNS isolated from consecutive blood
cultures were studied. The strains were identified with MicroScan
panels (6). Additional biochemical tests recommended in the
Manual of Clinical Microbiology (7),
susceptibilities to desferrioxamine (8), and cellular fatty
acid profile (15) were used whenever necessary. The isolates
were saved at 
70°C and were subcultured twice before testing.
For the OSAS method, plates were spot inoculated with a direct colony
suspension with a turbidity equivalent to that of a 0.5 McFarland
standard according to the recommendations of NCCLS (12). The
plates were incubated at 35°C and were read at 24 and 48 h. The
ODD test was performed as described by NCCLS (12), and the
zone diameters were determined at 24 and 48 h of incubation.
Oxacillin susceptibility testing with the MicroScan Gram Positive Combo
type 6 panels and Vitek GPS-SA cards were performed according to the
manufacturers' instructions. MicroScan panels were read at 24 and
48 h with an AutoScan 4 instrument. A Vitek 120 Reader/Incubator
was used to process the GPS-SA cards; the reader incubator read the
cards within 24 h whenever the growth was sufficient.
For PCR, an overnight growth of organisms in 1.5 ml of brain heart
infusion broth was pelleted and washed once with sterile saline. The
pellet was suspended in 1 ml of distilled water, and an equal volume of
Chelex 100 was added. The suspension was boiled for 7 min and
centrifuged at 18,000 × g for 15 min, and 1 µl of the supernatant was used in 20 µl of amplification mixture. The primers used to detect the mecA and femA genes
and the overlapping sequence of staphylococcal insertion element
IS431 have been published by Vannuffel et al.
(16). The last set of primers served as an internal control
for the amplification process. The PCR mixture and amplification
conditions were identical to those published by Vannuffel et al.
(16). The bands were detected with UV light after
electrophoresis of the amplified products through a 6% polyacrylamide gel containing ethidium bromide. The presence of a 309-bp band was
considered a positive result. A positive PCR product was confirmed by
digestion with the restriction enzyme AatII, which produced 219- and 85-bp fragments.
Of the 181 CoNS tested, 132 (72.9%) were S. epidermidis.
Other species represented were S. hominis (23 strains),
S. warneri (9 strains), S. capitis (7 strains),
S. haemolyticus (6 strains), S. lugdenensis (3 strains), and S. simulans (1 strain). Eighty-two of the 181 strains were methicillin susceptible and 99 were methicillin resistant,
as determined by the presence of the mecA gene. The OSAS
method, the ODD test, and the MicroScan and Vitek systems accurately
identified these mecA-negative strains as methicillin susceptible at both 24 and 48 h.
At 24 h, the Vitek system assigned the highest number of
mecA-positive strains to the category of methicillin
resistance and the OSAS method assigned the lowest number of strains to
this category. After 48 h of incubation, the rates of detection of methicillin-resistant strains improved for all three methods for which
results were evaluated at 24 and 48 h. The combination of Vitek
cards plus the OSAS method was the most sensitive of all combinations
at 24 h of incubation. The sensitivities of the four possible
combinations were identical at 48 h. The numbers of isolates identified as methicillin resistant by each method individually and in
combination after 24 and 48 h of incubation are indicated in Table
1. All non-PCR methods tested failed to
classify six mecA-positive S. epidermidis strains
as methicillin resistant.
Several studies have demonstrated that PCR is a sensitive method for
the detection of methicillin resistance in S. aureus and
strains of CoNS (4, 10, 16). However, most laboratories are
not in a position to perform the test. Phenotypically, the detection of
methicillin resistance in staphylococci is complicated by the
heterogeneous expression of the resistance. Although all cells possess
the mecA gene, only 1 in 105 to 107
organisms expresses the resistance phenotypically (1). The OSAS method is a sensitive and cheap technique for the detection of
methicillin-resistant S. aureus and CoNS. Commercially
available systems are less reliable. In one study that used a
mecA gene probe to identify MRSA, the MicroScan and Vitek
systems failed to detect 17 and 36 of 254 MRSA strains, respectively
(14). The reliability of the OSAS method for the detection
of PCR-proven mecA-positive strains of CoNS has been
demonstrated. However, for some strains 48 h of incubation is
required (18).
None of the individual test methods or various combinations were able
to detect all methicillin-resistant strains. However, six strains of
S. epidermidis were consistently susceptible to oxacillin by
all methods at 24 h and remained so after 48 h of incubation.
We suspect that these strains were unable to express the
mecA gene and are therefore phenotypically methicillin
susceptible. Strains that are unable to express the mecA
gene have been described previously (14). If the six strains
in our study are excluded from our analysis, then the sensitivity of
all combination methods was 100% at 48 h. The combination of
Vitek and an OSAS plate was able to identify 92 of 93 phenotypically
methicillin-resistant strains at 24 h.
We conclude that commercially available susceptibility test methods
should be used in combination with an ODD method or an OSAS plate.
Isolates of CoNS resistant by any method at 24 h can be reported
as such. The accuracy of the combination of Vitek cards and the OSAS
method was 98.9% (if the six strains mentioned above are excluded from
analysis); therefore, oxacillin susceptibility results could be
released at 24 h if this combination were used.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Infection Control, London Health Sciences Centre, Box 5375, 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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Journal of Clinical Microbiology, January 1998, p. 273-274, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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