Journal of Antimicrobial Chemotherapy (2002) 49, 197-200
© 2002 The British Society for Antimicrobial Chemotherapy
Brief report |
Prediction of mecA-positive coagulase-negative staphylococci: assessment of different phenotypic methods, breakpoints, culture media and culture conditions
a Department of Clinical Bacteriology, University Hospital of Umeå; b Department of Ecology and Environmental Science, University of Umeå; c Department of Infectious Diseases, University Hospital of Umeå, 90185 Umeå, Sweden
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Abstract |
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Sensitivity for the detection of mecA-positive coagulase-negative staphylococci (CNS) was evaluated for different breakpoints of resistance for oxacillin using three different susceptibility tests, either on Mueller–Hinton agar supplemented with 2% NaCl (MH-NaCl agar) or on paper disc method agar supplemented with 5% defibrinated blood (PDM-blood agar). The Etest, multipoint inoculation test and disc diffusion test showed comparable sensitivity (0.96, 0.96 and 0.95, respectively) using an oxacillin breakpoint of
0.5 mg/L or 
17 mm for the disc test, after incubation at 35°C for 24 h on MH-NaCl agar. The sensitivity decreased for breakpoints 1 mg/L and when PDM-blood agar was used instead of MH-NaCl agar. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Test conditions and choice of breakpoint for oxacillin resistance have a major effect on the detection of methicillin resistance in mecA-positive coagulase-negative staphylococci (CNS).1 In an attempt to improve the screening for oxacillin-resistant isolates of CNS, the NCCLS has recommended a reduction of the oxacillin breakpoint from
4 to 0.5 mg/L.2 The aim of the present study was to examine the usefulness of this breakpoint and the influence of two different culture media [Mueller–Hinton agar supplemented with 2% NaCl (MH-NaCl agar) or paper disc method agar supplemented with 5% defibrinated blood (PDM-blood agar)] for predicting the presence of mecA among unselected clinical isolates of CNS. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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A total of 551 consecutive clinical isolates of staphylococci were collected during a 1 month period at our laboratory. The isolates were classified as Staphylococcus aureus or CNS by standard methods and presence of the nuc gene.3 The PCR amplification for detection of mecA and nuc genes was performed as described previously.3
Susceptibly testing was performed on paper disc medium – antibiotic sensitivity medium (PDM-ASM agar; AB Biodisk, Solna, Sweden) supplemented with 5% defibrinated horse blood (PDM-blood agar), or Mueller–Hinton II agar (Becton Dickinson, Cockeysville, MD, USA) supplemented with 2% NaCl (MH-NaCl agar). PDM-blood agar is one of the media recommended by the Swedish Reference Group for Antibiotics (SRGA; http://www.srga.org). CNS isolates were tested for oxacillin susceptibility by Etest (AB Biodisk), disc diffusion test and multipoint inoculation technique.2,3 Bacterial isolates were suspended, as recommended by SRGA, in 0.1 M phosphate-buffered saline to a final concentration of 0.5 McFarland standard (A520 = 0.19 ± 0.1), corresponding to c. 1 x 108 cfu/mL. The agar plates were incubated in ambient air at 30 or 35°C for 24 and 48 h. S. aureus (ATCC 29213) was used as a control.
Confidence limits were used for testing the effects of different methods and culture conditions. Sensitivity and specificity were calculated according to standard procedures.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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A total of 551 clinical isolates of staphylococci were examined, of which 316 (57%) were identified as CNS and 235 (43%) as S. aureus. Of the CNS isolates, 140 (44%) were mecA positive, compared with one (0.4%) of the S. aureus isolates.
A sensitivity of 0.96 for detection of mecA-positive isolates of CNS was found for both the Etest and the multipoint test, using a breakpoint of 0.5 mg/L, and when applied on MH-NaCl agar after incubation at 35°C for 24 h (Table
, Figure). Under these conditions the specificity for the Etest and the multipoint test was 0.88 and 0.90, respectively. For both methods the sensitivity decreased for oxacillin breakpoints of 1 mg/L and when PDM-blood agar was used instead of MH-NaCl agar (Figure).
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, mecA positive, 
, mecA negative (<12 mm); 
, mecA positive, 
, mecA negative (
, mecA positive, 
, mecA negative (
, mecA negative (For disc testing we found a sensitivity of 0.95 and specificity of 0.85 using a breakpoint of
17 mm, after incubation for 24 h at 35°C on MH-NaCl agar (Table
). Use of a breakpoint of <12 mm, and of PDM-blood agar instead of MH-NaCl agar, was associated with decreased sensitivity (Table).
After prolonging the incubation period from 24 to 48 h, a marginal increase in sensitivity was found for testing on MH-NaCl agar, whereas sensitivity generally increased for PDM-blood agar (Table, Figure). Incubation at 30°C instead of 35°C did not significantly increase the sensitivity of testing on PDM-blood agar, whereas sensitivity decreased for the multipoint test on MH-NaCl agar (Figure).
Among the 176 mecA-negative isolates of CNS, 9–15% were classified as oxacillin resistant with the Etest, compared with 7–11% for the multipoint test and 9–40% for the disc test, depending on culture conditions and definition of breakpoint.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Our results indicate that a breakpoint for oxacillin resistance of
0.5 mg/L is better suited to predicting mecA- positive isolates of CNS in routine laboratory practice than 1 mg/L. The appropriate breakpoint for phenotypic detection of methicillin resistance in CNS is a matter of debate and different breakpoints are currently recommended by various national societies1,2 (see http://www.srga.org). The discrepancies between recommendations, which hampers comparison of results obtained in different countries, are probably related to the use of different methods for susceptibility testing and possibly also differences between CNS species.1 There are, however, presently several reports, including the present study, supporting the reduction in the oxacillin breakpoint to 0.5 mg/L for testing on Mueller–Hinton agar.4–7
The Etest, disc diffusion test and multipoint inoculation showed comparable sensitivity and specificity when performed on MH-NaCl agar. Applying a breakpoint of 0.5 mg/L for the Etest and the multipoint inoculation test we obtained an acceptable sensitivity (0.96) and specificity for both tests after incubation at 35°C for 24 h on MH-NaCl agar. Nearly identical results were obtained with the disc test using a breakpoint of 17 mm. The Etest seems to offer limited advantage over disc testing or the multipoint test for identification of mecA-positive CNS in routine practice when performed on MH-NaCl agar.
Culture media and test conditions, such as incubation temperature, have an important effect on the expression of oxacillin resistance.1,8 Our results indicate that the MH-NaCl agar is superior to PDM-blood agar for detection of mecA-positive CNS. The outcome for MH-NaCl agar was less dependent on breakpoints, culture conditions and a time of incubation. Despite being a well-defined growth medium, which is favourable when attempting to attain standardized testing procedures, PDM-blood agar appears sub-optimal for cultivation of CNS. Regarding choice of incubation temperature, we found, as in previous studies,7 that an incubation temperature of 35°C is adequate for oxacillin susceptibility testing of CNS in most instances.
Approximately 10% of our clinical isolates of mecA-negative CNS currently express phenotypic oxacillin resistance mediated by mechanisms other than the mecA gene. The presence of such strains is another inherent limitation of oxacillin susceptibility testing.6 These isolates, which must be considered as potentially oxacillin resistant,9 were identified irrespective of whether an oxacillin breakpoint of 0.5 or 2 mg/L was used. Detection of the mecA gene may be required for correct diagnosis in certain instances.
Addition of NaCl to culture media is generally recommended for susceptibility testing of staphylococci, except for disc testing, as it may reduce specificity, especially for S. aureus.1,8 Whether or not the addition of NaCl effects disc testing in CNS seems less certain.1,7 Comparative studies are required to better determine the effects of adding NaCl to Mueller–Hinton agar for oxacillin disc testing of CNS, especially when reducing the breakpoint of resistance to 17 mm.
In conclusion, the results of the present study support the present NCCLS recommendations for choice of oxacillin breakpoint, 0.5 mg/L, and a growth inhibition zone of 17 mm for predicting mecA-positive CNS. The Etest, disc diffusion test and multipoint inoculation showed comparable sensitivity and specificity when performed on MH-NaCl agar. Incubation at 35°C for 24 h may be recommended and MH-NaCl agar seems preferable to PDM-blood agar.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We would like to thank Stig Granström and Marianne Rönnmark for technical support. This work was supported by grants from the Förenade Liv, Mutual Group Life Insurance Company, Stockholm, Sweden and the Scandinavian Society for Antimicrobial Chemotherapy.
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Notes |
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* Corresponding author. Tel: +46-90-7851226; Fax: +46-90-7853591; E-mail: tor.monsen{at}climi.umu.se
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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1 . Brown, D. F. (2001). Detection of methicillin/oxacillin resistance in staphylococci. Journal of Antimicrobial Chemotherapy 48, Suppl. 1, 65–70. [Abstract]
2 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing: Eleventh Informational Supplement M100-S11. NCCLS, Wayne, PA.
3 . Monsen, T., Olofsson, C., Ronnmark, M. & Wistrom, J. (2000). Clonal spread of staphylococci among patients with peritonitis associated with continuous ambulatory peritoneal dialysis. Kidney International 57, 613–8. [Medline]
4 . Marshall, S. A., Wilke, W. W., Pfaller, M. A. & Jones, R. N. (1998). Staphylococcus aureus and coagulase-negative staphylococci from bloodstream infections: frequency of occurrence, antimicrobial susceptibility, and molecular (mecA) characterization of oxacillin resistance in the SCOPE program. Diagnostic Microbiology and Infectious Disease 30, 205–14. [Web of Science][Medline]
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Martineau, F., Picard, F. J., Lansac, N., Menard, C., Roy, P. H., Ouellette, M. et al. (2000). Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy 44, 231–8.
Received 28 June 2001; returned 21 August 2001; revised 8 October 2001; accepted 12 October 2001
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