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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Introduction |
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Materials and methods |
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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 MuellerHinton 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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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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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, 915% were classified as oxacillin resistant with the Etest, compared with 711% for the multipoint test and 940% for the disc test, depending on culture conditions and definition of breakpoint.
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Discussion |
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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 MuellerHinton 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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Notes |
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References |
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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, 6138. [ISI][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, 20514. [ISI][Medline]
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Hussain, Z., Stoakes, L., Massey, V., Diagre, D., Fitzgerald, V., El Sayed, S. et al. (2000). Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci. Journal of Clinical Microbiology 38, 7524.
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De Giusti, M., Pacifico, L., Tufi, D., Panero, A., Boccia, A. & Chiesa, C. (1999). Phenotypic detection of nosocomial mecA- positive coagulase-negative staphylococci from neonates. Journal of Antimicrobial Chemotherapy 44, 3518.
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Kohner, P., Uhl, J., Kolbert, C., Persing, D. & Cockerill, F., III (1999). Comparison of susceptibility testing methods with mecA gene analysis for determining oxacillin (methicillin) resistance in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus spp. Journal of Clinical Microbiology 37, 295261.
8 . Chambers, H. F. (1997). Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clinical Microbiology Reviews 10, 78191. [Abstract]
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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, 2318.
Received 28 June 2001; returned 21 August 2001; revised 8 October 2001; accepted 12 October 2001