1 National Center for Antimicrobials and Infection Control, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S; 2 Department of Clinical Microbiology, Hvidovre Hospital, Copenhagen, Denmark; 3 The Swedish External Reference Laboratory for Susceptibility Testing, Växjö; 4 The Swedish Institute for Infectious Disease Control, Solna, Sweden
Received 25 March 2003; returned 2 April 2003; revised 9 May 2003; accepted 9 May 2003
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Abstract |
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Methods: A total of 457 S. aureus, including 190 MRSA of several defined PFGE types and a number of low-level resistant isolates, were tested with a cefoxitin 30 µg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 3536°C. This method was compared with the standard SRGA (Swedish Reference Group for Antibiotics) method (oxacillin 1 µg disc on Iso-Sensitest agar supplemented with 5% defibrinated horse blood, confluent growth and 24 h incubation in ambient air at 30°C).
Results: The cefoxitin method was excellent, with a sensitivity of 100% and a specificity of 99% using an interpretative zone diameter of S 29 mm and R < 29 mm. Its performance was much better than the SRGA method, which with this collection of difficult strains had a sensitivity of only 78% using the current breakpoint of S
12 mm.
Conclusion: We suggest that the cefoxitin method should replace that currently recommended by the SRGA for the detection of MRSA, and that it would fit well into BSAC methodology.
Keywords: susceptibility testing, disc diffusion, MRSA, Iso-Sensitest Agar
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Introduction |
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Recently, two reports demonstrated the excellent performance of a cefoxitin 30 µg disc on MuellerHinton agar with standard inocula of 106 (semi-confluent) and 107 cfu/mL.4,9 We compared the method for detection of methicillin resistance recommended by the Swedish Reference Group for Antibiotics (SRGA), with a cefoxitin 30 µg disc on Iso-Sensitest agar, using a semi-confluent (106 cfu/mL) inoculum and overnight incubation at 3536°C, which are the SRGA standard conditions for susceptibility testing of staphylococci against other antibiotics.7 If accurate, this would eliminate the present need in the SRGA method for a separate test for MRSA. Furthermore, it would easily fit the BSAC recommendations for susceptibility testing.
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Material and methods |
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A total of 457 S. aureus, all tested for the presence of the mecA gene by the EVIGENE MRSA detection kit using the manufacturers instructions,10 were investigated. The isolates comprised 267 mecA-negative and 190 mecA-positive Danish and Swedish S. aureus isolates (see Table 1). One hundred and eight of the isolates were consecutive, routine blood culture isolates from the Department of Clinical Microbiology, Hvidovre University Hospital, Copenhagen, and the rest represented modern MRSA collections from the Danish National Staphylococcal Reference Laboratory at Statens Serum Institut, Copenhagen, Denmark and the Swedish Institute for Infectious Disease Control, Solna, Sweden.
The Danish collection represented MRSA sent to the laboratory in 2001 (repeat samples excluded, n = 105), including low-level resistant variants, i.e. strains with oxacillin MIC 2 mg/L (Etest on MuellerHinton agar supplemented with 2% NaCl), and 79 epidemiologically unrelated methicillin-susceptible S. aureus (MSSA). The Swedish collection contained 82 MRSA, including more than 22 different PFGE types (e.g. Berlin IV, Finland E7, France A and B, S German II, UK E1, E15 and E16, Spain E1),11 and 83 epidemiologically unrelated MSSA.
Susceptibility testing
All isolates were tested with a cefoxitin 30 µg disc (Oxoid, Basingstoke, UK), on Iso-Sensitest agar (ISA, Oxoid), using semi-confluent growth and overnight incubation in ambient air at 3536°C, and with an oxacillin 1 µg disc (Oxoid) on ISA supplemented with 5% defibrinated horse blood, confluent growth and 24 h incubation in ambient air at 30°C (the method recommended by the SRGA).7 Using callipers, inhibition zone diameters were measured to the nearest millimetre at the inner zone edge. For cefoxitin, a sensitivity of 100% was the criterion for determining the interpretive zone diameter of resistance. For oxacillin, the SRGA recommendation was followed.7
S. aureus ATCC 29213 was included on each occasion for quality control, and S. aureus ATCC 25923 was included in some of the test runs (Table 2). All values for the oxacillin 1 µg disc for S. aureus ATCC 29213 were within the published range.7
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Results |
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We further investigated whether the addition of 5% defibrinated horse blood to the ISA medium or using a confluent inoculum (McFarland 0.5) affected the cefoxitin inhibition zone ranges for MRSA (n = 106) or MSSA (n = 182) isolates. Blood supplementation did not alter the inhibition zone ranges for MRSA or MSSA isolates. For the variant using ISA with blood and confluent growth, an interpretive zone diameter of R < 25 mm and S 26 mm resulted in a sensitivity of 100% and a specificity of 98% (data not shown).
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Discussion |
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The present study showed how well the cefoxitin 30 µg disc performed on ISA using standard incubation conditions, with a sensitivity of 100%, a specificity of 99% and an interpretive zone diameter of S 29 and R < 29 mm, thereby confirming the results obtained by Felten et al. on MuellerHinton agar.4 The small difference in the zone diameter ranges in the two studies could be related both to the difference in agar type and to the inclusion of many difficult (low-level resistant) isolates in this study. The latter is underlined by the fact that 22% of the MRSA went undetected by the screening method currently recommended by the SRGA. A further advantage of the cefoxitin screening test is that it eliminates the need for separate inoculum, media, and incubation time and temperature. The cost and workload is thereby reduced.
Since our results were obtained on the medium and with the inoculum recommended by the BSAC Working Party on Antimicrobial Sensitivity Testing for S. aureus against other antibiotics, this method would fit the BSAC recommendations.2
The reliability of our findings and conclusions is strengthened both by the large number of MRSA PFGE types included in the study and by the use of very heterogeneous isolates. The results of testing consecutive blood culture isolates indicate that routine MSSA will rarely exhibit zones smaller than the proposed interpretive zone diameter of R < 29 mm. This is especially important in countries with a very low prevalence of MRSA, as is the case in Scandinavia, where it has been <1% for many years (national mandatory reporting systems).
One disadvantage of the cefoxitin method is that the zones reached are large, even for resistant isolates, and this could interfere with the inhibition zones of adjacent discs, at least when using the standard 9 cm agar plate. Furthermore, the gap between the inhibition zones of isolates with and without the mecA gene is very narrow (Figure 1). We are therefore in the process of investigating discs with a lower cefoxitin content. This should establish whether these problems can be solved by reducing the zone sizes and by introducing a larger difference in zone diameter between MRSA and methicillin-susceptible isolates. These studies will also reveal whether the cefoxitin disc test can be used for coagulase-negative staphylococci.
In conclusion, a cefoxitin 30 µg disc on ISA, using a semi-confluent inoculum and overnight incubation at 3536°C, performed significantly better than screening methods used hitherto for the detection of MRSA. In our hands, in two different laboratories, interpretive zone diameter breakpoints of S 29 mm and R < 29 mm resulted in a sensitivity of 100% and a specificity of 99%, despite the fact that difficult S. aureus strains were used in the study. We suggest that this method replaces that currently recommended by the SRGA for detection of methicillin resistance in S. aureus
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Footnotes |
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References |
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2
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8 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility TestsSeventh Edition: Approved Standard M2-A7. NCCLS, Villanova, PA, USA.
9 . Mougeot, C., Guillaumat-Tailliet, J. & Libert, J. M. (2001). Staphylococcus aureus: nouvelle détection de la résistance intrinsèque par la méthode de diffusion. Pathologie Biologie 49, 199204.[CrossRef][ISI][Medline]
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