1 National Center for Antimicrobials and Infection Control, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark; 2 The Swedish External Reference Laboratory for Susceptibility Testing, Växjö, Sweden
Received 10 June 2004; returned 30 July 2004; revised 22 October 2004; accepted 26 October 2004
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Abstract |
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Methods: Six hundred and forty-one Staphylococcus aureus (261 mecA-negative and 380 mecA-positive) and 344 coagulase-negative staphylococci (CoNS) (132 mecA-negative and 212 mecA-positive) were investigated. The CoNS represented nine species, Staphylococcus epidermidis being the most frequent (n=231). All isolates were tested using semi-confluent growth on Iso-Sensitest agar (ISA), and MuellerHinton agar (MH) using a 5 and a 10 µg cefoxitin disc and overnight incubation in ambient air at 3537°C.
Results: For S. aureus, both cefoxitin discs performed with high accuracy on both media. The sensitivity and specificity for the following proposed interpretive zone diameters were: ISA 5 µg, R < 14 mm (99.5% and 98.1%); ISA 10 µg, R < 22 mm (99.5% and 98.1%); MH 5 µg, R < 12 mm (99.7% and 98.1%); and MH 10 µg, R < 18 mm (99.5% and 98.9%), respectively. All four variants were superior to oxacillin using the former SRGA methodology. In CoNS, a substantial overlap was seen for all variants. However, by avoiding primary interpretation in the overlapping interval, highly accurate results could be obtained for 81%, 80%, 91% and 97% of the isolates, respectively.
Conclusion: For S. aureus, cefoxitin 5 and 10 µg discs performed with high accuracy on both ISA and MH using semi-confluent growth and standard incubation conditions. With the introduction of a defined interval in which primary interpretation should be avoided, the method could also be used for CoNS.
Keywords: MRSA , coagulase-negative staphylococci , susceptibility testing
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Introduction |
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Several breakpoint committees, such as BSAC (UK), NCCLS (USA), SRGA (Sweden) and the Danish reference group on antibiotic resistance (Denmark) have either already decided to recommend the use of cefoxitin for disc diffusion as superior to the hitherto used oxacillin disc screen methods or have ongoing projects in which the cefoxitin disc method is compared with currently recommended methods. Our investigation was undertaken to investigate the performance of lower strength cefoxitin discs hoping that these could be placed on the same plate as other antibiotic discs since our previous investigation with cefoxitin showed that inhibition zones around 30 µg discs were too large. We also investigated whether cefoxitin could reliably be used to detect methicillin resistance in CoNS.
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Materials and methods |
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A total of 641 S. aureus and 344 CoNS were investigated, all tested for the presence of the mecA gene by the EVIGENETM MRSA Detection Kit (where MRSA stands for methicillin-resistant S. aureus) using the manufacturer's instructions.6,7 The isolates were obtained in the following way (identical strains from the same patient were excluded):
S. aureus
Three sets of Danish clinical strains, either referred consecutively to the Staphylococcus National Reference Laboratory, Statens Serum Institut, Copenhagen, Denmark in 2001 (n=182) or in 2003 (n=199), or isolated from consecutive blood-cultures submitted in 2002 to the Department of Clinical Microbiology, Hvidovre University Hospital, Copenhagen, Denmark (n=104). One set of Swedish isolates consisting of a collection of epidemiologically unrelated recent MRSA obtained from the Swedish Institute for Infectious Disease Control, Solna, Sweden and clinical MSSA isolates from the Department of Clinical microbiology, Central Hospital, Växjö, Sweden (n=151). Furthermore, five MRSA which had previously been shown to be very difficult to detect by phenotypic methods were kindly supplied by P. Allouch, France (n=2); A. van Grietheysen, The Netherlands (n=1); and A. Sundsfjord, Norway (n=2). The collection included low-level resistant variants, i.e. isolates with oxacillin MIC2 mg/L [MIC testing carried out with Etest (AB Biodisk, Solna, Sweden) on MuellerHinton agar supplemented with 2% NaCl, data not shown].
CoNS
Altogether 344 invasive CoNS isolates from Denmark (n=184) and Sweden (n=160) were included. The isolates were speciated using RapidStaph32 (bioMérieux, Marcy-l'Étoile, France): Staphylococcus epidermidis (172 mecA-positive and 59 mecA-negative), Staphylococcus hominis (13 and 23), Staphylococcus haemolyticus (10 and 11), Staphylococcus warneri (9 and 12), Staphylococcus capitis (6 and 10), Staphylococcus lugdunensis (2 and 14) and other CoNS (0 mecA-positive and 3 mecA-negative).
PFGE
All MRSA isolates were typed by PFGE. Macrorestriction profiles were carried out according to the HARMONY protocol.8 Briefly, DNA was SmaI-digested and fragments were separated on a 1% agarose gel in a CHEF II or a CHEF III PFGE apparatus (Bio-Rad, Hercules, CA, USA). Lambda-octamer size markers (Bio-Rad) were included in every sixth lane for optimal normalization and NCTC 8325 was used as a reference strain.
The gels were analysed using Bionumerics version 3.0 (Applied Maths, Kortrijk, Belgium) and relatedness was determined by visual inspection of UPGMA dendrograms based on Dice coefficients (0.5% optimization and 2.0% position tolerance). Clusters were defined as pulse-types sharing more than 80% homology.9
Pulse-types were defined as non-identical isolates (1 visible band difference).
Susceptibility testing
All isolates were tested with a 5 and 10 µg cefoxitin disc (Oxoid, Basingstoke, UK), on Iso-Sensitest agar (ISA; Oxoid) and MuellerHinton BBL II agar (MH; Becton Dickinson, Cockeysville, MD, USA), using an inoculum yielding semi-confluent growth. Plates were incubated overnight (1620 h) in ambient air at 3537°C. Using callipers, inhibition zone diameters were measured to the nearest millimetre at the inner zone edge. All isolates were also tested with a 1 µg oxacillin disc (Oxoid) on ISA supplemented with 5% defibrinated horse blood, confluent growth and 24 h incubation in ambient air at 30°C using zone diameter breakpoints of S 12, R
11 mm, (i.e. the SRGA recommended method as described in the original reference from 1997).10
Two isolates obtained from Norway (numbered 9-8 and 10-22) were also tested using a 30 µg cefoxitin disc on both media using inocula and incubation conditions as described for the other cefoxitin discs.
S. aureus ATCC 29213 was included on each occasion for quality control.
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Results |
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The zone diameters for ATCC 29213 for each of the four methodological variants were (mean ± 2 S.D.): ISA cefoxitin 5 µg, 20 ± 3 mm; ISA 10 µg, 25 ± 3 mm; MH 5 µg, 15 ± 2 mm; MH 10 µg, 22 ± 2 mm. All values for the oxacillin 1 µg disc for S. aureus ATCC 29213 were within the published range.10
S. aureus
The results for each of the methodological variants (cefoxitin 5 and 10 µg on ISA and MH, respectively) are shown in Figure 1(ad) and Table 1. None of the methodological variants was able to completely separate mecA-positive from mecA-negative isolates.
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CoNS
None of the five methods could separate the mecA-negative from the mecA-positive isolates and a substantial overlap (representing as many as 20% of the strains) was seen with all methods (Table 1). However, if the overlap was used as an interval inside which interpretation was not permitted, as many as 81%, 80%, 91% and 97% of the isolates could be classified with high accuracy as either susceptible or resistant for the respective ISA cefoxitin 5 and 10 µg and MH cefoxitin 5 and 10 µg methodological variants (Table 3). The results and accuracy of the cefoxitin methods were comparable to the results obtained with the SRGA methodology using an interpretive zone diameter of S 21 mm and R < 16 mm.
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Discussion |
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As the use of a dense inoculum has hitherto been a prerequisite for detection of methicillin resistance using phenotypic assays, it is important to emphasize that the MRSA isolates used for this study were highly diverse representing as many as 31 different clusters with 107 pulse-types,9 including many of the internationally recognized clones.
For S. aureus, all four methodological variants of this study were comparable and as well or better than the cefoxitin 30 µg disc when strains used in both studies were compared.5
Based on calculations of sensitivity and specificity, we suggest the following interpretive zone diameters for cefoxitin 5 and 10 µg discs on ISA and MH with semi-confluent growth and 1620 h incubation in ambient air: ISA 5 µg, R < 14 mm; ISA 10 µg, R < 22 mm; MH 5 µg, R < 12 mm; and MH 10 µg, R < 18 mm (Table 2). These are 1 to 4 mm higher than we suggested in a preliminary report.11
Neither the 5, 10 or 30 µg discs nor the former recommended SRGA method using a 1 µg oxacillin disc on ISA could detect the two Norwegian MRSA isolates (9-8 and 10-22). Both exhibited low oxacillin and cefoxitin MIC values but were reported as being detected by a screening method incorporating 4 mg/L oxacillin in MH agar supplemented with 2% NaCl (A. Sundsfjord, personal communication).
Felten et al. have also reported one mecA-positive isolate which could not be detected by the cefoxitin disc method.12 However, the fact that disc diffusion using cefoxitin produces results that are highly accurate under routine susceptibility testing conditions, i.e. with standard media, a standard inoculum yielding semi-confluent growth and a standard incubation time and temperature of 1620 h and 3537°C, respectively, makes this method a very attractive phenotypic method for detection of methicillin resistance in S. aureus in routine laboratories.
In CoNS, the substantial overlaps between mecA-positive and mecA-negative isolates in all methodological variants were disappointing. However, by introducing a zone diameter interval within which a secondary test (PCR or PBP2-agglutination) would be carried out before interpretation of the mecA status of the strain, any of the methodological variants could be reliably used as an initial test for determination of methicillin susceptibility in these isolates. The alternative is to use the higher breakpoint and thus accept false resistance of 19%.
In conclusion, our data support the use of a cefoxitin disc on standard media, e.g. on ISA and MH under standard antimicrobial susceptibility testing conditions, as a reliable and robust method for detecting methicillin resistance in S. aureus. The 5 and 10 µg cefoxitin discs perform as well as the 30 µg cefoxitin disc, and offer the advantage of yielding smaller inhibition zones than the latter. Although CoNS are more problematic than S. aureus, a feasible strategy for using cefoxitin to detect methicillin resistance in the former was devised.
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References |
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