a Public Health and Clinical Microbiology Laboratory, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP; b Public Health Laboratory, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, UK
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Abstract |
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Introduction |
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The British Society for Antimicrobial Chemotherapy (BSAC) has provisionally recommended a method for susceptibility testing of CNS. To ensure accurate detection of methicillin resistance, it recommends achieving a semi-confluent growth and the incubation of isolates for 48 h.3 However, this results in delays to laboratory reports and patients may have commenced glycopeptide treatment before sensitivity to methicillin is confirmed. Susceptibility testing has been further complicated by the forthcoming withdrawal of methicillin and its replacement by oxacillin. These problems may encourage centres to use glycopeptides regardless of susceptibility results. We feel that such a practice should be avoided and that methods promoting more rapid determination of methicillin susceptibility should be pursued. We have evaluated the BSACrecommended methicillin disc method, oxacillin disc testing and Etest using a selection of clinically significant CNS and have compared the results with PCR detection of the mecA gene.
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Materials and methods |
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Forty-two clinically significant blood culture CNS isolates were analysed. The control strains Staphylococcus epidermidis NCTC 11047 (methicillin sensitive) and S. epidermidis NCTC 11964 (methicillin resistant) were examined in parallel.
Polymerase chain reaction (PCR)
DNA extraction was performed on isolates from blood agar. Two to five colonies were suspended in 50 µL lysostaphin (100 mg/L; SigmaAldrich Company Ltd, Dorset, UK) and incubated at 37°C for 10 min. Following the addition of 50 µL proteinase K (100 mg/L; Sigma Aldrich) and 150 µL 100 mM Tris pH 7.5, the suspension was incubated for 10 min at 37°C and then boiled for 5 min. After centrifugation at 13,000g for 2 min, the supernatant containing the extracted bacterial DNA was used in the PCR assay.
Multiplex PCR was performed for the detection of mecA and coagulase (coa) genes. A 214 bp fragment of the mecA gene and a 117 bp fragment of the coa gene were amplified using four oligonucleotide primers (Primer set NCL-SA-PS, Novocastra Laboratories, Newcastle upon Tyne, UK). The PCR was performed using a capillary air thermal cycler (BioGene Ltd, Kimbolton, Cambridgeshire, UK).4 The master mix consisted of 4 mM MgCl2/bovine serum albumin/Ficoll/Dye (BioGene), 200 µM dNTPs, 15 pmol of each primer and 0.25 U Taq polymerase. The final reaction mixture consisted of 1 µL DNA extract and 9 µL master mix. The cycling profile comprised an initial step at 94°C for 1 min, followed by 15 cycles of 94°C for 0 s, 72°C for 4 s, then 25 cycles of 94°C for 0 s, 60°C for 0 s, 72°C for 4 s and a final extension step of 72°C for 30 s. Appropriate positive, negative and extraction controls were included in each PCR run. Total assay time including DNA extraction was c. 1.5 h.
Susceptibility testing
Susceptibility testing was performed by the disc diffusion method on Columbia agar with and without 2% NaCl (Oxoid, Basingstoke, UK). A suspension of each isolate was made so that the turbidity was equal to a 0.5 McFarland standard; a 1/10 dilution was then prepared in sterile distilled water and agar plates were inoculated so as to obtain semi-confluent growth. A 5 µg methicillin disc and 1 µg oxacillin disc (MAST Diagnostics, Merseyside, UK) were applied to each plate and following incubation at 30°C, zone sizes were measured after 24 and 48 h. Zone sizes of 15 mm for methicillin and
16 mm for oxacillin were deemed to indicate susceptibility.5,6 Methicillin Etests (AB Biodisk, Solna, Sweden) were performed on Columbia agar without salt and MICs recorded after 24 and 48 h incubation at 30°C. A value > 4 mg/L was taken as resistant and
4 mg/L as sensitive.
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Results |
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Discussion |
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In general, the oxacillin disc susceptibility method, with and without 2% NaCl, compared well with methicillin disc testing on agar containing 2% NaCl. The Etest however, especially at 24 h, did not offer an advantage over disc testing and failed to detect resistance in two isolates at 48 h.
Currently available phenotypic methods for the detection of methicillin resistance are suboptimal. External quality assessment error rates of around 7% are encountered with easier strains of S. epidermidis, rising to 2250% with more difficult strains.8 On some occasions (e.g. with blood culture isolates), it could be 72 h from the time of detection of a positive culture until confirmation of susceptibility to methicillin. If the excessive use of glycopeptides is to be avoided, we must make use of molecular methods that facilitate rapid detection of strains likely to respond to ß-lactams. Detection of the mecA gene corresponds well with phenotypic resistance in Staphylococcus aureus and CNS.9 The mecA PCR method described here allows rapid detection of a resistant genotype. However, such technology is not available in all centres and cost remains an important issue. We believe that optimal and timely detection of resistance or susceptibility in CNS may require a combination of phenotypic and genotypic methods. Disc susceptibility testing is an acceptable initial method but if isolates appear susceptible at 24 h, then mecA detection could be performed on the same day and therapy modified accordingly.
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Notes |
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References |
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2 . York, M. K., Gibbs, L., Chehab, F. & Brooks, G. F. (1996). Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci. Journal of Clinical Microbiology 34, 24953.[Abstract]
3 . British Society for Antimicrobial Chemotherapy. (1998). BSAC Standardized Disc Sensitivity Testing MethodThe Newsletter of the British Society for Antimicrobial Chemotherapy.
4 . Kearns, A. M., Seiders, P. R., Wheeler, J., Freeman, R. & Steward, M. (1999). Rapid detection of methicillin resistant Staphylococci by multiplex PCR. Journal of Hospital Infection 43, 337.[ISI][Medline]
5 . Hedin, G. & Löfdahl, S. (1993). Detecting methicillin-resistant Staphylococcus epidermidisdisc diffusion, broth breakpoint or polymerase chain reaction? Acta Pathologica, Microbiologica et Immunologica Scandinavica 101, 3118.
6 . Hedin, G. & Hambraeus, A. (1991). Screening tests for the detection of methicillin resistance in Staphylococcus epidermidis. Journal of Antimicrobial Chemotherapy 28, 68194.[Abstract]
7 . Law, D., Megson, G. M., Keaney, M. G. L. & Ganguli, L. A. (1992). The influence of salt concentration on the detection of methicillin resistance in coagulase-negative staphylococci. Journal of Antimicrobial Chemotherapy 30, 60314.[Abstract]
8 . Snell, J. J. (1994). Problems in susceptibility testingsfindings of UK NEQAS for microbiology. Journal of Antimicrobial Chemotherapy 33, 14.[ISI][Medline]
9 . Wallet, F., Roussel-Delvallez, M. & Courcol, R. J. (1996). Choice of a routine method for detecting methicillin-resistance in staphylococci. Journal of Antimicrobial Chemotherapy 37, 9019.[Abstract]
Received 4 May 1999; returned 10 August 1999; revised 1 September 1999; accepted 20 September 1999