Journal of Antimicrobial Chemotherapy (2000) 45, 111-113
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Comparison of PCR detection of mecA with methicillin and oxacillin disc susceptibility testing in coagulase-negative staphylococci
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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The provisional BSAC method for the detection of methicillin sensitivity in coagulase-negative staphylococci (CNS) requires incubation of isolates for 48 h and raises the problem of timely reporting of susceptibility data. The forthcoming withdrawal of methicillin raises another difficulty. We evaluated 42 clinically significant CNS blood culture isolates by PCR, methicillin and oxacillin disc testing and by using methicillin Etests. Our results suggest that, although oxacillin disc susceptibility testing is a reasonable first line step, optimal and timely detection of resistance or susceptibility may require a combination of phenotypic and genotypic methods.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Coagulase-negative staphylococci (CNS) are a common cause of hospital-acquired infection, and are often isolated from neonates, the immunocompromised and patients with indwelling prosthetic devices. In many centres, when infections with CNS are suspected, patients are empirically commenced on glycopeptides. However, excessive use of these agents may select for resistant or tolerant organisms.1 Although the majority of clinically significant isolates are resistant to ß-lactams, these drugs offer the advantages of favourable pharmacokinetic properties and low associated costs and are thus recommended for the treatment of susceptible strains.2
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.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Microorganisms
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; Sigma–Aldrich 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.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The results are summarized in the Table.
The control strains were appropriately resistant or sensitive by all methods. The absence of coa gene was confirmed in all isolates. Of the 42 isolates studied, 33 (78.6%) were mecA positive and nine were mecA negative. One mecA-positive isolate was susceptible by all methods, presumably owing to non-expression of the mecA gene. One mecA-negative isolate had microcolonies detected at 24 h when 2% NaCl was used, and at 48 h by all methods including methicillin Etest. Despite the absence of mecA the isolate was considered to be phenotypically resistant. The remaining eight mecA-negative isolates were sensitive on disc testing to oxacillin and methicillin, both with and without 2% NaCl at 24 and 48 h. Of the remaining 32 mecA-positive isolates, all were resistant on disc testing at 24 h to oxacillin with and without 2% NaCl, and to methicillin disc testing on 2% NaCl. Methicillin disc testing without 2% NaCl failed to identify resistance in six of the 32 isolates at 24 h but the results were comparable to those of the other methods at 48 h.
|
Methicillin Etest failed to detect resistance in eight mecA-positive isolates at 24 h and three of these remained sensitive at 48 h. Performance of the test on Columbia agar (Mueller–Hinton agar is recommended) without 2% NaCl may have contributed to these results.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We have evaluated the BSAC method for the detection of methicillin resistance in 42 clinically significant CNS isolates. Although the addition of NaCl is not recommended for CNS, we found that it increased the rapidity of detection of resistance with methicillin discs, although no difference was observed with oxacillin discs. CNS are a heterogeneous group and the addition of salt to sensitivity test agar will affect members differently.7
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 22–50% 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.
|
Notes |
|---|
* Corresponding author. Tel: +44-191-232-5131; Fax: +44-191-201-0156; E-mail: J.C.Graham{at}ncl.ac.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 . Raad, I., Alrahwan, A. & Rolston, K. (1998). Staphylococcus epidermidis: emerging resistance and need for alternative agents. Clinical Infectious Diseases 26, 1182–7.[Web of Science][Medline]
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, 249–53.[Abstract]
3 . British Society for Antimicrobial Chemotherapy. (1998). BSAC Standardized Disc Sensitivity Testing Method—The 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, 33–7.[Web of Science][Medline]
5 . Hedin, G. & Löfdahl, S. (1993). Detecting methicillin-resistant Staphylococcus epidermidis—disc diffusion, broth breakpoint or polymerase chain reaction? Acta Pathologica, Microbiologica et Immunologica Scandinavica 101, 311–8.
6
.
Hedin, G. & Hambraeus, A. (1991). Screening tests for the detection of methicillin resistance in Staphylococcus epidermidis. Journal of Antimicrobial Chemotherapy 28, 681–94.
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, 603–14.
8
.
Snell, J. J. (1994). Problems in susceptibility testings—findings of UK NEQAS for microbiology. Journal of Antimicrobial Chemotherapy 33, 1–4.
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, 901–9.
Received 4 May 1999; returned 10 August 1999; revised 1 September 1999; accepted 20 September 1999
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Perazzi, M. R. Fermepin, A. Malimovka, S. D. Garcia, M. Orgambide, C. A. Vay, R. de Torres, and A. M. R. Famiglietti Accuracy of cefoxitin disk testing for characterization of oxacillin resistance mediated by penicillin-binding protein 2a in coagulase-negative staphylococci. J. Clin. Microbiol., October 1, 2006; 44(10): 3634 - 3639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. B. R. Ferreira, N. L. P. Iorio, K. L. Malvar, A. P. F. Nunes, L. S. Fonseca, C. C. R. Bastos, and K. R. N. Santos Coagulase-Negative Staphylococci: Comparison of Phenotypic and Genotypic Oxacillin Susceptibility Tests and Evaluation of the Agar Screening Test by Using Different Concentrations of Oxacillin J. Clin. Microbiol., August 1, 2003; 41(8): 3609 - 3614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N Cimolai and J E Carter Clinical validation for oxacillin susceptibility testing of coagulase negative staphylococci Arch. Dis. Child., June 1, 2002; 86(6): 446 - 447. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Graham, J. A. Phillips, O. M. Murphy, A. M. Kearns, F. K. Gould, and R. Freeman Use of Mastalex to detect methicillin resistance in coagulase-negative staphylococci J. Antimicrob. Chemother., November 1, 2000; 46(5): 850 - 850. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||