1 Department of Microbiology, Pacific Laboratory Medicine Services, Royal North Shore Hospital, St Leonards 2065; 2 Department of Microbiology and Infectious Diseases, The Canberra Hospital, Canberra, ACT 2606, Australia
Received 15 November 2004; returned 15 December 2004; revised 6 January 2005; accepted 7 January 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: Eight hundred and seventy-one strains of Staphylococcus aureus, collected from eight tertiary referral centres serving diverse socio-economic populations, were included in the study using NCCLS disc diffusion and the agar dilution methods.
Results: Using cefoxitin and NCCLS criteria for disc diffusion, the sensitivity and specificity for recognizing methicillin resistance were both 100%. Similar results were obtained when the strains were tested by the agar dilution method. The cefoxitin MICs for methicillin-susceptible strains were 4 mg/L.
Conclusions: Testing with cefoxitin as a surrogate marker for the detection of methicillin resistance was very accurate with both disc diffusion and agar dilution methods. Such testing clearly distinguished methicillin-resistant strains of S. aureus from methicillin-susceptible strains.
Keywords: community-onset MRSA infections , hospital-acquired infections , non-multiresistant Staphylococcus aureus , oxacillin resistance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Until recently, most strains of methicillin-resistant Staphylococcus aureus (MRSA) were isolated from hospitalized patients and were multidrug resistant. This feature was helpful in their recognition in the clinical laboratory. However, MRSA have now appeared in the community worldwide.917 These strains are not multidrug resistant (nmrMRSA). In addition, our experience (unpublished data) and other reports18 have noted strains of S. aureus that are not methicillin resistant, but which are still multiresistant to non-ß-lactam antibiotics. The mecA gene (which encodes methicillin resistance) does not appear to be present in these strains.
The gold standard for identifying MRSA is to detect the mecA gene,4 or its product, PBP2a, by latex agglutination.19,20 However, these tests are not within the scope of many clinical laboratories and are relatively expensive. Cefoxitin and moxalactam have been reported as surrogate markers for the detection of methicillin resistance.21,22 The NCCLS has recently reported cefoxitin zone diameter interpretive criteria for the prediction of mecA-mediated resistance.23 The objectives of this study were: (i) to compare the MIC distributions of cefoxitin and oxacillin in S. aureus strains with different resistance antibiograms; (ii) to establish a suitable concentration of cefoxitin that could be used in agar media which would distinguish methicillin-resistant strains from methicillin-susceptible strains; (iii) to evaluate the usefulness of the cefoxitin disc test in identifying MRSA in our local strains; and (iv) to determine the usefulness of cefoxitin in correctly identifying a collection of methicillin-susceptible strains of S. aureus that were multiresistant to non-ß-lactam antibiotics (RSA).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial strains were collected at eight major tertiary care centres. Each centre collected 100 consecutive isolates from individual patients. Cultures were identified using a set of conventional tests specified by AGAR24 and maintained at 70°C. Additional strains of nmrMRSA and also multiresistant but methicillin-susceptible strains of S. aureus (RSA), obtained from a collection maintained at one laboratory (RNSH), were also included in the study.
Susceptibility tests
General. Susceptibility tests were performed by the agar dilution method (NCCLS)25 or by the disc diffusion method (NCCLS).26 In these studies, MuellerHinton II Agar (BBL 211438) was used, and NCCLS methodology for testing and interpretation was followed.23,25,26 Methicillin-susceptible S. aureus (MSSA) and MRSA strains were categorized by phenotypic criteria: their susceptibility to oxacillin at 2 mg/L and, additionally, susceptibility to other anti-staphylococcal antibiotics.
Resistance surveillance. Testing was performed by the individual laboratories. All cultures were tested against 16 antibacterials by agar dilution at concentrations recommended by the NCCLS25 to define susceptibility.
Cefoxitin testing. Cefoxitin MIC by agar dilution at 35°C using a range of 0.25256 mg/L was also performed by each laboratory. In addition, five laboratories performed disc testing using a 30 µg cefoxitin disc on MuellerHinton agar (NCCLS).23,26 All media were prepared at one laboratory.
Further tests. Selected cultures, including all nmrMRSA strains, were forwarded to one laboratory (RNSH) for further testing. MICs of oxacillin and cefoxitin were determined using MuellerHinton II Agar (BBL 211438) with and without added 2% NaCl. Cefoxitin MICs were also determined using Oxoid MuellerHinton Agar (CM337) and in Oxoid Columbia Agar (CM 331). Oxacillin and methicillin MICs were determined only for nmrMRSA and RSA strains, and not for MSSA and MRSA strains.
Detection of mecA by PCR
The multiplex PCR procedure for the detection of mecA and nuc genes was carried out according to Brakstad et al.27,28 DNA was extracted by suspending portions of four to five colonies in 50 µL of lysostaphin (100 mg/L). After incubation for 10 min at 37°C, 50 µL of proteinase K (100 mg/L) and 150 µL of TE buffer (1 mM EDTA/10 mM Tris; pH 7.5) were added and incubated for a further 10 min at 37°C. The specimens were then incubated at 95°C for 10 min and centrifuged at 12 000 rpm for 3 min; 5 µL of the supernatant was used in each PCR mixture.
The PCR mixture of 25 µL consisted of 0.2 U of Amplitaq Gold, 2.5 µL of 10x PCR buffer, 2.5 µL of 25 mM MgCl2, 0.35 µL of 20 mM primers for mecA and 0.25 µL of 20 µM primers for nuc. The PCR was carried out in an OmniGene thermocycler using the following program: 10 min at 94°C, 35 cycles of 1 min at 94.5°C, 30 s at 53°C, 1 min at 72°C, followed by 5 min at 72°C. PCR products were analysed by gel electrophoresis; mecA product banded at 533 bp and the nuc at 267 bp. All nmrMRSA and RSA strains were confirmed for the presence or absence of the mecA gene by PCR.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cefoxitin MIC results are presented in Table 1. All 575 strains of MSSA were inhibited by cefoxitin at concentrations of 4 mg/L; 520 of these strains (90%) had an MIC of 4 mg/L. Similarly, all 24 RSA strains were also inhibited by 24 mg/L of cefoxitin (and were mecA-negative). However, nine of these 24 strains were resistant to oxacillin and methicillin by the NCCLS breakpoints using agar dilution testing. All MRSA strains were highly resistant to cefoxitin; 95% of the strains had an MIC between 128 and 256 mg/L. Cefoxitin MICs for the nmrMRSA strains were generally lower, and ranged between 8 and 256 mg/L. Only one of these strains had an MIC of 8 mg/L; this strain was mecA-positive but appeared oxacillin-susceptible (MIC 2 mg/L) and methicillin-susceptible (8 mg/L) on agar dilution testing.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There was a clear difference in the MICs of cefoxitin between the methicillin-susceptible strains (MSSA and RSA) and those that were methicillin resistant (nmrMRSA and MRSA). Although all strains of MSSA and RSA were inhibited by cefoxitin at 4 mg/L, 90% of strains had an MIC of 4 mg/L. MRSA strains showed high-level resistance to cefoxitin. MICs ranged from 8 to > 256 mg/L, with 170 of the 177 strains having an MIC of 128 > 256 mg/L. Against the nmrMRSA strains, cefoxitin MICs were distributed over a range of 8256 mg/L; for 88 of the 95 strains the cefoxitin MIC was between 32128 mg/L. For one strain, the cefoxitin MIC was 8 mg/L; this strain was mecA-positive but tested susceptible to oxacillin and to methicillin. Such strains are probably very heterogeneous in their expression of methicillin resistance and have been reported to be rare.4
Nine of the 24 RSA strains appeared resistant to oxacillin by standard testing with oxacillin agar dilution. Oxacillin MICs were 4 mg/L for seven of the strains and 8 mg/L for another two strains. All were mecA-negative and were inhibited by cefoxitin at 4 mg/L.
Our observations with the use of cefoxitin discs were very encouraging. Using the NCCLS disc diffusion criteria26
to define resistance (cefoxitin zone diameters of 19 mm for resistance and
20 mm for susceptibility), the sensitivity and specificity were 100% in the 598 strains tested in the study. Skov et al.22
tested cefoxitin 30 µg discs on Oxoid IsoSensitest agar and reported that a zone diameter of < 29 mm would be appropriate for methicillin-resistant strains. We have tested a few nmrMRSA strains on BBL MuellerHinton agar and on Oxoid IsoSensitest agar in parallel, and have noted that the zone diameters are generally larger on Oxoid IsoSensitest agar. If NCCLS recommendations26
are to be used, it would be necessary to adhere to the 20 mm cut-off recommendation.
NCCLS has not made recommendations for using cefoxitin to define methicillin resistance using agar dilution tests. The data presented here suggest that, if a breakpoint of cefoxitin 4 mg/L is used, then the sensitivity and specificity for detecting methicillin resistance would be 100% on the diverse selection of clinical isolates included in this study. The addition of 2% NaCl to MuellerHinton agar is recommended when testing oxacillin and methicillin,23,25 and has been shown to improve sensitivity when testing community-acquired strains.5 However, this does not appear to be necessary when testing using cefoxitin, and testing can be performed at standard temperatures (i.e. 3537°C). In addition, it may be possible to use such plates to identify the presence of methicillin-resistant S. aureus in specimens submitted for screening purposes as well.
![]() |
Acknowledgements |
---|
Participating AGAR laboratories: The Canberra Hospital (Peter Collignon & Susan Bradbury); Concord Repatriation General Hospital (Thomas Gottlieb & Glenn Funnell); John Hunter Hospital (Sue Tiley & Ian Winney); Nepean Hospital (James Branley & Sam Ryder); South West Area Pathology Service (Iain B. Gosbell & Helen Ziochos); Royal Hobart Hospital (Alistair McGregor & Rob Peterson); and Royal North Shore Hospital (Clarence J. Fernandes & Lorna A. Fernandes); Westmead Hospital (David Mitchell).
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Sakoulas, G., Gold, H. S., Venkataraman, L. et al. (2001). Methicillin-resistant Staphylococcus aureus: comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains. Journal of Clinical Microbiology 39, 394651.
3 . Bignardi, G. E., Woodford, N., Chapman, A et al. (1996). Detection of the mecA gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. Journal of Antimicrobial Chemotherapy 37, 5363.[Abstract]
4
.
Brown, D. F. J. (2001). Detection of methicillin/oxacillin resistance in staphylococci. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 6570.
5
.
Gosbell, I. B., Mercer, J. L. et al. (2003). Detection of intrinsic oxacillin resistance in non-multiresistant, oxacillin-resistant Staphylococcus aureus (NORSA). Journal of Antimicrobial Chemotherapy 51, 46870.
6 . Milne, L. M., Crow, M., Emptage, A. G. & Selkon, J. B. (1993). Effects of culture media on detection of methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci by disk diffusion methods. Journal of Clinical Pathology 46, 3947.[Abstract]
7 . Annear, D. I. (1968). The effect of temperature on resistance of Staphylococcus aureus to methicillin and some other antibiotics. Medical Journal of Australia 1, 4446.[Medline]
8 . Huang, M. B., Gay, T. E., Baker, C. N. et al. (1993). Two per cent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods. Journal of Clinical Microbiology 31, 26838.[Abstract]
9 . Collignon, P., Gosbell, I., Vickery, A. et al. (1998). Community-acquired methicillin-resistant Staphylococcus aureus in Australia. Lancet 352, 1456.[CrossRef][ISI][Medline]
10 . Collignon, P. (1999). Increased incidence of methicillin-resistant strains of Staphylococcus aureus in the community. Journal of Infectious Diseases 179, 1592.[CrossRef][ISI][Medline]
11 . Gosbell, I. B., Mercer, J. L., Neville, S. A. et al. (2001). Community acquired, non-multiresistant oxacillin-resistant Staphylococcus aureus ("NORSA") in South Western Sydney. Pathology 33, 20610.[CrossRef][ISI][Medline]
12 . Gosbell, I. B., Mercer, J. L., Neville, S. A. et al. (2001). Non-multiresistant and multiresistant methicillin-resistant Staphylococcus aureus in community-acquired infections. Medical Journal of Australia 174, 62730.[ISI][Medline]
13 . Diekema, D. J., Pfaller, M. A., Schmitz, F. J. et al. (2001). Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and in the Western Pacific Region for the SENTRY antimicrobial surveillance program, 19971999. Clinical Infectious Diseases 32, Suppl. 2, S11432.[CrossRef][ISI][Medline]
14 . Chambers, H. F. (2001). The changing epidemiology of Staphylococcus aureus? Emerging Infectious Diseases 7, 17882.[ISI][Medline]
15 . Hiramatsu, K., Okuma, K., Ma, X. X. et al. (2002). New trends in Staphylococcus aureus infections: glycopeptide resistance in hospital and methicillin resistance in the community. Current Opinion on Infectious Diseases 15, 40713.
16 . Salgado, C. D., Farr, B. M. & Calfee, D. P. (2003). Community-acquired methicillin-resistant Staphylococcus aureus: a meta analysis of prevalence and risk factors. Clinical Infectious Diseases 36, 1319.[ISI][Medline]
17 . Ellis, M. W., Hospenthal, D. R., Dooley, D. P. et al. (2004). Natural history of community-acquired methicillin-resistant Staphylococus aureus colonization and infection in soldiers. Clinical Infectious Diseases 39, 9719.[CrossRef][Medline]
18
.
Delpano, A., Panayotis, T. T., Glupczynski, Y. et al. (2000). In vivo deletion of the methicillin resistance mec region from the chromosome of Staphylococcus aureus strains. Journal of Antimicrobial Chemotherapy 46, 6179.
19 . Nakatomi, Y. & Sugiyama, J. (1998). A rapid latex agglutination assay for the detection of penicillin binding protein 2'. Microbiology and Immunology 42, 73943.[ISI][Medline]
20 . Gosbell, I. B., Neville, S. A., Mercer, J. L. et al. (2001). Evaluation of the MRSA-Screen test in detecting oxacillin-resistance in community and hospital isolates of Staphylococcus aureus. Pathology 33, 4935.[CrossRef][ISI][Medline]
21
.
Felten, A., Grandry, B., Lagrange, P. H. & Casin, I. (2002). Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA) a disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test. Journal of Clinical Microbiology 40, 276671.
22
.
Skov, R., Smyth, R., Clausen, M. et al. (2003). Evaluation of a 30 µg cefoxitin disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 52, 2047.
23 . National Committee for Clinical Laboratory Standards. (2004). Performance Standards for Antimicrobial Susceptibility TestingFourteenth Informational Supplement M100-S14. NCCLS, Wayne, PA, USA.
24 . Nimmo, G., Bell, J. & Mitchell, D. (2003). Antimicrobial resistance in Staphylococcus aureus in Australian teaching hospitals, 19881999. Microbial Drug Resistance 9, 15560.[CrossRef][ISI][Medline]
25 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow AerobicallySixth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
26 . National Committee for Clinical Laboratory Standards. (2003). Performance Standards for Antimicrobial Disk Susceptibility TestsEighth Edition; Approved Standard M2-A8. NCCLS, Wayne, PA, USA.
27 . Brakstad, O. G., Asabakk, K. & Maeland, J. A. (1992). Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. Journal of Clinical Microbiology 30, 165460.[Abstract]
28 . Brakstad, O. G., Maeland, J. A. & Tveten, Y. (1993). Multiplex polymerase chain reaction for detection of genes for Staphylococcus aureus thermonuclease and methicillin resistance and correlation with oxacillin resistance. Acta Pathologica Microbiologica et Immunologica Scandinavia 101, 6818.
|