Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, UK
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Abstract |
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Introduction |
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Conventional disc diffusion and dilution tests for the detection of methicillin resistance in S. aureus require overnight incubation, while rapid methodssuch as Vitek,2 Rapid ATB Staph2 and Crystal MRSA3still require several hours of incubation. Almost all MRSA have an additional penicillin-binding protein, PBP2a, mediated by the mecA gene.4 PCR methods detecting the mecA gene will provide results within a few hours, but are not available in most laboratories and test costs are relatively high. A rapid latex agglutination test based on detection of PBP2a has been described.5 The method employs latex particles coated with monoclonal antibodies to PBP2a, which is extracted from test colonies. With colonies of a wide range of MRSA grown on blood agar, a sensitivity of 98.5100% and a specificity of 100% have been reported.68 MRSA screening swabs are, however, commonly plated on selective media and the presence of selective agents such as NaCl, oxacillin or methicillin in these media may affect the reliability of the test with some strains, particularly those which are markedly heterogeneous and do not grow well on selective media.
In this study, we tested the performance of a latex agglutination method, the Mastalex MRSA screening test, with isolates from a wide variety of sources grown on blood agar and a variety of selective media used in screening for MRSA.
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Materials and methods |
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Fifty-two MRSA, including representatives of epidemic strains (EMRSA) 116, were from various sources in the UK, USA, France, Japan and Australia. Twenty-seven methicillin-susceptible isolates of S. aureus were recent clinical isolates or susceptible control strains. Resistance was confirmed by testing for mecA.9
Media
Colonies were grown on the following media: Columbia agar (Oxoid, Basingstoke, UK) with 5% horse blood (blood agar); blood agar containing oxacillin 2 mg/L; mannitol salt agar (MSA) from Mast (Bootle, UK); Mast MSA containing oxacillin 2 mg/L; MSA from Oxoid; Oxoid MSA containing oxacillin 2 mg/L; and BairdParker agar (Oxoid) containing ciprofloxacin 8 mg/L. Plates were incubated for 18 h in air at 37°C except for blood agar with oxacillin 2 mg/L, which was incubated for 18 h at 30°C.
Mastalex MRSA latex agglutination method
The Mastalex MRSA method (Mast) was used according to the manufacturer's instructions. Briefly, 10 colonies were suspended in 200 µL extraction reagent 1 and heated in a boiling water bath for 3 min. Tubes were cooled and 50 µL extraction reagent 2 was added. Tubes were centrifuged for 1 min in a microcentrifuge. Fifty microlitres of supernatant were mixed with 50 µL sensitized latex suspension and rotated manually for 3 min while looking for agglutination. The supernatant was tested simultaneously with a negative control latex suspension. The time at which agglutination was visible by eye was recorded.
Statistical analysis
Differences in detection of methicillin resistance with the Mastalex method when colonies were grown on different media were tested by the 2 test.
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Results and discussion |
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Tests with four (7.7%) and 16 (30.8%) of the 52 MRSA gave false-negative results with colonies from the Mast and Oxoid MSA media without oxacillin, respectively. These tests were significantly less reliable than tests on colonies from blood agar (P < 0.05 and P < 0.001 in comparisons with Mast and Oxoid MSA, respectively). Tests on colonies grown on Oxoid MSA without oxacillin were also significantly less reliable than tests on colonies grown on any other medium tested (P < 0.001 when compared with blood agar with or without oxacillin and Mast MSA with oxacillin; P < 0.01 when compared with other media). Colonies grown on MSA were frequently sticky, making it difficult to pick up colonies on a loop and to suspend growth evenly; these difficulties were more marked with Oxoid MSA, possibly because the Oxoid medium has 7% added NaCl, as opposed to 3% in the Mast medium.
There was no correlation between EMRSA type and false-negative results. Because of their current prominence, 20 of the 52 MRSA tested were EMRSA types 15 and 16. False-negative results with these types were recorded with two isolates grown on Mast MSA, one on Mast MSA with oxacillin, six on Oxoid MSA and two on Oxoid MSA with oxacillin. This was not significantly different from results with the 32 other MRSA isolates, where falsenegative results were recorded with two isolates grown on Mast MSA, none on Mast MSA with oxacillin, 10 on Oxoid MSA and three on Oxoid MSA with oxacillin.
Addition of oxacillin to MSA improved the reliability of tests, but false-negative results were still found in tests on colonies from one and five isolates grown on Mast and Oxoid media, respectively. Also the growth of two isolates on Mast MSA and one on Oxoid MSA was inhibited. In contrast, Willey et al.8 found results on MSA with oxacillin 2 mg/L to be reliable.
The mean times to read a positive result (Table) were markedly lower for tests on colonies from blood agar and BairdParker agar than from those on MSA. Agglutination times were also reduced on all media to which oxacillin was added. This is presumably related to induction of PBP2a production by growth in the presence of oxacillin, leading to a higher concentration of PBP2a in the agglutination reaction. The blood agar plates with and without oxacillin were incubated at 30 and 37°C, respectively, and the lower temperature of incubation of the plates with oxacillin may have contributed to better expression of PBP2a and consequently more rapid agglutination results with colonies from this medium.
BairdParker agar containing ciprofloxacin is a useful medium for isolation of MRSA in situations where ciprofloxacin-resistant strains are endemic.10 However, half of the strains tested in this study did not grow on the medium. With strains that did grow, growth was good and agglutination was rapid. The cause of the single false-negative result (confirmed on retesting) with colonies from BairdParker agar with ciprofloxacin is not obvious.
The Mastalex test procedure is straightforward but relatively labour intensive, especially the agglutination reaction. A single test took 10 min whereas a batch of 10 tests took 24 min. The use of a rotating platform or shaker might be considered, but this is likely to extend the agglutination time. Other factors that may affect the speed and reliability of the test are the density of the cell suspension, which varies depending on how many colonies are available, how good the growth is on the test medium and how easy it is to pick up colonies on the loop, particularly from media containing NaCl.
In conclusion, the Mastalex latex agglutination test for methicillin resistance in S. aureus is highly reliable and reasonably rapid if colonies are grown on blood agar, with or without added oxacillin. Caution is needed when the method is used with colonies grown on MSA or Baird Parker agar with ciprofloxacin. The method would be particularly useful when confirmation of resistance is urgently required.
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Acknowledgments |
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Notes |
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References |
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2
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Frebourg, N. B., Nouet, D., Lemee, L., Martin, E. & Lemeland, J. F. (1998). Comparison of ATB staph, rapid ATB staph, Vitek, and E-test methods for detection of oxacillin heteroresistance in staphylococci possessing mecA. Journal of Clinical Microbiology 36, 527.
3 . Knapp, C. C., Ludwig, M. D. & Washington, J. A. (1994). Evaluation of BBL crystal MRSA ID system. Journal of Clinical Microbiology 32, 25889.[Abstract]
4 . Matsuhashi, M., Song, M. D., Ishino, F., Wachi, M., Doi, M., Inoue, M. et al. (1986). Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus. Journal of Bacteriology 167, 97580.[ISI][Medline]
5 . 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]
6
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van Griethuysen, A., Pouw, M., van Leeuwen, N., Heck, M., Willemse, P., Buiting, A. et al. (1999). Rapid slide latex agglutination test for detection of methicillin resistance in Staphylococcus aureus. Journal of Clinical Microbiology 37, 278992.
7 . Vuopio-Varkila, J., Swenson, J., Killgore, G., Hill, B., McAllister, S. & Tenover, F. C. (1999). Evaluation of MRSA-ScreenTM to detect methicillin-resistant staphylococci. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, California, 1999. Abstract 874, p. 207. American Society for Microbiology, Washington, DC.
8 . Willey, B. M., Tennant, B., Moore, T. C., Pearce, L., McGeer, A., Low, D. E. et al. (1999). Evaluation of a simple, rapid methicillin-resistant Staphylococcus aureus identification protocol. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, California, 1999. Abstract 871, p. 206. American Society for Microbiology, Washington, DC.
9 . Bignardi, G. E., Woodford, N., Chapman, A., Johnson, A. P. & Speller, D. C. (1996). Detection of the mec-A gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. Journal of Antimicrobial Chemotherapy 37, 5363.[Abstract]
10 . Davies, S. & Zadik, P. M. (1997). Comparison of methods for the isolation of methicillin resistant Staphylococcus aureus. Journal of Clinical Pathology 50, 2578.[Abstract]
Received 29 March 2000; returned 31 July 2000; revised 5 October 2000; accepted 2 November 2000