Goessens, Wil

Leeuwen, Willem

Luijendijk, A

Pelt, Carina

Verbrugh, Henri

Undetermined

EUR Research Repository

EUR Research Information PortalRapid detection of methicillin resistance in staphylococcus aureus isolates by theMRSA-screen latex agglutination testPublished in:Journal of Clinical MicrobiologyPublication status and date:Published: 01/01/1999Document VersionPublisher's PDF, also known as Version of recordCitation for the published version (APA):Leeuwen, W., Pelt, C., Luijendijk, A., Verbrugh, H., & Goessens, W. (1999). Rapid detection of methicillin resistance instaphylococcus aureus isolates by the MRSA-screen latex agglutination test. Journal of Clinical Microbiology, 37(9), 3029-3030. http://hdl.handle.net/1765/9150Link to publication on the EUR Research Information PortalTerms and Conditions of UseExcept as permitted by the applicable copyright law, you may not reproduce or make this material available to any third partywithout the prior written permission from the copyright holder(s). Copyright law allows the following uses of this materialwithout prior permission:            • you may download, save and print a copy of this material for your personal use only;            • you may share the EUR portal link to this material.In case the material is published with an open access license (e.g. a Creative Commons (CC) license), other uses may beallowed. Please check the terms and conditions of the specific license.Take-down policyIf you believe that this material infringes your copyright and/or any other intellectual property rights, you may request itsremoval by contacting us at the following email address: openaccess.library@eur.nl. Please provide us with all the relevantinformation, including the reasons why you believe any of your rights have been infringed. In case of a legitimate complaint,we will make the material inaccessible and/or remove it from the website.JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/99/$04.0010Sept. 1999, p. 3029–3030 Vol. 37, No. 9Copyright © 1999, American Society for Microbiology. All Rights Reserved.Rapid Detection of Methicillin Resistance in Staphylococcus aureusIsolates by the MRSA-Screen Latex Agglutination TestWILLEM B. VAN LEEUWEN,* CINDY VAN PELT, AD LUIJENDIJK,HENRI A. VERBRUGH, AND WIL H. F. GOESSENSDepartment of Medical Microbiology and Infectious Diseases, Erasmus University MedicalCenter Rotterdam, Rotterdam, The NetherlandsReceived 19 January 1999/Returned for modification 27 February 1999/Accepted 9 June 1999The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecAPCR (“gold standard”) for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screentest detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSAstrains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecAPCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n 5106), as well as for Micrococcus spp. (n 5 10), members of the family Enterobacteriaceae (n 5 10), Streptococcuspneumoniae (n 5 10), and Enterococcus spp. (n 5 10) (specificity 5 100%). Producing the same PBP2a antigen,all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecAPCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain tothe species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity andsensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in themicrobiology laboratory.In most routine microbiological settings, the detection ofmethicillin resistance among Staphylococcus aureus isolates isbased on phenotypic assays. Genetic confirmation of positivefindings based on the detection of the mecA gene (4) is per-formed in reference laboratories only, as these techniques arenot yet generally available. Methicillin resistance in staphylo-cocci is mediated by the mecA gene, encoding the penicillin-binding protein 2a (PBP2a), which has a reduced affinity forbeta-lactam antibiotics. Denka Seiken has developed a simplelatex agglutination assay for the detection of methicillin resis-tance, which makes use of a specific monoclonal antibody di-rected toward the PBP2a antigen (5). In order to get insightinto the sensitivity of the assay, 90 genetically unrelated me-thicillin-resistant S. aureus (MRSA) strains were included aswell as 106 epidemiologically unrelated methicillin-susceptibleS. aureus (MSSA) strains to control the specificity of the assay.In order to test the extent of cross-reactivity of the MRSA-Screen assay, other species were also included: 10 methicillin-resistant Staphylococcus epidermidis (MRSE) strains and 10isolates each of species of the family Enterobacteriaceae, Mi-crococcus spp., Enterococcus spp., and Streptococcus pneu-moniae (Table 1).S. aureus strains were identified by the AccuProbe S. aureusculture identification test (Gen-Probe, Inc., San Diego, Calif.),performed in accordance with the manufacturer’s protocol.Methicillin resistance of all S. aureus (MRSA and MSSA) andS. epidermidis (MRSE) strains was analyzed by means of thedisk diffusion test in accordance with National Committee forClinical Laboratory Standards guidelines (6), and the mecAgene was detected by PCR as the “gold standard” (4). Coag-ulase-negative staphylococci were identified as S. epidermidisby Staphaurex (Murex Biotech Ltd., Dartford, United King-dom) and API-Staph (bioMérieux, Marcy l’Etoile, France).The complete panel of strains (Table 1) was tested withMRSA-Screen, and the test was performed in accordance withthe manufacturer’s protocol. In short, a loopful of bacterialcells was suspended in 200 ml of extraction reagent 1 andsubsequently lysed by boiling for 3 min. After cooling to roomtemperature, 50 ml of extraction reagent 2 was added to 200 mlof the lysate and samples were mixed well. After 5 min ofcentrifugation (1,500 3 g), 50 ml of the supernatant was usedfor testing agglutination with sensitized latex particles (1 drop)and another 50 ml of the supernatant was used for testing withthe control latex particles (1 drop). The test slides were mixedby rotating them for 3 min, after which agglutination was as-sessed visually. S. aureus ATCC 29213 was used as a negativecontrol in both the mecA PCR and the MRSA-Screen test. AmecA-positive clinical isolate was used as a positive controlstrain.The results are summarized in Table 2. For all 90 geneticallydiverse MRSA strains consisting of six different mecA types(2), the presence of the mecA gene was reconfirmed with PCR.As MRSA-Screen detects the PBP2a antigen in 87 of 90MRSA strains, the sensitivity is 97%. Repeated testing aftermultiple subculturing did not reconcile the results for the threeinitial agglutination-negative, mecA-positive S. aureus isolates.These discrepancies are probably due to the suppression ofmecA gene transcription (3). In order to bypass this problem,the three isolates were again incubated overnight in the pres-ence of a disk containing 5 mg of methicillin. The MRSA-Screen assay was repeated with colonies taken within the zoneof inhibition or at the border of the inhibition zone. All threeisolates became positive, whereas the control, i.e., coloniestaken from the same culture plate but not in the vicinity of themethicillin disk, remained negative. These findings actuallyindicate that the sensitivity of the assay can be improved byperforming the agglutination assay with isolates previously ex-posed to methicillin. None of the 106 genetically diverse MSSAstrains demonstrated positive results with MRSA-Screen or* Corresponding author. Mailing address: Erasmus University Med-ical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam,The Netherlands. Phone: 31 10 4633668. Fax: 31 10 4633875. E-mail:vanleeuwen@bacl.azr.nl.3029the mecA PCR, directing the specificity to 100%. The Enter-obacteriaceae strains, S. pneumoniae, Enterococcus spp., andMicrococcus spp. were identified with Microscan (Dade Beh-ring, Leusden, The Netherlands), optochin disk diffusion (Ox-oid, Basingstoke, United Kingdom), Vitek (bioMérieux), andAPI-Staph (bioMérieux), respectively. Among these genera,no false-positive results could be detected, ruling out the pres-ence of cross-reacting antigens. Since the genetic mechanismof methicillin resistance in coagulase-negative staphylococci isidentical to that in S. aureus (1), all MRSE strains (n 5 10)displayed positive results in both mecA PCR and the MRSA-Screen test.In our opinion, the assay should be applied in a routinesetting by using the following consecutive steps. After isolationof a suspect MRSA strain, it first needs to be identified to thespecies level to exclude coagulase-negative staphylococci. Sec-ond, the potential MRSA strain is subjected to the MRSA-Screen test. In the event of a positive result, the identificationis definitely MRSA. If a negative result is found, the assay hasto be repeated after the strain has been exposed to methicillinin order to induce possible methicillin resistance. If the MRSAstrain is then positive, MRSA has been identified. In the eventof a negative result, the mecA PCR should be used as the finaland conclusive confirmation assay.In conclusion, the MRSA-Screen latex test has the potentialfor the detection of MRSA in a routine microbiology setting asit combines high speed and excellent specificity and sensitivitywith limited requirements for special equipment or skilledpersonnel.REFERENCES1. Hiramatsu, K. 1995. Molecular evolution of MRSA. Microbiol. Immunol.39:531–543.2. Kreiswirth, B., J. Kornblum, R. D. Arbeit, W. Eisner, J. N. Maslow, A.McGeer, D. E. Low, and R. P. Novick. 1993. Evidence for a clonal origin ofmethicillin resistance in Staphylococcus aureus. Science 259:227–230.3. Kuwahara-Arai, K., N. Kondo, S. Hori, E. Tateda-Suzuki, and K. Hiramatsu.1996. Suppression of methicillin resistance in a mecA-containing pre-methi-cillin-resistant Staphylococcus aureus strain is caused by the mecI-mediatedrepression of PBP 29 production. Antimicrob. Agents Chemother. 40:2680–2685.4. Murakami, K., W. Minamide, K. Wada, E. Nakamura, H. Teraoka, and S.Watanabe. 1991. Identification of methicillin-resistant strains of staphylococciby polymerase chain reaction. J. Clin. Microbiol. 29:2240–2244.5. Nakatomi, Y., and J. Sugiyama. 1998. A rapid latex agglutination assay for thedetection of penicillin-binding protein 29. Microbiol. Immunol. 42:739–743.6. National Committee for Clinical Laboratory Standards. 1998. PerformanceNational Committee for Clinical Laboratory Standards for antimicrobial sus-ceptibility testing; eighth informational supplement, vol. 18. National Com-mittee for Clinical Laboratory Standards, Wayne, Pa.7. Tenover, F. C., R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G.Hancock, G. A. Hebert, B. Hill, R. Hollis, et al. 1994. Comparison of tradi-tional and molecular methods of typing isolates of Staphylococcus aureus.J. Clin. Microbiol. 32:407–415.8. van Leeuwen, W., A. van Belkum, B. Kreiswirth, and H. Verbrugh. 1998.Genetic diversification of methicillin-resistant Staphylococcus aureus as afunction of prolonged geographic dissemination and as measured by binarytyping and other genotyping methods. Res. Microbiol. 149:497–507.TABLE 1. Characterization of the strain collections used in this studySpecies No. of strains Origin Reference(s)MRSA 90 Geographically and temporally diverse strains (n 5 40) 2, 7Community-acquired strains from New York, N.Y., hospital (n 5 26) 8Strains from outbreak clusters in EMCRa (n 5 24)MSSA 106 Primary blood culture isolates from EMCR (n 5 91)Geographically and temporally diverse strains (n 5 15) 2, 7MRSE 10 Isolates from hematology department of EMCREnterobacteriaceae member 10 Primary clinical isolates from blood cultures at EMCRS. pneumoniae 10 Primary clinical isolates from blood cultures at EMCREnterococcus sp. 10 Primary clinical isolates from blood cultures at EMCRMicrococcus 10 Primary clinical isolates from blood cultures at EMCRa EMCR, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands.TABLE 2. Results obtained with diagnostic tests ondiverse strain collectionsaOrganism No. ofisolatesNo. (%) of isolates positive by:AccuProbe mecA PCR MRSA-ScreenMRSA 90 90 (100) 90 (100) 87 (97)MSSA 106 106 (100) 0 (0) 0 (0)MRSE 10 ND 10 (100) 10 (100)a Micrococcus spp., S. pneumoniae, Enterococcus spp., and Enterobacteriaceaestrains displayed no agglutination test results with the MRSA-Screen assay. Theother diagnostic tests were not done for these strains. ND, not done.3030 NOTES J. CLIN. MICROBIOL.

Rapid detection of methicillin resistance in staphylococcus aureus isolates by the MRSA-screen latex agglutination test

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Rapid detection of methicillin resistance in staphylococcus aureus isolates by the MRSA-screen latex agglutination test

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