This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Horstkotte, M. A.
Right arrow Articles by Mack, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Horstkotte, M. A.
Right arrow Articles by Mack, D.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 2001, p. 3700-3702, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3700-3702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Rapid Detection of Methicillin Resistance in Coagulase-Negative Staphylococci by a Penicillin-Binding Protein 2a-Specific Latex Agglutination Test

Matthias A. Horstkotte, Johannes K.-M. Knobloch, Holger Rohde, and Dietrich Mack*

Institut für Medizinische Mikrobiologie und Immunologie, Universitätsklinikum Hamburg-Eppendorf, D-20246 Hamburg, Germany

Received 29 May 2001/Returned for modification 4 July 2001/Accepted 15 July 2001


    ABSTRACT
Top
Abstract
Text
References

The detection of PBP 2a by the MRSA-Screen latex agglutination test with 201 clinical coagulase-negative staphylococci had an initial sensitivity of 98% and a high degree of specificity for Staphylococcus epidermidis strains compared to PCR for mecA. Determination of oxacillin MICs evaluated according to the new breakpoint (0.5 µg/ml) of the National Committee for Clinical Laboratory Standards exhibited an extremely low specificity for this population.


    TEXT
Top
Abstract
Text
References

Coagulase-negative staphylococci (CoNS) are major nosocomial pathogens (10, 12, 18, 22, 24), and treatment of infections caused by CoNS is increasingly problematic due to the frequent occurrence of isolates resistant to multiple antibiotics (2).

The majority of clinical CoNS harbor the mecA gene, which encodes an additional penicillin-binding protein (PBP), PBP 2a, essential for expression of methicillin resistance (3). Phenotypic detection of methicillin resistance in CoNS is difficult due to the heterogeneous expression of mecA (6, 16, 21, 27). mecA detection by PCR is very sensitive but is not feasible for the busy clinical microbiology laboratory.

Thus, rapid, sensitive, and specific procedures for the detection of methicillin resistance in CoNS are urgently needed. A latex agglutination (LA) test (MRSA-Screen) detects PBP 2a by using latex particles sensitized with monoclonal antibodies specific for PBP 2a of Staphylococcus aureus (17). This test was evaluated for S. aureus with overall favorable results for sensitive and specific detection of methicillin-resistant S. aureus (MRSA) (5, 11, 15, 17, 25, 26).

(Part of this work will appear in the doctoral thesis of M. A. Horstkotte, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.)

CoNS strains (n = 197) were consecutively collected at the University Hospital Hamburg-Eppendorf from November 1997 to January 1998, including blood culture (n = 77), infected catheter (n = 91), wound (n = 15), and urine (n = 14) isolates. A single isolate per patient was included. S. epidermidis 1457, 1057, 9225, and RP62A were included as reference strains (14). S. aureus ATCC 29213 and ATCC 25923 were used for quality control. The strains were kept at -80°C and were subcultured twice onto Columbia blood-agar (CBA) plates before testing. Species identification was performed by using Gram stain morphology, the catalase test, the clumping factor test, and the ID 32 Staph system (bioMérieux, Marcy l'Etoile, France).

Oxacillin MICs were determined by the broth microdilution method, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (9).

PCR detection of mecA was performed by using the conditions described previously (13, 20) with the primers mecAsense (181-5'-GAA ATG ACT GAA CGT CCG AT-3'-200) and mecAantisense (330-5'-GCG ATC AAT GTT ACC GTA GT-3'-311) (19). S. epidermidis 1457 and RP62A were included as negative and positive controls, respectively.

For the MRSA-Screen (Denka Seiken Co., Niigata, Japan), isolates were grown overnight on CBA. The manufacturer's instructions were essentially followed; however, a larger inoculum was used by suspending a loopful of bacteria to at least a McFarland no. 6 standard in extraction buffer. Agglutination results were read after 3 min.

A total of 201 isolates of CoNS (142 S. epidermidis, 15 S. haemolyticus, 10 S. hominis, 9 S. saprophyticus, 6 S. capitis, 4 S. lugdunensis, 4 S. warneri, 4 S. xylosus, 2 S. schleiferi, 2 S. cohnii, 1 S. chromogenes, 1 S. simulans, and 1 S. kloosii isolates) were tested. mecA was detected by PCR in 126 (62.7%) of all strains, of which 102 were S. epidermidis strains and 24 were non-S. epidermidis strains (Table 1). By the MRSA-Screen, 119 of 126 mecA-positive strains were LA positive. On initial testing 2 mecA-positive S. epidermidis strains were LA negative and 5 strains displayed weakly positive agglutination (Tables 1 and 2). Initially, 67 of 75 mecA-negative strains were LA negative, but 8 non-S. epidermidis strains displayed a positive (n = 1) or weakly positive (n = 7) agglutination result (Tables 1 and 2). When the weakly positive reactions were also counted as positive, PBP 2a was detected with sensitivities of 98.4, 98.0, and 100% and specificities of 89.3, 100, and 77.1% in all strains of CoNS, S. epidermidis strains only, and non-S. epidermidis strains, respectively.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Detection of methicillin resistance by latex agglutination of PBP 2a compared to mecA PCR


                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Strains with discrepant results between mecA PCR and the PBP 2a latex agglutination test

The 12 weakly positive strains and the 3 strains with LA results discordant with the mecA PCR result were retested. All seven mecA-positive strains were LA positive on retesting. On retesting, six of eight mecA-negative strains exhibited a negative result by LA; however, two S. warneri strains still displayed false-positive LA results (Tables 1 and 2). All mecA-positive strains retested were S. epidermidis (Table 2). Among the mecA-negative strains retested, only non-S. epidermidis species were observed (Table 2). Retesting of weakly positive strains resulted in PBP 2a detection with sensitivities of 98.4, 98.0, and 100% and specificities of 97.3, 100, and 94.3% in all strains of CoNS, S. epidermidis strains only, and non-S. epidermidis strains, respectively.

MIC testing revealed for 125 of 126 mecA-positive strains oxacillin MICs of 4 µg/ml and for 1 S. epidermidis isolate an oxacillin MIC of 2 µg/ml. For only 9 of 75 mecA-negative strains were oxacillin MICs <= 0.25 µg/ml, but for 66 strains oxacillin MICs were between 0.5 and 2 µg/ml. Compared to mecA PCR, all mecA-positive strains of CoNS were found to be oxacillin resistant by use of the actual oxacillin breakpoint (0.5 µg/ml) of NCCLS (9). However, 66 of 75 mecA-negative strains of CoNS, for which oxacillin MICs ranged from 0.5 to 2 µg/ml, would have been falsely identified as oxacillin resistant, indicating that the actual breakpoint has a low degree of specificity.

In the present study detection of PBP 2a by MRSA-Screen was highly sensitive and specific and at least equivalent to other phenotypic techniques for the detection of methicillin resistance in CoNS (6, 16, 21, 27). Compared to the results of PCR, MRSA-Screen was superior to the standard broth microdilution assay when the present oxacillin breakpoint (0.5 µg/ml) suggested by NCCLS (9) was used. The latter method misidentified 66 of 75 mecA-negative strains of CoNS as oxacillin resistant. In contrast, an excellent specificity of the new breakpoint was reported for S. epidermidis, S. hominis, and S. haemolyticus, but the breakpoint was less accurate when it was applied to other species of CoNS (8). A lack of specificity of the new breakpoint could jeopardize the efforts directed at curtailing the overuse of glycopeptide antibiotics. When we evaluated our MIC data with the old oxacillin breakpoint (4.0 µg/ml), one mecA-positive S. epidermidis isolate would have been misclassified as susceptible and one mecA-negative S. kloosii isolate would have been misclassified as resistant.

The principal difficulties of performance of the MRSA-Screen with CoNS were reported to be regarding sensitivity (7, 15) or specificity (1, 28), or both (4, 23). A low initial sensitivity was reported, with satisfactory results obtained only after induction of PBP 2a synthesis with an oxacillin disk during overnight subcultivation (7). In contrast, all 60 mecA-positive strains of CoNS were reported to be LA positive after 3 min without induction (28). Apparently, the sensitivity of the MRSA-Screen depends on the amount of bacteria used in the inoculum or additional enhancement of PBP 2a expression. We prefer using a rather heavy inoculum, as oxacillin induction of PBP 2a requires subcultivation and could delay the results by 24 h. Additionally, extended agglutination times for increased sensitivity (7) can lead to false-positive results (28).

When CoNS were evaluated, weakly positive LA results occurred (7, 28). In our study, retesting of weakly positive strains led to positive test results for all of the mecA-positive strains and to negative results for all but two mecA-negative strains. For use in the clinical laboratory, it seems reasonable to retest a weakly positive isolate and to interpret a concordant LA result (positive or weakly positive on retesting) as positive. If the second LA test gives a discordant (negative) result, either the species diagnosis could help in the decision, as all false-positive results occurred with non-S. epidermidis strains, or an independent reference method could be used in parallel.

In our study two S. warneri strains displayed false-positive LA test results. False-positive LA test results were reported with S. lugdunensis, S. warneri, S. simulans, and S. hominis (1, 7, 23, 28).

Apparently, the MRSA-Screen performs favorably with clinical isolates of CoNS, especially the most frequently encountered species, S. epidermidis. False-positive results occur primarily with non-S. epidermidis strains. With a turnaround time of about 30 min, this assay could replace other phenotypic methods for determination of methicillin resistance in CoNS in the clinical microbiology laboratory.


    ACKNOWLEDGMENTS

We thank Rainer Laufs for continuous support.

This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (to D.M.).


    FOOTNOTES

* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie und Immunologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. Phone: 49 40 42803 2143. Fax: 49 40 42803 4881. E-mail: dmack{at}uke.uni-hamburg.de.


    REFERENCES
Top
Abstract
Text
References

1. Andrews, J. M., F. J. Boswell, and R. Wise. 2000. Establishing MIC breakpoints for coagulase-negative staphylococci to oxacillin. J. Antimicrob. Chemother. 45:259-261[Free Full Text].
2. Archer, G. L., and M. W. Climo. 1994. Antimicrobial susceptibility of coagulase-negative staphylococci. Antimicrob. Agents Chemother. 38:2231-2237[Free Full Text].
3. Berger-Bächi, B. 1997. Resistance not mediated by beta -lactamase (methicillin resistance), p. 158-174. In K. B. Crossley, and G. L. Archer (ed.), The staphylococci in human disease. Churchill Livingston, New York, N.Y.
4. Cavassini, M., A. Wenger, K. Jaton, J. Bille, and D. S. Blanc. 2001. Further evaluation of the MRSA-Screen kit for rapid detection of methicillin resistance---reply. J. Clin. Microbiol. 37:3784.
5. Cavassini, M., A. Wenger, K. Jaton, D. S. Blanc, and J. Bille. 1999. Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus. J. Clin. Microbiol. 37:1591-1594[Abstract/Free Full Text].
6. Chambers, H. F. 1997. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin. Microbiol. Rev. 10:781-791[Abstract].
7. Hussain, Z., L. Stoakes, S. Garrow, S. Longo, V. Fitzgerald, and R. Lannigan. 2000. Rapid detection of mecA-positive and mecA-negative coagulase-negative staphylococci by an anti-penicillin-binding protein 2a slide latex agglutination test. J. Clin. Microbiol. 38:2051-2054[Abstract/Free Full Text].
8. Hussain, Z., L. Stoakes, V. Massey, D. Diagre, V. Fitzgerald, S. El Sayed, and R. Lannigan. 2000. Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci. J. Clin. Microbiol. 38:752-754[Abstract/Free Full Text].
9. Jorgensen, J. H., J. D. Turnidge, and J. A. Washington. 1999. Antibacterial susceptibility tests: dilution and disk diffusion methods, p. 1526-1543. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
10. Kloos, W. E. 1997. Taxonomy and systematics of staphylococci indigenous to humans, p. 113-137. In K. B. Crossley, and G. L. Archer (ed.), The staphylococci in human disease. Churchill Livingston, New York, N.Y.
11. Louie, L., S. O. Matsumura, E. Choi, M. Louie, and A. E. Simor. 2000. Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus. J. Clin. Microbiol. 38:2170-2173[Abstract/Free Full Text].
12. Mack, D., K. Bartscht, S. Dobinsky, M. A. Horstkotte, K. Kiel, J. K. M. Knobloch, and P. Schäfer. 2000. Staphylococcal factors involved in adhesion and biofilm formation on biomaterials, p. 307-330. In Y. H. An, and R. J. Friedman (ed.), Handbook for studying bacterial adhesion: principles, methods, and applications. Humana Press, Totowa, N.J.
13. Mack, D., K. Bartscht, C. Fischer, H. Rohde, C. de Grahl, S. Dobinsky, M. A. Horstkotte, K. Kiel, and J. K. M. Knobloch. 2001. Genetic and biochemical analysis of Staphylococcus epidermidis biofilm accumulation. Methods Enzymol. 336:215-239[Medline].
14. Mack, D., M. Haeder, N. Siemssen, and R. Laufs. 1996. Association of biofilm production of coagulase-negative staphylococci with expression of a specific polysaccharide intercellular adhesin. J. Infect. Dis. 174:881-884[Medline].
15. Marriott, D. J., T. Karagiannis, J. L. Harkness, and P. Kearney. 1999. Further evaluation of the MRSA-Screen kit for rapid detection of methicillin resistance. J. Clin. Microbiol. 37:3783-3784[Free Full Text].
16. McDonald, C. L., W. E. Maher, and R. J. Fass. 1995. Revised interpretation of oxacillin MICs for Staphylococcus epidermidis based on mecA detection. Antimicrob. Agents Chemother. 39:982-984[Abstract].
17. Nakatomi, Y., and J. Sugiyama. 1998. A rapid latex agglutination assay for the detection of penicillin-binding protein 2'. Microbiol. Immunol. 42:739-743[Medline].
18. Rupp, M. E., and G. L. Archer. 1994. Coagulase-negative staphylococci: pathogens associated with medical progress. Clin. Infect. Dis. 19:231-243[Medline].
19. Ryffel, C., W. Tesch, I. Birch-Machin, P. E. Reynolds, L. Barberis-Maino, F. H. Kayser, and B. Berger-Bächi. 1990. Sequence comparison of mecA genes isolated from methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Gene 94:137-138[CrossRef][Medline].
20. Schmitz, F. J., C. R. Mackenzie, B. Hofmann, J. Verhoef, M. Finken-Eigen, H. P. Heinz, and K. Kohrer. 1997. Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR. J. Med. Microbiol. 46:773-778[Abstract/Free Full Text].
21. Tenover, F. C., R. N. Jones, J. M. Swenson, B. Zimmer, S. McAllister, and J. H. Jorgensen. 1999. Methods for improved detection of oxacillin resistance in coagulase-negative staphylococci: results of a multicenter study. J. Clin. Microbiol. 37:4051-4058[Abstract/Free Full Text].
22. Thylefors, J. D., S. Harbarth, and D. Pittet. 1998. Increasing bacteremia due to coagulase-negative staphylococci: fiction or reality? Infect. Control Hosp. Epidemiol. 19:581-589[Medline].
23. Udo, E. E., E. M. Mokadas, A. Al Haddad, B. Mathew, L. E. Jacob, and S. C. Sanyal. 2000. Rapid detection of methicillin resistance in staphylococci using a slide latex agglutination kit. Int. J. Antimicrob. Agents 15:19-24[CrossRef][Medline].
24. U.S. Department of Health and Human Services. 1996. Public Health Service: National Nosocomial Infectious Surveillance (NNIS) report. Data summary from October 1986-April 1996, issued May 1996. Am. J. Infect. Control 24:380-388[CrossRef][Medline].
25. van Griethuysen, A., M. Pouw, N. van Leeuwen, M. Heck, P. Willemse, A. Buiting, and J. Kluytmans. 1999. Rapid slide latex agglutination test for detection of methicillin resistance in Staphylococcus aureus. J. Clin. Microbiol. 37:2789-2792[Abstract/Free Full Text].
26. van Leeuwen, W. B., C. van Pelt, A. Luijendijk, H. A. Verbrugh, and W. H. Goessens. 1999. Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-Screen latex agglutination test. J. Clin. Microbiol. 37:3029-3030[Abstract/Free Full Text].
27. York, M. K., L. Gibbs, F. Chehab, and G. F. Brooks. 1996. Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci. J. Clin. Microbiol. 34:249-253[Abstract].
28. Zbinden, R., M. Ritzler, E. Ritzler, and B. Berger-Bächi. 2001. Detection of penicillin-binding protein 2a by rapid slide latex agglutination test in coagulase-negative staphylococci. J. Clin. Microbiol. 39:412[Free Full Text].

Journal of Clinical Microbiology, October 2001, p. 3700-3702, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3700-3702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Join-Lambert, O. F., Clauser, S., Guillet, C., Jais, J.-P., Abachin, E., Quesnes, G., Carbonnelle, E., Le Monnier, A., Zahar, J.-R., Kayal, S., Berche, P., Ferroni, A. (2007). Comparison of cefoxitin and moxalactam 30 {micro}g disc diffusion methods for detection of methicillin resistance in coagulase-negative staphylococci. J Antimicrob Chemother 59: 763-766 [Abstract] [Full Text]  
  • Perazzi, B., Fermepin, M. R., Malimovka, A., Garcia, S. D., Orgambide, M., Vay, C. A., de Torres, R., Famiglietti, A. M. R. (2006). Accuracy of cefoxitin disk testing for characterization of oxacillin resistance mediated by penicillin-binding protein 2a in coagulase-negative staphylococci.. J. Clin. Microbiol. 44: 3634-3639 [Abstract] [Full Text]  
  • Miller, M. B., Meyer, H., Rogers, E., Gilligan, P. H. (2005). Comparison of Conventional Susceptibility Testing, Penicillin-Binding Protein 2a Latex Agglutination Testing, and mecA Real-Time PCR for Detection of Oxacillin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococcus. J. Clin. Microbiol. 43: 3450-3452 [Abstract] [Full Text]  
  • Horstkotte, M. A., Knobloch, J. K.-M., Rohde, H., Dobinsky, S., Mack, D. (2004). Evaluation of the BD PHOENIX Automated Microbiology System for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci. J. Clin. Microbiol. 42: 5041-5046 [Abstract] [Full Text]  
  • Kipp, F., Becker, K., Peters, G., von Eiff, C. (2004). Evaluation of Different Methods To Detect Methicillin Resistance in Small-Colony Variants of Staphylococcus aureus. J. Clin. Microbiol. 42: 1277-1279 [Abstract] [Full Text]  
  • Horstkotte, M. A., Knobloch, J. K.-M., Rohde, H., Dobinsky, S., Mack, D. (2002). Rapid Detection of Methicillin Resistance in Coagulase-Negative Staphylococci with the VITEK 2 System. J. Clin. Microbiol. 40: 3291-3295 [Abstract] [Full Text]  
  • Hussain, Z., Stoakes, L., John, M. A., Garrow, S., Fitzgerald, V. (2002). Detection of Methicillin Resistance in Primary Blood Culture Isolates of Coagulase-Negative Staphylococci by PCR, Slide Agglutination, Disk Diffusion, and a Commercial Method. J. Clin. Microbiol. 40: 2251-2253 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Horstkotte, M. A.
Right arrow Articles by Mack, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Horstkotte, M. A.
Right arrow Articles by Mack, D.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»