An Easy-to-Use, Rapid and Inexpensive Method to Determine Methicillin Resistance In Staphylococcus aureus

Abstract

Objectives: We aimed to report a new turbidimetric method to identify methicillin resistance in S. aureus strains just two hours after identification of the microorganism, and to analyze diagnostic and discrimination abilities of this new method. Methods: A total of 319 S. aureus isolates were included in the study. Identification of bacteria was done by the colony morphology, and conventional biochemical methods. The turbidimetric method we developed is based on different growth rates of S. aureus in two media, with or without oxacillin. The growth rates of MRSA and MSSA are similar in normal media; however, the MRSA grows significantly faster in the media containing oxacillin. Therefore, after 2 hours of incubation, the difference of turbidity produced by bacteria in the media with or without oxacillin is less in case of MRSA, and more in case of MSSA. The absorbances of the microplates were measured before incubation, and at 2nd and 3rd hours of incubation. The “absorbance rate” was calculated for each bacteria based on those absorbance values measured. The bacteria were classified as MRSA or MSSA based on the absorbance rate. Results: All MRSA and MSSA strains were correctly discriminated via our turbidimetric method, when an absorbance rate of 1.900 was taken as cut-off value. The new method could diagnose MRSA with 100% specificity and 100% sensitivity in just 2 hours. Conclusions: The turbidimetric method is a rapid, easy and cheap method that does not require any specific equipment. It can be easily performed in every microbiology laboratory. 

Keywords

• Turbidimetric method,MRSA,MSSA

References

1. 1. Becher K, von Eiff C. Staphylococcus, Micrococcus, anda other Catalase-positive Cocci. Versalovic J, ed. Annual of Clinical Microbiology. 10th ed. American Society for Microbiology, ASM Press, 1752 N St. NW, Washington, DC 20036-2904, USA, 2011; 308-330.
2. 2. Tille P M. Bailey and Scott's Diagnostic Microbiology. 13th ed. C.V. Mosby Company, St. Louis, MO, USA, 2014.
3. 3. Winn W, Allen S, Janda W, et al. Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Lippincott Williams & Wilkins Company, 351 West Camden Street. Baltimore , MD 21201, USA, 2006.
4. 4. Sancak B. Resistance Mechanisms of MRSA: Epidemiology in the World and Turkey. ANKEM J 2012; 26: 38-47.
5. 5. Klein E Y, Sun L, Smith D L, et al. The Changing Epidemiology of Methicillin-Resistant Staphylococcus aureus in the United States: A National Observational. Am J Epidemiol 2013; 177:666-674.
6. 6. Statistical Bulletin: Deaths Involving MRSA: England and Wales, 2008 to 2012.
7. 7. Isenberg, HD. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington D.C. 2010.
8. 8. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplements. CLSI document M 100- S19 (ISBN 1-56238-690-5). Clinical and Laboratory Standards Institute , 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2009.

9. 9. Swenson JM, Tenover FC. Results of disk diffusion testing with cefoxitin correlate with presence of mecA in Staphylococcus spp. J Clin Microbiol 2005;43:3818-3823.
10. 10. Smyth RW, Kahlmeter G. Mannitol salt agarcefoxitin combination as a screening medium for methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005; 43: 3797-3799.
11. 11. Iosipenko AD, Shchegolev SIu, Shenderov BA, et al. A spectroturbidimetric method of rapid evaluation of microbial sensitivity to antibiotics. Antibiot Med Biotekhnol 1985; 30: 208-212.
12. 12. Kolmert A, Wikstrom P, Hallberg KB. A fast and simple turbidimetric method for the determination of sulfate in sulfate-reducing bacterial cultures. J Microbiol Meth 2000; 41: 179-184.
13. 13. Metris A, George SM, Peck MW, et al. Distribution of turbidity detection times produced by single cellgenerated bacterial populations. J Microbiol Meth 2003; 55: 821-827.
14. 14. Galante LJ, Brinkley MA, Lodder RA. Bacterial monitoring in vials using a spectrophotometric assimilation method. Pharm Res 1992; 9: 357-364.
15. 15. Cekovska Z, Panovski N, Petrovska M. Methicillinresistant Staphylococcus aureus: comparison of susceptibility test methods with mecA gene analysis for determining oxacillin (methicillin) resistance in our clinical isolates. Bratisl Lek Listy 2005;106:163-167.
16. 16. Pottumarthy S, Fritsche TR, Jones RN. Evaluation of alternative disk diffusion methods for detecting mecA-mediated oxacillin resistance in an international collection of staphylococci: validation report from the SENTRY Antimicrobial Surveillance Program. Diagn Microbiol Infect Dis 2005; 51:57-62.
17. 17. Boutiba-Ben Boubaker I, Ben Abbes R, Ben Abdallah H, et al. Evaluation of a cefoxitin disk diffusion test for the routine detection of methicillinresistant Staphylococcus aureus. Clin Microbiol Infect 2004; 10: 762-765.
18. 18. Cauwelier B, Gordts B, Descheemaecker P, et al. Evaluation of a disk diffusion method with cefoxitin (30 microg) for detection of methicillin-resistant Staphylococcus aureus. Eur J Clin Microbiol Infect Dis 2004;23:867-868.
19. 19. Skov R, Larsen AR, Frimodt-Moller N, et al. Evaluation of different disk diffusion/media combinations for detection of methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci. APMIS 2003; 111: 905-914.
20. 20. Atay T, Gulay Z, Kocagoz S, et al. Comparison of routine sensitivity tests and mecA gene analysis for determination of methicillin resistance in Staphylococcus aureus isolates. Mikrobiyol Bul 2002;36:133-140.
21. 21. Kuzucu C, Dalgalar M, Durmaz R, et al. Comparison of methods used to detect methicillin resistance in staphylococci. Mikrobiyol Bul 2002;36:253-257.
22. 22. Araj GF, Talhouk RS, Simaan CJ, et al. Discrepancies between mecA PCR and conventional tests used for detection of methicillin resistant Staphylococcus aureus. Int J Antimicrob Agents 1999; 11:47-52.
23. 23. Ryffel C, Kayser FH, Berger-Bachi B. Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci. Antimicrob Agents Chemother 1992;36:25-31.
24. 24. Bignardi GE,Woodford N, Chapman A, et al. Detection of the mec-A gene and pbenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. J Antimicrob Chemother 1996; 37: 53-63.
25. 25. Derek FJ B, Edwards DI, Hawkey PM, et al. Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrob Chemother 2005; 56: 1000-1018.
26. 26. Kohner P, Uhl J, Kolbert C, et al. Comparison of Susceptibility Testing Methods with mecA Gene Analysis for Determining Oxacillin (Methicillin) Resistance in Clinical Isolates of Staphylococcus aureus and Coagulase-Negative Staphylococcus spp. J Clin Microbiol 1999; 37: 2952-2961.
27. 27. Rasooly R, Hernlem B. TNF as Biomarker for Rapid Quantification of Active Staphylococcus Enterotoxin A in Food. Sensors 2012; 12: 5978-5985.
28. 28. Grundmann H, Aires-de-Sousa M, Boyce J, et al. Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet 2006; 368: 874-885.
29. 29. Kampf G, Lecke C, Cimbal AK, et al. Evaluation of the BBL Crystal MRSA ID System for detection of oxacillin resistance in Staphylococcus aureus. J Clin Pathol 1999; 52:225-227.
30. 30. Diederen B, van Duijn I, van Belkum A, et al. Performance of CHROMagar MRSA Medium for Detection of Methicillin-Resistant Staphylococcus aureus. Clin Microbiol 2005; 43: 1925–1927.
31. 31. Hedin G, Fang H. Evaluation of Two New Chromogenic Media, CHROMagar MRSA and S. aureus ID for Identifying Staphylococcus aureus and Screening Methicillin-Resistant S. aureus. J Clin Microbiol 2005;43:4242–4244.
32. 32. Louie L, Matsumura S. O, Choi E, et al. Evaluation of Three Rapid Methods for Detection of Methicillin Resistance in Staphylococcus aureus. J Clin Microbiol 2000; 38:2170-2173.
33. 33. Levi K, Bailey C, Bennett A, et al. Evaluation of an Isothermal Signal Amplification Method for Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Patient-Screening Swabs. J Clin Microbiol 2003;41:3187-3191.