Comparison of PCR and phenotypic methods for the detection of methicillin resistant Staphylococcus aureus
-
Anitha Madhavan
,
Arun Sachu
,
Anukumar Balakrishnan
,
Anu Vasudevan
,
Sobha Balakrishnan
,
Jayalakshmi Vasudevapanicker
Abstract
Background and Objectives: Resistance to methicillin in methicillin resistant strains of Staphylococcus aureus (MRSA) is due to the presence of mec-A gene ,which encodes a low affinity penicillin binding protein (PBP)-2a or PBP2. Accurate and rapid identification of MRSA in clinical specimens is essential for timely decision on effective treatment. The aim of the study was to compare three different methods for detection of MRSA namely cefoxitin disc diffusion, CHROM agar MRSA and VITEK-2 susceptibility with PCR which is the gold standard reference method and to find the antibiotic susceptibility pattern of these isolates by VITEK-2.
Materials and Methods: A Total of 100 non-duplicate S. aureus isolates were collected from different clinical samples among both outpatient and inpatients. Detection of MRSA among these isolates was done by cefoxitin disc diffusion, VITEK-2, CHROM agar MRSA and PCR.
Results: The sensitivity and specificity of cefoxitin disc diffusion and Vitek was found to be 97.2% and 100%, while that of CHROM agar was found to be 100% and 78.6%. The overall prevalence of MRSA in our study by PCR was 72%.
Conclusion: Based on the findings in our study, isolates which show cefoxitin zone diameter < 22 mm can be reported as MRSA. However, those isolates which have a zone diameter between 22-24 mm, should ideally be confirmed by PCR.
1. Kirby WM. Extraction of a highly potent penicillin inactivator from penicillin resistant staphylococci. Science 1944;99:452-453.
2. KNOX R. A new penicillin (BRL 1241) active against penicillin-resistant staphylococci. Br Med J 1960;2: 690-693.
3. Jevons MP. “Celbenin” - resistant Staphylococci. Br Med J 1961;1:124-125.
4. Otto M. Community-associated MRSA: what makes them special? Int J Med Microbiol 2013;303:324-330.
5. Gopalakrishnan R, Sureshkumar D. Changing trends in antimicrobial susceptibility and hospital acquired infections over an 8 year period in a tertiary care hospital in relation to introduction of an infection control programme. J Assoc Physicians India 2010;58 Suppl:25-31.
6. Patel AK, Patel KK, Patel KR, Shah S, Dileep P. Time trends in the epidemiology of microbial infections at a tertiary care center in west India over last 5 years. J Assoc Physicians India 2010;58 Suppl:37-40.
7. Mbah AN, Isokpehi RD. Identification of functional regulatory residues of the β-lactam inducible penicillin binding protein in methicillin-resistant Staphylococcus aureus. Chemother Res Pract 2013;2013: 614670.
8. Berger-Bächi B, Rohrer S. Factors influencing methicillin resistance in staphylococci. Arch Microbiol 2002;178:165-171.
9. Chambers HF. Methicillin-resistant staphylococci. Clin Microbiol Rev 1988;1:173-186.
10. Hartman BJ, Tomasz A. Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrob Agents Chemother 1986;29:85-92.
11. Cooper BS, Stone SP, Kibbler CC, Cookson BD, Roberts JA, Medley GF, et al. Isolation measures in the hospital management of methicillin resistant Staphylococcus aureus (MRSA): Systematic review of the literature. BMJ 2004;329:533.
12. Towner KJ, Talbot DC, Curran R, Webster CA, Humphreys H. Development and evaluation of a PCR based immunoassay for the rapid detection of methicillin resistant Staphylococcus aureus. J Med Microbiol 1998;47:607-613.
13. Centers for Disease Control and Prevention (CDC). Staphylococcus aureus resistant to vancomycin--United States, 2002. MMWR Morb Mortal Wkly Rep 2002;51:565-567.
14. CLSI (2008). Performance Standards for Antimicrobial Susceptibility Testing, 15th informational supplement, M100-S15. Wayne, PA: Clinical and Laboratory Standards Institute.
15. Hussain Z, Stoakes L, Garrow S, Longo S, Fitzgerald V, Lannigan R. 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 2000;38:2051-2054.
16. Diederen B, van Duijn I, van Belkum A, Willemse P, van Keulen P, Kluytmans J. Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005;43:1925-1927.
17. Nonhoff CG, Mascart MJ, Struelens C, Van Den Borre, Denis O (2004). Detection of hetero-resistant MRSA: controlled comparison of oxacillin/cefoxitin susceptibility testing by disk diffusion, agar screen, Vitek-2 and BD Phoenix automated systems, abstr. P-1630, p. 460. Abstr. 14th European Congress of Clinical Microbiology and Infectious Diseases, Prague, Czech Republic.
18. Lee JH, Jeong JM, Park YH, Choi SS, Kim YH, ChaeJS, et al. Evaluation of the methicillin-resistant Staphylococcus aureus (MRSA)-Screen latex agglutination test for detection of MRSA of animal origin. J Clin Microbiol 2004;42:2780-2782.
19. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.
20. CLSI . Performance standards for antimicrobial susceptibility testing. 29th ed. Clinical and Laboratory Standards Institute; Wayne, PA: 2019. CLSI supplement M100.
21. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: a laboratory manual. New York: Cold Spring Harb. Lab. Press.
22. Unal S, Hoskins J, Flokowitsch JE, Wu CY, Preston DA, Skatrud PL. Detection of methicillin-resistant Staphylococci by using the polymerase chain reaction. J Clin Microbiol 1992;30:1685-1691.
23. Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, et al. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J Clin Microbiol 1995;33:2864-2867.
24. Baddour MM, AbuElKheir MM, Fatani AJ. Comparison of mecA polymerase chain reaction with phenotypic methods for the detection of Methicillin-resistant Staphylococcus aureus. Curr Microbiol 2007;55:473-479.
25. Pramodhini S, Thenmozhivalli PR, Selvi R, Dillirani V, Vasumathi A, Agatha D. Comparison of various phenotypic methods and mecA based PCR for the detection of MRSA. J Clin Diagn Res 2012;5:1359-1362.
26. Krishnan PU, Miles K, Shetty N. Detection of methicillin and mupirocin resistance in Staphylococcus aureus isolates using conventional and molecular methods: a descriptive study from a burns unit with high prevalence of MRSA. J Clin Pathol 2002;55:745-748.
27. Velasco D, Tomas MM, Cartelle M, Beceiro A, Perez A, Molina F, et al. Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus. J Antimicrob Chemother 2005;55:379-382.
28. Pillai MM, Latha R, Sarkar G. Detection of methicillin resistance in Staphylococcus aureus by polymerase chain reaction and conventional methods: a comparative study. J Lab Physicians 2012;4:83-88.
29. Pourmand MR, Hassanzadeh S, Mashhadi R, Askari E. Comparison of four diagnostic methods for detection of methicillin resistant Staphylococcus aureus. Iran J Microbiol 2014;6:341-344.
30. Panda RK, Mahapatra A, Mallick B, Chayani N. Evaluation of genotypic and phenotypic methods for detection of methicillin resistant Staphylococcus aureus in a tertiary care hospital of eastern Odisha. J Clin Diagn Res 2016;10:DC19-21.
31. Datta P, Gulati N, Singhla N, VasudevaH, Bala K, Chander J, et al. Evaluation of various methods for the detection of methicillin-resistant Staphylococcus aureus (MRSA) and their susceptibility pattern. J Med Microbiol 2011;60:1613-1616.
32. Stoakes L, Reyes R, Daniel J, Lennox G, John MA, Lannigan R, et al. Prospective comparison of a new chromogenic medium, MRSASelect, to CHROMagar MRSA and mannitol-salt medium supplemented with oxacillin or cefoxitin for detection of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2006;44:637-639.
33. Roisin S, Nonhoff C, Denis O, Struelens MJ. Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin. J Clin Microbiol 2008;46:2525-2528.
2. KNOX R. A new penicillin (BRL 1241) active against penicillin-resistant staphylococci. Br Med J 1960;2: 690-693.
3. Jevons MP. “Celbenin” - resistant Staphylococci. Br Med J 1961;1:124-125.
4. Otto M. Community-associated MRSA: what makes them special? Int J Med Microbiol 2013;303:324-330.
5. Gopalakrishnan R, Sureshkumar D. Changing trends in antimicrobial susceptibility and hospital acquired infections over an 8 year period in a tertiary care hospital in relation to introduction of an infection control programme. J Assoc Physicians India 2010;58 Suppl:25-31.
6. Patel AK, Patel KK, Patel KR, Shah S, Dileep P. Time trends in the epidemiology of microbial infections at a tertiary care center in west India over last 5 years. J Assoc Physicians India 2010;58 Suppl:37-40.
7. Mbah AN, Isokpehi RD. Identification of functional regulatory residues of the β-lactam inducible penicillin binding protein in methicillin-resistant Staphylococcus aureus. Chemother Res Pract 2013;2013: 614670.
8. Berger-Bächi B, Rohrer S. Factors influencing methicillin resistance in staphylococci. Arch Microbiol 2002;178:165-171.
9. Chambers HF. Methicillin-resistant staphylococci. Clin Microbiol Rev 1988;1:173-186.
10. Hartman BJ, Tomasz A. Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrob Agents Chemother 1986;29:85-92.
11. Cooper BS, Stone SP, Kibbler CC, Cookson BD, Roberts JA, Medley GF, et al. Isolation measures in the hospital management of methicillin resistant Staphylococcus aureus (MRSA): Systematic review of the literature. BMJ 2004;329:533.
12. Towner KJ, Talbot DC, Curran R, Webster CA, Humphreys H. Development and evaluation of a PCR based immunoassay for the rapid detection of methicillin resistant Staphylococcus aureus. J Med Microbiol 1998;47:607-613.
13. Centers for Disease Control and Prevention (CDC). Staphylococcus aureus resistant to vancomycin--United States, 2002. MMWR Morb Mortal Wkly Rep 2002;51:565-567.
14. CLSI (2008). Performance Standards for Antimicrobial Susceptibility Testing, 15th informational supplement, M100-S15. Wayne, PA: Clinical and Laboratory Standards Institute.
15. Hussain Z, Stoakes L, Garrow S, Longo S, Fitzgerald V, Lannigan R. 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 2000;38:2051-2054.
16. Diederen B, van Duijn I, van Belkum A, Willemse P, van Keulen P, Kluytmans J. Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005;43:1925-1927.
17. Nonhoff CG, Mascart MJ, Struelens C, Van Den Borre, Denis O (2004). Detection of hetero-resistant MRSA: controlled comparison of oxacillin/cefoxitin susceptibility testing by disk diffusion, agar screen, Vitek-2 and BD Phoenix automated systems, abstr. P-1630, p. 460. Abstr. 14th European Congress of Clinical Microbiology and Infectious Diseases, Prague, Czech Republic.
18. Lee JH, Jeong JM, Park YH, Choi SS, Kim YH, ChaeJS, et al. Evaluation of the methicillin-resistant Staphylococcus aureus (MRSA)-Screen latex agglutination test for detection of MRSA of animal origin. J Clin Microbiol 2004;42:2780-2782.
19. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.
20. CLSI . Performance standards for antimicrobial susceptibility testing. 29th ed. Clinical and Laboratory Standards Institute; Wayne, PA: 2019. CLSI supplement M100.
21. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: a laboratory manual. New York: Cold Spring Harb. Lab. Press.
22. Unal S, Hoskins J, Flokowitsch JE, Wu CY, Preston DA, Skatrud PL. Detection of methicillin-resistant Staphylococci by using the polymerase chain reaction. J Clin Microbiol 1992;30:1685-1691.
23. Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, et al. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J Clin Microbiol 1995;33:2864-2867.
24. Baddour MM, AbuElKheir MM, Fatani AJ. Comparison of mecA polymerase chain reaction with phenotypic methods for the detection of Methicillin-resistant Staphylococcus aureus. Curr Microbiol 2007;55:473-479.
25. Pramodhini S, Thenmozhivalli PR, Selvi R, Dillirani V, Vasumathi A, Agatha D. Comparison of various phenotypic methods and mecA based PCR for the detection of MRSA. J Clin Diagn Res 2012;5:1359-1362.
26. Krishnan PU, Miles K, Shetty N. Detection of methicillin and mupirocin resistance in Staphylococcus aureus isolates using conventional and molecular methods: a descriptive study from a burns unit with high prevalence of MRSA. J Clin Pathol 2002;55:745-748.
27. Velasco D, Tomas MM, Cartelle M, Beceiro A, Perez A, Molina F, et al. Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus. J Antimicrob Chemother 2005;55:379-382.
28. Pillai MM, Latha R, Sarkar G. Detection of methicillin resistance in Staphylococcus aureus by polymerase chain reaction and conventional methods: a comparative study. J Lab Physicians 2012;4:83-88.
29. Pourmand MR, Hassanzadeh S, Mashhadi R, Askari E. Comparison of four diagnostic methods for detection of methicillin resistant Staphylococcus aureus. Iran J Microbiol 2014;6:341-344.
30. Panda RK, Mahapatra A, Mallick B, Chayani N. Evaluation of genotypic and phenotypic methods for detection of methicillin resistant Staphylococcus aureus in a tertiary care hospital of eastern Odisha. J Clin Diagn Res 2016;10:DC19-21.
31. Datta P, Gulati N, Singhla N, VasudevaH, Bala K, Chander J, et al. Evaluation of various methods for the detection of methicillin-resistant Staphylococcus aureus (MRSA) and their susceptibility pattern. J Med Microbiol 2011;60:1613-1616.
32. Stoakes L, Reyes R, Daniel J, Lennox G, John MA, Lannigan R, et al. Prospective comparison of a new chromogenic medium, MRSASelect, to CHROMagar MRSA and mannitol-salt medium supplemented with oxacillin or cefoxitin for detection of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2006;44:637-639.
33. Roisin S, Nonhoff C, Denis O, Struelens MJ. Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin. J Clin Microbiol 2008;46:2525-2528.
| Files | ||
| Issue | Vol 13 No 1 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i1.5489 | |
| Keywords | ||
| Methicillin resistant Staphylococcus aureus; Cefoxitin; Polymerase chain reaction; Chrom agar; Vitek | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Madhavan A, Sachu A, Balakrishnan A, Vasudevan A, Balakrishnan S, Vasudevapanicker J. Comparison of PCR and phenotypic methods for the detection of methicillin resistant Staphylococcus aureus. Iran J Microbiol. 2021;13(1):31-36.
