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 ~  Abstract
 ~  Introduction
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Conclusion
 ~  References
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ORIGINAL ARTICLE
Year : 2009  |  Volume : 27  |  Issue : 1  |  Page : 27-29
 

Comparison of cefoxitin disc diffusion test, oxacillin screen agar, and PCR for mecA gene for detection of MRSA


Department of Microbiology, Armed Forces Medical College, Pune-440 040, India

Date of Submission13-Mar-2008
Date of Acceptance18-Aug-2008

Correspondence Address:
S Kumar
Department of Microbiology, Armed Forces Medical College, Pune-440 040
India
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PMID: 19172055

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 ~ Abstract 

Cefoxitin is a potent inducer of the mecA regulatory system. It is being recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. The aim of our study was to evaluate the efficacy of cefoxitin disc diffusion test to characterize MRSA and compare it with oxacillin agar screening and detection of mecA gene by PCR. Materials and Methods: Fifty strains of S. aureus isolated from clinical samples were used in the study. Routine antibiotic susceptibility testing was performed including oxacillin disk. Oxacillin screen agar plates with 4% NaCl and 6 ”g/ml of oxacillin were inoculated and interpreted as per standard guidelines. Cefoxitin disc diffusion test was performed using 30 ”g disc and zone sizes were measured. PCR for amplification of the mecA gene was performed. Results: Out of the 50 isolates, 28 were found to be methicillin resistant by oxacillin disc diffusion test, 30 were resistant by oxacillin screen agar method, and 32 were resistant with cefoxitin disc diffusion. For these 32 isolates mecA gene was positive. Conclusion: Results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene. Thus, the test can be an alternative to PCR for detection of MRSA in resource constraint settings.


Keywords: mecA gene, MRSA, cefoxitin


How to cite this article:
Anand K B, Agrawal P, Kumar S, Kapila K. Comparison of cefoxitin disc diffusion test, oxacillin screen agar, and PCR for mecA gene for detection of MRSA. Indian J Med Microbiol 2009;27:27-9

How to cite this URL:
Anand K B, Agrawal P, Kumar S, Kapila K. Comparison of cefoxitin disc diffusion test, oxacillin screen agar, and PCR for mecA gene for detection of MRSA. Indian J Med Microbiol [serial online] 2009 [cited 2014 Sep 19];27:27-9. Available from: http://www.ijmm.org/text.asp?2009/27/1/27/45164



 ~ Introduction Top


Methicillin resistant Staphylococcus aureus (MRSA) strains emerged soon after the introduction of methicillin into clinical practice. In addition to being a nosocomial pathogen, MRSA has become a community pathogen. Strains that possess mecA gene are either heterogeneous or homogeneous in their expression of resistance. The heterogeneous expression occasionally results in minimal inhibitory concentrations that appear to be borderline and consequently the isolates may be interpreted as susceptible. [1]

In the recent past, there have been multiple reports on the use of cefoxitin as a surrogate marker for detection of mecA- gene-mediated methicillin resistance. [2],[3],[4] Cefoxitin is a potent inducer of the mecA regulatory system. [4] The Clinical and Laboratory Standards Institute (CLSI) guidelines (2006) has recommended cefoxitin disc diffusion method for the detection of MRSA. This is performed by using a 30 ”g cefoxitin disc and an inhibition zone diameter of ≤19 mm is reported as methicillin resistant and ≥20 mm is considered as methicillin sensitive. [5]

The aim of our study was to evaluate the efficacy of cefoxitin disc diffusion test to detect methicillin resistance in S. aureus and compare it with oxacillin agar screening and detection of mecA gene by PCR, which is considered as the gold standard.


 ~ Materials and Methods Top


A total of 50 strains of S. aureus isolated from clinical samples were used in the study. Confirmation of the strains was done using standard tests like catalase, slide and tube coagulase, and growth on Mannitol salt agar. Routine antibiotic susceptibility testing was performed by Kirby-Bauer disc diffusion method for the following antibiotics: ampicillin (10 ”g), amoxicillin/clavulanic acid (20/10 ”g), ciprofloxacin (5 ”g), erythromycin (15 ”g), clindamycin (2 ”g), gentamicin (10 ”g), oxacillin (1 ”g), and vancomycin (30 ”g).

Oxacillin screen agar

Mueller-Hinton agar (MHA) plates containing 4% NaCl and 6 ”g/ml of oxacillin were prepared. Plates were inoculated with 10 ”L of 0.5 Mc Farland suspension of the isolate by streaking in one quadrant and incubated at 35 0 C for 24 h. Plates were observed carefully in transmitted light for any growth. Any growth after 24 h was considered oxacillin resistant. [6],[7]

Cefoxitin disc diffusion test

All the isolates were subjected to cefoxitin disc diffusion test using a 30 ”g disc. A 0.5 Mc Farland standard suspension of the isolate was made and lawn culture done on MHA plate. Plates were incubated at 37 0 C for 18 h and zone diameters were measured. An inhibition zone diameter of ≤ 19 mm was reported as oxacillin resistant and ≥20 mm was considered as oxacillin sensitive. [8]

Quality control strains - methicillin sensitive S. aureus (MSSA) ATCC 25923 and methicillin resistant S. aureus (MRSA) ATCC 43300 - were used as negative and positive controls, respectively. [5]

PCR amplification of the mecA gene

DNA extraction was performed by QIAamp DNA minikit (QIAGEN). The mecA gene was amplified using the primers as described by Geha et al , [9] and given in [Table 1]. A 50 ”l PCR reaction consisted of plus 45 ”l of master mix containing PCR buffer (1X), dNTP mix (0.2 mM of each), primer (0.5 ”M), Taq DNA polymerase (0.25 U), and MgCl 2 (1.5 mM) with 5 ”L of template DNA. Cycling conditions were - hot start at 94 0 C for 4 minutes followed by 30 cycles of denaturation at 94 0 C for 45 seconds, annealing at 50 0 C for 45 seconds, and extension at 72 0 C for 1 minute and final extension step at 72 0 C for 3 minutes. PCR products were visualized on 2% agarose gel with ethidium bromide dye under UV transilluminator. Amplicons of 310 bp were consistent with mecA gene amplification [Figure 1].


 ~ Results Top


Among 50 S . aureus strains, 28 were MRSA and 22 were MSSA by routine disc diffusion test using oxacillin disk. Thirty were MRSA and 20 were MSSA in oxacillin agar screening. Thirty two were resistant with cefoxitin disc diffusion test and in these 32 isolates mecA gene was detected.The results for inhibition zone diameters by cefoxitin disc diffusion are given in [Table 2]. The sensitivity and specificity of the three phenotypic tests as compared with genotypic test are given in [Table 3].


 ~ Discussion Top


Detection of mecA gene or its product, penicillin binding proteins (PBP2a), is considered the gold standard for MRSA confirmation. [10] Recent studies indicate that disc diffusion testing using cefoxitin disc is far superior to most of the currently recommended phenotypic methods like oxacillin disc diffusion and oxacillin screen agar testing and is now an accepted method for the detection of MRSA by many reference groups including CLSI. [11] The accurate and early determination of methicillin resistance is of key importance in the prognosis of infections caused by S. aureus . In this study, we attempted to evaluate different methods for detection of mecA .

During the last several years, the CLSI-AST has attempted to improve the accuracy of detecting mecA -positive strains of S. aureus . In our study, the observations using the CLSI disc diffusion criteria to define resistance (cefoxitin zone diameters of ≤19 mm for resistance and ≥20 mm for sensitivity), the sensitivity and specificity were 100% in the 50 strains tested in the study whereas the results of oxacillin screen agar were not so accurate. Results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene [Table 2], and thus the cefoxitin disk diffusion method is very suitable for detection of MRSA and the test can be an alternative to PCR for detection of MRSA in resource constraint settings.

From a clinical perspective, it is important to differentiate isolates that have mecA -positive resistance from the infrequently encountered isolates that have borderline resistance because it may affect therapy. Strains that possess mecA -classic resistance are either heterogenous or homogenous in their expression of resistance. It is the testing of heteroresistant isolates which may appear as susceptible. The presence of resistance in S. aureus isolates on an oxacillin screen agar plate generally means that the isolate is mecA positive. Occasionally, however, heteroresistant mecA -positive strain is not detected due to low expression of resistance. Oxacillin agar screen generally does not detect borderline resistant strains, when studies have included strains whose resistance is heterogeneous the test has been shown to perform less well. Also, agar dilutions and oxacillin disc diffusion method may be affected by various components of MHA, temperature, and duration of incubation. [12]


 ~ Conclusion Top


Although the number of isolates are less, this study provides an evidence that cefoxitin can be used as an accurate surrogate marker in routine susceptibility testing at 37 0 C for 18-24 hours. In addition, the results have shown 100% sensitivity and specificity as compared to mecA gene detection by PCR. Hence, it can be used as an alternative to the technically demanding PCR.

 
 ~ References Top

1.Swenson JM, Patel JB, Jorgensen JK. Special phenotypic methods for detecting antibacterial resistance, Chapter 74. In: Murray PR, Baron ES, Jorgensen JH, Landry ML Pfaller MA, editors. Manual of Clinical Microbiology, 9th ed. Washington DC: ASM Press; 2007. p. 1175-76.   Back to cited text no. 1    
2.Felten A, Grandy B, Lagrange PH, Casin I. Evaluation of three techniques for detection of low level methicillin-resistant Staphylococcus aureus (MRSA): A disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test. J Clin Microbiol 2002;40:2766-71.  Back to cited text no. 2    
3.Swenson JM, Tenover FC; the Cefoxitin Disk Study Group. Results of disk diffusion testing with cefoxitin correlate with presence of mecA in Staphylococcus spp. J Clin Microbiol 2005;43:3818-23.  Back to cited text no. 3    
4.Swenson JM, Lonsway D, McAllister S, Thompson A, Jevitt L, Patel JB. Detection of mecA-mediated Resistance Using Cefoxitin Disk Diffusion (DD) in a Collection of Staphylococcus aureus Expressing Borderline oxacillin MICs. Diagn Microbiol Infect Dis 2007;58:33-9.   Back to cited text no. 4    
5.Clinical and Laboratory Standards Institute / NCCLS Performance standards for Antimicrobial disc diffusion tests; Approved standards. 9th ed. CLSI Document M2-M9. Wayne Pa: Clinical and Laboratory Standards Institute; 2006.  Back to cited text no. 5    
6.Isenberg HD, editor. Clinical microbiology procedures hand book. 2 nd ed. Washington DC: ASM Press; 2004.  Back to cited text no. 6    
7.Brown DF, Edwards DI, Hawkey PM, Morrison D, Ridgway GL, Towner KJ, et al . Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphyloccocus aureus (MRSA). J Antimicrob Chemother 2005;56:1000-18.  Back to cited text no. 7    
8.Swenson JM, Spargo J, Tenover FC, FerraroM J. Optimal inoculation methods and quality control for the NCCLS oxacillin agar screen test for detection of oxacillin resistance in Staphylococcus aureus . J Clin Microbiol 2001;39:3781-4.  Back to cited text no. 8    
9.Geha DJ, Uhl JR, Gustaferro CA, Persing DH. Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory. J Clin Microbiol 1994;32:1768-72.  Back to cited text no. 9    
10.Skov R, Smyth R, Larsen AR, Bolmstrτm A, Karlsson A, Mills K, et al . Phenotypic detection of methicillin resistance in Staphylococcus aureus by disk diffusion testing and etest on Mueller-Hinton agar. J Clin Microbiol 2006;44:4395-9.  Back to cited text no. 10    
11.Skov R, Smyth R, Clausen M, Larsen AR, Frimodt-Møller N, Olsson-Liljequist B, et al. Evaluation of a cefoxitin 30 μg disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus . J Antimicrob Chemother 2003;52:204-7.  Back to cited text no. 11    
12.Fernandes CJ, Fernandes LA, Collignon P; Australian Group on Antimicrobial Resistance. Cefoxitin resistance as a surrogate marker for the detection of methicillin-resistant Staphylococcus aureus . J Antimicrob Chemother 2005;55:506-10.  Back to cited text no. 12    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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