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Indian Journal of Medical Microbiology, Vol. 27, No. 1, January-March, 2009, pp. 27-29 Original Article Comparison of cefoxitin disc diffusion test, oxacillin screen agar, and PCR for mecA gene for detection of MRSA Anand KB, Agrawal P, Kumar S, Kapila K Department of Microbiology, Armed Forces Medical College, Pune-440 040 Date of Submission: 13-Mar-2008 Code Number: mb09006 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 µg/ml of oxacillin were inoculated and interpreted as per standard guidelines. Cefoxitin disc diffusion test was performed using 30 µg 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 Introduction 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 µg 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 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 µg), amoxicillin/clavulanic acid (20/10 µg), ciprofloxacin (5 µg), erythromycin (15 µg), clindamycin (2 µg), gentamicin (10 µg), oxacillin (1 µg), and vancomycin (30 µg). Oxacillin screen agar Mueller-Hinton agar (MHA) plates containing 4% NaCl and 6 µg/ml of oxacillin were prepared. Plates were inoculated with 10 µL 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 µg 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 µl PCR reaction consisted of plus 45 µl of master mix containing PCR buffer (1X), dNTP mix (0.2 mM of each), primer (0.5 µM), Taq DNA polymerase (0.25 U), and MgCl 2 (1.5 mM) with 5 µL 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 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 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 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
Copyright 2009 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb09006t1.jpg] [mb09006f1.jpg] [mb09006t3.jpg] [mb09006t2.jpg] |
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