Mannitol Salt Agar-Cefoxitin Combination as a Screening Medium for MRSA

1. Mannitol Salt Agar-Cefoxitin Combination as a Screening Medium for MRSA SMYTH, RW and KAHLMETER, G Dept. of Clinical Microbiology, Central Hospital, S-351 85 Växjö, Sweden. The Swedish External Reference Laboratory for Antibiotic Susceptibility Testing SWE AST P 1478 Tel: +46 470 587460 Fax: +46 470 587455 robert.smyth@ltkronoberg.se Results Background Selective culture media for methicillin resistant Staphylococcusaureus (MRSA) has traditionally been based on blood agar, mannitol salt agar or Baird-Parker agar containing methicillin, oxacillin, ciprofloxacin or polymixin B, alone or in combination. An enrichment step consisting of nutrient broth with up to 7% sodium chloride has also been advocated, prior to inoculation of the above selective agars. The reported sensitivity of these media varies from 68% to 100%. For mannitol salt agar containing 1 mg/L oxacillin a sensitivity of 90.7% and a specificity of 96.0% was reported for a collection of mecA positive and negative strains 1. Recent reports 2,3 have shown that a cefoxitin disc may be more reliable for predicting resistance to methicillin in S. aureus. It would therefore seem reasonable to replace oxacillin with cefoxitin in a medium for screening patient specimens for MRSA. The development of such a medium is described. Table I Growth of 79 MRSA and 89 MSSA on MSA containing 4 mg/L cefoxitin after 18 and 48 hours incubation. Material and Methods Mannitol Salt Agar (MSA) DM160D, Mast Diagnostics, Merseyside, UK. Cefoxitin sodium salt, Sigma-Aldrich. E test, AB Biodisk, Solna, Sweden. The strains: 79 mecA positive S. aureus (MRSA) and 89 mecA negative S. aureus (MSSA). This collection consisted of more than 22 different PFGE types (e.g. Berlin IV, Finland E7, France A and B, S German II, UK E1, E15, and E16, Spain E1) and 14 BORSA strains. The development of a selective medium containing an appropriate cefoxitin concentration was carried out in three stages. Firstly the minimum inhibitory concentrations (MIC) of cefoxitin were determined for 75 mecA positive and 25 mecA negative S. aureus strains (fig 1). Secondly, MSA plates containing 2,3 or 4 mg/L cefoxitin were prepared and inoculated with the same collection of strains. Thirdly, and based on the results of stage two (fig 2) a medium containing 4 mg/L cefoxitin was prepared and a total of 79 MRSA strains and 89 MSSA strains were tested, each with two different inocula: 102 and 105 viable organisms (Table I). Incubation was carried out in air at 35-37ºC and for up to 48 hours. Fig 1 Minimum inhibitory concentrations of cefoxitin for 25 MSSA (blue bars) and 75 MRSA (red bars) using E test. References 1. Smyth RW, Kahlmeter G, Olsson Liljequist B, Hoffman B. Methods for identifying methicillin resistance in Staphylococcus aureus. J Hosp Inf (2001) 48: 103 2. Felton A, Grandry B, Lagrange H, Casin I. Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant MRSA. J Clin Microbiol (2002) 40: 2776 3. Skov R, Smyth R, Clausen M et al. Evaluation of a cefoxitin 30 mcg disc on IsoSensitest Agar for detection of MRSA. J Antimicrob Chemother (2003) 52: 204-207 Conclusion Mannitol Salt cefoxitin medium would seem to be superior both in terms of sensitivity and specificity, to screening media containing oxacillin. The majority of the strains tested produced visible colonies after 18 hours incubation, even with an inoculum of 100 cfu. However, an incubation of 48 hours is still required in order to exclude the presence of MRSA. An additional advantage over oxacillin-containing media is that the shelf-life of this medium is a full 30 days at a temperature of 2-8°C. Fig 2 Performance of 25 MSSA and 75 MRSA on MSA containing 2, 3 or 4 mg/L cefoxitin.