Mannitol Salt Agar-Cefoxitin Combination as a screening Medium for Methicillin-Resistant Staphylocuccus aureus.

TITLE

INTRODUCTION

In disk diffusion tests, cefoxitin is now considered a better indicator than oxacillin for the presence of the mecA gene in staphylococcus aureus.

A logical extension of this work is the incorporation of cefoxitin in to media selective for MRSA.

Culture media selective for MRSA have traditionally been based on blood agar , mannitol salt agar or Bird-Parker agar containing oxacillino or methicillin or combination with other antibiotics. These screening media for MRSA have never achieved the acceptable level of sensitivity and specificity.

The paper describes a selective media based on mannitol salt agar containing 4mg/l cefoxitin support the growth of unique collection of mecA positive S aureus and inhibit the growth of mecA- negative strain

Materials and Methods

The following groups of strain were The following groups of strain were tested in this study:tested in this study:

1.1. 100 strain collection of 75 MRSA 100 strain collection of 75 MRSA strain and 25 MSSA strain .strain and 25 MSSA strain .

2. 64 S aureus strain that tested negative 2. 64 S aureus strain that tested negative for the mecA gene ( 14 were for the mecA gene ( 14 were classified as BORSA strain on classified as BORSA strain on account of their elevated MICs for account of their elevated MICs for oxacillin) oxacillin)

3. 8 mecA positive strains which have proved 3. 8 mecA positive strains which have proved difficult or impossible to classify as such by the difficult or impossible to classify as such by the use of cefoxitin disks.use of cefoxitin disks.

4. An additional four MRSA strains that were 4. An additional four MRSA strains that were initially classify as BORSA strains but that were initially classify as BORSA strains but that were later found to be positive for the mceA gene.later found to be positive for the mceA gene.

The presence or absence of mecA gene and nuc gene were determined for all strain by PCR.

The MICs for the 100 strain collection were determined by using Etest on Iso-sensitest agar. (oxoid,Basingstoke,United Kingdom).

MSA plate were prepared with cefioxitin at concentration of 2,3,or 4mg/l.the test strains were applied at concentration of 102 and 105 viable organisms and incubation was carried out in air aerobically at 35C to 37C and the result was read at 18 and 48 h. The growth of any visible colonies after incubation was recorded as a positive result.

RESULT

The MIC result for the 100 collection were as The MIC result for the 100 collection were as follows:follows:

All 25 MSSA MIC of cefoxitin- All 25 MSSA MIC of cefoxitin- 2mg/l. 2mg/l.

All 68 MRSA MIC of cefoxitin- 4mg/l.All 68 MRSA MIC of cefoxitin- 4mg/l.

Seven mecA positive strains MIC of Seven mecA positive strains MIC of cefoxitin – cefoxitin – > 4mg/l.> 4mg/l.

MSA plate with 2mg/l cefoxitin allowed MSA plate with 2mg/l cefoxitin allowed the growth of all MSSA and MRSA strains the growth of all MSSA and MRSA strains after 48h incubation after 48h incubation

MSA plate with 3mg/l cefoxitin allowed MSA plate with 3mg/l cefoxitin allowed the growth of four MSSA strains and all the growth of four MSSA strains and all MRSA strains.MRSA strains.

MSA plate with 4mg/l cefoxitin allowed MSA plate with 4mg/l cefoxitin allowed growth of none of the 25 MSSA and all 75 growth of none of the 25 MSSA and all 75 MRSA strains. MRSA strains.

DISCUSSION Methicillin or oxacillin has been the agent of Methicillin or oxacillin has been the agent of

choice in selective media for mceA-positive for s choice in selective media for mceA-positive for s aureus over 20 years. aureus over 20 years.

The results of this study demonstrated that The results of this study demonstrated that cefoxitin is far superior to oxacillin when it is cefoxitin is far superior to oxacillin when it is used in this particular mannitol salt media . used in this particular mannitol salt media .

By the use of a 48h incubation and a By the use of a 48h incubation and a 10 210 2––CFU/ml inoculam, the sensitivity and specificity CFU/ml inoculam, the sensitivity and specificity of cefoxitin MAS plate method were 100%of cefoxitin MAS plate method were 100%(compared to 90.7% with oxacillin) and 100% (compared to 90.7% with oxacillin) and 100% ( compared to 96.0% for oxacillin) respectively.( compared to 96.0% for oxacillin) respectively.

How ever more frequently patient specimens How ever more frequently patient specimens contain methicillin resistant CoNS .This are capable contain methicillin resistant CoNS .This are capable to grown MAS plate and produce pink colonies to grown MAS plate and produce pink colonies which are easily distinguishable from the yellow which are easily distinguishable from the yellow colonies of mannitol positive S aureus. colonies of mannitol positive S aureus.

In highly prevalence area of MRSA cefoixtin plate In highly prevalence area of MRSA cefoixtin plate can easily detected MRSA and methicilin resistant can easily detected MRSA and methicilin resistant CoNS will not be perceived as a problem. CoNS will not be perceived as a problem.