E-test for Detection of Antimicrobial susceptibility of Vancomycin (Glycopeptides) in Methicillin Resistant Staphylococcus aureus (MRSA)

Abstract

E-test is a quantitative technique for determining the antimicrobial susceptibility of gram positive and gram negative bacteria. The system comprises a predefined antibiotic gradient which is used to determine the MIC (Minimum Inhibitory Concentration), in µg /ml of different antimicrobial agents against microorganisms as tested on agar media using overnight incubation. MIC of a given antibiotic in µg /ml that will inhibit the growth of a particular bacterium under defined experimental conditions. E-test directly quantifies antimicrobial susceptibility in terms of discrete MIC values. However in using a predefined stable and continuous antibiotic concentration gradient, E-test MIC values can be more precise and reproducible than results obtained from conventional procedures. E-test microbial concentration gradient is preformed, predefined and stable and is not dependent on diffusion. MIC of 0.5 µg /ml, 1 µg /ml and 1.5 µg/ml were observed when Vancomycin E-test was used to determine the glycopeptide susceptibility in MRSA (Methicillin Resistant Staphylococcus aureus). I. Introduction The principle of the epsilometer test was first described in 1988 and was introduced commercially in 1991 by AB Biodisk. The Etest method (AB Biodisk, Solna, Sweden) gives an MIC result and is affected by test conditions in a similar way to other MIC and diffusion methods. Evaluations comparing the Etest with dilution MIC and molecular methods have generally found good essential agreement. The Etest has an advantage over other MIC methods. In this it is easy to set up as a disc diffusion test. It is a quantitative test technique, for determining the minimum inhibitory concentration MIC of Anti microbial agents, against micro organisms and for detection of resistance mechanisms. The Etest gradient technology is based on a combination of the concepts of dilution and diffusion principles for susceptibility testing. As with other dilution methods, Etest directly quantifies antimicrobial susceptibility in terms of discrete MIC values. The Etest is basically an agar diffusion method. It consists of plastic strip calibrated with a MIC scale in µg/ml and codes to identify the antimicrobial agent. A predefined concentration gradient antibiotic across 15 two-fold dilutions of a conventional MIC method, is immobilized on the other surface of the carrier. When Etest strip is applied on to an inoculated agar surface the preformed exponential gradient of antimicrobial agent or reagents are immediately transferred to the agar matrix. MIC is read directly from the scale in terms of µg/ml at the point where the edge of the inhibition ellipse intersects the strip other growth patterns may also be seen for resistance detection methods. Although processed like the disc diffusion test i.e. similar inoculums preparation, choice of agar media and incubation conditions. The Etest is a preformed, predefined, stable and it is not dependent on diffusion. To obtain reproducible MICs from a gradient based system, a stability of the gradient must be maintained throughout the critical period when the position of the growth/inhibition edge for a particular bacterium/antibiotic combination is determined due to the stability and precision of the Etest predefined gradient MIC values have been shown to be reproducible and equivalent to those of dilution procedures. The Etest utilizes a rectangular strip that has been impregnated with the drug to be studied. A lawn of bacteria is spread and grown on an agar plate, and the Etest strip is laid on top; the drug diffuses out into the agar, producing an exponential gradient of the drug to be tested. There is an exponential scale printed on the strip. After 24 hours of incubation, an elliptical zone of inhibition is produced and the point at which the ellipse meets the strip gives a reading for the (MIC) of the drug.