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Antibiotic Susceptibility Testing: Epsilometer Tests to Determine MIC Values of Two Antibiotics and Evaluate Antibiotic Synergy

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Antibiotic susceptibility is defined as the sensitivity of a bacteria to antibiotics and can be measured using a broth dilution test or an Epsilometer test, also called an E-test.

In the broth dilution method, a standardized number of bacteria are added to a growth media containing serial antibiotic dilutions. If susceptible, the bacteria cannot grow at the higher antibiotic concentrations but continue to multiply at the lower antibiotic concentrations, causing media to turn turbid. The lowest antibiotic concentration at which the bacteria can no longer survive or multiply is referred to as the minimum inhibitory concentration, or MIC, value of the antibiotic for the given bacteria.

In an E-test, a plastic strip impregnated with a predefined gradient of antibiotic is applied over a freshly spread lawn of bacteria on a Mueller-Hinton agar, or MH-A, Petri plate. The antibiotic diffuses out into the agar media, where it is taken up by the bacteria. If susceptible, the bacteria cannot multiply and will die off, forming a clear zone around the E-strip, which is referred to as the growth inhibition zone. At the point where the growth intersects with the E-strip, the corresponding value on the scale gives the MIC value of the antibiotic.

Often antibiotics are used in combination to prevent the emergence of antibiotic resistant strains of bacteria. This often results in a synergistic, rather than additive, effect. Synergistic means that the combined effect of the two antibiotics is greater than the sum of their individual activities. However, the effect is considered significant only when the MIC value of the antibiotic combination decreases by at least two-fold. This criterion is evaluated by calculating the fractional inhibitory concentration, or FIC, index. By summing the ratio of the MIC of each antibiotic in combination with the MIC of each antibiotic individually, an FIC index less than 0.5 indicates synergy.

Antibiotic synergy can be measured using two E-test based methods: a non-cross test or a cross test. In a non-cross test, first, the E-strips for two different antibiotics with predetermined MIC values are applied to two separate plates. After the antibiotics have diffused into the medium, the original E-strips are removed and the E-strips for the alternate antibiotics are placed such that their MIC scales lay exactly over the MIC scales of the previous strips. In a cross test, which is a faster version of the non-cross test, the E-strips of the two antibiotics are placed together in a cross formation, such that the scales of their MIC marks form a 90 degree angle at the intersection. Following incubation in both techniques, the MIC value of each antibiotic in combination with the other antibiotic is read at the point where the growth inhibition zone intersects with the edge of the E-strip. Then, the FIC index is calculated.

This video will demonstrate how to determine the MIC value of a given antibiotic for a given bacteria using an E-test and a micro broth dilution test. You will also learn how to determine synergy between two antibiotics using a cross test and a non-cross test.

To begin, put on any appropriate personal protective equipment, including laboratory gloves and a lab coat. Next, sterilize the work space using 70% ethanol. Next, collect 15 milliliters of sterile Mueller-Hinton broth with 50% lysed horse blood and 20 milligrams per milliliter beta-nicotinamide. And five to eight Mueller-Hinton agar plates. Now, to prepare a McFarland turbidity standard number 0.5, measure out 9.95 milliliters of 1% sulfuric acid solution. Then, add 50 microliters of 1% barium chloride solution to the sulfuric acid solution. Vortex the solution well to obtain a turbid suspension. Cover the tube with aluminum foil and set it aside. Next, dispense one milliliter of saline solution into a 15 milliliter tube.

Use a sterile loop to scrape up a sample of the bacterial growth from your bacterial test plate, here, Streptococcus group G. Then, place the bacteria-laden loop into the saline solution, stir gently, and then vortex the tube well. Now, place the bacterial suspension and McFarland turbidity standards side by side and compare them for turbidity equivalence. Add either additional saline or bacterial colonies until the bacterial suspension's turbidity matches that of the standard. Once the desired turbidity is obtained, dip a sterile cotton tip applicator into the bacterial suspension. To inoculate the MH-A plate, swab the entire surface of the plate gently with a zigzag motion. Next, label the bottom sides of the plates with the name of the bacteria and the date.

To begin, take out a penicillin G E-test strip, holding it by the edge with forceps. Gently place strip into the center of the freshly swabbed MH-A plate and replace the lid. In this example, a second antibiotic, gentamicin, is also tested. Thus, the strip placement process is repeated with the second plate and a gentamicin E-test strip. To determine the results of the E-test, collect the first plate that contains the penicillin G E-test strip. Now, determine the point where the inhibition zone intersects with the antibiotic strip. Read the corresponding numerical value on the scale. This value represents the MIC value of penicillin G. Determine the MIC value for gentamicin in the same manner.

To begin, inoculate an MH-A plate with Streptococcus group G strain bacteria. Label the bottom of the plate with the name of the bacteria, antibiotics to be used, and the date. Now, place an E-test strip for the antibiotic of interest in the center of the plate. Then, hold the second test strip at a 90 degree angle to the first strip and locate its MIC mark. Gently lay the second E-strip over the first at the point where the two MIC values intersect. Once the strips are placed, do not move them. Next, incubate the plates at 37 degrees celsius for 18 to 20 hours.

After inoculating two MH-A plates, with Streptococcus group G strain bacteria, place an E-test strip for one antibiotic on the surface of one plate. Then, place an E-test strip for the other antibiotic on the second plate as demonstrated. Using a plastic inoculation loop, mark the MIC value of each antibiotic on the surface of its respective plate. Next, cover the plates and incubate them at room temperature for one hour. After this, use forceps to remove the E strips. Next, collect one of the plates and an E-test strip for the other antibiotic. Hold the E-test strip over the imprint left by the first strip and locate the point where the MIC value on the E strip aligns with the marked line. Gently place the strip at this intersecting point. Repeat this process for the second plate and incubate both plates at 37 degrees celsius for 18 to 20 hours.

First, obtain a bacterial suspension with an established bacterial concentration and dilute the culture in MHF broth to achieve an OD600 of 0.003. Next, weigh out 16 milligrams of penicillin G and 128 milligrams of gentamicin. Transfer each weighed dry antibiotic into 215 milliliter conical tubes. Add 10 milliliters of distilled water to each of the conical tubes and mix well by vortexing. Label the tubes with the antibiotic name and concentration.

Performing the assay in triplicate, add 400 microliters of the working bacterial solution into the first wells of three rows of a 96-well microtiter plate. Next, add 200 microliters of the working bacterial solution in MHF broth to the wells of the three rows. Now, to generate a two-fold serial antibiotic dilution, first add four microliters of antibiotic stock to the first well, generating a 100 fold dilution. Sequentially, transfer 200 microliters of bacteria-antibiotic solution to each well, beginning from the first well through the second to last well in each row, ensure proper mixing by pipetting two to three times after every transfer. Discard the final 200 microliters of bacteria-antibiotic solution.

To determine the results of the broth micro dilution test for penicillin G, first locate the wells that exhibit no visible bacterial growth, indicated by a lack of turbidity. From these wells, identify the well with the lowest antibiotic concentration. This represents the MIC value of penicillin G for the tested bacteria. The MIC value of gentamicin can be determined using the same assay and technique.

To determine the results of the non-cross test, collect the first plate, which contains a penicillin G E strip. Then, determine the point where the growth inhibition zone intersects with the antibiotic strip. The corresponding value on the scale represents the MIC value for penicillin G in combination with gentamicin. In this example, the MIC value in combination is 0.064 micrograms per milliliter.

Now, collect the second plate, which contains the gentamicin E strip, and determine the MIC value in combination as previously demonstrated. To evaluate the effect of combination, first calculate the fractional inhibitory concentration or FIC for penicillin G by dividing the MIC in combination by the MIC of the antibiotic alone. Repeat this process for gentamicin. Then, calculate the FIC index using the equation shown here. A two-fold reduction in the MIC value in combination yields an FIC index value that is less than or equal to 0.5 and demonstrates synergy between penicillin G and gentamicin. In this case, the calculated FIC value is 1.18 which is greater than 0.5. Thus, the results do not demonstrate synergy between penicillin G and gentamicin against the Streptococcus group G strain.

To determine the results of the cross test, first determine the point where the growth inhibition zones intersect with their respective E strips. Read the numerical value on each E-test strip that corresponds to this intersection point. These values represent the MIC value in combination for penicillin G and gentamicin. Next, to evaluate the effect of the combination, calculate the FIC index using the equation shown here. In this example, the calculated FIC value is 1.18, which is greater than 0.5. This means that penicillin G and gentamicin do not act synergistically against the Streptococcus group G strain.

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