Determination of the susceptibility of a bacterial pathogen to antimicrobial drugs can be done by one of the two principal methods: dilution or diffusion. It is important to use a standardized method that controls for all the factors that affect antimicrobial activity. In the United States, the tests are per formed according to the methods of the Clinical and Laboratory Standards Institute (CLSI).

Using an appropriate standard test organism and a known sample of drug for comparison, these methods can be used to estimate either the potency of antibiotic in the sample or the susceptibility of the microorganism.

Dilution Method

Graded amounts of antimicrobial substances are incorporated into liquid or solid bacteriologic media. Commonly, twofold (log₂) dilutions of the antimicrobial agents are used. The media are subsequently inoculated with test bacteria and incubated. The end point is taken as that amount of antimicrobial substance required to inhibit the growth of—or to kill—the test bacteria. Agar dilution susceptibility tests are time consuming, and their use is limited to special circumstances. Broth dilution tests were cumbersome and little used when dilutions had to be made in test tubes; however, the advent of prepared broth dilution panels for many different drugs in manual or automated microdilution plates (eg, Vitek 2 instrument and bioMerieux, Inc.) has greatly enhanced and simplified the method. The advantage of microbroth dilution tests is that they permit a quantitative result to be reported, indicating the amount of a given drug necessary to inhibit (minimum inhibitory concentration, MIC) or kill (minimum bactericidal concentration, MBC) the microorganisms tested.

Diffusion Method

A widely used method in smaller laboratories is the disk diffusion test. A filter paper disk containing a measured quantity of a drug is placed on the surface of a solid medium that has been inoculated on the surface with the test organism. After incubation, the diameter of the clear zone of inhibition surrounding the disk is taken as a measure of the inhibitory power of the drug against the particular test organism. This method is subject to many physical and chemical factors in addition to the simple interaction of drug and organisms (eg, the nature of the medium and diffusibility, molecular size, and the stability of the drug). Nevertheless, standardization of conditions permits determination of the susceptibility of the organism.

Interpretation of the results of diffusion tests must be based on comparisons between dilution and diffusion methods. Such comparisons have led to the establishment of reference standards. Linear regression lines can express the relationship between log of minimum inhibitory concentration in dilution tests and diameter of inhibition zones in diffusion tests.

Use of a single disk for each antibiotic with careful standardization of the test conditions permits the report of susceptible or resistant for a microorganism by comparing the size of the inhibition zone with a standard of the same drug. Inhibition around a disk containing a certain amount of antimicrobial drug does not imply susceptibility to that same concentration of drug per milliliter of medium, blood, or urine.

An alternative to using disks are plastic strips with a predefined gradient of antibiotic covering a continuous con centration range. Printed on the strip is a scale with values that correspond to the concentration of antibiotic forming the gradient. When placed on an agar dish inoculated with a lawn of test bacteria, the antibiotic diffuses out in a concentration dependent fashion from the plastic strip and growth is inhibited. Rather than producing a circular zone of inhibition, an elipse is formed, hence the name of the test, an Epsilometer test (or Etest). The MIC value is read from the scale in terms of µg/mL where the ellipse edge intersects the strip.

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مواضيع ذات صلة

Antimicrobial Chemoprophylaxis

Drug–Pathogen Relationships

Clinical Implications of Drug Resistance

Origin of drug resistance

Resistance to Sulfonamide

Control of Antibiotics Overuse

International Distribution of Antibiotics: A Scandinavian Example

Rediscovery of Penicillin by a Basic Scientific Approach

Penicillin: the First Antibiotic

Sulfonamide: the First Antibacterial Agent Acting Selectively

Drug elimination

Drug distribution