Antimicrobials

Antimicrobial Effectiveness and Sensitivity

Tests for Antimicrobial Effectiveness

The disk diffusion method, ETEST, dilution method, serum killing power, and new automated methods can be used to determine a microbe's sensitivity to particular antimicrobials.

Tests that assess the effectiveness of antimicrobial chemicals are useful clinical and research tools. Researchers need to know which microbial strains are susceptible to new drugs and need to determine the therapeutic dose, the minimum quantity of a drug necessary to treat a patient.

The disk diffusion method, or Kirby-Bauer test, allows for testing large numbers of antimicrobial chemicals against a microbial strain. Paper disks are infused with a set concentration of antimicrobial. A broth of the microbe is smeared across growth media in a petri dish, and disks with the antimicrobials are placed on top. The antimicrobials in the disks diffuse into the surrounding media. The whole dish is incubated to allow the microbe to grow, and effective antimicrobials create a circular area around the disk known as the zone of inhibition, where the microbe does not grow.
The disk diffusion method, or Kirby-Bauer test, is a method for determining the susceptibility of microorganisms to different antibiotic and disinfectant chemicals.
The epsilometer test (or ETEST) allows for the determination of the lowest concentration of an antimicrobial necessary to stop microbial growth, or the minimum inhibitory concentration (MIC). The ETEST uses a strip of plastic with a concentration gradient of antimicrobial that is placed on a petri dish prepared similarly to the disk diffusion method. After incubation, a zone of no growth is apparent around the ETEST strip, and the MIC is read off of the strip.
The ETEST strip contains a continuous range of antimicrobial chemical, from low concentration at the bottom of the strip to high concentration at the top.
Credit: Khan et al.; licensee BioMed Central Ltd.License: CC BY 2.0
The dilution method is also used to determine the minimum inhibitory concentration by diluting an antimicrobial in solution until the tested microbe is able to survive and reproduce. To perform the dilution method, the antimicrobial is added to tubes containing a liquid broth growth medium in a series of concentrations. The microbial strain is then added and incubated. Microbial growth produces a foggy appearance in the broth, allowing easy determination of the MIC. The dilution method can also be used to determine the minimum bactericidal concentration (MBC), or the lowest concentration of an antimicrobial necessary to kill 99.9% of bacterial cells. In this procedure a uniform quantity of bacterial cells are added to a dilution series of the antimicrobial, incubated, and then tested to determine how many cells retain the ability to grow when returned to an optimal growth environment. There are now several commercially available systems operating on similar principles to the dilution method that can automatically determine MIC or MBC. These systems reduce errors, allow testing of large numbers of samples, and free up clinicians or researchers.

While various tests are performed on microbes found in laboratory settings, testing the effectiveness of antimicrobials given to patients is equally important. A common test takes a blood sample from a patient on an antimicrobial course, and the serum containing the antimicrobial in the blood is separated. The serum is diluted many times, a uniform dose of the pathogen is added, and the samples are incubated. The lowest concentration of the patient's serum that effectively inhibits the pathogen's growth is the serum killing power.

Ideal Attributes of Antimicrobials

The ideal antimicrobial must be soluble in body fluids, be toxic to microbe but not host, not be allergenic, be stable under host body conditions, and have a toxicity that is not easily altered.

Effective antimicrobials must meet a wide range of conditions. In order to be effective in a patient, the chemical must be able to move through the patient's body to the sites of infection. With the exception of those used on topical infections, antimicrobials must have solubility in blood and other body fluids for transport to the infection. Once at the sites of infection, the antimicrobial must enter infected host tissues and cells to reach the microbial pathogens. In the process of transport through the body, the antimicrobial needs to remain chemically stable and intact.

Many chemicals are modified when circulated in the body. Antimicrobials that are modified in the patient lose effectiveness, and the modifications may then prove toxic to the patient. Therefore, chemical stability is imperative and needs selective toxicity. A chemical with selective toxicity must kill the microbe or inhibit microbial growth while exhibiting minimal toxicity to the patient. Antimicrobials should have few or no side effects to the patient and should not produce allergic responses. An effective antimicrobial should eliminate the entire pathogen population in the patient without any persister cells remaining after treatment.

A persister cell is a bacterial cell susceptible to an antibiotic that survives through a course of the antibiotic. Patients are advised to fully complete their course of antibiotics to guard against persister cells and recurring infections that are less susceptible to the antibiotic. Ideally, the antimicrobial treatment should not disturb the patient's resident microbial microflora, though very few meet this criterion, and some can make patients vulnerable to secondary infection. Additionally, the monetary cost of the antimicrobial should be low enough that it is accessible to patients.

The ideal antimicrobial does not exist. An antimicrobial that is ideal in one context for treatment of one pathogen will be different than in other circumstances. Clinicians and physicians must weigh the effectiveness of an antimicrobial for a given pathogen against potential side effects, allergic reactions, and cost.