14.6 Testing the Effectiveness of Antimicrobials

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing tells clinicians which antibiotics will actually work against a specific bacterial isolate. Without it, treatment is essentially a guess. These tests guide drug selection, proper dosing, and play a direct role in slowing antibiotic resistance by ensuring narrow-spectrum agents are used when possible.

Kirby-Bauer Disk Diffusion Test

The Kirby-Bauer test is the most widely used method for determining whether a bacterium is susceptible or resistant to specific antibiotics. It's relatively simple, inexpensive, and gives clinically useful results.

Here's how it works:

1. A standardized bacterial inoculum is spread evenly across the surface of a Mueller-Hinton agar plate.
2. Paper disks, each impregnated with a specific concentration of a different antibiotic, are placed on the agar surface.
3. The plate is incubated (typically 16–18 hours at 35°C).
4. During incubation, each antibiotic diffuses outward from its disk into the agar, creating a concentration gradient. The concentration is highest right next to the disk and decreases with distance.
5. Susceptible bacteria can't grow near the disk, producing a clear zone of inhibition around it. Resistant bacteria grow right up to the disk.

The diameter of each zone of inhibition is measured in millimeters and compared to standardized interpretive charts published by the Clinical and Laboratory Standards Institute (CLSI). Based on these breakpoints, the bacterium is categorized as:

• Susceptible (S) — the antibiotic is likely to be effective at standard doses
• Intermediate (I) — the antibiotic may work at higher doses or at body sites where the drug concentrates
• Resistant (R) — the antibiotic is unlikely to be effective

A larger zone generally means greater susceptibility. For example, a Staphylococcus aureus isolate might show a large zone around a penicillin disk (susceptible), while MRSA would show little or no zone around the same disk (resistant).

MIC vs. MBC in Antimicrobial Testing

These two values answer different clinical questions: Can we stop the bacteria from growing? And can we actually kill them?

Minimum Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial that prevents visible bacterial growth after overnight incubation. It tells you the minimum amount of drug needed to inhibit the organism. MIC values directly guide drug selection and dosing. For example, if a pathogen has a low MIC for vancomycin, that drug is a strong treatment candidate.

Minimum Bactericidal Concentration (MBC) is the lowest concentration required to kill 99.9% of the original bacterial inoculum. To determine the MBC, you take samples from the MIC tubes/wells that showed no visible growth and subculture them onto fresh agar. If colonies grow, the bacteria were inhibited but not killed at that concentration. The lowest concentration that yields no colony growth (or ≥99.9% killing) is the MBC.

The MBC is always equal to or higher than the MIC. The relationship between the two is clinically meaningful:

• If the MBC is close to the MIC (typically within a 4-fold difference), the drug is considered bactericidal — it kills the bacteria at concentrations close to those that inhibit them. Aminoglycosides like gentamicin and fluoroquinolones like ciprofloxacin tend to be bactericidal.
• If the MBC is much higher than the MIC, the drug is considered bacteriostatic — it stops growth but doesn't efficiently kill. Chloramphenicol is a classic example.

This distinction matters clinically. Bactericidal drugs are generally preferred for serious infections like endocarditis or meningitis, where the immune system alone may not clear inhibited bacteria.

Methods of Antimicrobial Susceptibility Determination

Several laboratory methods exist for determining MIC values. Each has trade-offs in terms of cost, labor, and throughput.

Macrobroth Dilution (Tube Dilution)

1. Prepare a series of test tubes containing broth medium with two-fold serial dilutions of the antimicrobial (e.g., 128, 64, 32, 16, 8, 4, 2, 1, 0.5 µg/mL).
2. Add a standardized bacterial inoculum to each tube.
3. Incubate overnight.
4. The MIC is the lowest concentration showing no visible turbidity (no bacterial growth).

This method is labor-intensive and uses large volumes of reagents, so it's more common in research settings or for confirmatory testing than in routine clinical labs.

Microdilution

1. Use a 96-well microtiter plate to set up serial dilutions of one or more antimicrobials in small volumes.
2. Add a standardized bacterial inoculum to each well.
3. Incubate overnight.
4. The MIC is the lowest concentration that inhibits visible growth.

Microdilution is the most commonly used method in clinical labs. The 96-well format allows testing of multiple antibiotics against the same isolate simultaneously, and the process is easily automated.

Etest (Antimicrobial Gradient Method)

1. Inoculate a standardized bacterial suspension onto an agar plate (similar to Kirby-Bauer setup).
2. Place a plastic strip impregnated with a continuous gradient of antimicrobial concentrations onto the agar.
3. Incubate overnight. The antibiotic diffuses into the agar, and an elliptical zone of inhibition forms around the strip.
4. Read the MIC where the edge of the elliptical zone intersects the concentration scale printed on the strip.

The Etest is particularly useful for fastidious or slow-growing organisms like Streptococcus pneumoniae, where broth-based methods can be unreliable. It combines the simplicity of a diffusion test with the quantitative precision of an MIC value.

Agar Dilution

1. Prepare a series of agar plates, each containing a different concentration of the antimicrobial mixed into the agar.
2. Inoculate a standardized bacterial suspension onto each plate.
3. Incubate and observe for growth.
4. The MIC is the lowest concentration that inhibits visible growth.

This method is useful for testing many bacterial isolates against a single antibiotic at once, but preparing the plates is time-consuming.

Antibiotic Resistance

Antibiotic resistance occurs when bacteria develop mechanisms that allow them to survive exposure to antibiotics that would normally inhibit or kill them. Understanding resistance is the reason susceptibility testing exists in the first place.

Resistance can be intrinsic (a natural property of the organism — for example, gram-negative bacteria are inherently resistant to vancomycin because the drug can't penetrate their outer membrane) or acquired through genetic mutations or horizontal gene transfer (transformation, transduction, or conjugation).

Common resistance mechanisms include:

• Enzymatic inactivation — bacteria produce enzymes that destroy or modify the drug (e.g., beta-lactamases breaking down penicillin)
• Target modification — the drug's binding site is altered so it no longer recognizes the target (e.g., altered PBPs in MRSA)
• Efflux pumps — bacteria actively pump the antibiotic out of the cell before it can act
• Decreased permeability — changes in porins or outer membrane structure reduce drug entry

Resistance contributes to treatment failures and the emergence of multidrug-resistant organisms (MDROs), making susceptibility testing and antimicrobial stewardship essential tools in clinical practice.