Post Antibiotic Effect: The Importance of Assessing Microbial Response After an Initial Antimicrobial Challenge
When people hear the words “antibiotic”, they often imagine a drug killing harmful bacteria when the two come into contact. But due to factors such as drug toxicity, host metabolism, etc. it is not possible to maintain continuous exposure of a drug against the target microorganism in the body, and multiple doses are required. How surviving bacteria respond after an initial antibiotic exposure, and how that informs subsequent drug dosing is called Post Antibiotic Effect (PAE).
Direct antimicrobial activity assays such as Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) only provide information on a single exposure of the bacteria to a drug. While this may provide information on an antibiotic’s efficacy, it provides little data on the effect of the drug on bacteria over time, also known as the antibiotic’s pharmacodynamics. PAE studies help to fill in the gaps related to an antibiotic’s pharmacodynamics and is an important factor in designing the clinical dosing regimen of antimicrobial drug. If bacteria grow back over a long period of time (i.e. long PAE) dosing is less frequent, while continuous dosing or frequent dosing is required for short PAE.
At Emery Pharma, PAE is determined in-vitro using the Standard Viable Count Method. A liquid culture with a fixed inoculum count is exposed to a concentration of drug for a specified time. After the given time, the drug is removed by dilution in fresh medium. The time it takes for the culture density to increase by 1 log10 CFU is measured. The post-antibiotic effect is the additional time needed by a culture that was treated with an antibiotic to increase in number (CFU) by 1 log10 relative to untreated controls. (see illustration below)
PAE = T – C
T = Time it takes drug exposed culture to increase in titer 1 log
C = Time it takes control culture to increase in titer 1 log
Figure legend. A sample post-antibiotic effect assay result is depicted. A bacteria culture is treated with Compound 1, Compound 2, or left untreated as a growth control. The bacteria are exposed to the test compounds for 1 hour, after which the compounds are removed by dilution in fresh media. The regrowth is monitored over a number of hours, after which the PAE is determined. In this example, the untreated growth control regrew to 1 log in 1 hour. Using the formula of PAE = T - C, Compound 1 has a PAE of (3h - 1h) = 2 hours. In contrast, Compound 2 has a PAE of (1h - 1h) = <1 hour. Compound 1 therefore has a better PAE as it took the bacteria longer to recover from the inhibitory effects of Compound 1 compared to Compound 2.
Some important factors to consider when starting a PAE study include strain selection and evaluating the role of serum in testing. Test strains used in PAE studies should be a mix of standard strains (for example, from ATCC) that reflect the behavior of naive, wild-type bacteria to the antibiotic. Relevant drug resistant strains should also be assayed to determine what effect antibiotic resistance has on PAE.
Serum is another important factor to consider in PAE testing. PAE testing is generally performed in a standardized bacteriological media such as Muller-Hinton broth (MHB) for reproducibility and consistency. However, the composition of bacteriological media is very different from human physiological makeup. PAE studies performed using pooled human serum can better reflect the interaction of an antibiotic with bacteria in a more physiologically relevant model. This can be taken a step further using heat-inactivated serum, in which heat-labile proteins such as the innate immune system’s complement factors are inactivated.
PAE studies contribute to clinical dosing decisions and are an important step in the antibiotic development pipeline. Emery Pharma has a bank of nearly 2,000 microbial strains, many of which are drug resistant strains, that could be used in the study of PAE. Contact us today and we can help you bring your product to the market!
Reference:
Lorian 4th edition (1996) Chapter 8, Post Antibiotic Effect in Antibiotics in Laboratory Medicine, pp 296-329. Williams and Wilkins, Baltimore, Md.
