In-situ headspace analysis of metabolic carbon dioxide of aerobic bacteria for assessing antimicrobial activity of natural products

Abstract

Headspace analysis of the volatile metabolites has been used as a good strategy for monitoring the microbial growth in several applications, but never been established for the evaluation of the activity of microbial inhibitor in the field of drug screening from natural products. We report on a new method for the determination of antimicrobial activity of drug compounds or crude extracts from natural products by measuring the amount of metabolic carbon dioxide produced in the drug-bacteria incubation system. 2 mL of medium containing bacteria and drug of interest was incubated at 37 °C for 24 h. The amount of metabolic carbon dioxide partitioned in the headspace was measured to evaluate the drug antimicrobial activity using headspace gas chromatography (HS-GC) coupled with thermal conductive detector (TCD). The principle and the standard procedure of the present method have been developed and verified. As a result, the precision of the present method was less than 4% (expressed as relative standard deviation), and an excellent agreement was found on both inhibition rate (R2 = 0.935) and the half inhibition concentration (R2 = 0.994) between the present method and a reference method (optical density method). By comparison, the present method is simpler and safer regarding the microorganism contamination due to the in-situ incubation and detection in the closed system, and suitable for the routine analysis of antimicrobial activity of natural products with high flexibility in both bacterial strains and sample properties.