A Microbiological Assay for Assessing the Applicability of Advanced Oxidation Processes for Eliminating the Sublethal Effects of Antibiotics on Selection of Resistant Bacteria

Subinhibitory levels of antibiotics in reservoirs highly affected by anthropogenic activity, e.g., wastewater treatment plants, have a profound impact on the development and spread of resistant bacteria in the biosphere. As an aid, advanced oxidation processes (AOPs) have been suggested to eliminate the antibacterial activity of several antibiotics, and this activity has been followed using conventional antibiotic susceptibility tests. While the antibacterial activity can hardly be monitored below the minimum inhibitory concentration (MIC) with these assays, the selective pressure on a bacterial population might remain in this concentration range. To assess the applicability of an AOP for eliminating the subinhibitory effects of antibiotics on selection of resistant bacteria, a microbiological assay is introduced. The test is based on the dynamics of a mixed bacterial population in response to the presence of antibiotics in a concentration range well below the MIC in a synthetic wastewater matrix. Sensitive and resistant subtypes of Staphylococcus aureus in a 1:1 ratio are added to the test medium, and the fraction of resistant mutants is determined after incubation for 24 h by simple colony counting. By using electron beam irradiation as an AOP, we show that the assay provides a simple tool for determining the optimal treatment stage.