Wastewater treatments minimize transmission of pathogens, and are required by the EPA with established treatment and monitoring requirements. The efficiency of treatment processes is determined by measuring the inactivation of indicator organisms (e.g., fecal coliforms) with culture-based techniques. Recently, Clostridium perfringens spores have been identified as excellent indicator organisms to assess the efficiency of wastewater disinfection processes, because they are ubiquitous in fecal matter (~104 CFU per gram) and are the resilient towards the sterilization regimens that readily kill water borne pathogens. Therefore, demonstration of C. perfringens spores inactivation verifies inactivation of less resilient disease-causing organisms. Currently, Clostridium spore inactivation is quantified by measuring the log reduction of culturability (MPN or CFU). These methods, however, require several days of incubation, before cloudy growth tubes or visible colonies can then be enumerated. Moreover, culture-based methods require manualsample handling and analysis and are not amenable for an automated, inline monitoring of wastewater treatment efficiency. In contract, fast germinable endospore biodosimetry (GEB) method was demonstrated by Ponce et al. at Caltech/JPL for monitoring sterilization of spacecraft (NASA funded) and pathogen decontamination efficiency (Department of Homeland Security funded) on the timescale of minutes. We have shown single spore detection, no false positives or negatives with GEB to enable sterility assessment. GEB was applied to monitor spore inactivation as a function of thermal, UV and vaporized H2O2 dosages in comparison to standard culture methods, which showed that GEB provides a 6-log sterility assurance level. §Here we will validate GEB for assessing C. perfringens spore viability in wastewater treatment facility samples. We anticipate that in Phase I, GEB will prove to be areliable for assessing C. perfringens spore viability in comparison to current state-of-practice methods (culturing). These results will serve as an excellent starting point for Phase II development and demonstration of a fully automated, inline GEB instrument intended for near real time monitoringof wastewater treatment efficiencies. Beyond the sterility assurance applications for NASA spacecraft bioburden reduction, DHS pathogen decontamination and EPA wastewater treatment efficiency monitoring, we envision that GEB will become and essential tool in the fight against microbial contamination in pharmaceutical, health care and food industries.
