Wastewater treatments minimize the transmission of pathogens and are required by 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 resilient toward the sterilization regimens that readily kill water-borne pathogens. Therefore, demonstration of C. perfringens spores inactivation verifies the 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 manual sample handling and analysis and are not amenable for an automated, inline monitoring of wastewater treatment efficiency. In contrast, the 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 [DHS] 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 a reliable tool 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 monitoring of 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 an essential tool in the fight against microbial contamination in pharmaceutical, health care and food industries.

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