Flow cytometry for rapid analysis of bacteriostatic versus bactericidal effects in Legionella pneumophila disinfection

Biocides, encompassing both oxidizing agents such as chlorine and non-oxidizing agents like quaternary ammonium compounds, are extensively employed to minimize the risk of Legionella pneumophila (L. pneumophila) outbreaks in evaporative cooling systems. Currently, biocide effectiveness to combat L. pneumophila is determined by cultivation test methods, which have the disadvantage of long testing times and fail to detect a viable but non-culturable status of bacteria. Cultivation-independent bacterial counting methods would, therefore, be superior for future analysis of biocide effects. Herein, we employed total cell counting and intact cell counting by flow cytometry (FCM) as rapid, cultivation-independent, and untargeted methods for effect-based analysis of biocide effectiveness. We used total cell counting and intact cell counting to detect DNA damage and membrane integrity changes in response to oxidizing sodium hypochlorite (NaClO) and non-oxidizing benzalkonium chloride (BAC). Biocide effect analysis over time was conducted on a dedicated cartridge-based flow cytometer for bacteria analysis, rqmicro.COUNT. The effects of NaClO and BAC on cell membranes and DNA could be well determined and distinguished. Process water contaminated by L. pneumophila was treated with different concentrations of NaClO and BAC, followed by regrowth experiments to determine bacteriostatic versus bactericidal effects. The results showed that a considerable reduction in intact cell count was related to the bacteriostatic effects of the treatment and the success of the disinfection of L. pneumophila according to the standard cultivation method. A significant decrease in total cell count indicating DNA damage was associated with bactericidal effects. Hence, we could show the potential of total cell counting and intact cell counting by FCM to be used as a rapid and effect-based analysis method for minimizing both health-related risks and biocide usage in process water.