Quantification of viable but nonculturable bacterial pathogens in anaerobic digested sludge

Enteric bacterial pathogens in sewage sludge easily become viable but nonculturable (VBNC) during anaerobic digestion, which escape detection by standard culture methods and pose a potential health risk. In this study, a method that is combining the standard culture method with the reverse transcription quantitative PCR (RT-qPCR) assay was developed for the quantification of bacterial pathogens in the VBNC state. The cycle threshold (CT) values from RT-qPCR assays were linear to the bacterial number in the range from 10⁹ to 10² most probable number (MPN) per reaction for Escherichia coli (R²=0.9964) and Salmonella typhimurium (R²=0.9938) and from 10⁹ to 10⁴ MPN per reaction for Shigella flexneri (R²=0.997), respectively. Mesophilic anaerobic digestion (MAD) caused the bacterial pathogens in sewage sludge entering into VBNC state with the incidence indexes of 0.01-1.12 for E. coli, 2.48-436.52 for S. typhimurium, and 4.17-6.61 for S. flexneri, respectively. Given different VBNC incidence indexes of bacterial pathogens in sewage sludge by MAD, the quantification results of VBNC pathogens using RT-qPCR could provide an improved evaluation of pathogen inactivation efficiency and biological safety in sludge anaerobic digestion.