title: Quantification of viable but nonculturable bacterial pathogens in anaerobic digested sludge.
abstract: 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.
authors.affiliation: Laboratory of Environmental Biotechnology, School of Environmental and Civil Engineering, Jiangnan University, Wuxi 214122, Jiangsu, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jiang
authors.forename: Bo
authors.initials: B
authors.lastname: Fu
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: He
authors.initials: H
authors.lastname: Liu
mesh_terms: D000693:Anaerobiosis; D058491:Bacterial Load / Q000379:methods*; D004926:Escherichia coli / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012486:Salmonella typhimurium / Q000302:isolation & purification*; D012722:Sewage / Q000382:microbiology*; D012763:Shigella flexneri / Q000302:isolation & purification*
chemical_list: D012722:Sewage