Time to revisit pathogen monitorization in full-scale wastewater treatment plants

Full scale wastewater treatment plants are usually considered to perform well in general removal of bacteria coming in with sewage. Therefore it is assumed that bacteria highly abundant in the process tanks and effluent comprise primarily of those that have active growth in the plant. However standard methods for detecting pathogenic bacteria in effluent are highly based on culture dependent methods, which may give erroneous results by underestimating the bacteria that are not removed. The aim of this study was to determine the fate of immigrating pathogenic bacteria with a specific focus on an underestimated pathogen group the genus Arcobacter. And investigate with culture independent methods if these are discharged with the effluent. Culture independent 16S rRNA gene amplicon sequencing was applied for the identification of bacteria. In influent, process tanks and effluent at 14 Danish full–scale WWTPs. Net growth rates and cell numbers were calculated for specific OTU’s using bacterial mass balances. Amplicon studies showed that the genus Arcobacter was abundant in all 14 investigated plants. Mass balances confirmed that Arcobacter did not grow in the plants indicating that cells of Arcobacter originated from the influent. The pathogenic species Arcobacter butzleri was isolated. Identity and presence of virulence genes were confirmed by genomics studies. Results indicates that specific genera stay in the water phase and are directly discharged undetected by the current culture-based methods. Other procedures and methods need to be implemented to successfully identify and remove bacteria like Arcobacter before they are discharged with the effluent.