Abstract
Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the "clean" effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock.IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.