Persistence of human- and cattle-associated Bacteroidales and mitochondrial DNA markers in freshwater mesocosms

Accurate identification of the origins of non-point source pollution is essential for the effective control of fecal pollution. Host-associated Bacteroidales and mitochondrial DNA (mtDNA) markers have been developed to identify the sources of human and cattle fecal pollution. However, the differences in persistence between these two types of markers under different environmental conditions are still poorly understood. Here, we conducted mesocosm experiments to investigate the influence of indigenous microbiota and nutrients on the decay of Bacteroidales and mtDNA markers associated with humans and cattle. Raw sewage or cattle feces were inoculated into mesocosms containing natural eutrophic water, sterile eutrophic water or artificial freshwater. The Bacteroidales markers HF183 (human) and CowM3 (cattle) and mtDNA markers HcytB (human) and QMIBo (cattle) were quantified using the quantitative polymerase chain reaction (qPCR) assays. All markers but HF183 decreased the fastest in the presence of indigenous microbiota. Nutrients caused a decrease in the persistence of HF183; however, no significant nutrient effects were observed for HcytB, CowM3, and QMIBo. The time to reach one log reduction (T₉₀) for HF183 and HcytB was similar; CowM3 reached T₉₀ earlier than QMIBo in all the treatments but eutrophic water. E. coli persisted longer than both Bacteroidales and mtDNA markers in the mesocosms regardless of inoculum type. Additionally, 16S rRNA gene amplicon sequencing was used to determine the changes in bacterial communities accompanying the marker decay. Analysis using the SourceTracker software showed that bacterial communities in the mesocosms became more dissimilar to those in the corresponding inoculants over time. Our results indicate that environmental factors are important determinants of genetic markers' persistence, but their impact can vary depending on the genetic markers. The cattle Bacteroidales markers may be more suitable for determining recent fecal contamination than cattle mtDNA.