Towards a better understanding of host-associated genetic markers for identification of fecal pollution sources in environmental waters

Abstract

Fecal contamination of surface water is a major public health concern and threatens ecosystem health. The water quality of many waterways in the United States has been declining due to pollution from both point and nonpoint sources from human and animal wastes. Quantitative polymerase chain reactions (qPCR) targeting host-associated genetic markers are commonly used to identify fecal contamination sources in microbial source tracking (MST) studies. Five studies were undertaken in this project to better understand the host-associated genetic markers for the identification of fecal pollution sources in environmental water. The first study evaluated the performance of bacterial and/or mtDNA markers associated with humans, cattle, Canada geese, and dogs in Alabama. Our results indicate that human- and cattle-associated bacterial markers and Canada goose- and dog-associated mtDNA markers provided superior performance characteristics for fecal samples collected from Alabama. The lower limits of detection (LLOD) and quantification (LLOQ) of each genetic marker were also determined for quantitative PCR (qPCR) assays to be used in further studies. In the second study, mesocosm experiments were conducted to investigate the influence of indigenous microbiota and nutrients on the decay of Bacteroidales and mtDNA markers associated with humans (HF183 and HcytB) and cattle (CowM3 and QMIBo) in freshwater mesocosms. Results indicate that the presence of indigenous microbiota accelerated the decay of all markers except HF183. While nutrients caused a decrease in the persistence of HF183, no significant impact was observed on HcytB, CowM3, and QMIBo. HF183 and HcytB took similar times to reach one log reduction (T90), while CowM3 reached T90 earlier than QMIBo in all the treatments but in 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 and 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. The third study aimed to determine the persistence of bacterial and mtDNA markers for humans (HF183 and HcytB), cattle (CowM3 and QMIBo), and chicken (LA35 and Chicken-ND5), as well as the general Bacteroidales marker AllBac in sediment microcosms using sentinel chambers. Results showed that host-associated bacterial markers decayed faster than mtDNA markers. Additionally, the general Bacteroidales marker AllBac remained stable throughout the study and showed greater persistence than host-associated bacterial markers. Cultivable E. coli concentrations significantly and positively correlated with gene copies of bacterial and mtDNA markers, except for cattle- and chicken-associated mtDNA markers. Furthermore, E. coli T90 values were significantly longer in cattle feces- and poultry litter-inoculated microcosms than those in sewage-inoculated ones. The fourth study investigated the impact of stream sediment texture (sand, sandy loam, loam, and clay loam) and varying dissolved organic carbon (DOC) contents (25 mg/L, 50 mg/L, and 75 mg/L) on the sedimentation of the human-associated bacterial marker HF183 in sedimentation cylinders. Results indicate that HF183 concentration remained stable in the sand treatment but declined rapidly within the first 6 hours in sandy loam, loam, and clay loam treatments. Subsequently, HF183 concentrations gradually increased from 12 to 24 hours, stabilizing until 48 hours. The impact of DOC on HF183 was consistent across DOC concentrations, with an initial sharp decrease within the first half an hour followed by a gradual increase. These findings highlight the importance of sediment composition, rather than dissolved organic carbon, in determining HF183 concentrations in the water column after sediment resuspension. The objective of the fifth study was to determine sources of fecal pollution in the Lake Martin watershed and the impact of significant rainfall events on microbial water quality. DNA extracted from water and sediment samples were subjected to quantitative qPCR analysis for human-, cattle- and chicken-associated genetic markers. The human- and cattle-associated markers were consistently present in water samples, regardless of weather conditions. Chicken markers were primarily detected after significant rainfall events. High concentrations of E. coli in water samples were observed after significant rainfall events, while sediment samples had even higher E. coli concentrations than the overlying water. Sediment samples, on the other hand, showed lower detection frequencies of the genetic markers. In summary, both bacterial and mtDNA markers were evaluated in this study. Our findings provide insight into the fate of FIB and genetic markers in the natural environment and can be used to improve watershed management and risk assessment.