Survival of Fecal Bacteroidales Cells and Persistence of Their DNA in Sediments as Quantified by PMA-qPCR

Microbial source tracking (MST) techniques using quantitative polymerase chain reaction (qPCR) are being increasingly applied to identify the sources of fecal pollution in water by detecting host-associated gene sequences of fecal identifiers. Members of the order Bacteroidales are the most widely used fecal identifiers in qPCR-based MST studies. While decay kinetics of Bacteroidales cells and DNA in natural waters have been thoroughly investigated, little is known about their survival and persistence in sediments that may contribute to water quality impairment upon perturbation.;Propidium monoazide (PMA)-qPCR, a method to inhibit DNA amplification from impaired cells or extracellular DNA during qPCR, was adapted to sediments in combination with an optimized method to detach Bacteroidales cells and DNA. Handshaking and 1% Tween 80/NaOH (pH 7.0) eluant was the most efficient technique to recover intact as well as total Bacteroidales cells in sediment samples with different particle sizes and salinities and after prolonged sediment-cell contact time.;The survival and persistence of host-associated Bacteroidales cells and DNA in fresh- and seawater sediments was investigated using two types of microcosm inoculated with human, cow and dog feces: a completely anaerobic microcosm of sediment mixed with feces for estimating the decay of Bacteroidales in oxygen-free sediments at two temperatures, and a core microcosm without mixing for analyzing the fate and transport of particle-based Bacteroidales settling on top of sediments.;In freshwater sediments, the survival and persistence of Bacteroidales cells and DNA in anaerobic microcosms were considerably extended, especially at the lower temperature of 6ºC, with decay rate constants of less than 0.09 d-1 (cells) and 0.03 d-1 (DNA). In core microcosms, the levels of Bacteroidales cells and DNA decreased approximately six times more slowly in the sediments than in the overlying water. The decay rate constants of host-associated Bacteroidales cells and DNA were significantly different at 6ºC but comparable at 20ºC. Various host markers decayed at similar rates at each condition tested. Cultivable Enterococcus and E. coli showed similar decay rate constants with Bacteroidales in core sediments, indicating the survival of anaerobic Bacteroidales was comparable to that of facultative fecal indicator bacteria in freshwater sediments.;In seawater sediments, the decay of Bacteroidales was positively related to temperature in anaerobic microcosms as observed in freshwater sediments. In core microcosms, Bacteroidales cells and DNA survived and persisted up to ten times longer in sediments than in the overlying water column, suggesting sediments can provide a favorable habitat for the fecal source identifiers. Bacteroidales cells decayed faster than DNA at 20ºC, but similarly at 6ºC, in sediments. Cultivable Enterococcus cells survived longer than Bacteroidales cells and DNA in seawater sediments, presumably due to different resistance levels to osmotic pressure based on cell envelope properties.;When compared in fresh- and seawater sediments, Bacteroidales cells survived longer in freshwater sediments than in seawater sediments at 20ºC while they showed similar decay in both sediments at 6ºC. Bacteroidales DNA persisted similarly in fresh- and seawater sediments, indicating that decay of DNA is less susceptible to salinity than is that of cells. Transport of Bacteroidales cells through sediment profiles was largely attributed to gravitational settling through the pore spaces of sediments. Sediment particle sizes significantly affected cell transport due to the filtration effect. Predictive analysis based on mass balance showed that Bacteroidales in sediments could account for a 30 to 200% increase in cells in the overlying water column upon sediment resuspension under the conditions tested. This finding demonstrates that sediment can act as a non-point source of fecal identifiers and that the detection of Bacteroidales in surface water may not necessarily indicate recent fecal pollution in the event of sediment resuspension.;The present study is the first to investigate the survival and persistence of Bacteroidales cells and DNA in fresh- and seawater sediments using PMA-qPCR. The prolonged persistence of host-associated Bacteroidales cells and DNA in sediments provides clear evidence that sediments should be considered in the practical application of MST in aquatic environments. Seasonal and geographical differences in Bacteroidales decay need to be considered for accurate interpretation of MST results. Understanding the fate and transport of Bacteroidales in sediments will advance our ability to determine their significance in MST monitoring and further contribute toward a more reliable health risk assessment in the field of microbial water quality.