Decay of host-associated Bacteroidales cells and DNA in continuous-flow freshwater and seawater microcosms of identical experimental design and temperature as measured by PMA-qPCR and qPCR

It is difficult to compare decay kinetics for genetic markers in an environmental context when they have been determined at different ambient temperatures. Therefore, we investigated the persistence of the host-associated genetic markers BacHum, BacCow and BacCan as well as the general Bacteroidales marker BacUni in both intact Bacteroidales cells and as total intracellular and extracellular marker DNA in controlled batch experiments at two temperatures using PMA-qPCR. Fecal Bacteroidales cells and DNA persisted longer at the lower temperature. Using the modified Arrhenius function to calculate decay constants for the same temperature, we then compared the decay of host-associated Bacteroidales cells and their DNA at 14°C in field-based flow-through microcosms containing human, cow, and dog feces suspended in freshwater or seawater and previously operated with an identical experimental design. The time for a 2-log reduction (T₉₉) was used to characterize host-associated Bacteroidales decay. Host-associated genetic markers as determined by qPCR had similar T₉₉ values in freshwater and seawater at 14°C when compared under both sunlight and dark conditions. In contrast, intact Bacteroidales cells measured by PMA-qPCR had shorter T₉₉ values in seawater than in freshwater. The decay constants of Bacteroidales cells were a function of physical (temperature) and chemical (salinity) parameters, suggesting that environmental parameters are key input variables for Bacteroidales survival in a predictive water quality model. Molecular markers targeting total Bacteroidales DNA were less susceptible to the variance of temperature, salinity and sunlight, implying that measurement of markers in both intact cells and DNA could enhance the predictive power of identifying fecal pollution across all aquatic environments. Monitoring Bacteroidales by qPCR alone rather than by PMA-qPCR does not always identify the contribution of recent fecal contamination because a signal may be detected that does not reflect a recent fecal event.