Fate and transport of pathogens in freshwater mesocosms : role of biofilms

We studied the fate and transport of common waterborne pathogens in freshwater systems using simple hydrodynamic modeling and their effect on mature biofilm communities using metagenomic sequencing. Pathogens were represented by four surrogates: Pseudomonas aeruginosa, Enterococcus faecalis and the bacteriophages P22 and GA. We ran a set of four experiments, at two different flow rates, 6 L min-1 (low) and 12 L min-1 (high). The flow rates represented conditions in urban canals during dry weather and the annual average. In each experiment, we operated four open channel flumes with partial recirculation (laminar flow range), and two of the flumes were spiked with the surrogates. Spiking was done once and concentrations of viable populations and their nucleic acids in water and biofilm were followed for 21 days. The remaining flumes received no surrogates. Additionally, microbial community assembly was monitored through metagenomic sequencing of the biofilms collected from flumes with and without surrogates. At low flow settling and decay terms alone could not explain bulk water concentrations in the presence of biofilms. Surrogates attached to biofilms within 24 hours and P. aeruginosa and E. faecalis were also found to grow within biofilms. Bacteriophage P22 was unable to multiply due to the absence of its host, but was retained in the biofilm throughout the experimental period. Bacteriophage GA was not detected in the flumes after spiking. Surrogates were also released into the overlying water column, thereby increasing the number of days required for complete removal of the organisms from the flumes. At low flow, P. aeruginosa was initially released continuously from the biofilm. Later, detachment was more random and occurred to a greater extent than expected based on erosion alone, suggesting sloughing via biological dispersal. P22 detachment was by erosion and E. faecalis did not detach. In comparison, at the high flow rate, the role of biofilms as a source and sink of surrogates remained the same. However, release of P. aeruginosa was through continuous erosion, as compared to random sloughing at the low flow rate. The two-log reduction times of surrogates in water were 24-57% higher and those in biofilms were 35-500% lower at high flow than at low flow. Concurrently, the detachment rates in biofilms were almost 80% higher and decay in biofilms was 22- 83% higher at high flow rate. Growth of P. aeruginosa was either absent or balanced by increased decay at high flow, whereas the growth rate of E. faecalis doubled at high flow. Community analysis revealed that the prokaryotic communities were not affected by surrogate addition at any flow rate. Eukaryotic communities showed similar trends. However, in two experiments, when Podocopida and Haplotaxida were present, the abundance of these organisms was related to the presence of surrogates, even though the latter were not pathogenic for any of them. Overall, nascent eukaryotic communities changed due to algal growth, while prokaryotic communities remained the same. In conclusion, freshwater microbial communities were resilient to pathogen exposure and surrogates survived in biofilms through co-existence and not invasion.Doctor of Philosophy (IGS