Water quality is frequently impacted by microbial pollution from human and animal
feces. Microbial source tracking (MST) can identify dominant pollution sources and
improve assessment of health risk compared to indicator bacteria alone. This study
aims to standardize and validate MST methods across laboratories in coastal Gulf of
Mexico states. Three laboratories evaluated library-independent MST methods for human
sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human
polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets
in human sewage seeded into buffer, freshwater or marine water (100% sensitivity).
The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay
(10(-5)-10(-6) dilution). LODs for HPyVs and M. smithii assays were similar to each
other (10(-3)-10(-4)), but were higher than Bacteroidales. The HPyVs assay was 100%
specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces
among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was
96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii
assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples.
An experts' workshop concluded that all methods showed sufficient accuracy and reliability
to move forward. SOPs will be distributed to collaborating laboratories for further
inter-laboratory comparison, and field validation will occur in year 2.