A Novel Microbial Source Tracking Microarray for Pathogen Detection and Fecal Source Identification in Environmental Systems

Pathogen detection and the identification of fecal contamination sources are challenging in environmental waters. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. A custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, and antibiotic resistance genes was tested against DNA obtained from whole genome amplification (WGA) of RNA and DNA from sewage and animal (avian, cattle, poultry, and swine) feces. Perfect and mismatch probes established the specificity Of the microarray in sewage, and fluorescence decrease of positive probes over a 1:10 dilution series demonstrated semiquantitative measurement. Pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21-33%) was lower than specificity (83-90%, percentage of true negatives). Next generation DNA sequencing revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources. Phyla and families not represented on the microarray are possible candidates for inclusion in subsequent array designs.