A
Novel Microbial Source Tracking Microarray for Pathogen
Detection and Fecal Source Identification in Environmental Systems
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Abstract
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, <i>Campylobacter fetus, Helicobacter
pylori</i>, <i>Salmonella enterica</i>, and <i>Giardia lamblia</i> 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 (21β33%) was lower than
specificity (83β90%, percentage of true negatives). Next generation
DNA sequencing revealed two dominant bacterial families that were
common to all sample types: <i>Ruminococcaceae</i> and <i>Lachnospiraceae</i>. 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