title: Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers.
abstract: In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.
authors.affiliation: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268, USA.
authors.forename: Hodon
authors.initials: H
authors.lastname: Ryu
authors.forename: Hiep
authors.initials: H
authors.lastname: Tran
authors.forename: Michael W
authors.initials: MW
authors.lastname: Ware
authors.forename: Brandon
authors.initials: B
authors.lastname: Iker
authors.forename: Shannon
authors.initials: S
authors.lastname: Griffin
authors.forename: Andrey
authors.initials: A
authors.lastname: Egorov
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Edge
authors.forename: Norman
authors.initials: N
authors.lastname: Newmann
authors.forename: Eric N
authors.initials: EN
authors.lastname: Villegas
authors.forename: Jorge W Santo
authors.initials: JW
authors.lastname: Domingo
mesh_terms: D001439:Bacteroides / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D004784:Environmental Monitoring / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D017422:Sequence Analysis, DNA; D012722:Sewage / Q000382:microbiology* / Q000469:parasitology; D014871:Water Microbiology; D060753:Water Quality
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012722:Sewage