title: Distribution of human-specific bacteroidales and fecal indicator bacteria in an urban watershed impacted by sewage pollution, determined using RNA- and DNA-based quantitative PCR assays.
abstract: The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as "naked DNA" in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.
authors.affiliation: Pegasus Technical Services, Inc., Cincinnati, Ohio, USA Department of Biomedical, Chemical, and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio, USA.
authors.forename: Vikram
authors.initials: V
authors.lastname: Kapoor
authors.affiliation: National Institute for Health and Welfare, Water and Health Unit, Kuopio, Finland Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio, USA.
authors.forename: Tarja
authors.initials: T
authors.lastname: Pitkänen
authors.affiliation: Pegasus Technical Services, Inc., Cincinnati, Ohio, USA.
authors.forename: Hodon
authors.initials: H
authors.lastname: Ryu
authors.affiliation: Pegasus Technical Services, Inc., Cincinnati, Ohio, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Elk
authors.affiliation: Department of Biomedical, Chemical, and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio, USA santodomingo.jorge@epa.gov wendeldw@uc.edu.
authors.forename: David
authors.initials: D
authors.lastname: Wendell
authors.affiliation: Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio, USA santodomingo.jorge@epa.gov wendeldw@uc.edu.
authors.forename: Jorge W
authors.initials: JW
authors.lastname: Santo Domingo
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D041963:Bacteroidetes / Q000235:genetics / Q000302:isolation & purification*; D002947:Cities; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D016983:Enterococcus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014876:Water Pollution
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial