title: Persistence and growth of fecal Bacteroidales assessed by bromodeoxyuridine immunocapture.
abstract: Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.
authors.affiliation: Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Sarah P
authors.initials: SP
authors.lastname: Walters
authors.forename: Katharine G
authors.initials: KG
authors.lastname: Field
mesh_terms: D041963:Bacteroidetes / Q000235:genetics / Q000254:growth & development* / Q000276:immunology; D001973:Bromodeoxyuridine / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D016133:Polymerase Chain Reaction; D012722:Sewage / Q000382:microbiology; D014871:Water Microbiology; D014876:Water Pollution
chemical_list: D004269:DNA, Bacterial; D012722:Sewage; D001973:Bromodeoxyuridine