title: Molecular detection of bacterial contamination in gnotobiotic rodent units.
abstract: Gnotobiotic rodents provide an important technique to study the functional roles of commensal bacteria in host physiology and pathophysiology. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches of culturing and Gram staining feces have inherent limitations, as many bacteria are uncultivable and fecal Gram stains are difficult to interpret. Thus, we developed and validated molecular methods to definitively detect and identify contamination in germ-free (GF) and selectively colonized animals. Fresh fecal pellets were collected from rodents housed in GF isolators, spontaneously contaminated ex-GF isolators, selectively colonized isolators and specific pathogen-free (SPF) conditions. DNA isolated from mouse and rat fecal samples was amplified by polymerase chain reaction (PCR) and subjected to quantitative PCR (qPCR) using universal primers that amplify the 16S rRNA gene from all bacterial groups. PCR products were sequenced to identify contaminating bacterial species. Random amplification of polymorphic DNA (RAPD) PCR profiles verified bacterial inoculation of selectively colonized animals. These PCR techniques more accurately detected and identified GF isolator contamination than current standard approaches. These molecular techniques can be utilized to more definitively screen GF and selectively colonized animals for bacterial contamination when Gram stain and/or culture results are un-interpretable or inconsistent.
authors.affiliation: Department of Medicine; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; Department of Microbiology and Immunology; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; Center for Gastrointestinal Biology and Diseases; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Packey
authors.affiliation: Department of Medicine; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; Center for Gastrointestinal Biology and Diseases; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Shanahan
authors.affiliation: Department of Medicine; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.
authors.forename: Sayeed
authors.initials: S
authors.lastname: Manick
authors.affiliation: The National Gnotobiotic Rodent Resource Center; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.
authors.forename: Maureen A
authors.initials: MA
authors.lastname: Bower
authors.affiliation: Department of Microbiology and Immunology; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Ellermann
authors.affiliation: Center for Gastrointestinal Biology and Diseases; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; College of Veterinary Medicine, North Carolina State University; Raleigh, NC USA.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Tonkonogy
authors.affiliation: Department of Medicine; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; Center for Gastrointestinal Biology and Diseases; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Carroll
authors.affiliation: Department of Medicine; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; Department of Microbiology and Immunology; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; Center for Gastrointestinal Biology and Diseases; University of North Carolina at Chapel Hill; Chapel Hill, NC USA; The National Gnotobiotic Rodent Resource Center; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.
authors.forename: R Balfour
authors.initials: RB
authors.lastname: Sartor
mesh_terms: D000821:Animal Feed / Q000032:analysis / Q000382:microbiology; D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005856:Germ-Free Life; D051379:Mice / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; PCR; RAPD PCR; commensal bacteria; gnotobiotic; microbiota; qPCR