6 - Temporal stability of faecal microbiota by Inter Spacer rDNA bacterial profiling (IS-pro)

Abstract

Gnotobiotic rodents facilitate the study of commensal bacteria and their roles in human physiology and pathophysiology. To ensure sterility, animals must be screened frequently for contamination by the traditional approach of culturing and Gram staining of feces. Yet many bacteria are uncultivable, fecal Gram stains can be difficult to interpret, and these methods are labor-intensive and time-consuming. Aims: To develop molecular methods of detecting contamination in gnotobiotic units.Methods:We collected fresh fecal pellets from mice housed in germ free (GF) isolators (N=8), a contaminated ex-GF isolator, an isolator colonized with 2 bacterial species, and a specific pathogen free (SPF) animal room (N=4). DNA from fecal samples was extracted using a phenol/chloroform extraction method combined with physical disruption of bacterial cells. Polymerase chain reactions (PCR) were carried out using templates of fecal DNA and primers that amplify the genes encoding the 16S rDNA from all bacterial groups. The numbers of 16S rDNA gene copies in each DNA sample were then quantified using quantitative Real-Time PCR (qPCR) with the same templates and primers and the appropriate set of standards. Finally, the 16S rDNA gene was amplified from fecal DNA from contaminated ex-GF mice, sequenced, and crossreferenced with a computerized data base to identify the bacterial species. Results: PCR yielded amplicons from isolated fecal DNA from, in descending order of intensity, all SPF mice, dual associated mice, and contaminated ex-GF mice, as well as from mice from one presumed GF isolator. Fecal DNA from mice from the other 7 GF isolators did not yield amplicons. qPCR confirmed that the level of bacteria in themouse feces from the questionable isolator, (4.0 X 108 copies 16S rDNA gene/μg feces), was 210-fold greater than the levels in the other 7 GF isolators, (1.9 X 106 copies/μg, p < 0.0001), and was comparable to the level in the known contaminated isolator, (3.0 X 108 copies/μg.) The source of DNA in mouse feces from GF isolators detected by qPCR was dead bacteria in autoclaved food. Upon review, both cultures and Gram stains from the isolator that we determined to be contaminated based on PCR and qPCR results had been deemed indeterminate by gnotobiotic facility staff. Sequencing the 16S rDNA gene revealed the contaminating bacterial species in the ex-GF isolator to be Bacillus simplex. Conclusions: We developed molecular methods to screen for contamination in gnotobiotic units that are more reliable and less timeconsuming than current traditional methods and can be used to identify the contaminating species.