Submitted to: Conference on Gastrointestinal Function
Publication Type: Abstract Only
Publication Acceptance Date: 4/18/2007
Publication Date: 4/28/2007
Citation: Ziemer, C.J., Wimmer, M. 2007. Application of quantitative PCR assays to detection of human Bacteroides species in the intestines of pigs [abstract]. In: Proceedings of Microbial Ecology in Health and Diseases, Conference on Gastrointestinal Function, April 17-18, 2007, Chicago, Illinois. 19:44. Interpretive Summary:
Technical Abstract: When evaluating the efficacy of probiotic bacteria, it is beneficial to know whether a fed bacterium reaches the appropriate location in the digestive tract. Use of quantitative PCR could detect specific bacteria in a sample with a complex microbial community. In order to determine whether three Bacteroides (isolated from human feces by scientists at Natick Soldier Center)fed to pigs reach the cecum and feces, we investigated the application of Bacteroides targeted quantitative polymerase chain reaction (PCR) primers/ probes developed by Layton et al. (2006). These PCR assays targeted human (HuBac) and all Bacteroides (AllBac) species. One concern for application of HuBac was the close relation among 16S rRNA gene sequences Bacteroides isolates and clones from pigs and humans. Concentrations of 24 h growth for Bacteroides isolates were determined by anaerobic growth on Wilkens-Chalgren agar (approximately 2 x 109 CFU/ml for all three isolates). Feces were obtained from two pigs, fed either standard diet or high fiber diet. Dilutions (0 through 10-7) were added to both fecal samples (1 ml dilution with 1 g feces) and mixed well (spiked feces). Using a MJ Research Opticon thermocycler, DNA extracted from pure cultures and spiked feces was used to determine detection limits and optimize standard curves for both AllBac and HuBac. DNA from pig fecal and cecal samples was use to determine whether HuBac amplified native Bacteroides present in the pig gut. The AllBac was more sensitive, detecting 102 CFU/ml compared to 104-105 CFU/ml using the HuBac with all three isolates. No amplification occurred when HuBac was applied to any pig samples. Both AllBac and HuBac detected the addition of human Bacteroides isolates to fecal samples; AllBac detected the increased concentration of Bacteroides while the HuBac detected isolate additions of at least 104 CFU/ml. In summary, both HuBac and AllBac Bacteroides quantitative PCR primers/probes can detect intestinal survival of fed human Bacteroides isolates in pigs.