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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Dairy and Functional Foods Research » Research » Research Project #431039

Research Project: Microbial Culture Studies to Develop a Stable Human Microbiota Community

Location: Dairy and Functional Foods Research

Project Number: 8072-41000-102-08-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Jul 1, 2016
End Date: Dec 31, 2019

Objective:
Development of an in vitro model of the human small intestinal microbiota that will allow the investigation of the fundamental principals that govern the composition and dynamic response of this important, yet virtually unexplored microbial community. JUSTIFICATION FOR ADDITIONAL FUNDS: Continue to systematically examine the effect of the following environmental factors on community structure (assessed by 16S tagged sequencing) and bacterial biomass (assessed by 16S copy number PCR): Then composition of feed, pH (2 vs. 6.5), pancreatic juice, and bile acids.

Approach:
Beginning with a stable fecal community, UPenn investigators will systematically examine the effect of the following environmental factors on community structure (assessed by 16S tagged sequencing) and bacterial biomass (assessed by 16S copy number PCR): Then composition of feed, pH (2 vs. 6.5), pancreatic juice, and bile acids. We hypothesize, that in composite, all of these factors will be insufficient to reproduce the composition of the human small intestinal microbiota in vitro. Based on unpublished results using phosphorescence imaging of the gut (Albenberg, L Gastroenterology 2014), we believe that the introduction of oxygen into the environment of the cultivar will be essential to modify the composition of the microbiota so that it more faithfully represents that community described in the human small intestine. Using the exquisite control of the BioFlo 320 as well as infra-red phosphorescence imaging, UPenn investigators will regulate oxygen levels in the cultivar model to establish a microaerobic environment. UPenn will assess community structure in response to this perturbation by 16S tagged sequencing with and bacterial biomass assessed by 16S copy number PCR. In parallel, metabolomic profiling will be performed on: a) The cultivar feeds; b) Pancreatic juice; c) Bile acids; d) Cultivar contents. There are two alternative approaches to the use of fecal material as the initial inoculum: a) Use fluid/tissue collected from the human small intestine (part of the Penn Small Intestinal Microbiome Program) and b) Combine pure strains of bacteria purchased from commercial sources (i.e. ATCC and/or DMZ) based on the current literature describing the composition of the human small intestinal microbiota. Using the USDA instrument, the microbiota composition will be measured using 16S rRNA gene sequencing. SCFA and other metabolites will be quantified using LC-Mass and GC-Mass in the USDA instrument.