Establishing a mucosal gut microbial community in vitro using an artificial simulator

Abstract

The Twin Simulator of the Human Intestinal Microbial Ecosystem (TWINSHIME®) was initially developed to study the luminal gut microbiota of the ascending (AC), transverse (TC), and descending (DC) colon regions. Given the unique composition and potential importance of the mucosal microbiota for human health, the TWINSHIME was recently adapted to simulate the mucosal microbiota as well as the luminal community. It has been previously demonstrated that the luminal community in the TWINSHIME reaches a steady state within two weeks post inoculation, and is able to differentiate into region specific communities. However, less is known regarding the mucosal community structure and dynamics. During the current study, the luminal and mucosal communities in each region of the TWINSHIME were evaluated over the course of six weeks. Based on 16S rRNA gene sequencing and short chain fatty acid analysis, it was determined that both the luminal and mucosal communities reached stability 10–20 days after inoculation, and remained stable until the end of the experiment. Bioinformatics analysis revealed the formation of unique community structures between the mucosal and luminal phases in all three colon regions, yet these communities were similar to the inoculum. Specific colonizers of the mucus mainly belonged to the Firmicutes phylum and included Lachnospiraceae (AC/TC/DC), Ruminococcaceae and Eubacteriaceae (AC), Lactobacillaceae (AC/TC), Clostridiaceae and Erysipelotrichaceae (TC/DC). In contrast, Bacteroidaceae were enriched in the gut lumen of all three colon regions. The unique profile of short chain fatty acid (SCFA) production further demonstrated system stability, but also proved to be an area of marked differences between the in vitro system and in vivo reports. Results of this study demonstrate that it is possible to replicate the community structure and composition of the gut microbiota in vitro. Through implementation of this system, the human gut microbiota can be studied in a dynamic and continuous fashion.

Following the community development of SIHUMIx – a new intestinal in vitro model for bioreactor use

“…13 Liu et al (2018) followed the development of their fecal community in the TWIN-SHIME by 16S rRNA and SCFA analyses. 17 In their study, the community structure became constant on day 10, whereas the communities' SCFA metabolism became constant not before day 17. Furthermore, in contrast to the complex fecal community, SIHUMIx develops simultaneously on the structural and metabolic level during adaptation.…”

Selection of fast and slow growing bacteria from fecal microbiota using continuous culture with changing dilution rate

“…The De-stat culture has not been applied for studies of complex microbial consortia earlier, while chemostat gut models have been used by several groups. It has been shown that major shifts in variability of fecal microbiota take place within the first 3–4 residential times while slight fluctuations occur even after 30 residential times [13,20,30] because of complex nature of the microbiota. Hence, the De-stat presented in the current paper is a promising approach for analyzing of the growth rate in microbial consortia with potential impact on health.…”

Metagenomic assessment of the Cebus apella gut microbiota

“…Before use, the samples were thawed at 37°C for 45 min in an anaerobic incubator and 250 µl of the sample was extracted using diethyl ether (1:2, v/v), 2‐methylhexanoic acid as the internal standard, sulfuric acid, and sodium chloride. The samples were well mixed and then centrifuged at 3,000 rpm (Centrisart A‐14C; Sartorious, Germany) to facilitate phase separation (Liu et al, ). The organic layer was transferred into a GC vial and 1 µl was injected into a gas chromatography/mass spectrometry (GC/MS) (Shimadzu QP2010 Ultra; Shimadzu, Columbia, MD), with a Stabilwax‐DA column, 30 m, 0.25 mm ID, 0.25 µm (Restek Corporation, Bellefonte, PA) with the following settings: injection port temperature set to 260°C, a split ratio of 1:20, a flow rate of 1.00 ml/min of helium.…”

“…The gut microbiota is a complex community of microorganisms found within the intestinal tract (Clayton et al, ; Daliri, Tango, Lee, & Oh, ; Lloyd‐Price, Abu‐Ali, & Huttenhower, ). The composition of the gut microbiota changes biogeographically, forming region specific communities adapted to each microenvironment (Liu et al, ). Within each region, the community will grow in the lumen, however, some of the community members will preferentially colonize the loose mucus layer lining the intestinal cells (Lavelle et al, ; Zhang et al, 2014a).…”

“…However, data collected from fecal samples do not accurately represent the distributed communities of the gut microbiota through the intestinal tract and, importantly, fail to accurately represent the mucosal communities (Mottawea et al, ). Knowledge of the composition of the site and phase‐specific microbiota is critical to elucidate dynamic changes associated with the gut microbiota (Liu et al, ).…”