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

Abstract

Diverse intestinal microbiota is frequently used in in vitro bioreactor models to study the effects of diet, chemical contaminations, or medication. However, the reproducible cultivation of fecal microbiota is challenging and the resultant communities behave highly dynamic. To approach the issue of reproducibility in in vitro models, we established an intestinal microbiota model community of reduced complexity, SIHUMIx, as a valuable model for in vitro use. The development of the SIHUMIx community was monitored over time with methods covering the cellular and the molecular level. We used microbial flow cytometry, intact protein profiling and terminal restriction fragment length polymorphism analysis to assess community structure. In parallel, we analyzed the functional level by targeted analysis of short-chain fatty acids and untargeted metabolomics. The stability properties constancy, resistance, and resilience were approached both on the structural and functional level of the community. We show that the SIHUMIx community is highly reproducible and constant since day 5 of cultivation. Furthermore, SIHUMIx has the ability to resist and recover from a pulsed perturbation, with changes in community structure recovered earlier than functional changes. Since community structure and function changed divergently, both levels need to be monitored at the same time to gain a full overview of the community development. All five methods are highly suitable to follow the community dynamics of SIHUMIx and indicated stability on day five. This makes SIHUMIx a suitable in vitro model to investigate the effects of e.g. medical, chemical, or dietary interventions.

The Activation of Mucosal-Associated Invariant T (MAIT) Cells Is Affected by Microbial Diversity and Riboflavin Utilization in vitro

“…The extended simplified intestinal human microbiota (SIHUMIx) community was used to investigate the interaction of intestinal bacterial communities and MAIT cells. This model community shows major features of a human intestinal community (Becker et al, 2011) and allows the reproducible cultivation (Krause et al, 2020). SIHUMIx comprises of eight bacterial species Anaerostipes caccae (DSM 14662), Bacteroides thetaiotaomicron (DSM 2079), Bifidobacterium longum (NCC 2705), Blautia producta (DSM 2950), Clostridium butyricum (DSM 10702), Clostridium ramosum (DSM 1402), Escherichia coli K-12 (MG1655), and Lactobacillus plantarum (DSM 20174).…”

“…To investigate the stimulation capacity of SIHUMIx under normal and stress conditions the SIHUMIx community was continuously cultivated in a Multifors 2 bioreactor (Infors, Switzerland, N = 2) as described in Krause et al (2020) using complex intestinal medium [CIM, Supplementary Table S2 ( Krause et al, 2020)]. In addition, we cultivated human fecal microbiota alike the SIHUMIx community in triplicate bioreactors.…”

“…Measurement was performed as described (Krause et al, 2020). 5 µg peptide lysate was injected into nanoHPLC (UltiMate 3000 RSLCnano, Dionex, Thermo Fisher Scientific, Waltham, United States).…”

“…The fecal and colonic bacteria were homogenized by ultra-sonication and stained with 0.68 µM 4 ,6-di-amidino-2-phenyl-indole (DAPI, Sigma-Aldrich, St-Louis, United States) overnight. Measurement and data analysis were performed as in Krause et al (2020).…”

“…We aimed to investigate if microbial stress can affect the MAIT cell activating potential of microbiota and thereby can directly influence MAIT cell activation. Since patients with active ulcerative colitis can show a lower colonic pH as part of the disease (Nugent et al, 2001;Moco et al, 2014), we cultivated the SIHUMIx model community in vitro and induced an acute acid stress of pH 5.5 (Krause et al, 2020), experimental set-up: Supplementary Figure S5). Thereafter, we compared the MAIT cell activating potential of the unstressed and the acid stressed SIHUMIx community.…”