Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Summary

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Abstract

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Introduction

Gut microbiota play an important role in the functioning of human intestines and in host health. Consequently, gut microbiota have recently become an important target for disease prevention and therapy¹. Moreover, gut bacteria interact with host intestinal cells and regulate fundamental host processes, including metabolic activities, nutrient availabilities, immune system modulation, and even brain function and decision-making^2,3. Endobiotics have considerable potential to influence the bacterial composition and diversity of gut microbiota. Thus, interactions between endobiotics and human gut microbiota have attracted increasing research attention^4,5,6,7,8,9.

It is difficult to evaluate interactions between endobiotics and human gut microbiota in vivo due to bioethics and economic constraints. For example, experiments investigating the interactions between endobiotics and human gut microbiota cannot be performed without permission of the Food and Drug Administration, and recruitment of volunteers is expensive. Consequently, animal models are often used for such investigations. However, the use of animal models is limited due to different microbiota compositions and diversity in animal- vs. human-associated communities. An alternative in vitro method to explore the interactions between endobiotics and human gut microbiota is through the use of batch culture experiments.

Exopolysaccharides (EPSs) are prebiotics that significantly contribute to the maintenance of human health¹⁰. Distinct EPSs that consist of different monosaccharide compositions and structures can exhibit distinct functions. Previous analyses have determined the composition of Bif EPSs, which are the representative xenobiotic targeted in the current study¹¹. However, host-associated metabolic effects have not been considered with regard to EPS composition and diversity.

The protocol described here uses the fecal microbiota from 12 volunteers to ferment Bif EPSs. Thin-layer chromatography (TLC), 16S rRNA gene high-throughput sequencing, and gas chromatography (GC) are then used in combination to investigate the interactions between EPSs and human gut microbiota. Distinct advantages of this protocol compared to in vivo experiments are its low cost and avoidance of interfering effects from the host’s metabolism. Furthermore, the described protocol can be used in other studies that investigate interactions between endobiotics and human gut microbiota.

Protocol

This protocol follows the guidelines of the ethics committee of Hunan University of Science and Engineering (Hunan, China), and the Zhejiang Gongshang University (Zhejiang, China).

1. Preparation of bacteria

1. Preparation of bifidobacterium medium broth
   1. Combine the following components in 950 mL of distilled water: meat extract, 5 g/L; yeast extract, 5 g/L; casein peptone, 10 g/L; soytone, 5 g/L; glucose, 10 g/L; K₂HPO₄, 2.04 g/L; MgSO₄·7H₂O, 0.22 g/L; MnSO₄.H₂0, 0.05 g/L; NaCl, 5 g/L; Tween 80, 1 mL; salt solution, 40 mL (CaCl₂.2H₂O, 0.25 g/L; KH₂PO₄, 1 g/L; NaHCO₃, 10 g/L; NaCl, 2 g/L); and resazurin, 0.4 mL (2.5 mg/L). Adjust the pH to 6.8 with 2 M NaOH.
   2. Autoclave at 121 °C for 15 min and allow the broth to cool to room temperature (RT) under anaerobic conditions (10% H₂, 10% CO₂, 80% N₂). Add filter-sterilized cysteine-HCl (0.5 g/L) and mupirocin (5 mg/L) to the medium.
2. Add an aliquot (50 μL) of frozen Bifidobacterium longum to a culture tube with 5 mL of bifidobacterium medium broth under anaerobic conditions, then culture in an anaerobic incubator for 24 h at 37 °C.

2. Preparation of bifidobacterial EPSs

1. Preparation of PYG agar medium
   1. Combine the following: peptone, 20 g/L; yeast extract, 10 g/L; glucose, 5 g/L; NaCl, 0.08 g/L; CaCl₂, 0.008 g/L; MgSO₄·7H₂O, 0.008 g/L; K₂HPO₄, 0.04 g/L; KH₂PO₄, 0.04 g/L; NaHCO₃, 0.4 g/L; agar, 12 g/L. Adjust the pH to 7.2 using 10 M NaOH.
   2. Autoclave the media at 121 °C for 15 min and cool to ~50 °C. Then, per 1 L of medium, add 0.5 mL of filter-sterilized vitamin K₁ solution (1 g of vitamin K₁ dissolved in 99 mL of 99% ethanol), 5 mL of haemin solution (0.5 g of haemin dissolved in 1 mL of 1 mol/L NaOH, then brought up to 100 mL with distilled water), and 0.5 g of cysteine-HCl.
   3. Before pouring the PYG plates, add filter-sterilized 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal, 0.06 g/L), LiCl·3H₂O (5.7 g/L) and mupirocin (5 mg/L) to the medium.
      NOTE: X-Gal and LiCl.3H₂O allow the identification of B. longum colonies on plates via coloration changes.
2. Inoculate 20 μL of B. longum strains (step 1.2) to PYG plates and place in an anaerobic incubator at 37 °C for 72 h.
3. Collect mucoid bacterial colonies from the PYG plates using a weighing scoop, then completely resuspend in 10 mL of phosphate-buffered saline (PBS) using a vortex oscillator.
   NOTE: The bacterial and EPS mixtures should be resuspended completely by vortexing or pipetting up and down repeatedly until the fibers are completely dissolved in PBS.
4. Centrifuge the suspension at 6,000 x g for 5 min.
5. Carefully transfer the supernatants to a new centrifuge tube and mix completely with three volumes of cold 99% ethanol by repeated inversion and blending.
6. Centrifuge the mixture at 6,000 x g for 5 min and completely remove the supernatants.
7. Remove the precipitates from the centrifuge tubes by scraping and drying the EPS extracts overnight using a speed vacuum.

3. Preparation of fermentation medium

1. Preparation of basic culture medium VI
   1. Combine the following: peptone, 3 g/L; tryptone, 3 g/L; yeast extract, 4.5 g/L; mucin, 0.5 g/L; bile salts No. 3, 0.4 g/L; NaCl, 4.5 g/L; KCl, 2.5 g/L; MgCl₂·6H₂O, 4.5 g/L; 1 mL Tween 80; CaCl₂.6H₂O, 0.2 g/L; KH₂PO₄, 0.4 g/L; MgSO₄·7H₂O, 3.0 g/L; MnCl₂·4H₂O, 0.32 g/L; FeSO₄·7H2O, 0.1 g/L; CoSO₄·7H₂O, 0.18 g/L; CaCl₂·2H₂O, 0.1 g/L; ZnSO₄·7H₂O, 0.18 g/L; CuSO₄·5H₂O, 0.01 g/L; and NiCl₂·6H₂O, 0.092 g/L. Adjust the pH to 6.5 with 1 M HCl.
   2. Prepare haemin and cysteine as done in section 2.1 and add after autoclaving and cooling.
2. Prepare culture media that contains different carbon sources with a VI base media. Prior to autoclaving, add 8 g/L of Bif EPS fibers to medium VI, comprising group VI_Bif. In addition, add 8 g/L starch to medium VI to represent group VI_Starch. Finally, medium VI without addition of a carbon source is used as the control (group VI).
   NOTE: Bif EPS and starch are first dissolved in hot water using a magnetic agitator, then mixed with prepared VI medium.
3. Autoclave all media at 121 °C for 15 min and allow to cool to RT.
4. Transfer a subsample (5 mL) of each medium to culture tubes in an anaerobic incubator, and store the remaining media at 4 °C.

4. Human fecal sample preparation

1. Collect fresh fecal samples immediately following fresh defecation from healthy adult human volunteers using feces containers, and subsequently use for slurry preparation.
   NOTE: Prior to sample collection, all the volunteers should be screened to ensure no receiving of antibiotics, probiotics, or prebiotic treatments for at least 3 months prior to donating samples. In addition, all donors must provide informed, written consent.
2. Transfer a fresh fecal sample (1 g) to 10 mL of 0.1 M anaerobic PBS (pH 7.0) into glass beakers, then use glass rods to prepare a 10% (w/v) slurry.
3. Use a 0.4 mM sieve to filter the fecal slurry. Then, use a subsample of the filtered slurry to inoculate batch culture fermentation experiments, and store the remainder at -80 °C for further analyses.
   NOTE: Steps 4.2–4.3 are conducted in an anaerobic chamber.

5. In vitro batch fermentation

1. Add filtered fecal slurry (500 μL) to the fermentation medium prepared in step 3.2 within an anaerobic chamber, then incubate at 37 °C.
2. Collect 2 mL of fermented samples at 24 h and 48 h in the anaerobic chamber and then centrifuge outside of the chamber at 6,000 x g for 3 min.
3. Carefully transfer the supernatants to a new centrifuge tube that will be used for polysaccharide degradation analysis and short-chain fatty acids (SCFAs) measurements.
4. Store the centrifugation pellets at -80 °C and subsequently use for bacterial genomic DNA extraction.

6. EPS degradation by human fecal microbiota

1. Load 0.2 μL of fermented supernatants onto pre-coated silica gel-60 TLC aluminum plates, then dry using a hair drier.
2. Develop the plates in 20 mL of a formic acid/n-butanol/water (6:4:1, v:v:v) solution and dry using a hair drier.
3. Soak the plates in the orcinol reagent to dye, then dry using a hair drier.
   1. Prepare orcinol reagents by dissolving 900 mg of Lichenol in 25 mL of distilled water then adding 375 mL of ethanol. Subsequently, concentrated sulfuric acid should be slowly added and the solution thoroughly mixed.
4. Heat plates at 120 °C for 3 min in a baking oven and evaluate degradation of EPS by measuring TLC bands.

7. Effects of EPS on human intestinal microbiota

1. Freeze-thaw the original fecal samples prepared in step 4.3 and fermented samples prepared in step 5.4.
2. Extract bacterial genomic DNA (gDNA) from all the samples using a stool bacterial genomic DNA extraction kit following the manufacturer’s instructions.
3. Determine DNA concentrations, integrities, and size distributions using a micro-spectrophotometer and agar gel electrophoresis.
4. Conduct PCR of bacterial 16S rRNA genes from the extracted gDNA using the following previously described forward and reverse primers¹²:
   -forward primer (barcoded primer 338F): ACTCCTACGGGAGGCAGCA
   -reverse primer (806R): GGACTACHVGGGTWTCTAAT
   Use the following thermal cycler conditions:
   1. 94 °C for 5 min.
   2. 94 °C for 30 s.
   3. 55 °C for 30 s.
   4. 72 °C for 1 min.
   5. Repeat 2–4 for 35 cycles
   6. 72 °C for 5 min.
   7. 4 °C hold until removal from thermal cycler.
5. Conduct high-throughput sequencing of PCR products at a DNA sequencing company using ultra-high throughput microbial community analysis.
6. Obtain clean, high quality sequences using the Quantitative Insights into Microbial Ecology (QIIME) sequence analysis pipeline¹³.
7. Define operational taxonomic units (OTUs) for 16S rRNA gene sequences with greater than 97% nucleotide similarity using bioinformatics tools such as the Mothur software suite¹⁴.
8. Choose a representative sequence from each OTU and use the RDP classifier along with the SILVA taxonomic database to classify representative sequences¹⁵.
9. Calculate Good’s coverage, alpha diversity metrics (including Simpson and Shannon index), and richness (observed number of OTUs) using bioinformatics tools¹⁶.

8. Effects of EPS on SCFA production by human intestinal microbiota

1. Add fermented supernatants (1 mL) prepared in step 5.3 to 2 mL centrifuge tubes.
2. Add 0.2 mL of 25% (w/v) metaphosphoric acid to each of the samples and thoroughly mix the solutions by vortexing.
3. Centrifuge the mixtures at 13,000 x g for 20 min and transfer the supernatants to fresh tubes.
4. Concomitantly, prepare solutions of 120 mM acetic, propionic, butyric, isobutyric, valeric and isovaleric acids. Then, add 1 mL of each prepared acid to 1.2 mL of 25% (w/v) metaphosphoric acid and use as the standard cocktails.
5. Filter the samples using a 0.22 μM membrane.
6. Detect SCFA concentrations using high performance gas chromatography according to previously described protocols^11,17.
   NOTE: An InertCap FFAP column (0.25 mM x 30 m x 0.25 μM) is used for gas chromatography (GC). SCFA concentrations are then quantified based on peak areas using the single-point internal standard method in the GC Solution software package.

Results

The production of mucoid EPS could be observed in B. longum cultures on PYG plates after anaerobic incubation for 72 h (Figure 1A). Centrifugation of culture scrapes, followed by ethanol precipitation and drying, resulted in the collection of cellulose-like EPS (Figure 1B). Dried EPS and soluble starch were then used as carbon sources for fermentation cultures. TLC was used for oligosaccharide separation and purity analysis due to its low cost and rapid...

Discussion

Significant progress has been made towards understanding human gut microbiota composition and activities over the last decade. As a consequence of these studies, the holobiont concept has emerged, which represents the interactions between hosts and associated microbial communities, such as in between humans and their gut microbiota^19,20. Furthermore, humans are even now regarded as superorganisms²¹, wherein the gut microbiota have been rec...

Materials

Name	Company	Catalog Number	Comments
0.22 µm membrane filters	Millipore	SLGP033RB	Use to filter samples
0.4-mm Sieve	Thermo Fischer	308080-99-1	Use to prepare human fecal samples
5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal)	Solarbio	X1010	Use to prepare color plate
Acetic	Sigma-Aldrich	71251	Standard sample for SCFA
Agar	Solarbio	YZ-1012214	The component of medium
Anaerobic chamber	Electrotek	AW 400SG	Bacteria culture and fermentation
Autoclave	SANYO	MLS-3750	Use to autoclave
Bacto soytone	Sigma-Aldrich	70178	The component of medium
Baking oven	Shanghai Yiheng Scientific Instruments Co., Ltd	DHG-9240A	Use to heat and bake
Beef Extract	Solarbio	G8270	The component of medium
Bifidobacterium longum Reuter	ATCC	ATCC® 51870™	Bacteria
Bile Salts	Solarbio	YZ-1071304	The component of medium
Butyric	Sigma-Aldrich	19215	Standard sample for SCFA
CaCl2	Solarbio	C7250	Salt solution of medium
Capillary column	SHIMADZU-GL	InertCap FFAP (0.25 mm × 30 m × 0.25 μm)	Used to SCFA detection
Casein Peptone	Sigma-Aldrich	39396	The component of medium
Centrifuge	Thermo Scientific	Sorvall ST 8	Use for centrifugation
CoSO4.7H2O	Solarbio	C7490	The component of medium
CuSO4.5H2O	Solarbio	203165	The component of medium
Cysteine-HCl	Solarbio	L1550	The component of medium
Ethanol	Sigma-Aldrich	E7023	Use to prepare vitamin K1
FeSO4.7H2O	Solarbio	YZ-111614	The component of medium
Formic Acid	Sigma-Aldrich	399388	Used to TLC
Gas chromatography	Shimadzu Corporation	GC-2010 Plus	Used to SCFA detection
Glass beaker	Fisher Scientific	FB10050	Used for slurry preparation
Glucose	Solarbio	G8760	The component of medium
Haemin	Solarbio	H8130	The component of medium
HCl	Sigma-Aldrich	30721	Basic solution used to adjust the pH of the buffers
Isobutyric	Sigma-Aldrich	46935-U	Standard sample for SCFA
Isovaleric Acids	Sigma-Aldrich	129542	Standard sample for SCFA
K2HPO4	Solarbio	D9880	Salt solution of medium
KCl	Solarbio	P9921	The component of medium
KH2PO4	Solarbio	P7392	Salt solution of medium
LiCl.3H2O	Solarbio	C8380	Use to prepare color plate
Meat Extract	Sigma-Aldrich-Aldrich	70164	The component of medium
Metaphosphoric Acid	Sigma-Aldrich	B7350	Standard sample for SCFA
MgCl2.6H2O	Solarbio	M8160	The component of medium
MgSO4.7H2O	Solarbio	M8300	Salt solution of medium
MISEQ	Illumina	MiSeq 300PE system	DNA sequencing
MnSO4.H20	Sigma-Aldrich	M8179	Salt solution of medium
Mupirocin	Solarbio	YZ-1448901	Antibiotic
NaCl	Solarbio	YZ-100376	Salt solution of medium
NaHCO3	Sigma-Aldrich	792519	Salt solution of medium
NanoDrop ND-2000	NanoDrop Technologies	ND-2000	Determine DNA concentrations
NaOH	Sigma-Aldrich	30620	Basic solution used to adjust the pH of the buffers
n-butanol	ChemSpider	71-36-3	Used to TLC
NiCl2	Solarbio	746460	The component of medium
Orcinol	Sigma-Aldrich	447420	Used to prepare orcinol reagents
Propionic	Sigma-Aldrich	94425	Standard sample for SCFA
QIAamp DNA Stool Mini Kit	QIAGEN	51504	Extract bacterial genomic DNA
Ready-to-use PBS powder	Sangon Biotech (Shanghai) Co., Ltd.	A610100-0001	Used to prepare the lipid suspension
Resazurin	Solarbio	R8150	Anaerobic Equipment
Speed Vacuum Concentrator	LABCONCO	CentriVap	Use to prepare EPSs
Starch	Solarbio	YZ-140602	Use to the carbon source
Sulfuric Acid	Sigma-Aldrich	150692	Used to prepare orcinol reagents
T100 PCR	BIO-RAD	1861096	PCR amplification
TLC aluminium sheets	MerckMillipore	116835	Used to TLC
Trypticase Peptone	Sigma-Aldrich	Z699209	The component of medium
Tryptone	Sigma-Aldrich	T7293	The component of medium
Tween 80	Solarbio	T8360	Salt solution of medium
Valeric	Sigma-Aldrich	75054	Standard sample for SCFA
Vitamin K1	Sigma-Aldrich	V3501	The component of medium
Vortex oscillator	Scientific Industries	Vortex.Genie2	Use to vortexing
Yeast Extract	Sigma-Aldrich	Y1625	The component of medium
ZnSO4.7H2O	Sigma-Aldrich	Z0251	The component of medium