Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Summary

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations' survivability.

Abstract

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Introduction

Humans cohabit with bacteria, which are present at the same number as human cells¹. Hence, it is of crucial important to obtain a comprehensive understanding of the human microbiome. The oral cavity is a unique environment in that it is divided into several smaller habitats, thus containing a large variety of bacteria and biofilms in those different locations. Being an open ecosystem, some species in the mouth may be transient visitors. However, certain microorganisms colonize soon after birth and form organized biofilms². These are found in the teeth surface above the gingival crevice, the subgingival crevice, tongue, m....

Protocol

1. Strains and Culture Conditions

NOTE: The synthetic oral community was composed by strains commonly present in the oral microbiome³.

1. Obtain the following strains from the American Type Culture Collection (ATCC): Aggregatibacter actinomycetemcomitans (ATCC 43718), Fusobacterium nucleatum (ATCC 10953), Porphyromonas gingivalis (ATCC 33277), Prevotella intermedia (ATCC 25611), Streptococcus mutans (ATCC 25175), Streptococcus sobrinus (ATCC 33478), Actinomyces naeslundii (ATCC 51655), Str....

Results

This protocol leads to the generation of a model suitable for elucidating the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. The counts of intact cells from individual strains is approximately 10⁸ cells mL^-1 prior the creation of the synthetic community, while the multispecies microcosm contained above 90% of viable cells during the establishment of the community (Figure 1A

Discussion

The oral microbiome is a key element in human health as recently reported by several authors^20,21. Previous findings suggest that the ingestion of saliva containing large loads of bacteria can influence the microbial ecosystem of the small intestine, which is one of the main sites for immune priming. The combination of a static upper gastrointestinal digestion model with the host interface represented by intestinal epithelial and mucus-secreting cells, served to .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
STRAINS
Aggregatibacter actinomycetemcomitans	American Type Culture Collection	ATCC 43718
Fusobacterium nucleatum	American Type Culture Collection	ATCC 10953
Porphyromonas gingivalis	American Type Culture Collection	ATCC 33277
Prevotella intermedia	American Type Culture Collection	ATCC 25611
Streptococcus mutans	American Type Culture Collection	ATCC 25175
Streptococcus sobrinus	American Type Culture Collection	ATCC 33478
Actinomyces viscosus	American Type Culture Collection	ATCC 15987
Streptococcus salivarius  TOVE-R
Streptococcus mitis	American Type Culture Collection	ATCC 49456
Streptococcus sanguinis	BCCM/LMG Bacteria Collection	LMG 14657
Veillonella parvula	Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures	DSM 2007
Streptococcus gordonii	American Type Culture Collection	ATCC 49818
CELL LINES
Caco-2 cells	European Collection of Authenticated Cell Cultures	86010202
HT29-MTX cells	European Collection of Authenticated Cell Cultures	12040401
REAGENTS AND CONSUMABLES
Brain Heart Infusion (BHI) broth	Oxoid	CM1135
Blood Agar 2	Oxoid	CM0055	Blood Agar medium
Menadione	Sigma	M9429
Hemin	Sigma	H9039
5% sterile defibrinated horse blood	E&O Laboratories Ltd,	P030
InnuPREP PCRpure Kit	Analytik Jena	845-KS-5010250	PCR purification kit
Big Dye	Applied Biosystems	4337454	Dye for sequencing
ABI Prism BigDye Terminator v3.1 cycle sequencing kit	Applied Biosystems	4337456
SYBR Green I	Invitrogen	S7585
Propidium Iodide	Invitrogen	P1304MP
T25 culture flasks uncoated, cell-culture treated, vented, sterile	VWR	734-2311
Trypsin-EDTA solution	Sigma-Aldrich	T3924-100ML
Trypan Blue solution 0.4%, liquid, sterile-filtered	Sigma-Aldrich	T8154
PBS	Gibco	14190250
DMEM cell culture media, with GlutaMAX and Pyruvate	Life technologies	31966-047
Corning Transwell polyester membrane cell culture inserts	Sigma-Aldrich	CLS3450-24EA
Mucin from porcine stomach Type II	Sigma-Aldrich	M2378
Inactivated fetal bovine serum	Greiner Bio One	758093
Antibiotic-Antimycotic (100X)	Gibco	15240062
Triton X 100 for molecular biology	Sigma-Aldrich	T8787
DPBS without calcium, magnesium	Gibco	14190-250
Pierce LDH Cytotoxicity Assay Kit	Thermo Fisher Scientific	88953
Corning HTS Transwell-24 well, pore size 0.4 µm	Corning Costar Corp	3450
Nuclease-free water	Serva Electrophoresis	28539010
EQUIPMENT
Neubauer counting chamber improved	Carl Roth	T729.1
BD Accuri C6 Flow cytometer	BD Biosciences	653118
PowerLyzer 24 Homogenizer	MoBio	13155
T100 Thermal Cycler	BioRad	186-1096
Flush system	Custom made	-
InnOva 4080 Incubator Shaker	New Brunswick Scientific	8261-30-1007	Shaker for 2.10
Memmert CO2 incubator	Memmert GmbH & Co.	ICO150med
Millicell ERS (Electrical Resistance System)	EMD Millipore, Merck KGaA	MERS00002
Millipore Milli-Q academic, ultra pure water system	Millipore, Merck KGaA	-
Shaker (ROCKER 3D basic)	IKA	4000000	Shaker for 6.10