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.
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.
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, mucosal surfaces and dental prosthetics and fillings3. Bacteria can also be present as flocs and planktonic cells in the lumen of the tooth canal, either intermixed with necrotic pulp tissue or suspended in a fluid phase.
There is active, continuous cross-talk between host cells and the resident microbiota4. Bacteria communicate within and between species, and only a small proportion of the natural colonizers can adhere to tissues, while other bacteria attach to these primary colonizers. For instance, cell-cell binding between microorganisms is key for integrating secondary colonizers into oral biofilms, and building complex networks of interacting microbial cells4. Around 70% of bacterial aggregates in a saliva sample are formed by Porphyromonas sp., Streptococcus sp., Prevotella sp., Veillonella sp., and unidentified Bacteroidetes. F. nucleatum is an intermediate colonizer in the subgingival biofilm and aggregates with the late colonizers P. gingivalis, T. denticola, and Tannerella forsythia, which are implicated in periodontitis5. In addition, Streptococcus mitis occupies both mucosal and dental habitats, while S. sanguinis and S. gordonii prefer to colonize teeth3. Thus, S. sanguinis is present in lower incisors and canines, while Actinomyces naeslundii has been found in upper anteriors6.
In addition, the indigenous microbiome plays a role in maintaining human health2. Resident microbiota participates in immune education and in preventing pathogen expansion. This colonisation resistance occurs because the native bacteria may be better adapted at attaching to surfaces, and more efficient at metabolising the available nutrients for growth. Although probiotic strains survive the gastrointestinal passage and remain active, the persistence of autochthonous bacteria swallowed from an upper location of the gastrointestinal tract has not been fully described. Thus, we subjected an artificial community, representative of the oral ecosystem, to simulated gastrointestinal transit conditions. Viability of bacterial cells was assessed using a multicompartment model resembling the gut epithelium. Current gut simulators offer suitable reproducibility in terms of analysis of the luminal microbial community7. However, bacterial adhesion and host-microbe interaction are separately addressed, as combining cell lines with microbial communities is challenging8. In contrast, we present a framework that provides potential mechanistic explanation of successful colonization events reported on the gut interface. Indeed, this model can be jointly used with a static gut model to evaluate the impact of microbial communities on host surface signalling.
1. Strains and Culture Conditions
NOTE: The synthetic oral community was composed by strains commonly present in the oral microbiome3.
2. Development of a Multispecies Community Representative of the Oral Microbiome
NOTE: Generate a synthetic community to simulate the potential adhesion capacity of oral bacteria to the in vitro gut epithelium. Grow bacteria on blood agar plates supplemented with 5 μg/mL hemin, 1 μg/mL menadione, and 5% sterile defibrinated horse blood, as described in Hernandez-Sanabria et al. (2017)11. In brief:
3. General Cell Culture Practices
NOTE: For general aspects of cell culture, the authors refer to Master and Stacey (2007)15. Obtain cell lines used from the European Collection of Authenticated Cell Cultures (Caco-2 ECACC 86010202 and HT29-MTX-E12 ECACC 12040401, Public Health England, UK). Reagents for cell culture can be purchased (see Table of Materials), unless otherwise specified.
4. Assembly of a Multicompartment Cell Model Simulating the Gut Host-microbe Interface
NOTE: Complete the co-culture in double chamber wells (diameter 24 mm, pore size 0.4 μm; see Table of Materials).
5. Bacterial Survival Following In Vitro Gastrointestinal Transit Conditions
NOTE: Prepare simulated digestion fluids following the protocol of Minekus et al. (2014)17, with the modifications described below. Prepare all the dilutions with ultrapure water. Filter-sterilize all solutions through a 0.22 µm filter and perform the digestion procedure under sterile conditions.
6. Bacterial Colonization Ability Following In Vitro Gastrointestinal Transit Conditions
7. Sample Analysis
NOTE: Analyse collected samples (S1-S7) to evaluate bacterial viability and adhesion potential to the intestinal cells, following a simulated gastrointestinal digestion.
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 and 1B). Based on the Live/Dead quantification, bacterial viability decreases after each digestion step (Figure 2). This can be a result of acid pH during the gastric passage and of the bile salts contained in the small intestine digestion fluids, as occurring under physiological conditions. Viability during the transit through both in vitro and in vivo gastrointestinal digestion may depend on variations in the environment (e.g., fed vs. fasted conditions), or even on bacteria protection processes (spore formation or enteric coatings).
Flow cytometric counts must be compared with suitable controls, such as heat-killed bacteria or background samples, to perform accurate viable cell quantification18. The harsh conditions of the stomach and small intestine digestion can switch bacteria to viable but non-culturable cells, which are live bacteria that do not either grow or divide. For this reason, plate counts differ from viable cell counts obtained with flow cytometry. For instance, in Figure 3, viable cells recovered from the mucosal interface are coloured in blue in the flow cytometry plot. However, no growth was observed when these mucus samples were plated (results not shown).
The fraction on which bacteria show higher viability rates is the cellular debris (S7), as revealed by plate counting (Figure 4). The number of colonies may be variable, but presence of metabolically active bacteria is found in the less diluted samples.
Figure 1: Cell viability of the bacteria composing the multispecies microbial community at the beginning of the in vitro gastrointestinal digestion. (A) Viable cell counts (log units) quantified by live/death staining and flow cytometry (average ± standard deviation, n = 3). (B) Representative flow cytometry plot of the multispecies microbial community after 48 h of co-culture. Dots in green, red, and black represent viable, damaged and background or non-classified bacteria, respectively. Please click here to view a larger version of this figure.
Figure 2: Cell viability of the bacteria after the simulated gastrointestinal digestion. Bars represent the number of viable cells (log units) after the oral, gastric, and small intestinal digestion steps (average ± standard deviation, n = 3). Please click here to view a larger version of this figure.
Figure 3: Representative plot of viable bacterial cells in the mucosal interphase. Red dots represent the background and blue dots the viable cells. Flow cytometry conditions were established based on Props et al. (2016)19. Mucosal samples were diluted 1:100 (v/v) and stained with Sybr Green/Propidium Iodide for 15 min, and 100 µL of sample was measured at FL1: 533/30 nm, FL2: 585/40 nm, FL3: >670 nm long pass, and FL4: 675/25 nm. Please click here to view a larger version of this figure.
Figure 4: Colony forming units (CFU) grown from the cellular debris in modified BHI plates. Three biological replicates were used for the bacterial adhesion assay and three technical replicates were performed for the CFU quantification. Two replicate plates containing -1 to -6 dilutions of the cell debris (S7) are shown in the figure. Please click here to view a larger version of this figure.
Cell culture | Adherent/ Suspension | Culturing Medium | General supplements | Specific supplements | Doubling time | Incubation conditions |
Caco-2 (ECACC 86010202) | Adherent | Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose | Inactivated fetal bovine serum (10% v/v) Penicillin (100 U/mL)* Streptomycin (0.1 mg/mL)* Amphotericin (0.0025 mg/mL)* |
Glutamax (4 mM) Non essential aminoacids (1% v/v) Pyruvate (1 mM) |
65–72 h | 95% humidity and 10% CO2 CO2 incubator (see Table of Materials) |
HT29-MTX (ECACC 12040401) | Adherent | HEPES (1 mM) | 20–24 h | |||
* Antibiotics and antifungals were removed from the cell culture media 2 days before starting the assays. |
Table 1: Description of the cell lines and media composition for cell culture maintenance.
The oral microbiome is a key element in human health as recently reported by several authors20,21. 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 unravel the impact of the microbial component on the host.
In vitro models are basic tools for research, allowing for conducting mechanistic studies and achieving background knowledge intended to reduce, make more efficient, and better target in vivo studies. For this reason, it is critical to ensure purity, viability, and optimal growth of axenic cultures prior to establishing the synthetic microbial community. We recommend evaluating the viability and composition of bacterial communities before exposure to cell cultures, to avoid bias in the analysis. High number of damaged bacteria may hinder the adhesion and further impact the integrity of the cell model. In addition, the use of a trustable source of cell lines will minimize cell line misidentification, contamination, and poor annotation, enhancing experimental reproducibility22.
Including mucus-producing cells enables resemblance to the small intestine, as mucus and mucins provide the first defence line of the gastrointestinal tract23. In addition, the mucosal layer is suitable for bacterial colonisation, allowing for characterising bilateral transport of bacterial metabolites. Moreover, simulated gastrointestinal conditions increase the adhesion ability of Lactobacillus paracasei strains to Caco-2 and mucin24, supporting the relevance of incorporating digestive processes in our model.
Further improvements in the model can be introduced to incorporate the immune host component, as previously reviewed25. However, current co-culture systems of epithelial and immune cells have not been used for host-microbe interaction applications (e.g., evaluation of food bioactive compounds or probiotics), which might indicate that those systems may lack reproducibility25.
Previous research with Caco-2, HT29-MTX or co-cultures assessed the adhesion of probiotic or pathogenic strains to mammalian cells26,27. Using single stains or associations of few microorganisms may provide a limited overview on the host-microbe interactions, and it is not representative of the complexity of the human gastrointestinal tract. Although the use of natural microbial communities can be more representative of the in vivo scenario, they are difficult to characterise and to study. In contrast, our methodology offers the opportunity of assessing multiple host-microbe interactions, using a complex and representative microcosm. The use of a defined synthetic microbial community allows for the generation of a system with reduced complexity28. Changes in the community can be a result of the microenvironmental conditions in the context of individual experiments. Thus, the microbial component of this model can be easily scaled up or down for deconstructing the impact of environment as a selective force. Finally, sampling accessibility guarantees further characterisation of the functional activities of both human cells and the associated microbial community.
In the past years, new in vitro models based on organoid technology have been developed. Organoids are 3D cell cultures that incorporate some of the key features of the represented primary tissue. Although intestinal organoids are a near-physiological system for studying adult stem cells and tissues, such model also has some limitations. Intestinal organoids have limited use in resembling inflammatory responses to infection or drugs, as they lack immune cells. Additionally, organoids are heterogeneous regarding viability, size, and shape, impeding phenotype screening and rendering standardization complex. Despite the limitation of immortalized cell lines for in vitro modelling of healthy tissues, the use of well-characterized and stable cell lines also offers advantages as repeatability, reproducibility, and low cost. These benefits allow for simultaneous screening of several conditions, but the translational use of the data is controversial. Primary cells may be more representative of in vivo physiology; however, they have high inter-individual variability, are not fully characterized, are heterogeneous, and have a limited time span. These factors are a drawback for including multiple conditions or replicates in one assay.
As combining cell lines with complex microbial communities may represent a challenge, we focused on developing a reproducible and repeatable model with high flexibility and applicability in laboratories with basic cell equipment, until more representative models became available and well characterised. We have evaluated the functionality of diverse bacterial communities using these multicompartment models, for instance, for evaluating probiotic behaviour in the upper gastrointestinal tract and for characterising xenobiotic impact on the gut interface. Thus, we propose this model as a base for further assays with a wide variety of probiotics, drugs, or food compounds, within a relevant and defined in vitro ecosystem.
The authors have nothing to disclose.
The authors gratefully acknowledge financial support from the Flanders Research Foundation to Marta Calatayud Arroyo (FWO postdoctoral fellowship-12N2815N). Emma Hernandez-Sanabria is a postdoctoral fellow supported by Flanders Innovation and Entrepreneurship (Agentschap voor Innovatie door Wetenschap en Technologie, IWT).
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 |