Another gap in the oral microbiome model research is the effect of preservation on oral microbiomes and laboratory oral cultures. The lack of research in this area is perhaps unsurprising given the newness of this subfield, though the paucity by no means implies that preservation of microbiomes is by unimportant, especially when we consider the potential of preserving oral communities for inoculating in vitro cultures or the potential of preserving oral samples for therapeutic applications. While preservation experiments have been extensively performed on single bacteria species, preservation of microbiome samples has only been studied recently, with the primary emphasis on the gut microbiome. Research on the gut microbiome has provided evidence that storing samples at various temperatures with different preservatives does not significantly impact the structures of the bacterial communities, though sampling method matters; that cryopreservation, especially using glycerol and inulin, greatly helps maintain the composition and functionality of artificial gut microbiota; that cryopreservation is less detrimental to sensitive strains than lyophilization; that intra- and inter-subject variability outweighs the technical variability, blueberries in containers including variations between sequencing runs and as a consequence of long-term storage; and that after long term storage, freeze-dried fecal samples could still be used for transplantation treatment of C. difficile infections.
However, there has not been much research on the effects of preserving an oral bacterial community and even less on oral bacterial communities generated in vitro. The importance of studying the effects of preserving an in vitro oral bacterial community is two-fold: First, to examine the effects of preservation on an in vitro oral bacterial community; and second, to investigate the feasibility of preserving a universal inoculum for seeding in vitro oral microbiome cultures, wherein the inoculum is extracted from as simple of a model as possible. A universal oral microbiome inoculum would help bring a great degree of control into the generation of in vitro oral microbial models, potentially in oral disease research where a healthy baseline oral community needs to be established.As mentioned above, what would benefit the investigation of oral microbiome members and their roles in health and disease – specifically, the correlations and relationships of the organisms, their effects on one another during development in both healthy and disease, and the effects of various conditions and agents on the community – is the ability to reliably study reproducible versions of this community of organisms. This ability necessitates the development of a complex, stable, and generalizable in vitro model community.
While the construction of lab models of the bacterial oral microbiota has received a fair amount of attention, and some such models have produced communities that resemble the human oral microbiome at least in membership if not in relative abundances, most such models still require specialized incubators and fastidious surfaces and exhibit somewhat poor repeatability across both technical replicates and biological replicates. There is clear a need for a model of the human oral bacterial microbiome that is relatively simple to cultivate, cost-effective, easy to maintain for longer growth, and reproducible to the extent allowable by statistical margins of error from technical replicates. To this end, we intended for our work to contribute to the foundation for this model, by constructing and characterizing an in vitro culture that encompasses the initial stages of the oral community development. We began by focusing on the dental plaque community, as it requires the least fastidious surface and can help refine the methodological details needed for more complex in vitro oral models. The goals of this project were sequential in nature. First, we investigated the feasibility of generating an oral bacterial community in vitro with the resources available to our particular group. Then, we examined the temporal behavior of the community to see how closely it adhered to the time evolution of other models and to the development of the human oral community in vivo.
Thirdly, we determined whether the community would be fitting to serve as the basis for the preservation experiment. Lastly, we developed an approach that would help elucidate the apparent correlations and covariances discovered among the microorganisms during the preservation experiment. It was our hope that at its conclusion, the project would have provided us with not only data on how a minimally complex multi-organism in vitro oral microbiome performs under basic culturing conditions, but also data on how such a community responds to preservation and subsequent propagation. This data would help determine the degree of representation and generalizability of our model, and help create a “baseline” inoculum for in vitro oral bacterial microbiome models.To investigate the feasibility of the project and establish foundational procedures, we performed preliminary experiments to cultivate a complex in vitro supragingival dental microbiome. The community requires plaque from healthy hosts to serve as inoculum; the model is based on previously published research on in vitro dental communities, specifically, communities generated in 24-well culture plates without the use of additional substrata, such as removable hydroxyapatite disks that partially mimic dental enamel. We chose this basis for the model for its versatility in the initial stages of community formation, facile operation, and low cost. The goal for this phase of the project was to test whether the on-site facilities and conditions would allow us to generate a community that reasonably resembles the initial colonization stages of the host oral community. Should the community exhibit reasonable resemblance, it could then serve as the basis upon which to build subsequent parts of the model. In addition to investigating the feasibility and quality of the procedure to generate a simplified community, we also aimed to assemble and test reliable tools for this project, including DNA extraction for sequencing, potential quantitative determination of different types of cells by spike-ins, and bio-informatics analysis. While DNA extraction in this phase of the project was ultimately performed by the sequencing center at UC Davis , we decided to examine the extraction efficiency of two different commercial kits that were most commonly used in microbiome research at the time, with the goal of bringing this part of the process into the lab in later phases of the project. In terms of quantitative determination of the number of cells, we used E. coli as “doping” or “spiking-in” cells. We chose an E. coli strain that was easy to grow and manipulate, such that we could roughly specify the number of live cells – approximated by colony-forming units – for spiking the dental plaque cultures after incubation. It was our goal to use the known number of E. coli cells to estimate the number of sequencing reads, and then to use the number of E. coli reads, the number of reads from other OTUs, and 16S copy numbers for all OTUs from sequencing to estimate the number or concentration of cells of dental plaque bacteria in the model communities. To achieve these goals, we needed a solid bio-informatics foundation using either the QIIME or mothur pipeline, planting blueberries in pots so we also examined the results from both pipelines to determine which one better suited our purposes.A single healthy volunteer host was used for dental plaque sample collection during this phase of the project.
The host was a 29-year-old female in good health with no systemic diseases or family history of systemic diseases. The host routinely practiced good dental hygiene and had no periodontal diseases, but did have two fillings on two back molar teeth. Sample collection took place under Protocols 3-18-0189 and 3-19-0119, approved by the UCSB Human Subjects Committee. Sample collection took place as follows: Supragingival plaque of molar teeth, cheek side, was obtained using a sterilized Gracey curette; on average, plaque was collected from 5 molar teeth. Prior to plaque collection, hosts had abstained from food, non-water liquids, and dental hygiene for 12 hours. Collected plaque was immediately suspended in sterile SHI medium, gently mixed, and divided equally among wells in a sterile 24-well plate such that each well received 1.98mL of the mixture. Prior to receiving sterile or inoculated medium, wells were preconditioned with clarified human saliva , which was supplied as frozen pooled fractions from healthy human volunteers and clarified in the lab. Clarification consisted of centrifuging defrosted saliva at 6,000 x g for 3 minutes, mixing with 1X PBS in a 1:1 ratio, and passing the mixture through 0.2µm filters. Conditioning was performed by adding 150µL clarified saliva to each well, allowing saliva to air dry without the plate lid at 37°C for 60 minutes, and sterilizing with short-wave UV light for 60 minutes.After sterile and inoculated medium had been dispensed into the wells, each well received 20µL of 0.5% sucrose prior to being covered with the well-plate lid. Controls and cultures were incubated in triplicates or quadruplicates to test for repeatability. Lidded well plates were place in a closed chamber , and the chamber was flushed three times with an anaerobic gas mixture consisting of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide, and then filled to positive pressure with the same gas mixture. During flushing and filling, gas from the cylinder was passed to the chamber through a 0.2µm filter to minimize potential microbial contamination. The chamber was then placed in an incubator set at 37°C and allowed to incubate for 20 to 24 hours. Some cultures were also incubated for four days to test the efficacy of commercial DNA extraction kits. The approximately one-day cultures were not fed again during incubation; each well of the four-day cultures was fed with 20µL of 0.5% sucrose every 24 hours before harvesting, with reestablishment of the anaerobic atmosphere after each feeding. After incubation, we observed for bacterial cell presence by looking for sedimented cells in the wells. From the wells with clear presence of cells , we harvested the wells by first aspirating 1.0mL of the liquid above the sedimented cultures into sterile microfuge tubes, and then mixing and pipetting the rest of the well into separate sterile microfuge tubes. For wells with no substantial cell presence , we gently and thoroughly mixed the wells and aspirated 1.0mL of the well contents into sterile microfuge tubes. Three samples from each type – controls, liquid above, and sedimented culture – were kept free of E. coli cells. We will henceforth refer to the E. coli cells as spike-ins and refer to the mixing with E. coli cells as “spiking”. Other samples received spike-ins at specified volumetric ratios, detailed in the E. coli Standard Curve and Spike-In subsection below. For controls and liquids with no spike-ins, the aforementioned 1.0mL aliquot in microfuge tubes was flash-frozen in liquid nitrogen and kept at -80°C until processing at the UC Davis Host Microbe Systems Biology Core . For sedimented cultures with no spike-ins, the 1.0mL of the culture was pelleted by centrifugation at 13,000 x g, the supernatant discarded, and the pellet flash-frozen and kept at -80°C until processing at the HMSB. Constitution and processing of spiked samples are described in the subsection titled E.coli Standard Curve and Spike-In.To observe the layers of sedimented cells, we reserved wells for microscopy without disturbing the layers, by carefully aspirating the liquid above and adding a commercial stain, which consisted of 200µL of a SYTO 9 and propidium iodide mixture from the FilmTracer LIVE/DEAD Biofilm Viability Kit . Dyes were allowed to penetrate the cells at room temperature for 30 minutes. To determine whether the surface of the bottoms of the wells affected the apparent viability of the cultured cells, we incubated cells in two different types of culture plates: standard tissue-culture-treated and surface-modified . Standard tissue-culture-treated plates have a net-negative surface charge while surface modified plates contain a mixture of positive and negative surface charges. Two plates of each type were used for this part of the preliminary experiments. Controls and cultures in both types of plates received identical treatment in terms of the volume of media, concentration of nutrients, and duration of incubation time. Results were visualized by staining and fluorescence microscopy procedures as described above.To determine the membership and abundances of the bacteria in these preliminary cultures using HTS, we attempted to develop an internal cell-counting standard using the E. coli K12 ER2738 strain . To do so, we first constructed a standard curve of colony-forming units vs. optical density at 600nm for ER2738 cells in the growth phase. For this curve, we grew overnight cultures of ER2738 and measured their optical density.