Uncovering complex microbiome activities via metatranscriptomics during 24 hours of oral biofilm assembly and maturation
For most of us, dental plaque (a.k.a. oral biofilms of bacteria that grow on our teeth) are something we only think about roughly twice a year. Once our teeth are cleaned we promise our dentist we will start to floss every day but this promise is quickly forgotten. I, however, am very interested in what this complex microbial universe is doing and how it impacts our health so I teamed up with a group of researchers from the J. Craig Venter Institute, the Forsyth Institute, the University of California Los Angeles, and the University of Washington to investigate and understand how oral bacteria behave when they grow together in naturally complex biofilm communities.
Oral bacteria growing as dental plaque have been known for several decades to catabolize dietary sugars and convert them into highly acidic metabolites (e.g. lactic acid), which cause tooth enamel erosion and eventually caries disease. With frequent consumption of carbohydrates, particularly when concurrent with a lack of oral hygiene, increased bacterial production of a sticky glucan matrix is favored, enmeshing cells and preventing diffusion of acidic metabolites. This results in a feed forward selection of acidogenic (acid producing) and aciduric (acid thriving) bacteria such as those belonging to mutant streptococci and lactobacilli. These bacteria dominate the dental plaque by producing copious amounts of lactic acid, delivered directly on the tooth enamel, which worsens the disease state and prevents reestablishment of health-associated community members.
Our first challenge was to develop a growth medium which captured growth of hundreds of bacterial plaque-growing species simultaneously. After iterations of media optimization, we succeeded in developing a novel growth medium that allows us to obtain a mixture of over a hundred of plaque bacteria by seeding as little as ten microliters of saliva in a total volume of one milliliter of growth medium. By using live/dead staining, confocal imaging and deep sequencing technologies (16S rRNA gene fragment sequencing and metagenomics) we were excited to discover that ~130 oral bacterial species were thriving in the sticky biofilm communities. To confirm our observations, we repeated the same experiments several times, seeded biofilm at different days in different 24-well plates, and confirmed there were no batch effects or biological biases – we realized it was possible to obtain the same community over and over again – like a perpetual machine!
We made a major discovery when we found that many of the biofilm community members were previously uncultivated and belonged to the enigmatic Candidate Phyla Radiation, such as TM7 and SR1. By using the complex community as a selection platform our team continued the isolation and domestication of the first TM7 member, TM7x, and its bacterial host, an Actinomyces odontolythicus strain, XH001.
Next, we set out to answer questions about how health-associated communities can recover from the recurring pH drop after eating a sugary snack, and which metabolites and signaling molecules are produced during sugar catabolism that could possibly be used for bacterial interspecies, as well as bacterial-host communication.
The results from these studies lead to the opportunity to follow the growth expansion and bloom of the cariogenic pathogen Lactobacillus fermentum, as highlighted our study in the Microbiome journal. This represents the first study that addresses bacterial behaviors and ecosystem functions as a complex oral biofilm community initially assembles and matures. We captured species-unique gene expression patterns related to biofilm community invasion resistance, cell-to-cell signaling, cell attachment mechanisms, iron sequestration, low pH stress responses, and unique secondary metabolite biosynthetic pathways throughout 24 hours of growth.
With this new knowledge, we can prioritize studies of clinically and ecologically important bacterial taxa and molecular mechanisms, which hopefully will lead to future developments of novel therapies to prevent and treat caries disease.