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.

Published in Microbiology

Uncovering complex microbiome activities via metatranscriptomics during 24 hours of oral biofilm assembly and maturation
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BioMed Central
BioMed Central BioMed Central

Uncovering complex microbiome activities via metatranscriptomics during 24 hours of oral biofilm assembly and maturation - Microbiome

Background Dental plaque is composed of hundreds of bacterial taxonomic units and represents one of the most diverse and stable microbial ecosystems associated with the human body. Taxonomic composition and functional capacity of mature plaque is gradually shaped during several stages of community assembly via processes such as co-aggregation, competition for space and resources, and by bacterially produced reactive agents. Knowledge on the dynamics of assembly within complex communities is very limited and derives mainly from studies composed of a limited number of bacterial species. To fill current knowledge gaps, we applied parallel metagenomic and metatranscriptomic analyses during assembly and maturation of an in vitro oral biofilm. This model system has previously demonstrated remarkable reproducibility in taxonomic composition across replicate samples during maturation. Results Time course analysis of the biofilm maturation was performed by parallel sampling every 2–3 h for 24 h for both DNA and RNA. Metagenomic analyses revealed that community taxonomy changed most dramatically between three and six hours of growth when pH dropped from 6.5 to 5.5. By applying comparative metatranscriptome analysis we could identify major shifts in overall community activities between six and nine hours of growth when pH dropped below 5.5, as 29,015 genes were significantly up- or down- expressed. Several of the differentially expressed genes showed unique activities for individual bacterial genomes and were associated with pyruvate and lactate metabolism, two-component signaling pathways, production of antibacterial molecules, iron sequestration, pH neutralization, protein hydrolysis, and surface attachment. Our analysis also revealed several mechanisms responsible for the niche expansion of the cariogenic pathogen Lactobacillus fermentum. Conclusion It is highly regarded that acidic conditions in dental plaque cause a net loss of enamel from teeth. Here, as pH drops below 5.5 pH to 4.7, we observe blooms of cariogenic lactobacilli, and a transition point of many bacterial gene expression activities within the community. To our knowledge, this represents the first study of the assembly and maturation of a complex oral bacterial biofilm community that addresses gene level functional responses over time.

At the time, nobody knew much about this complexity at all. We realized that most of our knowledge of the human microbiome derive from studies of single bacterial species growing in a cultivation flask in the research laboratory, yet the human microbiome consists of thousands of interacting microbial species, some of which mean a lot to our health. With this in mind we decided to challenge this knowledge gap and develop an oral in vitro biofilm model system representative of highly complex human dental plaque. The initial most burning questions to answer were; What if bacteria, just like humans, behave differently when they’re social and interact with their kind? Which natural microbiological processes are involved in pathogen suppression vs. expansion; and what are the roles of metabolic activity and signaling in biofilm community development?


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.





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