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There is still an unknown environment on earth, and it’s closer to home than you’d think. Full of wild and fantastic life, this environment is full of possibility – and maybe some of this morning’s breakfast.

You read that right. This unexplored environment is the human oral cavity.

As of 2018, approximately one third of the more than 700 bacteria species in the human oral cavity, a more complete way to describe the human mouth, have yet to be cultured by scientists. Though efforts to study this “dark microbiota,” or unknown microbe community, have become prolific in the past decade, progress can be slow.

Many of the bacteria present in the mouth are anaerobic, meaning they don’t need oxygen to survive. They live deep in oral tissue or mucus-like bacteria layers called biofilms, and they often actually die in the presence of oxygen. That means removing the microorganisms from the mouth and keeping them alive long enough to study can be a real challenge.

But it’s a challenge that Karissa Cross, a doctoral candidate studying microbiology made a central focus of her life four years ago when she started her job as a graduate research assistant at Oak Ridge National Laboratory.

Since that time, Cross isolated and cultured the oral microbe Desulfobulbus oralis—an accomplishment that will give scientists important information about both gum disease and the relationships between different microbes.

Cross, who “doesn’t like easy things,” originally embraced the problem while working on a National Institute of Health-funded grant under the supervision of Mircea Podar, a distinguished scientist at ORNL.

“I had always wanted to come back to studying human health and disease,” Cross said, “and so it just seemed like a perfect serendipitous fit that he [Podar] had just gotten this grant and said he needed a microbiologist.”

According to ORNL’s website, the grant was given for researchers to study the “novel microbial minority of the human oral cavity and how these organisms may contribute to the progression of periodontal disease.”

Cross said that from the beginning, the team of researchers she joined chose their particular focus on D. oralis because an earlier paper had indicated that the microorganism often showed up in the mouths of people with periodontal disease, a common inflammatory illness of the gums.

However, the team needed further research to make science-based conclusions about whether or not D. oralis might actually cause the disease.

Isolating D. Oralis

To find out more about Desulfobulbus in the human mouth, Cross set out to isolate the microorganism, or remove it from the presence of other bacteria. That meant she first had to learn how to grow it in the lab. D. oralis, a “finicky bug,” resisted Cross’ efforts.

Besides the fact that it dies in the presence of oxygen, Cross said D. oralis exhibits qualities that make it difficult to cultivate. For one, the microorganism is not abundant in the human mouth, sometimes making up as little as 1% or less of the oral microbial presence. Even in individuals with periodontal disease, it’s not plentiful. It also grows very slowly and will not grow on an agar plate, the traditional method for cultivating a colony of bacteria.

“A lot of my first year was just trying to optimize medium,” Cross said.

In other words, she spent countless hours testing different chemical combinations to find conditions where the microbe could live and multiply.

“If you look at it wrong, it will die,” Cross said, an introspective smile spreading across her face. “It’s a handful, but I guess it’s my handful.

Another complicating factor was that the microbe relies on the byproducts of another bacteria, known as Fusobacterium nucleatum, to survive. This poses a serious problem for a scientist trying to get D. oralis to thrive on its own.

“The thing about this type of project is that it’s what you can consider ‘high-risk high-reward,’” Cross said. “So, there was always the possibility that it was never going to happen.”

To overcome the problem, she developed a method to harvest F. nucleatum’s byproducts. Cross then added them to a mixture of sulfate, lactate and salts. After years of perfecting her D. oralis cultivation, she started noticing results.

“We knew we were getting close – more and more Desulfobulbus every day showing up in our medium, less of other things,” Cross said.

Cross and her team successfully isolated D. oralis in August of 2016.

“After many years of not getting it, I was very skeptical when we did get the organism isolated,” Cross said. “Science is filled with a lot of disappointment and setbacks, and it’s really hard to come into this field being optimistic when more times than not, things fail, and you really have to learn how to deal with failure.”

The defining moment came when her research team sequenced the genome of the microbe, she said. That allowed them to determine that their samples did not contain the DNA of any other bacteria, meaning D. oralis had truly been isolated.

Because she isolated the microbe, Cross got to name it. D. oralis is a member of the Desulfobulbus genus, so that part of the name was already designated, but she gave it the species name “oralis” because it is the only Desulfobulbus from the human oral cavity.

“I toyed with the idea of naming it something like, relating to my name,” Cross said, laughing. “But at the end of the day, I was so tired after all of this I didn’t even care what it was named.”

Finding medical meaning

With D. oralis isolated and cultured, Cross’ team could begin testing its effects on human hosts. Cross doesn’t think of her work in terms of fighting bacteria—she said she isn’t a clinical microbiologist.

Rather, she thinks of herself more as a microbial ecologist. But of course, evaluating D. oralis’ role in human disease was always a goal of the project.

Payal Chirania, a first-year doctoral student in UT’s genome science and technology program, joined the research team with that goal in mind.

To test D. oralis’ potential disease causing capacity, Chirania and her fellow scientists incubated the microbe with a sample of human epithelial cells, which essentially act as the lining of mouth tissue.

“So one of the markers for any disease causing agent is that when you infect with it, or incubate cells with it…the cells react,” Chirania said. “The ways cells react is that they create something called an autoimmune response.”

She explained that the isolation enabled the team to observe that D. oralis itself produced toxins that did spark a pro-inflammatory state in the mouth.

“The immune response can be inflammatory…that means that the organism—in this case D. oralis—is acting as a pathogen, that it’s causing the cells to defend themselves,” Chirania said.

This led researchers to believe that D. oralis was an important organism that they should keep studying as a potential disease causing agent in periodontal disease, along with other bacteria in the mouth.

“The mouth creates an immune response that leads to a pro-inflammatory environment of immune cells that are trying to fight. They’re letting out all of their immune molecules,” Cross said. “That leads to extended inflammation that then leads to the bone and tissue loss.”

According to the Center for Disease Control, periodontal disease affects nearly half of adults age 30 and older in the United States. That number rises with age and factors like poverty. In the developing world, the disease is more widespread and can be deadly.

Both Cross and Chirania are proud that their work might be able help people.

“Once you know what’s causing a disease, you can actually go to try to find a cure more appropriately,” Chirania said.

The other implication in terms of microbial interactions, Cross said, is the likelihood that multiple bacteria contribute to the same disease. The interdependent nature of microbes like D. oralis and F. nucleatum suggests that some maladies of the mouth are caused by not just one, but many bacteria at the same time.

Moving forward

For Cross, her results were worthwhile, and they will be a large part of her dissertation: she hopes to complete her doctorate in the summer of 2019.

When her paper detailing her research finally completed its tedious review process and achieved publication in academic journals, it also ended up making her the subject of local fame. The Knoxville News Sentinel ran a front-page story featuring her work.

To see her face peeping out of newsstands around town, she said, was strange indeed. But it’s a point of pride, too.

“We’re currently the only lab in the world that can grow it, so, and possibly I’m the only person,” Cross said, before quickly noting that her advisor likely could as well.

But Cross’ future might not include much more work with D. oralis. She is excited about one set of genomes she was able to gain from the project but is eager to move on to other things. Her work will contribute to a quickly growing body of research about the human oral microbiome, but Cross knows firsthand that science is slow and can be trying: she learned that during three and a half years hovering over cultures of D. oralis.

“The changes that we’re beginning to implement now, we might not even get to live to see the final results,” Cross said. “For example…developing targets to cure diseases that are going to take many, many more years of research.”

The lack of immediate gratification doesn’t bother her, she said.

“We get to be the starting steps to do that.”

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