Studying bacteria in a petri dish or test tube has yielded insights into how they function and, in some cases, contribute to disease. But this approach leaves out crucial details about how they act in the real world.
Taking a translational approach, researchers at the University of Pennsylvania School of Dental Medicine and the Georgia Institute of Technology imagined the bacteria that cause tooth decay in three dimensions in their natural environment, dental plaque formed on the teeth of toddler affected by cavities.
The researchers found that Streptococcus mutans, a major bacterial species responsible for tooth decay, is encased in a protective multilayered community of other bacteria and polymers forming a unique spatial organization associated with the location of the disease onset.
“We started with these clinical samples, extracted teeth from children with severe tooth decay,” said Hyun (Michel) Koo, DDS, MS, PhD, senior coauthor and professor of Penn Dental’s Department of Orthodontics divisions of Community Oral Health and Pediatric Dentistry.
“The question that popped in our minds was how these bacteria are organized and whether their specific architecture can tell us about the disease they cause,” said Koo.
To address this question, the researchers used a combination of super-resolution confocal and scanning electron microscopy with computational analysis to dissect the arrangement of S mutans and other microbes of the intact biofilm on the teeth.
These techniques let the researchers examine the biofilm layer by layer, gaining a three-dimensional picture of the specific architectures.
“It’s clear that identifying the constituents of the human microbiome is not enough to understand their impact on human health,” said senior coauthor Marvin Whiteley, PhD, Georgia Tech Bennie H. and Nelson D. Abell Chair in Molecular and Cellular Biology.
“We also have to know how they are spatially organized. This is largely under-studied, as obtaining intact samples that maintain spatial structure is difficult,” said Whiteley, who also is the Georgia Research Alliance Eminent Scholar codirector in Emory-Children’s CF Center at the Georgia Institute of Technology.
In the current work, the researchers discovered that S mutans in dental plaque most office appeared arranged in a mound against the tooth’s surface. But it wasn’t alone.
When S mutans formed the inner core of the rotund architecture, other commensal bacteria such as S oralis formed additional outer layers precisely arranged in a crownlike structure. Supporting and separating these layers was an extracellular scaffold made of sugars produced by S mutans, effectively encasing and protecting the disease-causing bacteria.
“We found this highly ordered community with a dense accumulation of S mutans in the middle surrounded by these ‘halos’ of different bacteria and wondered how this could cause tooth decay,” said Koo.
To learn more about how structure impacted the function of the biofilm, the researchers attempted to recreate the natural plaque formations on a toothlike surface in the lab using S mutans, S oralis, and a sugar solution.
The researchers successfully grew the formations with rotund-shaped architecture and crown-like structure and then measured levels of acid and demineralization associated with them.
“What we discovered, and what was exciting for us, is that the rotund areas perfectly matched with the demineralized and high acid levels on the enamel surface,” said Koo.
“This mirrors what clinicians see when they find dental caries: punctuated areas of decalcification known as ‘white spots.’ The crown-like structure could explain how cavities get their start,” said Koo.
In a final set of experiments, the researchers applied an antimicrobial treatment. When the crown-like structures were intact, the S mutans in the inner core largely avoided dying. Only breaking up the scaffolding material holding the outer layers together enabled the antimicrobial to penetrate and effectively kill the cavity-causing bacteria.
The findings may help researchers more effectively target the pathogenic core of dental biofilms but also have implications for other fields, the researchers said.
“It demonstrates that the spatial structure of the microbiome may mediate function and the disease outcome, which could be applicable to other medical fields dealing with polymicrobial infections,” said Koo.
“It’s not just which pathogens are there but how they’re structured that tells you about the disease that they cause,” said Whiteley. “Bacteria are highly social creatures and have friends and enemies that dictate their behaviors.”
The field of microbial biogeography is young, the researchers said. However, extending this demonstration, which links community structure with disease onset, opens up a vast array of possibilities for future medically relevant insights, they said.
The study, “Spatial Mapping of Polymicrobial Communities Reveals a Precise Biogeography Associated With Human Dental Caries,” was published by the Proceedings of the National Academy of Sciences of the United States of America.