Live oral bacteria found in arterial plaque

March 22, 2005

GAINESVILLE, Fla. — Gum disease has been linked to hardening of the arteries for nearly a decade, and scientists have long fingered a gang of oral bacteria as the obvious suspects behind many cases of the vessel-clogging killer.

Now University of Florida researchers have cornered the bacterial ringleaders of gum disease inside human artery-clogging plaque — the first concrete evidence to place the pathogens at the heart of the circulatory crime scene. Their report appears in the current issue of Atherosclerosis, Thrombosis and Vascular Biology.

“Our finding is important because it has proved there are live periodontal bacteria in human atherosclerotic tissue,” said study investigator Ann Progulske-Fox, a professor of oral biology at the UF College of Dentistry. “Now we can begin to understand how these bacteria contribute to the disease process.”

The oral bacteria UF researchers found in the plaque, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, are two of the most aggressive offenders in periodontal disease, the leading cause of adult tooth loss. Because of the strong association between periodontal and cardiovascular diseases, scientists have postulated for years that oral pathogens contribute to arterial damage that leads to heart attack or stroke, which kill nearly a million Americans a year. In fact, a recent study conducted elsewhere found a direct correlation between the amount of periodontal bacteria in the mouth and the formation of blockages in the carotid artery in the neck.

To reach the circulatory system, the bacteria have to breech the barrier between tissues in the mouth and the bloodstream, Progulske-Fox said. For patients with periodontal disease, whose gums are inflamed and bleed easily, bristles from even the softest toothbrush can tear tiny blood vessels in the compromised gum tissues, leaving the door wide open for bacteria to enter.

But could the bacteria elude the body’s protective immune response once within the bloodstream?

Researchers worldwide have sought to empirically nab oral bacteria — dead or alive — in atherosclerotic tissues. They have found remnants of bacterial DNA in arteries, signaling that bacteria had entered the bloodstream. Yet scientists have never been able to grow periodontal bacteria isolated from arterial plaque in Petri dishes, even though the same species of bacteria swabbed from oral plaque can be cultured that way. Therefore, researchers could not be sure the DNA was from bacterial trespassers destroyed by the immune system in the bloodstream, or if live bacteria were actually directly involved in plaque formation within the vessel walls.

“It makes sense that those periodontal bacteria most invasive in the mouth could be able to adapt to the vascular situation,” said study project leader Emil Kozarov, an adjunct associate professor of oral biology at UF and a faculty member at Nova Southeastern University.

Probably only a handful of periodontal bacteria have been successful in making the jump of being infectious to both the oral and vascular tissues. Identifying these bacteria would give researchers the inside scoop on how the bacteria may contribute to cardiovascular disease, said Kozarov.

To find them, Progulske-Fox’s team took the unusual approach of attempting to grow bacteria from arterial plaque directly on human artery cells. They obtained a section of a diseased carotid artery from a 74-year-old, partially toothless male patient undergoing surgery to remove an 80 percent blockage at Shands at UF in Gainesville. After removal, the sample was rinsed and placed on ice, then rushed to Progulske-Fox’s nearby lab in a sealed, sterile container.

Within six hours of leaving the operating room, researchers pureed plaque from the artery and set it to incubate in a brew of healthy arterial cells and liquid growth medium. After 24 hours, the cells were separated from the slurry in the flask, washed several times, then subjected to a series of fluorescent baths containing antibodies sensitive to P. gingivalis and A. actinomycetemcomitans bacteria.

Finally, the cells were fixed to a glass slide and placed under a microscope to search for the presence of invasive periodontal bacteria within the cell structures. If any of the artery cells were infected with the bacteria, fluorescent antibodies would light them up like Alcatraz in lockdown.

Progulske-Fox and her team found the endothelial cells were infected with both P. gingivalis and A. actinomycetemcomitans, proving live bacteria had been present in the atherosclerotic plaque.

“This report certainly provides a smoking gun that live bacteria have become seeded from the oral cavity to become inhabitants of the vessel wall,” said Dr. Steve Offenbacher, distinguished professor of periodontology at the University of North Carolina at Chapel Hill School of Dentistry. “The exciting implications focus on the known ability of these bacteria to destroy connective tissue in the mouth, suggesting that when infecting the vessel wall they may contribute to the instability of the atherosclerotic plaque — leading to acute events such as heart attack or stroke.”

Progulske-Fox plans to study atherosclerotic tissue samples from 50 to 60 more patients to better understand how bacteria infect arterial cells. She suspects some strains of the bacteria may be more successful in breaching the barriers separating oral tissues from the bloodstream. These bad bugs would become “most wanted” in the fight against periodontal and cardiovascular disease.

“More study samples will show us which strains are implicated in the disease process, so we can design simple diagnostic technology that could be used in a dental office to identify specific bacteria the patient is carrying and whether that bacteria is known to cause atherosclerotic disease,” said Progulske-Fox.

She envisions those diagnostic tests would be the first step in the war against periodontal and cardiovascular diseases, eventually leading to the development of a vaccine that would prevent oral bacteria from ever gaining a stronghold in the mouth. Antibiotic or antimicrobial treatments that could kill the bacteria after they have entered the circulatory system might also someday be possible.

For now, however, she advises people to practice good oral hygiene.

“It is important for these patients to have very good dental hygiene,” said Progulske-Fox, “because losing a tooth may not be a big deal to some people, but it can become a life-threatening situation.”