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Stanford Report, December 8, 1999

New analysis reveals human mouth carries more germs than expected

BY KRISTIN WEIDENBACH

The human mouth is awash with bacteria. Mostly neighborly bugs, they live on our teeth and gums, helping to digest food and to ward off attack by less friendly, disease-causing bacteria that can steal their way in.

Stanford researchers have now shown that more of these oral inhabitants exist than previously thought. Using a combination of old and new scientific methods to study a scraping of plaque from a healthy human mouth, the researchers found evidence of 37 unique bacteria that microbiologists had never before recorded. Some were closely related to bacteria that scientists are familiar with, but others were very different.

Knowing more about the bacteria that reside in a normal, healthy mouth may help physicians understand changes in the bacterial population that can lead to gingivitis, periodontitis and tooth decay.

"Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well-studied and familiar microbial environment," said David Relman, MD, assistant professor of medicine and of microbiology and immunology at Stanford, and lead author of the study published in the December 7 issue of the Proceedings of the National Academy of Sciences. Relman and colleagues conducted the research in his lab at the Veterans Affairs Palo Alto Health Care System.

According to Relman, the subgingival crevice -- the deep gum pocket cradling each tooth -- has been repeatedly scrutinized in the search for microbes. Even though almost 500 bacterial strains have been identified already, Relman believes this may be only a fraction of the bacteria living in this oral groove.

Oral bacteria have traditionally been studied by taking a scraping or sample from inside the mouth, growing the bugs in the laboratory and then identifying different species according to biochemical tests and the type of food source that each bacteria prefers. Using this method, the Relman team identified bacteria found in a sample of plaque taken from the subgingival crevice.

They also searched the same sample using molecular techniques. Instead of nurturing the bacteria in the lab, they prepared DNA directly from the plaque and studied each genetic sequence that had a bacterial signature. Comparing the results, they found that the molecular method yielded many new bacteria. Not only did the method reveal bugs that had never before been found in the mouth, many were bugs that had not yet been documented by microbiologists.

The team discovered 31 bacteria using the molecular method. In contrast, the traditional approach, which only identifies bacteria that can be cultivated in the lab, uncovered only six new bugs.

"Sequence-based environmental microbial surveys have taught us that cultivation methods woefully underrepresent the true extent of bacterial diversity," said Relman.

Although the cultivation method traditionally used by clinical microbiology labs did not uncover the bacterial diversity revealed by the molecular technique, the researchers found that some bacteria were more readily recovered the old way, indicating that DNA analysis and cultivation both have a role in the comprehensive study of human microbial populations.

Characterization of previously undocumented oral bacteria is part of the Relman team's larger effort to identify rare and unusual microbes that make their home in the human body -- many cohabiting benignly but some causing mysterious human disease.

Ian Kroes, MD, lead author of the study, was a medical student in Relman's lab at the time the research was conducted. Paul Lepp, PhD, a postdoctoral fellow in the lab and the third member of the research team, conducted much of the DNA analysis.

The research was funded by the Donald E. and Delia B. Baxter Foundation and the Lucille P. Markey Charitable Trust. SR