The Bug Stops Here

March 13, 1998

Researchers at Forsyth Dental Center are tracing the identity of unknown and emerging microbes, some of which have foiled the efforts of investigators around the country. Using the newest molecular methods, Floyd Dewhirst and Bruce Paster, associate professors in the Harvard School of Dental Medicine's Department of Oral Biology, are identifying mysterious bacterial species that may be the cause of dental root caries and periodontitis. But the search is not limited to the oral cavity. Dewhirst and Paster have received funding from the Centers for Disease Control in Atlanta to ID 1,000 bacterial cultures on the CDC's most wanted list.

When clinical microbiology laboratories come across an unknown bacterium, they often send it to a state microbiology laboratory for identification. If the state laboratory cannot identify it, the sample is sent to the CDC. If the CDC is stumped, the sample will be sent to Forsyth. "If there's some unknown organism--we are the ones who are going to identify it," says Paster.

The traditional phenotypic methods of bacterial identification rely on how a bacterium behaves, its shape, and what food sources it uses for growth. The molecular approach, which zeroes in on genetic material, can be quicker and more specific. And identified DNA sequences provide phylogenetic information about how each bacterium is related to other members of its species.

Dewhirst and Paster are masters of microbial phylogeny and characterization. They have identified bacteria from such varied sites as mouse intestines, 57-year-old Dead Sea water, bird feces, and termite guts. The "flashlight" they use to illuminate the shadowy phylogenetic relationships between microorganisms is the sequence of the 16S ribosomal RNA gene.

"This gene has conserved regions and very variable regions," says Dewhirst. The conserved, unchanging sections allow the researchers to zoom in on the informative gene. From there, they can analyze the variable sections to determine which species the bacterium belongs to and how it is related to other microorganisms.

"This group is one of the premier groups in the world," says Doug Smith, section leader at PE Applied Biosystems, Foster City, Calif. Smith and his colleagues have harnessed this expertise by forming a collaboration with Paster and Dewhirst. The company provides reagents and sequencing software to the researchers, and receives accurate microbial sequences in return. The aim is to produce an integrated and user-friendly system for bacterial identification that can be used in many applications, says Dewhirst.

Scientists from other academic institutions have also joined the collaboration. The result, which has been partially released, is PE Applied Biosystems" MicroSeq" package. It contains a sequence database, report-writing software, and reagent kits.

The major application of these products will be testing for sterility in pharmaceutical manufacturing, says Smith. "Clean rooms have associated labs that have to identify any bugs that they pick up," he says. These are mostly environmental bacteria isolated from the air, water, or shedding human skin, which are hard to identify because their sequences may not be in publicly available databases. The other expected users will be clinical laboratories trying to identify "fall-through" samples'those that are too difficult to identify by traditional methods.

The greatest advantage with the molecular method is that it circumvents the need for bacterial cultivation -- a stumbling block in the identification of some microorganisms. So-called uncultivable organisms can still be analyzed because this technique uses genomic DNA as the starting material -- DNA that can be prepared directly from a clinical sample such as saliva or dental plaque. "Up to a half of the microorganisms present in the oral cavity can't be grown and we know nothing about them," says Paster. But the two researchers are poised to change that.

Using the information they have collected from 16S sequencing of bacteria inhabiting the surface of teeth and gums, Paster and Dewhirst have designed an advanced "checkerboard" assay to identify oral streptococci suspected of being associated with root caries.

DNA probes -- short gene fragments from known bacteria -- are attached at one end to a solid support. The remainder of the fragment floats freely, able to bind unknown bacterial DNA from a clinical sample applied to the support. If the bacterial DNA is a perfect match with the probe, they will bind together and activate a fluorescent reaction. Fluorescence forms a black patch on unexposed photographic film, informing the researchers that the bacterium they are searching for is present.

The pattern of black squares signifying each positive result gives the checkerboard assay its name. It was originally developed by this group of investigators in 1994, but the 1998 version, described in the March issue of Methods in Cell Science, is faster, more specific, and has a larger capacity. "You can look at a bunch of things at a time, which is more than most formats can handle," says Dewhirst. It is also better at identifying closely related bacterial subspecies.

Probes are designed based on variable regions of the 16S rRNA gene. As new bacteria are identified, probes to detect them are added to the checkerboard assay. "Once we know what we're looking for, by using the checkerboard method, one technician can process many samples and have the results within 24 hours," says Paster.

Analysis of 20,000 samples obtained from dental plaque of people with root caries is about to begin. Investigation of periodontal disease is also under way. Scientists still don't know which bacteria cause refractory periodontitis -- a persistent form of periodontal disease that does not respond to antibiotics or conventional therapy. Using the checkerboard technique, Paster and Dewhirst can quickly scan many samples to identify the microbial culprits. Then investigation of virulence factors and possible custom-made treatments can be initiated.

Harvard Medical School

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