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Biomedicine

Oral Ecology

Dental researchers are making significant strides in identifying the microorganisms that colonize the mouth. To head off serious decay and disease, scientists are developing tools that prevent these microbes from gaining a foothold.

The next time you kiss someone, think about this: in your mouth, and in the mouth of every adult, live more than 400 different species of microorganisms, mostly bacteria. Billions and billions of them grow in layers, crowded together and wrapped cozily around each other, on every slimy surface, dark nook, and inviting cranny. It’s enough to make a body want to keep lips permanently pursed.

With an average temperature of about 95 degrees, a saliva-induced humidity of 100 percent, and regular stoking with sugar and other simple carbohydrates-manna from bacterial heaven-the mouth provides a home for such a diversity of species that it could be called the tropical rainforest of the body. “In one mouth, the number of bacteria can easily exceed the number of people who live on earth,” says Sigmund Socransky, a dental researcher at the Forsyth Dental Center in Boston, Mass. “In a clean mouth, 1,000 to 100,000 bacteria live on each tooth surface. A person who doesn’t have a terribly clean mouth can have 100 million to 1 billion bacteria growing on each tooth.”

These facts are more useful than fodder for cocktail party chatter. An entire branch of dental research has grown up around “oral ecology”-the study of the relationships among the inhabitants of this minute jungle ecosystem-to develop the next generation of weapons in the fight against tooth and gum disease.

Since 1959, when scientists isolated a species of infectious bacteria that causes most cavities, a national campaign to reduce tooth decay has focused on brushing, flossing, and adding fluoride to water supplies, toothpaste, and mouthwashes. Fluoride, a chemical that appears naturally in groundwater in many areas of the world, quickly bonds with the tooth’s enamel to maintain its smooth crystalline surface and deter bacteria from gaining a toehold.

These dental hygiene methods have worked so well that today 51 percent of U.S. children under 12 have no tooth decay. However, many of the remaining 49 percent have severe forms of cavities that are difficult to control, even with the best dental hygiene. And other problems challenge dental researchers. Periodontal disease-infection of the gums that is caused by about a half-dozen bacterial species-affects millions of adults and children. People with Sjogren’s syndrome, an auto-immune disease of unknown origin that causes severe drying of the mouth, eyes, and other mucosal surfaces, have serious problems with tooth decay, as do many people whose saliva glands stop functioning after certain medical procedures.

Over the last 20 years, modern biotechnology, including genetic engineering and techniques to study anaerobic bacteria-those that live without oxygen and cause most periodontal disease-have enabled oral ecologists such as Socransky to identify some of the organisms. They have not only pinpointed about a dozen species of bacteria living in the mouth that can cause infections in the teeth and gums, but they also have made significant strides in understanding how these organisms colonize the mouth and how they are transmitted from one person to another.

Researchers are now applying their new knowledge to develop techniques that prevent the organisms from gaining residence in the first place, or to force them out using innocuous strains or new antibiotics once they’ve already settled in. They are also attempting to create artificial saliva for people with compromised saliva glands to sluice harmful germs out of the mouth and into the digestive system before they can stick to the teeth and gums.

Microscopic Ecosystem

Researchers have determined that the mouth’s microorganisms have evolved along with the human species, probably for as long as it has existed. In exchange for living in their tropical paradise, the mostly beneficial bacteria help fend off disease-producing germs that attempt to infiltrate the mouth from the outside world. For example, some beneficial bacteria produce organic acids, such as proprionic and buteric acid, that kill organisms responsible for a number of intestinal problems.

Yet when human babies pop into the world, their wails of greeting burst from sterile mouths. “Within minutes to hours, however, they are colonized with organisms that stay with them until they die,” says Socransky. These bacteria, yeast, viruses, and protozoa, most of which are harmless, enter from anything that makes contact with a baby’s mouth: air, breast, bottle nipple, thumb, and other objects.

The growth of organisms in the mouth follows the classical pattern of ecological succession-the way bare land eventually turns into thick jungle. A few pioneer species settle first, creating a habitat friendly to other species, which then, too, move in. When the first baby teeth push through the gums, another set of species-including the dreaded Streptococcus mutans, the bacteria believed responsible for most tooth decay-takes hold. During puberty, the composition of saliva changes, so that still another group of organisms immigrate and flourish. By the time humans reach adulthood, their mouths harbor what’s known as a climax community-a complex group of organisms, each with its own preferred microhabitat.

Although diets vary the world over, dental researchers have found the same organisms in human mouths no matter where people live. Some species live only on the cheeks. Others prefer the back of the tongue versus the front, especially the group of anaerobic bacteria that live in the crevices of the tongue and emit hydrogen sulfide, the origin of most severe bad breath. Another group will survive only on the palate. And the teeth themselves provide a plethora of living options-surfaces open to the outside world, sides facing the back of the mouth, a strip along the edge of the gums, and the gloomy, wet, oxygen-deprived spaces between the gums and the teeth.

Saliva, the amazing fluid that keeps this ecosystem in balance, harbors its own collection of bacteria, as well as a host of other substances. Bicarbonate ions buffer the tooth-decaying acids produced by harmful bacteria such as S. mutans. Phosphate and calcium ions supersaturate saliva and continually repair the microscopic chinks made in the teeth by the bacteria’s acid.

Saliva also contains antibacterial agents, such as lysozyme, which kill bacteria by opening up their cell walls. About 60 proteins float around in saliva. Some of them actually provide nutrients for bacterial growth while others lubricate the mouth and cause bacteria to stick together in such large clumps that they can’t adhere to tooth surfaces and are easily washed away. Saliva even contains antiviral components. In fact, researchers at the National Institute for Dental Research, who have found that the AIDS virus does not live in saliva, are trying to isolate a substance that they believe may be effective against the AIDS virus.

Most of the time, the inhabitants of the mouth live in more or less perfect harmony. “Congress should take lessons from the mouth,” says Yolanda Bonta, manager of clinical research at Colgate Oral Pharmaceuticals in Piscataway, N.J. But, like breakdowns in budget talks that sometimes lead to government shutdowns, sometimes things go to hell in a handbasket in the mouth, too.

Indeed, ecological conditions in the mouth are never stable. People change their diets, lose teeth, have crowns or false teeth put in, or take drugs that affect certain microorganisms. “For example, an epilepsy medication causes overgrowth of gums,” says Socransky, “and that changes the microbiota.” Radiation of the head and neck for cancer treatment as well as a host of medications also cause a drastic drop in saliva production, allowing bacteria to run rampant.

Changes also occur after every meal, after every brushing and flossing, even every time we swallow, as millions of bacteria lose their grip on tooth surfaces and tumble down the throat. During a night’s sleep, when saliva production drops to near zero, bacteria, like the minions in the “Fantasia” version of Moussorgsky’s “A Night on Bald Mountain,” revel in their freedom and multiply with abandon until the dawn.

Abundant sugar can power S. mutans into a frenzy of activity. In fact, while some strains of S. mutans produce natural antibiotics against the bacteria that cause strep throat, and were probably useful in the mouths of people of primitive cultures, the preponderance of refined sugars in the modern diet changed the oral landscape so drastically that S. mutans is now more harmful than helpful. As it gobbles up sugar, S. mutans produces much more acid than saliva can buffer, and the excess eats away at the minerals of tooth enamel. Without adequate brushing and flossing, plaque grows, producing calcified deposits and a cozy home for more species that do more damage. Sticky opportunistic bacteria grab hold in the newly formed holes and crevices, causing tooth decay, and no amount of salival flow will wash them off.

Preventing Colonization

In the last few years, researchers have made significant strides not only in understanding how bacteria settle into their particular niches but also in developing methods to prevent certain harmful strains from doing so. At the University of Alabama School of Dentistry, Page Caufield, a professor of oral biology, found that humans are colonized by S. mutans-the cavity-causing bacteria-during a “window of infectivity,” around two years of age. At that time, S. mutans is usually passed from the primary caregivers probably as they spew saliva droplets while talking into the face of a child whose teeth are emerging. “The window opens and closes,” says Caufield. “If children aren’t infected by S. mutans then, another bacteria species moves in and uses that niche. People don’t exchange S. mutans as adults.”

In an attempt to prevent the transmission of S. mutans to children, Caufield and his team will soon begin clinical trials on 250 mothers who carry particularly harmful strains of the bacteria. Their teeth will be treated with Chlorzoin, an antimicrobial dental sealant, that can effectively block colonization of S. mutans for up to six months, during their children’s window of infectivity. The researchers say that the procedure will have no effect on the other bacteria-beneficial or otherwise-that began colonizing the babies’ mouths from the moment they were born. Thus they hope that the trials will prove this a safe means of allowing 250 children to live a life free of the S. mutans strains that have plagued their mothers.

Chlorzoin is also undergoing clinical trials in several other countries. The largest clinical trial involves more than 1,300 school-age children in Dundee, Scotland. The children, who have been identified as at high-risk for S. mutans, are having their teeth painted with the sealant several times over three years. Researchers hope that it will reduce S. mutans populations to a level so low that cavities will be prevented.

In the meantime, Chlorzoin, which was developed by researchers at the University of Toronto and approved as a prescription drug in Canada in 1993, is already being sold by Oralife, a Toronto-based company that has teamed up with a Canadian dental insurance company to train dentists to use Chlorzoin. The dentists will include Chlorzoin as part of a decay-prevention program in children and adults who have especially virulent strains of S. mutans or conditions such as dry mouth that exacerbate tooth decay. If the program proves successful, Ross Perry, president of Oralife, anticipates that dental sealants will soon be used in the United States.

According to Perry, approximately 10 percent of North Americans are at high risk for tooth decay, while another 10 to 15 percent are at medium risk. The amount spent on restorative dentistry by these two groups accounts for 60 percent of the total dollars spent on dentistry. “By doing this treatment over two years, S. mutans can be reduced, and adults can move down a notch in their risk factor,” he says. “Before this program, insurance companies never paid for prevention. They always paid after a person was infected.”

Dental Vaccines

Martin Taubman and Daniel Smith, dental immunologists at the Forsyth Dental Center, have been working on an alternate method of preventing S. mutans from colonizing children’s teeth. The researchers know that humans develop antibodies to mutans streptococci (the group of bacteria containing S. mutans) when they’re around three years old, after the bacteria have already colonized their teeth. People retain these antibodies as adults but never develop enough to completely wipe out the bacteria. The team’s goal, therefore, is to develop a vaccine that can be given to children before their mouths are colonized by S. mutans. That way, when the bacteria infect them, the antibodies would already be present.

A vaccine that Taubman and Smith have developed thus far induces the body to produce antibodies against an enzyme produced by mutans streptococci. The enzyme-glucosyltransferase, or GTF-breaks down the sugar in food into more simple sugars-glucose and fructose. The resulting simple-sugar molecule is critical because it is the substance to which bacteria bind, like dust bunnies to Velcro. Thus the enzyme helps create a mass of bacteria large enough to metabolize other carbohydrates and produce lactic acid. “You get enough bugs, you get enough acid, you produce a hole,” says Taubman.

The antibodies produced by the vaccine interfere with the site on the enzyme that cleaves the complex sugars, preventing them from being broken down into the components to which the bacteria like to adhere. The vaccine was first tested on animals, who, after immunization, developed fewer cavities. It was then given to adult humans, whose immune systems have been shown to produce antibodies.

Taubman and Smith are in the process of refining the vaccine so that it can be made artificially-that is, not from the live mutans streptococci themselves, but from molecular components-but do not immediately anticipate testing it on children simply because the Forsyth Dental Center lacks the necessary medical infrastructure. They anticipate working with centers that specialize in vaccines for children to piggyback their vaccine onto those developed for other diseases.

Researchers are also developing vaccines to help prevent periodontal disease. Scientists originally thought the task would be daunting because, unlike tooth decay, the disease appeared to involve the complex interactions between many species of bacteria: some bacteria live in the periodontal pocket-the space between the gums and the teeth-and interact with a second set that colonizes the tooth above the pocket.

But oral ecologists have recently discovered that most periodontal disease may be caused by just three bacteria. One species is responsible for most juvenile periodontia, while two other species cause most infection in adults. At the University of Washington, researchers are developing a vaccine for one of the species of bacteria, Porphyromonas gingivalis, that infects adults. The research began several years ago when Roy Page, professor of periodontics at the University of Washington School of Dentistry, created a vaccine from deactivated Porphyromonas cells that were harmless but could still initiate an immune response. He then added an adjuvant-a mix of oils and other ingredients-that helps the antigen last longer and makes the immune cells more responsive-and administered the vaccine to macaque monkeys.

Since most animals do not normally get periodontal disease-the only species it shows up in other than humans is beagles-Page wrapped threads around the bottom of the monkeys’ teeth to encourage bacteria to colonize. He then infected the monkeys with all the types of bacteria that cause periodontal disease in adult humans. After 36 weeks, he performed a computer analysis of dental x-rays taken before and after the study to measure the difference in bone destruction, the usual result of gum infection caused by Porphyromonas gingivalis. The analysis showed that a control group experienced significant bone decay while the vaccinated monkeys had virtually none. The results suggested that the antibodies developed by the monkeys against Porphyromonas were also effective against the other species of bacteria.

The U.S. Food and Drug Administration requires that vaccines be developed from specific proteins that induce an immune response, instead of from killed cells that may induce unpleasant side effects in humans. One likely candidate is a protein on the surface of Porphyromonas called cystine protease that induces an immune response to the bacteria, and seems to cause no side effects. The FDA also prefers proteins that are made from pure antigen-that is, antigen derived directly from DNA-to reduce the chance that they will contain “impurities,” or other cell components, that may cause unwanted reactions. To that end, Page is working with Marilyn Lantz, a genetics researcher at the University of Indiana, to make a recombinant version of the protein from pure DNA for further testing in monkeys. “If we get protection from cystine protease,” Page says, “we’ll ask the FDA to do clinical trials for safety in humans.”

Displacing Bacteria

Other new research is aimed at ridding the mouth of harmful organisms that have already found comfortable habitats. At the University of Florida at Gainesville, molecular biologist and dentist Jeffrey Hillman has been using genetic engineering to develop a harmless strain of S. mutans that will replace the acid-producing varieties that occupy and cause cavities in most mouths.

In the early 1980s, Hillman and his research team isolated a type of S. mutans that metabolizes sugar but doesn’t produce acid as a waste product. But no matter what he and his colleagues tried-for example, eliminating the original strain with antibiotics and painting the teeth with iodine-the S. mutans that already occupied a person’s mouth would not be budged. “They have hiding places,” he says. “Nobody found a way to wipe them out entirely.”

So Hillman examined hundreds more strains of S. mutans to find one that produced an antibiotic-like molecule that killed all other strains of S. mutans. Using genetic engineering, he modified it so that it would no longer produce acid. This so-called effector strain can colonize tooth surfaces, he says, and wipe out native S. mutans.

Within the next few months, Hillman will begin trials on laboratory rats, infecting their mouths with the bacteria. If the bacteria perform as intended, he will then conduct trials on humans. He anticipates that, eventually, dentists will apply the bacteria during a typical cleaning. “In theory, the new strain should stay with people the rest of their lives,” says Hillman. “And since S. mutans normally is transmitted from mother to child, this effector strain will also be transmitted, and will prevent tooth decay in the children of those treated.”

Root Problems

Other attempts to oust offensive germs from their comfortable dwellings are taking aim at the bacteria, yeast, and protozoa that dive down tiny cracks in a tooth to infect the blood- and nerve-rich tooth roots and cause ice-pick-like stabbing pain. If root infections in certain teeth in the upper jaw are not treated, the bacteria can cause serious eye infections, even blindness. “That’s why they’re called eye teeth,” says Kathleen Olender, an endodontist at the University of California-San Francisco Dental School.

Similarly, if a tooth in the lower jaw becomes severely diseased, the bacteria can travel to the throat and cause a condition called Ludwig’s angina in which the larynx can swell so much that the person can’t breathe, says Craig Baumgartner, chair of the Department of Endodontology at Oregon Health Sciences University in Portland. Bacteria from a tooth in an upper jaw can also spread into the pterygoid plexus, a locus of nerves and blood vessels in the face. “The bacteria can back up the veins into the brain,” he says, and even cause dementia.

Baumgartner says that while endodontists see these severe infections only occasionally in the United States, they are still common in developing countries where people don’t have access to antibiotics and aggressive care to surgically remove infected roots. Moreover, although root-canal procedures have been performed for hundreds of years, and today about 10 million teeth a year undergo endodontic procedures, it was not until antibiotics were developed after World War II that dentists were allowed to perform root canals in the United States because the bacterial infection in the root was often difficult to contain.

Researchers aiming to control these infections have recently identified eight species of bacteria that seem to cause most of the infections that occur in tooth roots. Their goal is to eliminate all of the bacteria in an infected root, since it is normally sterile. But because there seems to be a complex interplay among the species of infecting bacteria-a byproduct of one is used as a nutrient by another-researchers such as Baumgartner at the Oregon Health Sciences University believe that identifying and focusing on killing one or two species would upset the ecosystem to such a degree that the rest would die off. Toward that end, he and other researchers are now testing the effectiveness of individual antibiotics on targeted bacteria species.

Mimicking Saliva

Saliva provides perhaps the most effective means of ridding the mouth of harmful bacteria. Indeed, without enough saliva, the 2 million people with Sjogren’s syndrome, which causes severe drying of mucosal surfaces, suffer a variety of oral infections. So, too, do millions of people whose saliva glands are affected by radiation treatments for head and neck cancers, bone-marrow transplants, some chemotherapy, or the more than 400 prescription and over-the-counter drugs that have been reported to cause mouth dryness.

Unfortunately, a quest to develop artificial saliva has proven largely unsuccessful. The main reason is that many of its components have not even been identified, says Philip Fox, clinical director of the National Institute of Dental Research (NIDR). These include the viral and bacterial components, as well as the myriad substances that aid in chewing, initial digestion, and swallowing. Researchers therefore haven’t been able to replicate its physical and chemical properties, nor have they been able to identify all the ways in which it contributes to the functioning of the mouth as an ecosystem.

Until they can say exactly what saliva is, NIDR researchers such as Fox are focusing instead on other options. For people who retain some part of their saliva gland, researchers have had success with a drug, pilocarpine, that stimulates the gland to produce more saliva. They are also searching for ways to control Sjogren’s syndrome through the use of steroids and other drugs that aim to calm down the immune response that shuts down saliva glands.

“We’re also attempting to reengineer the saliva gland,” says Fox, by using gene-transfer technology to try to reconstitute a damaged gland. In one method, NIDR researchers Brian O’Connell and Bruce Baum are using standard gene-transfer technology in which a virus that infects a cell is used to carry a gene that will cause the cell to produce a substance that its own DNA is not programmed to produce. When salivary glands have been damaged by radiation or disease, cells may still be there, but they are not water-secreting cells. In animal studies, the researchers have been able to transfer genes into cells and get them to produce water. Fox says that within two to three years, the same techniques may be able to be used on humans.

Because the field of oral ecology is at its beginning stages and the fruits of the new research still need to go through the long process of clinical trials, most new techniques will not see the inside of people’s mouths for 5 to 10 years, say researchers. Until then, Bonta of Colgate Oral Pharmaceuticals advises that “although you can afford not to brush for 72 hours, if you pass that threshold, the bacteria will have taken hold and multiplied to such concentrations that the acid they produce has begun making holes in the teeth. Moreover, you may not be able to remove the plaque and bring the infected parts of the teeth back to health.”

It’s also best to brush for more than a minute, says Ernest Newbrun, a dental researcher in the University of California at San Francisco’s Department of Oral Biology. That’s the effort required to clean the 150 tooth surfaces found in most people’s mouths and to bring the bacteria count down to a manageable and healthy 1,000 to 100,000 per tooth.

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