The Center for Biofilm Engineering

A potpourri of probing and treating biofilms of the oral cavity 

Microbe Magazine, October 2009 by Carol Potera _____________________________________ Decades before biofilms were named, microbiologists studying bacteria in the oral cavity realized that complex microbial communities form plaque on tooth surfaces-perhaps the prototype for much of biofilm research today. Some of that oral cavity-focused biofilm research is leading to new products to combat plaque and bad breath, while other efforts are providing insights about how microorganisms behave within these complex oral communities. Here are some highlights from the 25th annual Biofilm Science & Technology (BST) meeting, convened at the Center for Biofilm Engineering (CBE) at Montana State University (MSU) in Bozeman last July.
	Colored scanning electron micrograph of dental plaque, consisting of bacterial biofilm embedded in a glycoprotein matrix. Oral biofilms are responsible for tooth decay and bad breath, and researchers are looking for ways to make biofilm removal easier—or their formation more difficult. (Magnification: x380; image © Steve Gschmeissner/Photo Researchers, Inc.)

More than 700 types of microorganisms grow in the mouth, many of them helping to form plaque along tooth surfaces. Within minutes after a professional hygienist removes plaque, however, it begins reforming. One candidate for impeding plaque is ficin, a cysteine protease derived from the sap of Ficus glabrata trees, says microbiologist Harsh Trivedi from Colgate- Palmolive Company in Piscatawny, New Jersey, who spoke during BST. U. S. Food and Drug Administration officials long ago assigned ficin, which is used widely as a meat tenderizer, to the "generally regarded as safe" (GRAS) list-a status that lowers regulatory hurdles for other uses.

Ficin potently prevents biofilms from forming in an anti-adhesion assay as well as on extracted human teeth treated in flow chambers, Trivedi says. It also penetrates with up to 90% efficiency a three-species biofilm containing Actinomyces naeslundii, Streptococcus gordonii, and Streptococcus oralis, all early colonizers of dental surfaces. In other tests involving human volunteers, use of a ficin-containing toothpaste decreases Porphyromonas gingivalis and changes levels of Fusobacterium and Porphyromonas species in plaque specimens. Although brushing with a ficin-containing toothpaste reduces plaque by 45% initially, after 6 weeks that plaque reduction drops to only 12% compared to individuals who use ordinary toothpaste, making ficin's commercial future uncertain, he says.

Another biofilm-related oral health issue involves bad breath. "No one wants bad breath or to have to smell bad breath," says dentist Alessandra Agostinho of the University of Sao Paulo, Brazil, a visiting scientist at the CBE and another participant at the BST meeting. About half of adults in the United States suffer from persistent halitosis, which is considered a clinical disorder. One likely source of halitosis is anaerobic gram-negative bacteria in oral biofilms that producevolatile sulfur compounds (VSC), including hydrogen sulfide and methyl mercaptan, she says.

To develop more precise information about the source of halitosis, Agostinho scrapes biofilms off tongues of volunteers, applies them to hydroxyapatite-coated glass slides, and then measures VSC concentrations. Halitosis is considered clinically significant when VSC levels rise above 1,000 ppb; the lab-tested biofilms give average readings of 1,490 ppb. When such biofilms are treated with a commercial toothpaste, mouthwash, sodium triphosphate (STP), or decapinol, the latter substance reduces plate counts by 99%, whereas the mouthwash reduced them by about 80%, and STP by 50%, she reports.

Robert Palmer, a microbiologist at the National Institute of Dental and Craniofacial Research at the National Institutes of Health in Bethesda, Md., is focusing on metabolic interactions that favor coaggregation of biofilm communities. The earliest colonizers of plaque include Streptococcus and Veillonella, and coaggregation, based on recognition between such cells, is suspected to play a role in biofilm development.

Palmer grew S. oralis and V. atypica PK1910 individually or together. Both types of microbe grow better in combination than as monocultures, especially V. atypica PK1910, whose growth is spurred by lactic acid from S. oralis. When V. atypica PK1910 nears S. oralis, it signals the latter to express amylase, which degrades stored glycogen to lactic acid via glycolysis. "That's the benefit Veillonella gains," says Palmer. "Coaggregation is important in the formation of biofilm communities."

Mechanical procedures can scrape biofilms off teeth, but making those biofilms either softer or more brittle could speed their removal from teeth or other surfaces, points out Eric Brindle, a mechanical engineering graduate student at MSU. He is systematically testing how treatments with a range of agents, including urea, dispersin B, chlorhexidine, and iron chloride, can change the viscoelastic properties of biofilms. The extracellular polymeric substance (EPS) that holds biofilms together allows them "to act like rubber bands that stretch and return to the original position," he says. Treatments with urea or dispersin B, an enzyme that chips away at the EPS matrix, renders Staphylococcus epidermis biofilms dramatically more viscous, he finds. In contrast, chlorhexidine- an ingredient in contact lens solutions and mouthwash-and iron chloride makes them stiffer. "Biofilm mechanical properties can be manipulated in desirable ways," he says.

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The CBE meeting held in July 2009 was the latest in a 25-year tradition that brings together academic researchers and industrial associates who are impacted by biofilms, including manufacturers of household and healthcare products, oil and chemical companies, and environmental cleanup firms. The industrial partners help to fund research at the CBE. "Despite the dismal economic climate, we've had a successful year, in good part because of steady industrial support," says Phil Stewart, CBE's director. Newly funded projects at the CBE range from finding remedies for chronic wound infections to sequestering carbon dioxide underground by plugging pores with biofilms to mitigate global warming.

This last paragraph was added back to the Microbe version with permission of the author.