Bruce J. Paster, PhD

More than 700 different species of bacteria may be able to live in the human mouth, though probably not all of them are present in the same mouth at the same time. Many of these oral bacteria have not been identified because they are impossible to grow in culture outside the mouth. Using the techniques of molecular biology, the Paster laboratory has been developing new tools, which do not depend on traditional culturing approaches, to hunt for oral micro-organisms. Since the causative agents of oral diseases are not fully known, it is likely that some, or even many, of the novel bacteria identified by these new methods may play important roles in disease.

The major research objective of the Paster laboratory is to develop methods for the rapid identification and enumeration of oral microorganisms so that we may elucidate their roles in oral diseases. We utilize a number of molecular techniques, such as nucleic acid sequencing, gene amplification via polymerase chain reaction, gene cloning, DNA probe development, DNA hybridization, in situ hybridization, and, more recently, DNA microarrays.

Phylogenetic Identification of Bacterial Species

Bacterial isolates have traditionally been characterized on the basis of biochemical and other phenotypic traits. However, these methods are labor intensive and costly. In collaboration with the Dewhirst laboratory, we have determined the complete 16S rRNA gene sequences for about 2,000 species of bacteria and have submitted over 1100 of these 16S rRNA sequences to GenBank. Most of our efforts have been concentrated within the following phylogenetic groups: Pasteurellaceae, including Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus; Neisseriaceae, including Eikenella corrodens and Kingella orale; Campylobacter/Wolinella/Helicobacter, including C. concisus, C. rectus, C. curvus, H. pylori, and H. mustelae; Selenomonas/Veillonella, including all named selenomonads, Centipeda periodontii, and several species of Veillonella; spirochetes, including all named oral treponemes; Bacteroides/Cytophaga/Flavobacterium, including all prevalent oral and nonoral species of Bacteroides, Prevotella, Porphyromonas, and Capnocytophaga; Fusobacterium, including F. nucleatum, F. periodonticum and Leptotrichia buccalis; and the oral streptococci. Based on their 16S rRNA sequences, phylogenetic trees of these and related species have been constructed. In a previous collaboration with the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, we used 16S rRNA sequence comparisons to identify emerging and unusual pathogenic bacterial isolates sent to the CDC for characterization.

Identification of Uncultivable Bacterial Species

Using gene amplification via polymerase chain reaction (PCR) and gene cloning, we are able to determine the 16S rRNA sequences from microorganisms that are not presently cultivable. Briefly, 16S rRNA genes of uncultivable strains are PCR-amplified from samples, using bacterial-universal and selective primers, and are subsequently cloned into E. coli plasmid vectors. Partial sequences (approximately 500 bases) of cloned inserts are used to initially determine the phylogenetic identity of a species or its closest relatives by comparison with known sequences. Clinical samples have been analyzed from patients with a range of oral diseases, including caries, periodontitis, refractory periodontitis, acute necrotizing ulcerative gingivitis (ANUG), necrotizing ulcerative periodontitis in HIV+ subjects, and noma (a gangrenous stomatitis that primarily affects malnourished children in developing countries). Of the more than 20,000 clones analyzed in these studies, we have identfied over 400 novel species and some of these appear to be associated with oral health or disease. Based on our microbial data and the findings of other investigators, we estimate that there are about 700 different bacterial species that can inhabit the human oral cavity.

Determination of Bacterial Population Distribution via DNA Hybridization

To provide a rapid, accurate and inexpensive means for characterizing microbial populations in clinical specimens, we have designed DNA probes based upon 16S rRNA sequences. Utilizing our extensive 16S rRNA database, we have developed over 400 DNA probes to identify organisms at the family, genus, species and sub-species level. We have used a PCR-based, reverse-capture checkerboard hybridization methodology to rapidly detect and quantify oral species of Streptococcus and other genera. This assay works well for identifying pure cultures and for detecting and enumerating specific species, higher taxa and the newly identified "uncultivated" species directly in clinical samples. Many of these probes are currently being used for the rapid identification and enumeration of bacteria in our own clinical studies at Forsyth. We are currently transitioning the checkerboard to a glass microarray system, termed the Human Oral Microbe Identification Microarray, or HOMIM. Presently, we can simultaneously test for 300 key oral species on a single microscope slide.

Selected Publications

Lockhart PB, Brennan MT, Sasser HC, Fox PC, Paster BJ, Bahrani-Mouget FK. 2008. Bactermia associated with toothbrushing and dental extraction. Circulation. 117(24):3118-25.

Bahrani-Mougeot FK, Baster BJ, Coleman S, Ashar J, Knost S, Sautter RL, Lockhart PB. 2008. Identification of oral bacteria in blood cultures by conventional versus molecular methods. Oral Surg Oraal Med Oral Pathol Oral Radiol Endod.
105:(6):720-4.

Kawai T, Paster BJ, Komatsuzawa H, Ernst CW, Goncalves RB, Sasaki H, Ouhara K, Stashenko PP, Sugai M, Taubman MA. 2007. Cross-reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent periodontal bone resorption in a mouse model. Oral Microbiol Immunol. 22(3):208-15.

Aas JA, Barbuto SM, Alpagot T, Olsen I, Dewhirst FE, Paster BJ. Subgingival plaque microbiota in HIV positive patients. 2007. J Clin Periodontol. 34(3):189-95.

Tanner ACR, Paster BJ, Lu SC, Kanasi E, Kent R, Van Dyke T, Sonis ST. 2006. Subgingival and tongue microbiota of adults with early periodontitis. J. Dent. Res. 85 (4) :318–323.

Fox JG, Taylor NS, Howe S, Tidd M, Xu S, Paster BJ, Dewhirst FE. 2006. Helicobacter anseris sp. nov. and Helicobacter brantaesp. sp. nov., isolated from feces of resident Canada geese in Greater Boston. Appl. Environ. Microbiol. 72(7) :4633–4637.

Paster BJ, Olsen I, Aas JA, Dewhirst FE. 2006. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol. 2000 42(1):80–87.

Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. 2005. Defining the normal bacterial flora of the oral cavity. J. Clin. Microbiol. 43(11):5721–5732.

Corby PM, Lyons-Weiler J, Bretz WA, Hart TC, Aas A, Boumenna T, Goss J, Corby AL, Junior HM, Weyant RJ, Paster BJ. 2005. Microbial risk indicators of early childhood caries. J. Clin. Microbiol. 43(11) :5753–5759.

Dewhirst FE, Shen Z, Scimeca MS, Stokes LN, Boumenna T, Chen T, Paster BJ, Fox JG. 2005. Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: Implications for phylogenetic inference and systematics. J. Bacteriol. 187(17):6106–6118.

Wyss C, Dewhirst FE, Paster BJ, Thurnheer T, Luginbühl A. 2005. Guggenheimella bovis gen., sp. nov., isolated from lesions of bovine dermatitis digitalis. Int. J. Syst. Evol. Microbiol. 55(Pt. 2): 667–671.

Shen Z, Xu S, Dewhirst FE, Paster BJ, Pena JA, Modlin IM, Kidd M, Fox JG. 2005. A novel enterohepatic Helicobacter species 'Helicobacter mastomyrnus' isolated from the liver and intestine of rodents. Helicobacter 10(1) :59–70.

Eribe ERK, Paster BJ, Caugant DA, Dewhirst FE, Stromberg VK, Lacy GH, Olsen I. 2004. Genetic diversity of Leptotrichia and description of Leptotrichia goodfellowii sp. nov., Leptotrichia hofstadii sp. nov., Leptotrichia shahii sp. nov. and Leptotrichia wadei sp. nov. Int. J. Syst. Evol. Microbiol. 54(2):583–592.

Wyss C, Moter A, Choi BK, Dewhirst FE, Xue Y, Schupbach P, Gobel UB, Paster BJ, Guggenheim B. 2004. Treponema putidum sp. nov., a medium-sized proteolytic spirochaete isolated from lesions of human periodontitis and acute necrotizing ulcerative gingivitis. Int. J. Syst. Evol. Microbiol. 54(4):1 117–1122.

Correia FF, Plummer AR, Paster BJ, Dewhirst FE. 2004. Genome size of human oral Treponema species by pulsed field gel electrophoresis. Oral. Microbiol. Immunol. 19(2):129–131.

Miklossy J, Khalil K, Gern L, Ericson RL, Darekar P, Bolle L, Hurlimann J, Paster BJ. 2004. Borrelia burgdorferi persists in the brain and causes dementia in chronic Lyme neuroborreliosis. J. Alzheimers Dis. 6:639–649.

Correia FF, Plummer AR, Ellen RP, Wyss C, Boches SK, Galvin JL, Paster BJ, Dewhirst FE. 2003. Two paralogous families of a two-gene subtilisin operon are widely distributed in oral treponemes. J. Bacteriol. 185(23) :6860–6869.

Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ. 2003. New bacterial species associated with chronic periodontitis. J. Dent. Res. 82(5) :338–344.

Kazor CE, Mitchell PM, Lee AM, Stokes LN, Loesche WJ, Dewhirst FE, Paster BJ. 2003. Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J. Clin. Microbiol. 41 (2):558–563.

Harper CG, Xu S, Rogers AB, Feng Y, Shen Z, Taylor NS, Dewhirst FE, Paster BJ, Miller M, Hurley J, Fox JG. 2003. Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica. Dis. Aquat. Organ. 57(1–2):1–9.

Staff

Research Technicians

Sara Barbuto, B.S.
Susan Boches, B.S.
Sean Cotton, B.S.
Erin Klein, B.S.