Faculty Bibliography

Understanding of the human microbiome continues to grow rapidly; however, reports on changes in the microbiome after HIV infection are still limited. This review surveys the progress made in methodology associated with microbiome studies and highlights the remaining challenges to this field. Studies have shown that commensal oral, gut, vaginal, and penile bacteria are vital to the health of the human immune system. Our studies on crosstalk among oral and gastrointestinal soluble innate factors, HIV, and microbes indicated that the oral and gut microbiome was altered in the HIV-positive samples compared to the negative controls. The importance of understanding the bacterial component of HIV/AIDS, and likelihood of "crosstalk" between viral and bacterial pathogens, will help in understanding the role of the microbiome in HIV-infected individuals and facilitate identification of novel antiretroviral factors for use as novel diagnostics, microbicides, or therapeutics against HIV infection.

Oral Diseases (2011) doi: 10.1111/j.1601-0825.2011.01848.x Objective: Oral infection is considered to play a critical role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ), and antibiotic therapy has become a mainstay of BRONJ therapy. This study was aimed to investigate the effect of antibiotics on bacterial diversity in BRONJ tissues. Materials and methods: The bacterial profile from soft tissues associated with the BRONJ lesion was determined using 16S rRNA-based denaturing gradient gel electrophoresis (DGGE) and sequencing. Twenty BRONJ subjects classified as stage 0-2 were enrolled in this study, and patient groups were divided into an antibiotic cohort (n = 10) treated with systemic antibiotic and a non-antibiotic cohort (n = 10) with no prior antibiotic therapy. Results: The DGGE fingerprints indicated no significant differences in bacterial diversity of BRONJ tissue samples. Patients on antibiotics had higher relative abundance of phylum Firmicutes with bacterial species, Streptococcus intermedius, Lactobacillus gasseri, Mogibacterium timidum, and Solobacterium moorei, whereas patients without antibiotics had greater amounts of Parvimonas micra and Streptococcus anginosus. Thirty percent of bacterial populations were uncultured (yet-to be cultured) phylotypes. Conclusion: This study using limited sample size indicated that oral antibiotic therapy may have a limited efficacy on the bacterial population associated with BRONJ lesions

AIM: The effect of photodynamic antimicrobial chemotherapy (PACT) on oral biofilms was evaluated. METHODS: Biofilms formed in vitro were treated with sensitizer (S) and/or light (L) in the test (S+L+) and control (S-L-, S+L-, S-L+) groups. Additionally, 21 volunteers wore intraoral devices and the biofilms formed on each side of the device were referred to as S+L+ or S-L- groups. RESULTS: Significant decreases in the viability of in vitro biofilms were observed after PACT. PACT was ineffective in inhibiting multi-species biofilms formed in situ. CONCLUSION: PACT was effective on in vitro biofilms and was ineffective on in situ biofilms.

Bisphosphonates (BPs) are the standard of care for patients with metastatic cancer and multiple myeloma to prevent skeletal complications (e.g., severe bone pain, pathologic fracture, etc.) and to treat osteoporosis. The cause and effect relationship between BPs and BP-related osteonecrosis of the jaws (BRONJ) is not well established. Current research suggests that bacterial biofilms may play a significant role in the pathogenesis of BRONJ. Recently, we have shown that BRONJ lesions are heavily colonized by oral bacteria and present many clinical challenges as they are difficult to culture and antibiotic resistance may result in misguided antibiotic therapy. Here we highlight the crosstalk among the oral bacteria and host immune response in BRONJ subjects. Using 16S rDNA molecular technique we characterize the total bacterial profile of BRONJ, BP and control subjects. Denaturing gradient gel electrophoresis cluster analysis revealed three clusters each representing the three groups, control, BP and BRONJ indicating that the microbiome present in tissue samples was distinct to each group. DGGE band pattern indicated that the BRONJ group had less bacterial diversity as compared to control indicating that high abundance of specific bacteria colonizing the BRONJ lesion. 16S sequencing and clonal analysis showed 6 phyla in all three groups. The phylum Firmicutes was predominant in BRONJ group (72%) followed by BP group (70%) as compared to control group (59%). The Chi-square test also showed significant differences in percent relative distribution of phyla, between control/BP groups (p<0.001), control/BRONJ (p<0.001) and BP/BRONJ (p<0.05). There was significantly increase in the gram positive bacteria in BRONJ group. PCR Array analysis indicated that the host genes responsible for antibacterial response such as MPO, CTSG, and NOD2 were significantly down regulated. Deficient innate immune responses to microorganisms together with poor healing and repair provide continuous opportunities for expanding!

A nonthermal low temperature air plasma jet is characterized and applied to study the plasma effects on oral pathogens and biofilms. Experiments were performed on samples of six defined microorganisms' cultures, including those of gram-positive bacteria and fungi, and on a cultivating biofilm sample of Streptococcus mutans UA159. The results show that the plasma jet creates a zone of microbial growth inhibition in each treated sample; the zone increases with the plasma treatment time and expands beyond the entire region directly exposed to the plasma jet. With 30s plasma treatment twice daily during 5 days of biofilm cultivation, its formation was inhibited. The viability of S. mutans cells in the treated biofilms dropped to below the measurable level and the killed bacterial cells concentrated to local regions as manifested by the fluorescence microscopy via the environmental scanning electron microscope. The emission spectroscopy of the jet indicates that its plasma effluent carries an abundance of reactive atomic oxygen, providing catalyst for the observed plasma effect. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3606486]

In oral cavity chronic inflammation has been observed at various stages of oral squamous cell carcinomas (OSCC). This inflammation could result from persistent mucosal or epithelial cell colonization by microorganisms. There is an increasing evidence of the involvement of oral bacteria in inflammation and warrant further studies on the association of bacteria in the progression of OSCC. The objective of this study was to evaluate the diversity and relative abundance of bacteria in the saliva of subjects with OSCC. Using 454 parallel DNA sequencing, approximately 58,000 PCR amplicons that span the V4-V5 hypervariable region of ribosomal RNAs from 5 subjects were sequenced. Members of 8 phyla (divisions) of bacteria were detected. The majority of classified sequences belonged to phyla, Firmicutes (45%) and Bacteroidetes (25%). Further, a total of 52 different genera containing approximately 860 (16.51%) known species were identified, 1077 (67%) sequences belonged to various uncultured bacteria or unclassified group. The species diversity estimates obtained with abundance-based coverage estimators (ACE) and Chao1 were greater than published analyses of other microbial profiles from the oral cavity. Fifteen unique phylotypes were present in all three OSCC subjects

OBJECTIVE: To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. METHODS: Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. RESULTS: Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. CONCLUSION: The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

Protease inhibitor cocktails are routinely added to clinical samples used for proteomic studies to inactivate proteases. As these same samples are often used for microbial studies, we determined whether the addition of protease inhibitors could affect the quantitative or qualitative assessment of microbial profiles. Twenty-two saliva samples were collected and processed immediately with or without the addition of a protease inhibitor cocktail. Conventional cultivation methods were used to evaluate total bacterial growth. Total genomic DNA was isolated and a specific 16S rRNA gene-targeted region was PCR-amplified and separated by denaturing gradient gel electrophoresis. A combination of 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis and LC-MS/MS methods was used to determine the effect of the protease inhibitors on the integrity of salivary proteins and peptides. Interestingly, no significant differences were observed in either the bacterial growth and composition or the integrity of salivary proteins between the two groups. Correlation coefficients between the paired samples for total cultivable microbiota (r(2) =0.847), total mutans streptococci (r(2) =0.898), total oral lactobacilli (r(2) =0.933), and total Streptococcus mutans (r(2) =0.870) also exceeded expected values. The results suggest that the addition of a protease inhibitor cocktail in saliva samples does not impact the growth of oral microbiota or compromise the ability to characterize its composition.

BACKGROUND AND OBJECTIVE: The most common risk factor for bisphosphonate-related osteonecrosis of the jaws (BRONJ) is dentoalveolar surgery. It has been suggested that reduced serum C-terminal telopeptide (CTX) can determine the degree of osteoclast suppression and may predict the development of BRONJ after dentoalveolar surgery. Although there are many radiographic appearances associated with BRONJ, there are little data that describes changes preceding dentoalveolar surgery. The objective of this retrospective study was: 1) to investigate if reduced serum CTX values (i.e., <150 pg/mL) were associated with BRONJ after dentoalveolar surgery; and 2) to determine if specific radiographic changes are associated with teeth that develop BRONJ after extraction. STUDY DESIGN: A retrospective review of radiographic and/or serum CTX data was performed for 68 patients with a history of bisphosphonate therapy who either underwent dental extraction or were diagnosed with BRONJ in the Department of Oral and Maxillofacial Surgery during the period 2007-2009. Postoperative healing was assessed for 26 patients with reduced serum CTX levels (<150 pg/mL) who either underwent dental extraction or treatment for BRONJ. Preoperative radiographs were evaluated for 55 patients who either healed normally or developed BRONJ after dental extraction. RESULTS: All 26 patients (100%) who had serum CTX levels <150 pg/mL healed successfully after dentoalveolar surgery (20 patients) or after treatment for BRONJ (6 patients). Among the 55 patients who underwent radiographic evaluation, 24 patients (83%) with BRONJ exhibited periodontal ligament (PDL) widening associated with extracted teeth, whereas only 3 patients (11%) who healed normally demonstrated PDL widening. CONCLUSION: These data suggest that radiographic PDL widening may be a more sensitive indicator than CTX testing in predicting risk of BRONJ. Current guidelines that recommend minimal surgical intervention may need to be revised to include alternative strategies for the elimination or management of this pathology.