Prem K. Sreenivasan

Development and characterization of a simple perfused oral microcosm

Aims:  To validate perfused, inline, filter‐based fermentation systems (multiple Sorbarod devices, MSD) for their ability to maintain stable oral bacterial communities. MSD enable replicate (n = 5) microcosm biofilms (BF) to be established and sampled, together with their perfusates (PA, cells in eluted medium).

Antimicrobial efficacy of 0·05% cetylpyridinium chloride mouthrinses

This study evaluated the antimicrobial activity of two commercially available 0·05% cetylpyridinium chloride (CPC) mouthrinses with or without alcohol and examined its antimicrobial activity on oral bacterial species including fresh clinical isolates compared to a chlorhexidine mouthrinse and a control fluoride mouthrinse without CPC. Two different approaches were used to evaluate antimicrobial activity. First, the minimum inhibitory concentration (MIC) was determined for each mouthrinse against a panel of 25 micro‐organisms including species associated with dental caries, gingivitis and periodontitis. Second, supragingival dental plaque obtained from 15 adults was incubated with the four mouthrinses to evaluate antimicrobial activity on micro‐organisms in oral biofilms. Both CPC mouthrinses exhibited lower MICs, that is, greater antimicrobial activity, against oral Gram‐negative bacteria especially periodontal pathogens and species implicated in halitosis such as Aggregatibacter actinomycemcomitans, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia and Solobacterium moorei than the control mouthrinse. Ex‐vivo tests on supragingival plaque micro‐organisms demonstrated significantly greater antimicrobial activity by the CPC mouthrinses (>90% killing, P < 0·001) and the chlorhexidine rinse (>98% killing, P < 0·05) compared to the control fluoride mouthrinse. Whilst the chlorhexidine mouthrinse was most effective, mouthrinses containing 0·05% CPC formulated with or without alcohol demonstrated broad‐spectrum antimicrobial activity against both laboratory strains and supragingival plaque bacteria compared to a control mouthrinse without CPC.

Characterization and prevalence of Solobacterium moorei associated with oral halitosis

Previous studies suggest that Solobacterium moorei is associated with oral halitosis. In the present study, we examined the prevalence of S. moorei on the dorsal surface of the tongue in 57 adults (21 with and 36 without halitosis) by bacterial culture and direct amplification of nucleic acids. We also examined the S. moorei type strain and four clinical isolates for 16S ribosomal nucleic acid sequence, H(2)S and enzyme production, and antibiotic susceptibility. S. moorei was found on the dorsal surface of the tongue in 100% of the subjects with halitosis and 14% of subjects without halitosis. Infection with S. moorei was correlated with organoleptic measures of halitosis and with volatile sulfur compound levels. Nucleic acid probe detection of S. moorei as a test for halitosis exhibited 100% sensitivity and 86% specificity. The S. moorei type strain and all four clinical isolates showed >98% 16S rDNA sequence similarity, produced H(2)S, demonstrated acid phosphatase, beta-galactosidase, alpha-glucosidase, esterase, leucine arylamidase and naphthol phosphohydrolase enzyme activities, and were sensitive to all antibiotics tested except gentamicin, kanamycin, nalidixic acid and rifampin. This study supports the hypothesis that S. moorei is associated with halitosis.

Individual microflora beget unique oral microcosms.

Aims:  To examine the efficacy of the multiple Sorbarod device (MSD) for the reproduction of inter‐individual variations in oral microbiotas. The MSD supports sessile growth on parallel cellulose filters, perfused with artificial saliva. This enables biofilms (BF) to be grown and sampled, together with released cells in eluted medium (perfusates, PAs).

A 6‐month study of the effects of 0.3% triclosan/copolymer dentifrice on dental implants

AIM Supportive therapy to maintain dental implants is increasingly important. This study examined the effect of a 0.3% triclosan/2% copolymer dentifrice on oral biofilms and gingival inflammation (GI) on dental implants and peri-implant tissues. MATERIALS AND METHODS One hundred and twenty adults with a dental implant and contra-lateral tooth were enrolled in this 6 month, double-blind, two-treatment, parallel group study. Sixty subjects were randomly assigned to a triclosan/copolymer dentifrice test group and 60 subjects to a fluoride dentifrice control group and instructed to brush twice daily for 6 months. At baseline, 3, and 6 months, a calibrated dentist assessed dental plaque, GI and collected supragingival dental plaque for microbiological analysis. RESULTS Subjects in the triclosan/copolymer group demonstrated significantly lower levels of dental plaque, gingivitis, and bleeding on probing at 3 and 6 months at both the implant and contra-lateral tooth compared with the fluoride group (p<0.05). There were significantly fewer Gram-negative anaerobes in the triclosan/copolymer group (p<0.05) including >90% reductions in Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eubacterium saburreum, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella melaninogenica, Solobacterium moorei, and Tannerella forsythia. CONCLUSIONS Twice daily use of a triclosan/copolymer dentifrice may enhance dental implant maintenance by reducing dental plaque and GI.

Multiparameter Assessments To Determine the Effects of Sugars and Antimicrobials on a Polymicrobial Oral Biofilm

ABSTRACT Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of Actinomyces viscosus, Streptococcus sanguinis, Streptococcus mutans, Neisseria subflava, and Actinobacillus actinomycetemcomitans cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (P < 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (P < 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (P < 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (N-acetyl-l-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (P < 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.

An in vitro evaluation of hydrolytic enzymes as dental plaque control agents.

The plaque-control potential of commercially available amylase, lipase and protease was evaluated by observing their effects on coaggregation and on bacterial viability within various plaque microcosms. A quantitative coaggregation assay indicated that protease significantly inhibited the extent of coaggregation of Actinomyces naeslundii and Streptococcus oralis (P <0.05) and of Porphyromonas gingivalis and S. oralis. Amylase significantly (P <0.05) increased the coaggregation of A. naeslundii versus Fusobacterium nucleatum and A. naeslundii versus P. gingivalis. Concomitant challenge of constant-depth film fermenter-grown plaques with the enzymes did not result in detectable ecological perturbations (assessed by differential culture and denaturing gradient gel electrophoresis). Similar dosing and analysis of multiple Sorbarod devices did not reveal increases in bacterial dispersion which could result from disaggregation of extant plaques. A short-term hydroxyapatite colonization model was therefore used to investigate possible enzyme effects on early-stage plaque development. Whilst culture did not indicate significant reductions in adhesion or plaque accumulation, a vital visual assay revealed significantly increased aggregation frequency following enzyme exposure. In summary, although hydrolytic enzymes negatively influenced binary coaggregation, they did not cause statistically significant changes in bacterial viability within plaque microcosms. In contrast, enzyme exposure increased aggregation within extant plaques.

Evaluation of the specificity and effectiveness of selected oral hygiene actives in salivary biofilm microcosms.

The microbiological effects of biocidal products used for the enhancement of oral hygiene relate to the active compound(s) as well as other formulation components. Here, we test the specificities of selected actives in the absence of multiple excipients. Salivary ecosystems were maintained in tissue culture plate-based hydroxyapatite disc models (HDMs) and modified drip-flow biofilm reactors (MDFRs). Test compounds stannous fluoride (SF), SDS, triclosan (TCS), zinc lactate (ZL) and ZL with SF in combination (ZLSF) were delivered to the HDMs once and four times daily for 6 days to MDFRs. Plaques were characterized by differential viable counting and PCR-denaturing gradient gel electrophoresis (DGGE). TCS and SDS were the most effective compounds against HDM plaques, significantly reducing total viable counts (P<0.05), whilst SF, ZL and ZLSF were comparatively ineffective. TCS exhibited specificity for streptococci (P<0.01) and Gram-negative anaerobes (P<0.01) following a single dosing and also on repeated dosing in MDFRs. In contrast to single exposures, multiple dosing with ZLSF also significantly reduced all bacterial groups, whilst SF and ZL caused significant but transient reductions. According to PCR-DGGE analyses, significant (P<0.05) reductions in eubacterial diversity occurred following 6 day dosing with both TCS and ZLSF. Concordance of MDFR eubacterial profiles with salivary inocula ranged between 58 and 97%. TCS and ZL(SF) exhibited similar specificities to those reported for formulations. TCS was the most potent antibacterial, after single and multiple dosage regimens.

A randomized, double-blind clinical study to assess the antimicrobial effects of a cetylpyridinium chloride mouth rinse on dental plaque bacteria.

BACKGROUND Studies with cetylpyridinium chloride (CPC) mouth rinses that range from 1 use to 6 months of use have documented the clinical efficacy of these formulations on supragingival plaque and gingivitis. OBJECTIVE The objective of the present study was to compare the effects of a commercially available mouth rinse containing 0.05% CPC versus a fluoride mouth rinse on the anaerobic bacteria found in dental plaque. Antimicrobial effects on the organisms of the supragingival plaque, a natural biofilm, were determined after 1 use and after 14 days of use of each mouth rinse. METHODS After enrollment, adult subjects from China completed a 1-week washout period and provided baseline samples of supragingival plaque for analysis of anaerobic bacteria. Subjects were randomly assigned to receive a commercially available mouth rinse formulated with 0.05% CPC or a fluoride mouth rinse. Subjects were assigned to each group according to a computer-generated randomization sequence. They were instructed to rinse with 20 mL of either the CPC or the fluoride mouth rinse for 30 seconds. Microbiologic analyses of dental plaque samples were conducted 12 hours after the first use of assigned mouth rinse. Subjects were instructed to continue twice-daily rinsing with their assigned mouth rinse for the next 14 days in addition to brushing their teeth with a commercial fluoride toothpaste. Dental plaque samples for microbiologic analyses were collected on day 15; this was done 12 hours after the final use of the assigned mouth rinses. A dentist conducted oral examinations before each sample collection to evaluate hard and soft tissue health over the course of the study. RESULTS The study included 117 adults (62 females, mean age, 28.70 years; 55 males, mean age, 30.41 years). Subjects rinsing with the CPC mouthwash (n = 58; mean age, 29.41 years) reported significant reductions in anaerobic bacteria versus those issued the fluoride rinse (n = 59; mean age, 29.61 years) 12 hours after 1 use and 12 hours after 14 days of use (P < 0.001). The mean percent reduction in anaerobic bacteria between the CPC mouth rinse and the fluoride mouth rinse was 29.98% after 1 use and 57.90% after 14 days of use. All enrolled subjects completed the study without any adverse events. CONCLUSION Use of the CPC mouth rinse was associated with significant reductions in the anaerobic bacteria of supragingival plaque compared with fluoride mouth rinse use in these adult subjects.

Antibacterials as anti-inflammatory agents: dual action agents for oral health.

Background Inflammatory processes with a range of specialized cells and biochemical mediators form a complex network of inter-related signal transducing pathways that relay information to preserve normal functions. Advances in molecular analyses of the information relay pathways for their constituents and principal ligands along with mechanisms utilized by the host for microbial recognition have stimulated interest in therapeutic agents with dual functionalities i.e. antibacterial and anti-inflammatory effects. Aim This review examines clinically tested agents for oral health applications with both antimicrobial and anti-inflammatory effects to include antibiotics, antimicrobials and phenolics. Results Bis-phenols such as triclosan, representing a unique dual functional therapeutic for routine oral hygiene, with its demonstrated clinical effects on inhibiting the dental plaque biofilm, reducing inflammation (gingivitis) and subsequent periodontitis is described. Cyclines, comprising another class of approved anti-inflammatory agents used at the patient level for oral health is discussed. Dual active agents in current clinical practice for systemic conditions are highlighted to summarize the clinical validity of dual function agents as an emerging therapeutic strategy. Conclusions Clinical studies demonstrate therapeutic benefits of agents with dual functionality with their effects on microorganisms and the concomitant host inflammatory response. Advances in microbial pathogenesis and resultant inflammation will facilitate progress in this emerging area poised to be a significant milestone for dental therapeutics