Clinical Antiplaque and Antigingivitis Efficacy of Two Dentifrices:

Objective: In vitro testing of antimicrobial agents is an important tool in the testing hierarchy, and may provide interesting insights into their potential clinical efficacy. Agents with demonstrable in vitro antimicrobial activity may be effective against the same micro organisms in vivo, whereas agents without demonstrable in vitro antimicrobial activity are unlikely to exhibit in vivo antimicrobial activity. In addition, these methods may also be useful in screening antimicrobial agents in product formulations because such agents with both in vitro and in vivo activity may have reduced antimicrobial effects when formulated into a dentifrice. Accordingly, this study examined the in vitro and ex vivo antimicrobial activity of three commercial dentifrices: one formulated with 0.243% sodium fluoride (Crest Cavity Protection Toothpaste-Regular); one with 0.454% stannous fluoride, sodium hexametaphosphate, and zinc lactate (Crest Pro-Health), and one with 0.3% triclosan, 2.0% PVM/MA copolymer, and 0.243% sodium fluoride (Colgate Total). • Methods: The minimum inhibitory concentration (MIC) of each dentifrice was determined for resident oral bacterial species, including bacteria that are associated with dental caries, periodontitis, and oral halitosis. Evaluations were performed on individual laboratory strains, and on oral bacteria from supragingival plaque samples obtained from 10 adults and from oral rinse samples obtained from 18 adults. • Results: The lowest MICs against the oral strains and human samples, i.e., greatest antimicrobial activity, were seen for the triclosan/ copolymer dentifrice. There was, in general, a four-fold difference in MICs between the triclosan/copolymer dentifrice and the stannous fluoride/sodium hexametaphosphate/zinc lactate dentifrice. The triclosan/copolymer dentifrice significantly inhibited periodontal pathogens, such as Aggregatibacter actinomycetemcomitans, Eikenella corrodens, and Fusobacterium nucleatum. In ex vivo tests measuring antimicrobial effects, the triclosan/copolymer dentifrice substantially inhibited bacterial growth after 30-, 60-, and 120second exposures compared to the sodium fluoride or stannous fluoride/sodium hexametaphosphate/zinc lactate dentifrices. Similarly, in ex vivo tests measuring antimicrobial effects on supragingival plaque biofilms, the triclosan/copolymer dentifrice substantially inhibited bacterial growth compared to the other test dentifrices. • Conclusion: Different in vitro and ex vivo analyses show that the triclosan/copolymer dentifrice has significant antimicrobial activity on oral bacteria, including species causing dental caries, periodontitis, and oral halitosis, and it provides superior efficacy compared to the stannous fluoride/sodium hexametaphosphate/zinc lactate dentifrice. (J Clin Dent 2010;21[Spec Iss]:96–100) 96 Introduction A variety of microbiological techniques have been used to identify and characterize the microorganisms residing in the human oral cavity. This is an important activity in order to further knowledge of the microorganisms that colonize the human body, the microbiome, because oral microorganisms can cause dental caries and periodontal disease, the most common infectious diseases in man. Ninety-two percent of people in the US 65 years of age and older have dental caries in their permanent teeth, 50.3% of people in the US population 30 years or older have gingivitis, and 26% have destructive periodontitis. Microscopy, bacterial culture, and, most recently, nucleic acid sequencing are routinely used to identify microorganisms. Microbial susceptibility to antimicrobial agents is routinely evaluated by disk diffusion assays (Kirby-Bauer), broth and agar dilution assays, and combination assays, such as the spiral gradient endpoint method and E test. Microbial susceptibility tests have obvious clinical applications in the prevention and treatment of infectious diseases. Of particular interest is the correlation between in vitro antimicrobial testing and in vivo efficacy. An agent that does not exhibit in vitro antimicrobial activity is unlikely to demonstrate in vivo anti microbial activity. On the other hand, an antimicrobial agent that demonstrates significant in vitro antimicrobial activity may or may not exert similar levels of in vivo antimicrobial activity. These levels are typically expressed as the minimum inhibitory concentration (MIC) or the minimum bactericidal concentration (MBC). The MIC is the lowest concentration of a drug that inhibits growth, while the MBC is the lowest concentration that kills a microorganism. MICs are used to determine susceptibility or resistance of microorganisms to an antimicrobial agent, i.e., what kind and how much of an antimicrobial agent to use in a particular clinical situation. Antibiotic breakpoints are defined based on the MIC and on the pharmacokinetics in healthy volunteers. Vol. XXI, No. 4 The Journal of Clinical Dentistry 97 slurry for 30, 60, or 120 seconds, and distributed onto solid media containing defibrinated sheep blood. The study protocol was approved by the Health Sciences Institutional Review Board at the University at Buffalo. Statistical Analysis Viable microorganisms recovered after antimicrobial treatments were evaluated by ANOVA and Tukey multiple comparison tests, with subjects and dentifrice in the model. Treatment effects are reported as significant at p < 0.05. Results The TCN/C dentifrice demonstrated significantly higher anti microbial activity (Table I) than the other two dentifrices, with MICs to oral bacteria ranging from less than 0.94 μg/mL to 30 μg/mL. By comparison, there were higher MICs for the SnF 2 dentifrice, ranging from 1.8 to 75 μg/mL. There was especially notable antimicrobial activity for the TCN/C dentifrice toward perio dontal pathogens, including Aggregatibacter actinomycetem comitans, Campylobacter, Eikenella corrodens, and Fusobacterium nucleatum. For the SnF 2 dentifrice, the highest MICs were to Capnocytophaga gingivalis and Actinomyces meyerii, but were otherwise similar to the F dentifrice for most of the gram-positive and gramnegative test microorganisms. In this study, the MICs for commercial dentifrices formulated with stannous fluoride/sodium hexametaphosphate/zinc lactate, triclosan/copolymer/sodium fluoride, and sodium fluoride were determined for microorganisms commonly found in the human oral cavity, using both laboratory strains and samples obtained from adult subjects to determine the effects of different treatment durations on microbial viability. Materials and Methods Media, Chemicals, and Reagents Bacteriological media were obtained from Becton-Dickinson (Sparks, MD, USA) and formulated in accordance with manufacturer’s instructions. Buffers, chemicals, and laboratory reagents were obtained from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise indicated. Bacterial Strains Oral bacteria were obtained from either the American Type Culture Collection (Manassas, VA, USA) or from the University at Buffalo School of Dental Medicine (Buffalo, NY, USA), and included oral and non-oral bacteria that can cause periodontal disease, dental caries, or oral halitosis. All bacteria were cultured on enriched tryptic soy agar, supplemented with 5% defibrinated sheep blood, 5.0 μg/mL hemin, and 0.5 μg/mL vitamin K 1 . Dentifrices Commercially available dentifrices for this investigation included a 0.243% sodium fluoride toothpaste (Crest Cavity Protection Toothpaste-Regular, Procter & Gamble, Cincinnati, OH, USA; henceforth F), a 0.454% stannous fluoride, sodium hexametaphosphate, and zinc lactate toothpaste (Crest Pro-Health, Procter & Gamble, Cincinnati, OH, USA; henceforth SnF 2 ), and a toothpaste containing 0.3% triclosan, 2.0% polyvinylmethyl ether maleic acid (PVM/MA) copolymer, and 0.243% sodium fluoride (Colgate Total, Colgate-Palmolive Company, New York, NY; henceforth TCN/C). Laboratory Tests Bacteria were tested against the F, SnF 2 , and TCN/C toothpastes, each dispersed in sterile water. Various dilutions of toothpaste slurries were incubated with bacteria cultured in liquid media, and the MIC was defined as the lowest concentration (highest dilution) in which the bacteria failed to grow. Positive controls included bacteria without toothpaste slurry, and negative controls included toothpaste slurry without added bacteria. Ex Vivo Tests Supragingival plaque was collected from 10 adults, dispersed by sonication, and distributed onto solid media containing defibrinated sheep blood with different concentrations of toothpaste. Following anaerobic incubation at 37°C for five days, the number of viable bacteria (CFU/mL) were enumerated from the solid media. Oral rinse samples were obtained from 18 adults follow ing informed consent and a one-week “washout” with a commercially available fluoride dentifrice. The subjects rinsed with 10 mL of sterile water for 10 seconds and expectorated into sterile tubes. The oral rinse samples were mixed with toothpaste Table I Minimum Inhibitory Dentifrice Concentrations Crest Crest Cavity Strain Colgate ProProtectionBacterial Species Number Total Health Regular Oral and Non-oral Microorganisms Actinomyces meyerii ATCC 33972 15 75 30 Actinomyces viscosus ATCC 43146 7.5 7.5 30 Bacillus cereus ATCC 11778 7.5 15 30 Bacillus subtilis ATCC 6051 15 >150> 75 Candida albicans ATCC 90028 30 150 75 Escherichia coli ATCC 4157 7.5 150 >150> Moraxella catarrhalis ATCC 8176 .< 0.94> 3.5 7.5 Staphylococcus aureus ATCC 6538 15 30 30 Veillonella dispar ATCC 17748 15 30 75 Veillonella atypica ATCC 27215 7.5 7.5 15 Periodontal Pathogens Aggregatibacter. actinomycetemcomitans ATCC 43717 < 0.94> 3.5 30 Aggregatibacter actinomycetemcomitans ATCC 43718 1.8 3.5 30 Capnocytophaga gingivalis ATCC 33124 3.5 75 15 Campylobacter rectus ATCC 33238 1.6 7.5 15 Eikenella corrodens ATCC 23834 .< 0.94> 15 30 Fusobacterium nucleatum ATCC 25586 1.8 7.5 15 Porphyromonas gingivalis ATCC 53977 1.8 1.8 15 Prevotella intermedia ATCC 25611 3.5 3.5 15 Prevotella melanogenica ATCC 25845 3.5 3.5 30 Prevotella nigresence NCTC 9336 1.8 1.8 15