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FINAL REPORT (LLUSD-STRL-02336) Evaluation of Antimicrobial Potential of BreathRx Anti-Bacterial Mouth Rinse Lorna Linda, CA 92350 November 18, 2002 FINAL REPORT Evaluation of Antimicrobial Potential of BreathRx Anti-Bacterial Mouth Rinse Purpose The purpose of this study was to determine the antimicrobial potential of BreathRx Antibacterial Mouth Rinse in eight microbes using the standard Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays (Baron et aI., 1994), following the guidelines of the Oral Health Care Products for Over-the-Counter Human Use Tentative Final Monograph (FDA, 1994). Materials and Methods The eight test organisms used for the assays were specified by the FDA in the Oral Health Care Products for Over-the-Counter Human Use Tentative Final Monograph (FDA, 1994). The included: 1.) Actinomyces viscosus (ATCC 19246); 2.) Fusobacterium nucleatum (ATCC 10953); 3) Porhyromonas gingivalis (ATCC 33277); 4) Prevotella intermedia (ATCC 49046); 5) Bacteroides forsyth us (A TCC 43037); 6) Candida albicans (A TCC 18804); 7) Streptococcus mutans (A TCC 25175); and, 8) Escherichia coli (A TCC 11775). The microbes were obtained from the American Type Culture Collection (Manassas, VA). BreathRx Anti-Bacterial Mouth Rinse (BRX ACT 001, 0724/02) was provided by the study sponsor. The material was received on July 30, 2002 and was kept in original bottle at 4°C until tested. PRAS Brucella plates and fluid thioglycollate medium were obtained from Anaerobes Systems (Morgan Hill, CA). Bacto Todd Hewitt broth, Difco Tryptic Soy broth and agar plates, BBI GasPak disposable anaerobic indicator, and BBL GasPak Plus anaerobic system envelopes with Palladium catalyst were purchased from Becton Dickinson Microbiology Systems (Sparks, MD). Brain heart infusion medium and chopped meat broth were from Difco Laboratories (Detroit, MI). Facultative bacteria, including E. coli, S. mutans were cultured with Tryptic Soy broth and Tryptic Soy agar, while fluid thioglycoflate broth and PRAS Brucella plates were used to culture A. viscosus, F. nucleatum, P. gingivalis, P. intermedia and B. forsythus for both the MIC and MBC experiments. Brain heart infusion broth and chopped meat broth were used for growth purposes only. Briefly, dilutions of the test rinse were prepared (1 ml total volume), starting from undiluted, 1:2 dilution up to 1 :512 dilution with sterile distilled water. An equal volume (1 ml) of the standardized cells was added to the diluted test rinse to make a total volume of 2 ml. The control was a mixture of 1 ml of sterile water and 1 ml of standardized test organism suspension. The mixtures were incubated overnight at 37°C. The turbidity of the culture was measured after 24 hours to determine the MIC of the test agent. If the mixture was clear, a volume of 100 III was plated on the appropriate agar plates, which were incubated overnight, and colonies, if any, were then counted to determine the MBC of the test agent. The MIC and MBC experiments were repeated using the same procedures to ensure the accuracy and reliability of the results. Results The results obtained from the first MIC and MBC experiments are presented in Tables 1 and 2, respectively. No evidence of microbial growth, as indicated, by the Clear media, was observed in tubes of S. mutans, P. gingivitis, P. intermedia and B. forsythus with the highest dilution (1 :512) of BreathRx Anti-Bacterial Mouth Rinse (Table 1). No growth of C. abacas and A. viscosus was detected at the dilution of 1: 128, while the media was clear at 1 :32 dilution in F. nucleate. E Coli, a none-oral bacterium, showed no evidence of growth at the 1:8 dilution of BreathRx Anti-Bacterial Mouth Rinse. The MBC results showed no colony growth of P. gingivitis, P. inter media and B. forsythus at 1:512 dilution of BreathRx Anti-Bacterial Mouth Rinse (Table 2), and no colony was observed in plates with S. mutans, A. viscosus, F. nucleate and C .. albicans at dilutions of 1: 128, 1:64, 1:32 and 1: 16, respectively. For E Coli, the bactericidal concentration of BreathRx Anti-Bacterial Mouth Rinse was 1:2 dilution (Table 2). The results of the repeat assay (Tables 3 and 4) were essentially the same as those obtained from the initial experiments. The only difference was found in C. albacons in the MIC assay; the no growth (clear media) was observed at 1 :64, which was a one-step higher concentration than that observed in the first assay. The MBC results were the same as those of the first assay. In both the first and repeat of the MIC and MBC assays, test microbes grew normally in control tubes or plates, and no microbial growth was detected in the media control Conclusion It is concluded that, under conditions of the present study, BreathRx Anti-Bacterial Mouth Rinse at highly diluted concentrations has significant bacteriostatic and bactericidal effects on S. mutans, A. viscosus, C. albicans, P. gingivatis, P. intermedia, F. nucleatum, B. forsythus as determined using the MIC and MBC methods. BreathRx Anti-Bacterial Mouth Rinse also effectively inhibits the growth of E. Coli at 1:8 dilution and kills E. Coli at 1:2 dilution. |
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