EVALUATION OF TWO ORAL PROBIOTIC PRODUCTS FOR MICROBIAL VIABILITY AND IN VITRO INHIBITION OF SELECTED PERIODONTAL BACTERIAL PATHOGENS.

Abstract

Objectives: One potential impact of oral probiotic products involves use of known bacterial antagonisms to alter the ecologic environment in periodontal pockets from one inhabited by pathogenic dental plaque microorganisms to one more favorable to colonization by non-pathogenic species (bacterial replacement). Until recently, the ability to introduce such beneficial effector bacteria into the oral cavity of periodontitis patients has been limited by the lack of specifically-formulated available commercial probiotic products. PerioBalance (Sunstar GUM), with two strains of the gram-positive, aerobic species Lactobacillus reuteri, and EvoraPlus (Oragenics), with freeze-dried strains of the gram-positive, aerobic species Streptococcus oralis, Streptococcus uberis, and Streptococcus rattus, are two recently-introduced commercial oral probiotic products proposed to have beneficial effects against periodontal disease. However, it is not known if the microbial species contained in these two oral probiotics are viable after the manufacturing process, and have the capability to exert inhibitory effects against putative periodontal bacterial pathogens when reconstituted in the oral cavity. Thus, the objective of the present study was to determine whether PerioBalance lactobacilli and EvoraPlus streptococci are viable upon product use, and possess in vitro inhibitory effects against fresh clinical strains of the putative periodontal bacterial pathogens, Tannerella forsythia and Prevotella intermedia/nigrescens, in the presence of anaerobic growth conditions. Methods: Commercial lots of PerioBalanceÒ and EvoraPlusÒ tablets were aseptically removed from the product packaging with sterile forceps, dissolved into Möller’s VMG I anaerobic dispersion solution, plated onto pre-reduced, enriched Brucella blood agar, and subjected to overnight anaerobic incubation at 35ºC in a culture cabinet containing 85% N2-10% H2-5% CO2, and to overnight aerobic incubation in a 5% CO2-95% air atmosphere. All culture plates were then visually examined under magnification for microbial colony growth. In vitro solid media competition assays were used to assess the in vitro inhibition capability of the two oral probiotics against T. forsythia and P. intermedia/nigrescens. Pioneer PerioBalance lactobacilli and EvoraPlus streptococci colonies were first grown on enriched Brucella blood agar media, followed by secondary spotting of T. forsythia and P. intermedia/nigrescens isolates immediately next to the established pioneer EvoraPlus and PerioBalanceÒ bacterial colonies such that they almost touched each other. After an additional overnight anaerobic incubation period, growth inhibition of the putative periodontal bacterial pathogens by the pioneer PerioBalance and EvoraPlus colonies was noted as the visual presence without magnification of a proximal zone of inhibition at the intersection of the pioneer colonies and the T. forsythia and P. intermedia/nigrescens colonies. Results: PerioBalance lactobacilli grew readily and in abundance in vitro on anerobically and anaerobically-incubated EBBA, with no other colony types or contaminating organisms. In contrast, EvoraPlus product samples purchased over-the-counter from drug stores in Maryland and Pennsylvania failed to exhibit any in vitro microbial growth under anaerobic and aerobic incubation conditions, with only EvoraPlus tablets obtained directly from the manufacturer yielding in vitro streptococcal growth. No in vitro inhibition was noted under anaerobic conditions of established PerioBalance lactobacilli and EvoraPlus streptococci pioneer colonies against subsequent growth of clinical isolates of T. forsythia and P. intermedia/nigrescens, with no zone of inhibition developing between their colonies and the immediately-adjacent established oral probiotic pioneer colonies. Conclusions: The two commercial oral probiotics evaluated varied considerably in the viability of their microbial constituents, with abundant growth of PerioBalance lactobacilli found in over-the-counter product material, and the lack of any EvoraPlus streptococci growth in product tablets obtained from sources other than directly from the manufacturer. Both oral probiotic products failed in vitro, in solid media competition assays, to inhibit growth of fresh clinical isolates the putative periodontal bacterial pathogens T. forsythia and P. intermedia/nigrescens under anaerobic growth conditions. These findings question the potential effectiveness of the two oral probiotic products to alter the subgingival ecology in periodontal pockets when anaerobic environmental conditions are present. Additional research is needed to assess the inhibitory potential of PerioBalance lactobacilli and EvoraPlus streptococci against additional isolates of subgingival bacterial species, and in circumstances where microaerophilic or aerobic environmental conditions are found.