Reduction of Streptococcus mutans With Reuterin, Produced by Lactobacillus reuteri

Objectives: The purpose of this study was to evaluate bacterial sensitivity and antimicrobial activity of reuterin, produced by L. reuteri-ATCC55730 on S. mutans-ATCCUA130 growth.
Methods: L. reuteri was cultured in 250mL-Erlenmeyer flask containing 150mL of MRS-broth medium. Samples were taken in exponential growth phase at 14, 16, 18, 20h, then samples were treated with 250mM-glicerol, and incubated for 2-4 h at 37°C. Reuterin was then isolated by poly-prep chromatography column for each one of samples, quantification was performed by a colorimetric method with acrolein as standard.
For the Kirby-Bauer method, Petri dishes containing Brain Hearth Infusion (BHI)-agar were inoculated with 100mL (1x10⁴ cells/mL) of S. mutans at exponential phase. A filter paper-disk was soaked with 20mLof reuterin. Petri dishes were incubated in anaerobic-conditions for 48h at 37°C. The results were based on the size of the halo around the disk(mm).
For Minimal Inhibitory Concentration (MIC)-performance, glass-tubes were prepared with BHI-broth-medium, reuterin and 1x10⁴ cells/mL of S. mutans strain (1mL final volume). Results were evaluated after 24 h of incubation at 37°C with anaerobic conditions. Data were analyzed by measuring DO600_nm, cellular concentration and pH. For both methods Chlorexidine-0.12% and distilled water were used as control solutions and Escherichia coli-ATCC43895 was used as control of pathogen onto Luria Bertani-agar/broth media. All experiments were performed by triplicate. Mean values and SD were calculated.
Results: Reuterin obtained was 30.73, 65.40, 49.20 and 49.60mg/mL. Results of both techniques showed that reuterin produced by L. reuteri has the ability to interrupt the growth of S. mutans. Halos-sizes were bigger, when reuterin was used (4.3mm) compared to those produced by CHX (3.2mm). Reuterin MIC was 64.50mg/mL (purified at 16h of L. reuteri late logarithmic-growth, OD600nm=0.323). Microscopically, L. reuteri stood-up regarding the pathogens in an approximate ratio 3:1 at 16h.
Conclusions: Reuterin may have potential as a biologic antimicrobial agent. Further studies are in progress.