Zoocin A and lauricidin in combination selectively inhibit Streptococcus mutans in a biofilm model

The oral cavity contains many different microbial species growing in a biofilm. Dental caries is the localised destruction of the tooth by organic acids produced from the bacterial fermentation of dietary carbohydrates. The mutans streptococci, in particular Streptococcus mutans, have been proposed as the main etiological agents of dental caries and high levels of mutans streptococci in the plaque is correlated with a higher risk for dental caries. A range of broad-spectrum antimicrobials are used to inhibit plaque formation. However, current research is focussed on more targeted approaches. The protein zoocin A has high activity against Streptococcus mutans, while the monoglyceride lauricidin is active against gram-positive bacteria. Both are therefore potential antimicrobials for anti-caries therapy. The aims of this study were to produce zoocin A at sufficient concentrations to use as an antimicrobial in a biofilm model and to develop a simple biofilm model for use studying the effects of zoocin A and lauricidin upon an oral biofilm. Zoocin A was produced as a recombinant protein in Escherichia coli. Zoocin A production of 165 mg per litre of culture was achieved by the use of a benchtop fermentor. Neither optimisation of codon usage or expression of zoocin A in E. coli BL21 CodonPlus-(DE3)-RIL resulted in increased yields of zoocin A. A triple-species biofilm model was developed where S. mutans, Streptococcus oralis and Actinomyces viscosus were grown on glass beads and supplied 1/3 strength brain heart infusion (BHI) supplemented with 1% sucrose as a nutrient source. Zoocin A and lauricidin were added directly to the feed medium, both individually and in combination. Biofilms were incubated for 24, 48 or 72 hours, then each species was enumerated and biofilm formation and the pH were measured. The addition of 40 µg/ml zoocin A to the biofilm model specifically reduced the levels of S. mutans in the biofilm by approximately two logs and was accompanied by an increase in the levels of S. oralis. Lauricidin reduced S. mutans by a similar amount when applied at 10 µg/ml. The use of zoocin A and lauricidin in combination resulted in a four log decrease in the level of S. mutans, while levels of S. oralis and A. viscosus remained elevated. Zoocin A and lauricidin also reduced biofilm formation and maintained the pH above 7.0, while the pH of control biofilms decreased to 4.3 after 72 hours. The effects of chlorhexidine on biofilm formation and pH were similar. However, S. mutans was reduced by approximately five logs and a reduction in the colony forming units of all three species was seen. The application of zoocin A and lauricidin to the biofilm model gave a targeted reduction in S. mutans. While the reduction was not as large as that seen with chlorhexidine, zoocin A and lauricidin did not reduce other bacterial species in the biofilm and gave a similar reduction in biofilm formation and maintenance of pH. There was little or no development of resistance to zoocin A or lauricidin by biofilm cells. This study suggests that antimicrobial agents can be used in combination in the oral cavity to achieve a targeted inhibition of S. mutans. Further research is required to optimise the dosage and the best route of delivery