Masayuki Kumada¹, Mizuho Motegi², Ryoma Nakao², Hideo Yonezawa², Hideki Yamamura³, Junji Tagami^1,4, Hidenobu Senpuku^2
1Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo; ²Department of Bacteriology, National Institute of Infectious Diseases, Tokyo; ³Research & Development Department, Biofermin Pharmaceutical Co, Ltd, Kobe; and ⁴Center of Excellence Program for Frontier Research on Molecular, Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, Japan

Received: October 22, 2007     Revised: January 18, 2008     Accepted: February 20, 2008

Corresponding author: Dr. Hidenobu Senpuku, Department of Bacteriology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail: This e-mail address is being protected from spam bots, you need JavaScript enabled to view it

Background and purpose: Enterococcus faecium is a normal bowel commensal and lactic acid bacterium that is rarely found in the oral cavity. This study investigated whether a non-pathogenic and non-biofilm strain of E. faecium functioned as a probiotic strain toward biofilm formation by Streptococcus mutans, which is an etiological agent for dental caries.

Methods: The effects of E. faecium on streptococcal biofilm was evaluated by absorbance of safranin-stained biofilm at 492 nm in a 96-well microtiter plate.

Results: The E. faecium strain demonstrated cell-number–dependent inhibition of biofilm in dual cultures with 4 laboratory and 16 clinical S. mutans strains, as well as laboratory strains of Streptococcus sobrinus and Streptococcus sanguinis, in 96-well microtiter plates. The inhibiting effects of E. faecium were not dependent on the production of bacteriocin from streptococci and E. faecium, low pH after mix culture, or biofilm formation levels of S. mutans. A culture supernatant sample of more than 10 kDa from E. faecium showed direct inhibiting effects toward S. mutans biofilm formation. Treatment of heat, butanol, and phenol to a supernatant sample restored biofilm formation in culture of S. mutans with the sample. Moreover, the tendencies of inhibition levels by the supernatant sample were associated with those by bacterial cells of E. faecium to S. mutans strains.

Conclusion: The E. faecium non-biofilm strain produced an inhibiting protein to streptococci biofilm formation, showed various susceptibilities to inhibit streptococcal biofilm, and acted as a probiotic bacterial inhibitor of streptococcal biofilm formation.

Key words: Biofilms; Cell communication; Colony count, microbial; Enterococcus faecium; Streptococcus mutans

J Microbiol Immunol Infect. 2009;42:188-196.