title: [Effects of immA and immB coding putative bacteriocin immunity proteins on the antimicrobial sensitivity in planktonic Streptococcus mutans and biofilm formation].
abstract: OBJECTIVE
  To investigate the effects of putative bacteriocin immunity proteins on the growth mode of Streptococcus mutans (Sm). To observe the differences of antimicrobial sensitivity in planktonic Sm wild-type strains and mutant strains caused by the inactivation of bacteriocin immunity proteins and their influence on the biofilm formation.
  
  
  METHODS
  Sm wild-type strains (WT) and its knockout mutants defective in immA and immB (ΔimmA(-) and ΔimmB(-) mutants) coding putative bacteriocin immunity proteins were cultured in brain heart infusion (BHI) and selected by erythromycin at the concentration of 10 mg/L. Optical density was detected by spectrophotometer every hour and growth curve was drawn. WT, ΔimmA(-) and ΔimmB(-) mutants were treated with ampicillin (0.04, 0.05, 0.06, 0.07, 0.08 mg/L), sodium fluoride (50, 100, 150, 200, 250 mg/L) and sodium hypochlorite (0.078%, 0.156%, 0.313%, 0.625%, 1.250%) for 24 hours. Optical density was detected by multifunctional micro plate reader. WT and the mutants were cultured in MBEC(TM) P&G Assay for 24 hours. The minimum biofilm eradication concentration (MBEC) of chlorhexidine against Sm was determined by serial dilution method. Confocal laser scanning microscopy (CLSM) was used to visualize the biofilm architecture, depth and ratio of live to dead bacteria.
  
  
  RESULTS
  Growth curve showed that it took about 3 hours to reach exponential phase and about 7 hours to stationary phase for WT, while 4 hours to exponential phase and 8 hours to stationary phase for mutants. Optical density of mutants were lower than WT in the presence of various antimicrobial agents (P < 0.01). In 0.06 mg/L ampicillin group, optical density value of WT, ΔimmA(-) and ΔimmB(-) mutants were 0.334 ± 0.016, 0.027 ± 0.016 and 0.047 ± 0.018. In 150 mg/L sodium fluoride group, optical density value of WT and mutants were 0.254 ± 0.018, 0.129 ± 0.011 and 0.167 ± 0.010. In 0.313% sodium hypochlorite group, optical density value of WT and mutants were 0.467 ± 0.008, 0.017 ± 0.006 and 0.050 ± 0.006. The MBEC of chlorhexidine against Sm WT, ΔimmA(-) and ΔimmB(-) mutants were 6.25, 1.57, and 3.13 mg/L. The results by CLSM showed a noticeable difference in biofilm architecture. The depth of WT biofilm was higher than the mutants biofilm (P < 0.01). The ratio of live to dead bacteria of WT biofilm was higher than ΔimmA(-) mutants in all layers (P < 0.05) and ΔimmB(-) mutants in the outer and intermedium layer (P < 0.01). There is no significant different between the inner layers of WT and ΔimmB(-) mutants (P = 0.191).
  
  
  CONCLUSIONS
  Putative bacteriocin immunity proteins have influence on the growth mode of Sm. The antimicrobial sensitivity of planktonic Sm can be up-regulated by the inactivation of immA or immB. The MBEC of chlorhexidine against ΔimmA(-) and ΔimmB(-) mutants is lower than WT. The inactivation of immA or immB affects the biofilm formation.
authors.affiliation: Department of Operative Dentistry and Endodontics, Sun Yat-sen University, Guangzhou, China.
authors.forename: Yong-biao
authors.initials: YB
authors.lastname: Huo
authors.forename: Jun-ni
authors.initials: JN
authors.lastname: Mai
authors.forename: Jun-qi
authors.initials: JQ
authors.lastname: Ling
authors.forename: Li-jun
authors.initials: LJ
authors.lastname: Huo
mesh_terms: D000667:Ampicillin / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001430:Bacteriocins / Q000235:genetics* / Q000276:immunology; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D002327:Cariostatic Agents / Q000494:pharmacology; D002710:Chlorhexidine / Q000494:pharmacology; D004202:Disinfectants / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010933:Plankton / Q000187:drug effects; D012969:Sodium Fluoride / Q000494:pharmacology*; D012973:Sodium Hypochlorite / Q000494:pharmacology*; D013295:Streptococcus mutans / Q000187:drug effects* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D002327:Cariostatic Agents; D004202:Disinfectants; D000667:Ampicillin; D012969:Sodium Fluoride; D012973:Sodium Hypochlorite; D002710:Chlorhexidine