Huo Yong-biao, Mai Jun-ni, Ling Jun-qi, Huo Li-jun
Department of Operative Dentistry and Endodontics, Sun Yat-sen University, Guangzhou, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2011 Aug;46(8):478-83.
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.
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.
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).
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.
研究假定的细菌素免疫蛋白对变形链球菌（Sm）生长模式的影响。观察因细菌素免疫蛋白失活导致的浮游Sm野生型菌株和突变菌株抗菌敏感性差异及其对生物膜形成的影响。
将编码假定细菌素免疫蛋白的Sm野生型菌株（WT）及其在immA和immB基因缺失的突变体（ΔimmA(-)和ΔimmB(-)突变体）在脑心浸液（BHI）中培养，并用浓度为10 mg/L的红霉素进行筛选。每小时用分光光度计检测光密度并绘制生长曲线。WT、ΔimmA(-)和ΔimmB(-)突变体分别用氨苄西林（0.04、0.05、0.06、0.07、0.08 mg/L）、氟化钠（50、100、150、200、250 mg/L）和次氯酸钠（0.078%、0.156%、0.313%、0.625%、1.250%）处理24小时。用多功能微孔板读数仪检测光密度。WT和突变体在MBEC(TM)宝洁检测中培养24小时。采用系列稀释法测定洗必泰对Sm的最低生物膜清除浓度（MBEC）。用共聚焦激光扫描显微镜（CLSM）观察生物膜结构、厚度及活菌与死菌比例。
生长曲线显示，WT约3小时达到指数期，约7小时达到稳定期；突变体4小时达到指数期, 8小时达到稳定期。在各种抗菌剂存在下，突变体的光密度低于WT（P < 0.01）。在0.06 mg/L氨苄西林组中，WT、ΔimmA(-)和ΔimmB(-)突变体的光密度值分别为0.334±0.0l6、0.027±0.016和0.047±0.018。在150 mg/L氟化钠组中，WT和突变体的光密度值分别为0.254±0.018、0.129±0.011和0.167±0.010。在0.313%次氯酸钠组中，WT和突变体的光密度值分别为0.467±0.008、0.017±0.006和0.050±0.006。洗必泰对Sm WT、ΔimmA(-)和ΔimmB(-)突变体的MBEC分别为6.25、1.57和3.13 mg/L。CLSM结果显示生物膜结构存在显著差异。WT生物膜厚度高于突变体生物膜（P < 0.01）。WT生物膜各层的活菌与死菌比例均高于ΔimmA(-)突变体（P < 0.05），外层和中层高于ΔimmB(-)突变体（P < 0.01）。WT和ΔimmB(-)突变体的内层之间无显著差异（P = 0.191）。
假定的细菌素免疫蛋白对Sm的生长模式有影响。immA或immB失活可上调浮游Sm的抗菌敏感性。洗必泰对ΔimmA(-)和ΔimmB(-)突变体的MBEC低于WT。immA或immB失活影响生物膜形成。
