细菌素免疫蛋白调控变形链球菌抗菌敏感性及生物膜形成的实验研究

Effects of immA and immB coding putative bacteriocin immunity proteins on the antimicrobial sensitivity in planktonic Streptococcus mutans and biofilm formation

目的观察细菌素免疫蛋白相关基因对变形链球菌抗菌敏感性及生物膜形成的影响，探讨细菌素免疫蛋白与细菌抗菌剂耐受性的关系，为生物膜抗菌敏感性的研究提供基础数据。方法筛选培养细菌素免疫蛋白基因突变株，绘制生长曲线。酶标仪检测不同质量浓度氨苄青霉素（0．04、0．05、0．06、0．07及0．08mg／L）、氟化钠（50、100、150、200及250mg／L）及不同质量分数的次氯酸钠（0．078％、0．156％、0．313％、0．625％及1．250％）作用下变形链球菌标准株、△immA-和△immB-突变株菌液的吸光度值。应用最小生物膜清除浓度（minimal biofilm eradicatin concentration，MBEC）桩钉96孔板以连续稀释法检测醋酸氯己定对3种菌株生物膜的MBEC。应用激光共聚焦扫描显微镜（confocal laser scanning microscope，CLSM）定量分析标准株和突变株生物膜结构。结果△immA-和△immB-突变株的迟缓期和稳定生长期均比标准株延时1h。氨苄青霉素为0．06ms／L时，标准株、△immA-突变株和△immB-突变株菌液吸光度值分别为0．334±0．016、0．027±0．016及0．047±0．018；氟化钠质量浓度为150mg／L时，3种菌株菌液吸光度值分别为0．254±0．018、0．129±0．011及0．167±0．01；当次氯酸钠质量分数为0．313％时，3种菌株菌液吸光度值分别为0．467±0．008、0．017±0．006及0．050±0．006，以上各组抗菌剂中，标准株与突变株吸光度值差异均有统计学意义（P〈0．01）。醋酸氯己定对3种菌株的MBEC分别为6．25、1．57及3．13mg／L。标准株生物膜厚度显著高于△immA-和△immB-突变株（P〈0．01）；标准株各层活菌比例均高于△immA-突变株（P〈0．05）；标准株中、外层活菌比例高于△immB-突变株（P〈0．01），但内层活菌比例差异无统计学意义（P=0．191）。结论细菌素免疫蛋白参与调控浮游细菌生长，尤其在生长初期；细菌素免疫蛋白相关基因缺陷使浮游态变形链球菌抗菌敏感性提高、抗菌剂MBEC降低及生物膜结构不成熟。

Objective To investigate the effects of putative bacteriocin immunity proteins on the growth mode of Streptococcus mutans （ Sm ） . To observe the differences of antimierobial 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 （ △ immA- and △ immB- 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, △immA- and △immB- mutants were treated with ampicillin （0. 04, 0.05, 0. 06, 0.07, 0. 08 mg/L） , sodium ilnoride（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 MBECTM 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 ofWT, △immA- and △immB- 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, △immA- and △immB- 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 △ immA- mutants in all layers （ P 〈 0. 05 ） and △immB- mutants in the outer and intermedium layer （ P 〈 0. 01 ） . There is no significant different between the inner layers of WT and △ immB- 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 △immA- and △immB- mutants is lower than WT. The inactivation of immA or immB affects the biofilm formation.