Antibacterial effect of sodium hypochlorite on in vitro biofilm formation of Enterococcus faecalis in different phases

doi:10.3969/j.issn.2095-4344.2014.52.026

Abstract

Abstract:

BACKGROUND: Studies have reported Enterococcus faecalis can exist in environment which is lack of nutrients and antimicrobials to cause re-infection of the root canal. Sodium hypochlorite solution is shown to have strong scavenging action as a root canal flushing fluid to eliminate residual Enterococcus faecalis.

OBJECTIVE: To explore the antibacterial effect of sodium hypochlorite on in vitro biofilm formation of Enterococcus faecalis in different phases.

METHODS: Biofilm models of Enterococcus faecalis were established at exponential phase, stationary phase and starvation phase, and then the biofilms were exposed to various concentrations of sodium hypochlorite solutions (1.0%, 2.5%, 5.25%). The contact time was 30 seconds, 5 minutes and 10 minutes. The direct optical observation of Enterococcus faecalis was conducted under confocal laser scanning microscope.

RESULTS AND CONCLUSION: In the same concentration of sodium hypochlorite solutions, the biofilms of starved cells were more resistant to sodium hypochlorite than those formed in the exponential phase and stationary phase (P < 0.05). When the concentration of sodium hypochlorite was 1%, the reduction of viable cells showed significant difference between the exponential phase and stationary phase (P < 0.05). When the concentration of sodium hypochlorite was 2.5% and 5.25%, the reduction of viable cells in the exponential phase and stationary phase had no statistical difference (P > 0.05). For the biofilms at the same phase, 5.25% sodium hypochlorite reduced more viable cells than 2.5% and 1% sodium hypochlorite (P < 0.05), and 2.5% sodium hypochlorite also could reduce more viable cells than 1% sodium hypochlorite, but it only exhibited a significant difference when the contact time was 30 seconds (P < 0.05). These results show that under the same concentration and same contact time, the Enterococcus faecalis biofilms at the starvation phase are more resistant to sodium hypochlorite than those at the exponential phase and stationary phase. Under the same contact time, 5.25% sodium hypochlorite exhibits the best antibacterial effect on the Enterococcus faecalis biofilms at the starvation phase.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: sodium hypochlorite, Enterococcus faecalis, biofilms

CLC Number:

R318

Wang Shan-shan, Dong Ming, Wang Li-na, Niu Wei-dong. Antibacterial effect of sodium hypochlorite on in vitro biofilm formation of Enterococcus faecalis in different phases [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(52): 8509-8514.

Figures/Tables 1

2.1 粪肠球菌生长曲线图图1所示：0-8 h，细菌大量繁殖快速增长，为对数期；8-24 h，细菌活性达到高水平后并保持相对稳定状态，为稳定期；48 h后，细菌开始大量死亡，细菌活性降低，但并没有完全死亡，而是在缺乏营养的环境中保持相应活性，为饥饿期。 2.2 次氯酸钠溶液对不同时期粪肠球菌生物膜的药物结果统计分析表明，各时期粪肠球菌生物膜在次氯酸钠溶液用药实验中，数据符合正态分布，相同作用时间内不同生理时期粪肠球菌生物膜对不同浓度次氯酸钠药物敏感性差异有显著性意义(P < 0.05)。药物作用30 s(表1)：①组间比较：在1%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义 (P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)；在2.5%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)；在5.25%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)。②组内比较：对数期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠作用下的活菌降低量比较差异有显著性意义(P < 0.05)。稳定期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠作用下的活菌降低量比较差异有显著性意义(P < 0.05)。饥饿期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠作用下的活菌降低量比较差异有显著性意义(P < 0.05)。药物作用5 min(表2)：①组间比较：在1%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)；在2.5%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)；在5.25%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)。 ②组内比较：对数期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠作用下的活菌降低量比较差异有显著性意义(P < 0.05)。稳定期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠作用下的活菌降低量比较差异有显著性意义(P < 0.05)。饥饿期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠作用下的活菌降低量比较差异有显著性意义(P < 0.05)。药物作用10 min(表3)：①组间比较：在1%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)；在2.5%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)；在5.25%次氯酸钠作用下，对数期与稳定期活菌降低量比较差异无显著性意义(P > 0.05)，对数期、稳定期与饥饿期活菌降低量比较差异有显著性意义(P < 0.05)。 ②组内比较：对数期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠作用下的活菌降低量比较差异有显著性意义(P < 0.05)。稳定期组中，1%与2.5%次氯酸钠作用下的活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠药物作用下的活菌降低量比较差异有显著性意义(P < 0.05)。饥饿期组中，1%与2.5%次氯酸钠药物作用下活菌降低量比较差异无显著性意义(P > 0.05)；1%、2.5%与5.25%次氯酸钠药物作用下活菌降低量比较差异有显著性意义(P < 0.05)。 2.3 不同时期粪肠球菌生物膜药物作用效果观察药物作用30 s、5 min、10 min后，3个不同时期粪肠球菌生物膜结构破坏，失去原有生物膜棉絮状结构特点，松脱、解离，失去生物膜保护后的细菌大量死亡，但各时期生物膜中仍有细菌存活。在药物实验过程中的数据分析结果中也同样得以证明，因此选取最大浓度(5.25%次氯酸钠)作用10 min后的生物膜样本进行观察。激光共聚焦显微镜下观察生物膜中的细菌绿色为活细菌，红色为死细菌，桔黄色和橙色一般认为是由死菌和活菌重叠造成的。图1显示，次氯酸钠溶液作用10 min后，发现各时期粪肠球菌生物膜表面死亡细菌严重脱膜、解离，但仍可见部分残存的细菌团块未被完全杀死，松散的黏附在玻片上(箭头所示)，饥饿期残留活细菌量密度明显较其他两个时期多，密集。"

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