表皮葡萄球菌附属基因调节子C特异结合多肽对聚氯乙烯材料表面细菌生物膜形成作用的体外研究

The inhibition of accessory gene regulator C specific binding peptides on biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride in vitro

目的探讨表皮葡萄球菌附属基因调节子C(accessory gene regulator C,agr C)特异结合多肽(简称为N1)对聚氯乙烯(polyvinyl chloride,PVC)材料表面表皮葡萄球菌生物膜形成作用的体外研究。方法以表皮葡萄球菌ATCC35984(生物膜表型阳性)和ATCC12228(生物膜表型阴性)作为研究对象。首先将两菌株分别加入浓度为100、200、400、800、1 600μg/mL的N1培养,以相同浓度agrC特异结合无关肽(简称为N0)培养作为对照;24 h后测定吸光度(A)值,确定N1最佳抑菌浓度。两菌株与最佳抑菌浓度的N1及N0分别培养,6、12、18、24、30、48 h测定A值,观察N1对表皮葡萄球菌生物膜形成能力的影响;在此基础上与PVC材料片培养6、12、18、24、30 h,扫描电镜观察PVC材料表面生物膜的表面结构;另取与ATCC35984菌株培养6、12、18、24 h的PVC材料片,激光共聚焦显微镜观察生物膜厚度。结果 A值测定显示,N1对ATCC35984菌株最佳抑菌浓度为800μg/mL。ATCC12228菌株与N1及N0培养后均未形成明显生物膜;ATCC35984菌株与N1及N0培养12 h时,生物膜形成能力差异有统计学意义(P<0.05),6、18、24、30、48 h时差异无统计学意义(P>0.05)。扫描电镜观察,ATCC35984菌株与N1及N0培养后,随时间延长均见成熟生物膜结构;而ATCC12228菌株培养后均未见生物膜形成。激光共聚焦显微镜观察,ATCC35984菌株与N1培养12 h时细菌量明显少于与N0培养,且以死细菌居多;6、18、24 h时两种培养条件下细菌数量未见明显差别,均以活细菌居多;同时,N1培养12、18 h时生物膜厚度明显小于N0培养(P<0.05)。结论 N1抑制表皮葡萄球菌生物膜形成的强度有量效关系;其在聚集期阻碍细菌的增殖和聚集,抑制生物膜形成,对成熟生物膜抑制作用不明显。

Objective To investigate the effect of accessory gene regulator C(agr C) specific binding peptides(named N1) on the biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride(PVC) materials in vitro. Methods Firstly, the two strains(ATCC35984, ATCC12228) were cultured with N1 at concentrations of 100,200, 400, 800, and 1 600 μg/mL, respectively. The control group was cultured with agrC specific binding unrelated peptides(named N0) at the same concentrations and the absorbance(A) value was measured after 24 hours to determine the optimal bacteriostatic concentration of N1. The two strains were cultured with N1 and N0 of the optimal concentration, respectively. The A values were measured at 6, 12, 18, 24, 30, and 48 hours to observe the effect of N1 on the biofilm formation ability of Staphylococcus epidermidis. On this basis, the surface structure of the biofilm on the surface of PVC material was observed by scanning electron microscopy after 6, 12, 18, 24, and 30 hours of incubation with PVC material sheet. The thickness of the biofilm was observed by laser confocal microscopy after 6, 12, 18, and 24 hours of incubation with ATCC35984 strain. Results The optimal bacteriostatic concentration of N1 was 800 μg/mL. ATCC12228 strain did not form obvious biofilm after being cultured with N1 and N0. When ATCC35984 strain was cultured with N1 and N0 for 12 hours, the difference in biofilm formation ability between groups N1 and N0 was statistically significant(P<0.05), but there was no significant difference at 6, 18, 24, 30, and 48 hours(P>0.05). Scanning electron microscopy examination showed that mature biofilm structure was observed in ATCC35984 strain and was not observed in ATCC12228 strain. Laser confocal microscopy observation showed that the number of bacteria in the group N1 was significantly lower than that in the group N0 at 12 hours, and the most of bacteria were dead bacteria. There was no significant difference in the number of bacteria at 6, 18, and 24 hours, and the most of them were live bacteria. The biofilm thickness of group N1 was significantly lower than that of group N0 at 12 and 18 hours(P<0.05). Conclusion The intensity of N1 inhibiting the formation of Staphylococcus epidermidis biofilm is dose-dependent. During the aggregation period, N1 can inhibit the biofilm formation by hindering the bacterial growth and aggregation. The inhibition effect on mature biofilm is not obvious.