乳酸和过氧乙酸对蜡样芽孢杆菌生物膜的抑制作用及其机制

Inhibitory effects and mechanisms of lactic acid and peroxyacetic acid on the biofilm formation of Bacillus cereus

摘要:

【背景】 近年来，蜡样芽孢杆菌(Bacillus cereus)侵袭力不断增强，感染宿主范围不断扩大，而其生物膜的形成与侵袭力和毒力息息相关。【目的】 对蜡样芽孢杆菌的生物膜形成能力进行了鉴定，并探究了乳酸(lactic acid, LA)和过氧乙酸(peroxyacetic acid, PAA)对B. cereus生物膜的抑制机理。【方法】 通过分析生物膜抑制生长曲线、生物膜代谢活性、胞外基质成分(多糖和蛋白质)含量、形态及相关基因表达，来评估LA和PAA对蜡样芽孢杆菌生物膜形成作用的影响。【结果】 蜡样芽孢杆菌可生成生物膜。LA和PAA对B. cereus的最小抑菌浓度(minimum inhibitory concentration, MIC)分别为0.252%、0.030%。生长曲线表明，1/2 MIC、MIC浓度的LA、PAA可显著抑制B. cereus生物膜的形成。由CCK-8法检测生物膜代谢活性可知，MIC浓度的LA和PAA使B. cereus生物膜的代谢活性分别降低了61.02%和69.27%。同时，由硫酸-苯酚法及考马斯亮蓝法对蜡样芽孢杆菌胞外多糖及胞外蛋白检测结果可知，MIC浓度的LA、PAA处理组的胞外多糖分别降低了64.34%和79.14%，胞外蛋白质分别降低了78.96%和80.52%。通过结晶紫染色对生物膜进行显微镜下观察，结果显示1/4 MIC下的LA、PAA即可抑制蜡样芽孢杆菌生物膜的生成。另外，通过实时定量PCR确定，MIC下的LA、PAA显著抑制了蜡样芽孢杆菌生物膜相关基因(gapB, pgm, flgE, SigB, abrB, calY, sipW, tasA)的表达。【结论】 LA、PAA可以作为安全的抗生物膜剂来抑制蜡样芽孢杆菌的生物膜形成。

Abstract:

[Background] In recent years, Bacillus cereus has demonstrated an escalating invasive capacity and a broadening spectrum of hosts, with its biofilm formation being intricately linked to its invasive potential and virulence. [Objective] To characterize the biofilm-forming capability of B. cereus and elucidate the inhibitory mechanisms of lactic acid (LA) and peroxyacetic acid (PAA) on the biofilm formation. [Methods] The impact of LA and PAA on the biofilm formation of Bacillus cereus was comprehensively evaluated by analyzing biofilm inhibition growth curves, biofilm metabolic activity, extracellular matrix components (including polysaccharides and proteins), morphological characteristics, and the expression levels of biofilm-related genes. [Results] B. cereus could form biofilms. The minimum inhibitory concentration (MIC) for LA and PAA against B. cereus were 0.252% and 0.030%, respectively. The biofilm growth curves indicated that LA and PAA at 1/2 MIC and MIC levels significantly curtailed the biofilm formation of B. cereus. The CCK-8 assay demonstrated that LA and PAA at MIC levels reduced the biofilm metabolism activity of B. cereus by 61.02% and 69.27%, respectively. The sulfuric acid-phenol and Coomassie brilliant blue staining results revealed that MIC levels of LA and PAA diminished extracellular polysaccharides by 64.34% and 79.14% and extracellular proteins by 78.96% and 80.52%, respectively. Microscopic examination following crystal violet staining confirmed that even at 1/4 MIC levels, LA and PAA impeded the biofilm formation of B. cereus. Furthermore, real-time quantitative PCR indicated that the MIC levels of LA and PAA markedly downregulated the expression of biofilm-related genes (gapB, pgm, flgE, SigB, abrB, calY, sipW, and tasA) in B. cereus. [Conclusion] Collectively, our data suggest that LA and PAA serve as effective and safe anti-biofilm agents, capable of inhibiting the biofilm formation of B. cereus.