花青素降低肺炎克雷伯菌生物膜的作用及机制研究

Study on the Effects and Mechanism of Cyanidin on the Biofilm of Klebsiella Pneumoniae

目的:分析花青素降低肺炎克雷伯菌生物膜的作用及其机制。方法:选取临床分离的碳青霉烯类耐药以及敏感肺炎克雷伯菌各3株。采用微量肉汤稀释法检测对亚胺培南、美罗培南和厄他培南的最低抑菌浓度(MIC),质控菌株为大肠埃希菌ATCC25922;生长曲线法检测花青素对肺炎克雷伯菌生长能力的影响;结晶紫染色法测定花青素对生物膜形成能力的影响;应用实时荧光定量PCR(qRT-PCR)检测花青素存在与不存在条件下与生物膜形成相关基因(luxS、mrkA、wbbM、pgaA和wzm)的表达量。用GraphPad Prism 8.0绘图并进行数据分析。结果:6株菌株中3株肺炎克雷伯菌均对3种碳青霉烯类药物耐药,MIC≥64μg·ml^(-1),其余3株肺炎克雷伯菌为敏感菌。花青素可以明显降低肺炎克雷伯菌的生长能力以及生物膜形成能力;qRT-PCR结果显示花青素主要可以降低生物膜形成相关基因mrkA和wbbM的表达量。结论:花青素可以降低肺炎克雷伯菌生物膜形成能力,其机制主要通过降低mrkA和wbbM基因的表达。

Objective: To investigate the effects and mechanism of cyanidin on the biofilm of Klebsiella pneumoniae.Methods: Three clinical carbapenem resistant and three sensitive K. pneumoniae strains were selected. Minimal inhibitory concentration(MIC) of imipenem, meropenem and ertapenem were tested by broth micro-dilution method. Escherichia coli ATCC25922 was served as quality control strain. The growth curve method was used to detect the effects of cyanidin on the growth of K. pneumoniae. Crystal violet staining method was further used to determine the effects of cyanidin on the formation of biofilm. Real-time quantitative PCR(qRT-PCR) was used to detect the expressions of biofilm-related genes(luxS, mrkA, wbbM, pgaA and wzm) in the presence and absence of cyanidin. Experimental data was analyzed using GraphPad Prism 8.0. Results: Among the 6 K. pneumoniae strains, three strains were resistant to carbapenems, and MIC were all ≥64 μg·ml^(-1). The remaining 3 strains were sensitive strains. Cyanidin could reduce the growth ability and biofilm formation ability of K. pneumoniae significantly. The results of qRT-PCR showed that cyanidin could mainly reduce the expressions of biofilm formation related genes mrkA and wbbM in K. pneumoniae.Conclusion: Cyanidin can reduce the biofilm formation ability of K. pneumoniae, and its mechanism is mainly related to diminishing the expressions of mrkA and wbbM.