单宁酸抑制肺炎克雷伯菌生物膜形成的作用及其机制

doi:10.3969/j.issn.1673-8640.2021.010.017

摘要/Abstract

摘要：

目的分析单宁酸对肺炎克雷伯菌生物膜形成的抑制作用及其机制。方法收集3株耐碳青霉烯类肺炎克雷伯菌（CRKP）和1株碳青霉烯类敏感肺炎克雷伯菌（KP 13883）临床分离株。采用微量肉汤稀释法检测菌株对亚胺培南和美罗培南的最小抑菌浓度（MIC）;采用96孔板结晶紫染色法测定单宁酸对菌株生物膜形成能力的影响;通过扫描电子显微镜观察单宁酸对菌株生物膜形态结构的影响;采用实时荧光定量聚合酶链反应（PCR）分析单宁酸对生物膜形成相关基因（mrkA、luxS和wbbM）表达量的影响。结果 3株CRKP均对亚胺培南和美罗培南耐药,MIC≥64 μg/mL。单宁酸可以明显抑制肺炎克雷伯菌的生长和生物膜形成能力（P<0.05）,与扫描电子显微镜观察结果一致;实时荧光定量PCR结果显示,单宁酸可以降低群体感应相关基因luxS和脂多糖相关基因wbbM的表达量。结论单宁酸可以通过降低luxS和wbbM的表达来抑制肺炎克雷伯菌的生物膜形成能力。

关键词: 肺炎克雷伯菌, 生物膜, 单宁酸, 脂多糖

Abstract:

Objective To study the inhibition and its mechanism of tannic acid on biofilm formation in Klebsiella pneumoniae. Methods Totally,3 isolates of carbapenem-resistant Klebsiella pneumoniae（CRKP） and 1 sensitive isolate KP 13883 were chosen. Minimum inhibitory concentrations（MIC） of imipenem and meropenem were determined by broth micro-dilution method,and the biofilm formation ability was evaluated using 96-well plate crystal violet staining method with or without tannic acid. The morphological differences of biofilm were obtained by scanning electron microscopy before and after the treatment of tannic acid. Real-time fluorescence quantitative polymerase chain reaction（PCR） was used to determine the expression of biofilm-related genes（mrkA,luxS and wbbM） in the presence and absence of tannic acid. Results The 3 CRKP isolates showed decreased susceptibilities to imipenem and meropenem,and the MIC were ≥64 μg/mL. Tannic acid could decrease the growth and biofilm formation ability of CRKP（P<0.05）,which was further confirmed by scanning electron microscopy. The results of real-time fluorescence quantitative PCR showed that tannic acid could reduce the expression of quorum sensing related gene luxS and lipopolysaccharide related gene wbbM. Conclusions Tannic acid could decrease the biofilm formation ability of CRKP.

Key words: Klebsiella pneumoniae, Biofilm, Tannic acid, Lipopolysaccharide

中图分类号:

R446.1

周燕霞, 金东芳. 单宁酸抑制肺炎克雷伯菌生物膜形成的作用及其机制[J]. 检验医学, 2021, 36(10): 1070-1073.

ZHOU Yanxia, JIN Dongfang. Inhibition and mechanism of tannic acid on biofilm formation in Klebsiella pneumoniae[J]. Laboratory Medicine, 2021, 36(10): 1070-1073.

图/表 5

表1 qRT-PCR引物序列

引物名称	引物序列（5'~3'）	引物大小/bp
mrkA-F	ACGTCTCTAACTGCCAGGC	115
mrkA-R	TAGCCCTGTTGTTTGCTGGT
luxS-F	AGTGATGCCGGAACGCGG	148
luxS-R	CGGCGTACCAATCAGGCTC
wbbM-F	ATGCGGGTGAGAACAAACCA	124
wbbM-R	AGCCGCTAACGACATCTGAC
rpoB-F	AGGCGAATCCAGCTTGTTCAGC	147
rpoB-R	TGACGTTGCATGTTCGCACCCATCA

图1 单宁酸对4株肺炎克雷伯菌生长能力的影响

图2 单宁酸对4株肺炎克雷伯菌生物膜形成能力的影响注：与各菌株对照比较,*P<0.05、**P<0.01、***P<0.001。

图3 单宁酸对肺炎克雷伯菌生物膜形成能力的影响电镜观察结果（×2 000）注：（a）未加入单宁酸;（b）加入单宁酸。

图4 单宁酸对肺炎克雷伯菌生物膜形成相关基因表达量的影响注：与未加入单宁酸组比较,*P<0.05;**P<0.01。

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