肺炎克雷伯菌对表没食子儿茶素没食子酸酯的适应性代价

Adaptive cost of Klebsiella pneumoniae on epigallocatechin gallate

  • 摘要:
    目的 探讨肺炎克雷伯菌在亚抑菌浓度表没食子儿茶素没食子酸酯(EGCG)中传代14 d适应性代价变化, 为EGCG的临床转化提供实验支持。
    方法 采用全基因组测序技术对肺炎克雷伯菌的耐药基因进行分析;结晶紫染色法检测生物膜形成能力的变化;荧光定量聚合酶链式反应(PCR)检测群体感应基因luxS及其调控基因(lsrK和lsrR)和毒力基因(aerobactinrmpA)转录水平差异;大蜡螟生存实验检测肺炎克雷伯菌致病性。
    结果 通过全基因组测序发现非黏液性CRKP菌株1和黏液性CRKP菌株2是ST11型, 都携带blaKPC-2基因;在亚抑菌浓度EGCG中处理后的传代其生长曲线无变化;生物膜形成能力减低;luxS基因表达量低于未处理组;铁载体aerobactin的转录水平随着传代天数和亚抑菌浓度的增加而受到抑制;毒力基因黏液表型调节基因rmpA转录能力也受到抑制;大蜡螟生存率提高, 差异均有统计学意义(P<0.05)。
    结论 亚抑菌浓度EGCG处理肺炎克雷伯菌后, 其生长速度无变化, 生物膜形成、毒力基因和群体感应基因的转录水平及致病性均受到抑制, 为EGCG的临床应用提供实验基础。

     

    Abstract:
    OBJECTIVE To explore the change of adaptive cost of Klebsiella pneumoniae in subinhibitory concentration of Epigallocatechin gallate (EGCG) for subculture of 14 days so as to provide experimental support for clinical transformation of EGCG.
    METHODS The drug resistance genes in the K. pneumoniae strains were analyzed by whole genome sequencing technology. The change of biofilm formation ability was detected by crystal violet staining, the transcriptional levels of quorum sensing gene luxS and its regulatory genes(lsrK and lsrR) as well as virulence genes(aerobactin and rmpA) were detected by means of fluorescent quantitative polymerase chain reaction (PCR). The pathogenicity of the K. pneumoniae was tested through survival assay of greater wax moth.
    RESULTS The whole genome sequencing showed that 1 strain of non-mucous CRKP and 2 strains of mucous CRKP were ST11, all of which carried blaKPC-2 gene. There was no change in the growth curve of subculture after treatment with subinhibitory concentration of EGCG; the biofilm forming ability was reduced. The expression level of luxS gene was higher in the non-treatment group. The transcriptional level of ionophore aerobactin was inhibited with the increase of subculture days and subinhibitory concentration; the transcription ability of the virulence gene mucous phenotype regulatory gene rmpA was inhibited as well. The survival rate of greater wax moth increased, and there was significant difference (P < 0.05).
    CONCLUSIONS The growth rate of K. pneumoniae strains dose not change after the treatment with the subinhibitory concentration of EGCG. The biofilm formation and the transcriptional levels of virulence genes and quorum sensing genes as well as the pathogenicity are inhibited, which may provide experimental bases for clinical application of EGCG.

     

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