Gerber Michael, Walch Christiane, Löffler Birgit, Tischendorf Kristin, Reischl Udo, Ackermann Grit
Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University of Leipzig, Liebigstraße 24, 04103 Leipzig, Germany.
Institute of Medical Microbiology and Hygiene, University Hospital of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
J Med Microbiol. 2008 Jun;57(Pt 6):776-783. doi: 10.1099/jmm.0.47739-0.
Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5x MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.
艰难梭菌是医院获得性感染性腹泻的主要病因。已知几种抗菌药物可诱发并促进艰难梭菌相关性腹泻（CDAD）。本研究调查了甲硝唑（MTR）、万古霉素（VAN）、克林霉素（CLI）和利奈唑胺（LZD）对艰难梭菌生长、毒素基因转录及毒素产生的影响。四株艰难梭菌菌株分别在含有和不含低于最低抑菌浓度（sub-MIC）的MTR、VAN、CLI和LZD（0.5倍MIC）的条件下培养，通过菌落计数测量生长情况。使用酶联免疫吸附测定法（ELISA，用于毒素A）和细胞毒性测定法（用于毒素B）检测培养上清液及超声破碎细胞中的毒素产生情况。采用实时定量聚合酶链反应（Real-time PCR）测量毒素A和B基因的转录情况。本研究的目的是将通过ELISA或细胞培养测定法对毒素A和B产生情况的分析与转录组分析相结合。在无抗生素条件下，这四株菌株表现出相似的生长情况及不同水平的毒素产生。无抗生素对照组在细菌生长达到平台期的后期显示出毒素产生，而暴露于抗生素的菌株则较早出现毒素产生。除CLI外，所有使用的抗生素均提高了毒素基因的转录率。本研究结果表明，低于最低抑菌浓度的抗生素可导致艰难梭菌主要毒力因子的基因转录发生变化。本研究描述了一种用于艰难梭菌毒素基因转录组分析的新方法。
