Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China; Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning Commission, Shanghai, China.
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China; Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning Commission, Shanghai, China.
Int J Antimicrob Agents. 2022 May;59(5):106570. doi: 10.1016/j.ijantimicag.2022.106570. Epub 2022 Mar 13.
Decreased effectiveness of metronidazole for the treatment of Clostridioides difficile infection has been documented. One reason for this is that levels of metronidazole in the colon are generally low; therefore, a modest increase in the minimum inhibitory concentration of metronidazole for C. difficile may result in an insufficient therapeutic concentration. Due to the lack of efficient genetic manipulation tools for C. difficile strains, the resistance mechanism is largely unknown. In this study, a metronidazole-resistant strain (SH182IR) was acquired by in-vitro induction with metronidazole from a clinical metronidazole-heteroresistant strain (SH182), and the genomic and transcriptional changes were investigated through whole-genome sequencing and RNA-seq. The morphology of the two strains was studied by transmission electron microscopy, and the roles of drug efflux pumps in metronidazole resistance were determined by inhibition assay. Genomic analysis showed that the ferrous iron transporter feoB3 was truncated in SH182IR, indicating that feoB3 contributed to the metronidazole resistance of C. difficile. RNA-seq analysis showed that genes involved in peptidoglycan synthesis, efflux pumps and metronidazole reductive action were expressed differentially between the two strains. Further cell imaging confirmed that cell wall thickness was significantly greater in SH182IR. The efflux pump inhibitor test showed that addition of reserpine or cyanide 3-chlorophenylhydrazone reduced metronidazole resistance in SH182IR, thus proving the role of efflux pumps in metronidazole resistance. These results found an association between genomic variation and metronidazole resistance in C. difficile, and show that metronidazole resistance in C. difficile is multi-factorial, involving metronidazole metabolism, cell wall thickness and efflux pumps. These findings will help improve knowledge and understanding of metronidazole resistance of C. difficile.
甲硝唑治疗艰难梭菌感染的疗效降低已有报道。原因之一是结肠中甲硝唑的水平通常较低；因此，甲硝唑对艰难梭菌的最低抑菌浓度略有增加，可能导致治疗浓度不足。由于缺乏有效的艰难梭菌遗传操作工具，其耐药机制在很大程度上尚不清楚。在这项研究中，通过体外诱导，从临床甲硝唑异质性耐药株（SH182）中获得了甲硝唑耐药株（SH182IR），并通过全基因组测序和 RNA-seq 研究了基因组和转录组的变化。通过透射电子显微镜研究了两株菌的形态，通过抑制试验确定了药物外排泵在甲硝唑耐药中的作用。基因组分析表明，SH182IR 中截短了亚铁转运蛋白 feoB3，表明 feoB3 有助于艰难梭菌对甲硝唑的耐药性。RNA-seq 分析表明，两株菌中涉及肽聚糖合成、外排泵和甲硝唑还原作用的基因表达存在差异。进一步的细胞成像证实，SH182IR 的细胞壁厚度明显增加。外排泵抑制剂试验表明，加入利血平或氰化物 3-氯苯腙可降低 SH182IR 对甲硝唑的耐药性，从而证明了外排泵在甲硝唑耐药中的作用。这些结果发现艰难梭菌中基因组变异与甲硝唑耐药之间存在关联，并表明艰难梭菌对甲硝唑的耐药性是多因素的，涉及甲硝唑代谢、细胞壁厚度和外排泵。这些发现将有助于提高对艰难梭菌甲硝唑耐药性的认识和理解。
