Department of Epidemiology and Health Statistics, College of Public Health, Zhengzhou University, Zhengzhou, 450001, Henan, China.
People's Hospital of Henan University of Chinese Medicine, Zhengzhou, 450003, Henan, China.
FEMS Microbiol Lett. 2022 Feb 22;369(1). doi: 10.1093/femsle/fnac015.
The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.
产碳青霉烯酶的大肠埃希菌已引起全球越来越多的关注，尤其是在亚胺培南（IMP）耐药方面。IMP 耐药的分子机制尚不清楚。本研究旨在探索大肠埃希菌中 IMP 的耐药机制。通过适应性实验室进化诱导敏感 Sx181-0-1 菌株产生耐药菌株。采用纸片扩散和微量肉汤稀释法测定药物耐药谱。全基因组测序和重测序用于分析主要敏感株和耐药株之间非同义单核苷酸多态性（nsSNP）。通过实时定量 PCR（RT-qPCR）鉴定具有 nsSNP 的这些基因的表达水平。在诱导的第 15 代（诱导时间=183 小时）出现耐药表型。在连续亚培养过程中，分别在暴露于递增浓度的 IMP 时分离出 IMP 最小抑菌浓度为 8 和 64µg ml-1 的 Sx181-32 和 Sx181-256。在 Sx181-32 和 Sx181-256 中均观察到 19 个 nsSNP，分布在 rpsU、sdaC、zwf、ttuC、araJ、dacC、mrdA、secF、dacD、lpxD、mrcB、ftsI、envZ 和两个未知功能基因（orf01892 和 orf01933）中。在这 15 个基因中，有 5 个基因（dacC、mrdA、lpxD、mrcB 和 ftsI）主要参与细胞壁合成。mrdA（V338A、L378P 和 M574I）和 mrcB（P784L、A736V 和 T708A）分别有三个氨基酸取代。与 Sx181-0-1 相比，rpsU、ttuC 和 orf01933 在 Sx181-32 和 Sx181-256 中的表达水平均升高。除了 araJ 之外，这些基因在 Sx181-256 中的表达水平均升高。细菌通过调节各种生物过程来产生对抗微生物药物的耐药性，其中涉及最多的是细胞壁合成（dacC、mrdA、lpxD、mrcB 和 ftsI）。mrdA、envZ 和 ftsI 基因的组合突变可能会增加对 IMP 的耐药性。本研究可以提高对大肠埃希菌中 IMP 耐药机制的认识。
