革兰氏阴性菌对大环内酯类的固有抗性主要归因于外膜的低渗透性,尽管潜在的遗传和分子机制仍有待完全阐明。这里,我们使用转座子定向插入位点测序(TraDIS)来鉴定与大肠杆菌对大环内酯类抗生素的内在抗性有关的染色体非必需基因,替米考星.我们在临床肠产毒性菌株(ETEC5621)和实验室菌株(K-12MG1655)中构建了两个>290,000和>390,000个独特Tn5插入的高度饱和转座子突变文库,分别。TraDIS分析鉴定了ETEC5621和MG1655在替米考星的1/8MIC(分别为n=15和16)和1/4MIC(分别为n=38和32)下生长所需的基因。对于这两种菌株,23个与脂多糖生物合成相关的基因,外膜组件,托尔帕尔系统,外排泵,和肽聚糖代谢在抗生素的存在下富集。野生型菌株中基因的个体缺失(n=10)导致替米考星的MIC降低了64至2倍,红霉素,和阿奇霉素,验证Tradis分析的结果。值得注意的是,删除surA或waaG,损害外膜,导致ETEC5621中所有三种大环内酯类药物的MIC下降最显著。我们的发现有助于全基因组理解大肠杆菌中固有的大环内酯抗性,揭示肽聚糖层的潜在作用。它们还提供了体外的概念证据,即大肠杆菌可以通过靶向维持外膜的蛋白质(如SurA和WaaG)而对大环内酯类敏感。
Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC (n = 15 and 16, respectively) and 1/4 MIC (n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes (n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG, which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli, shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.