Escherichia coli Metagenome

Introduction: The rampant misuse and overuse of antibiotics have precipitated the emergence of multidrug-resistant and pan drug-resistant bacteria, constituting a formidable global health threat. Antibiotic adjuvants that potentiate the efficacy of existing antibiotics represent a particularly promising avenue to address this challenge.Methods: We performed genome-wide CRISPRi screening to identify homologous recombination pathway as broad-spectrum potential targets for antibiotic adjuvants. Additionally, we employed three distinct strategies to limit expression and function of recA, a key protein in homologous recombination pathway, including dCas9-sgRNA system delivered via conjugation, RecX-20 peptide fused with cell-penetrating motif, and an inhibitor cisplatin, validated by surface plasmon resonance.Results: Inhibition of homologous recombination pathway significantly increased bacterial susceptibility to multiple antibiotic classes (quinolones, beta-lactams, aminoglycosides, and nitrofurantoin) while reducing horizontal gene transfer of antibiotic resistance. RecA deficiency led to membrane damage, and impaired efflux pump activity, resulting in higher intracellular antibiotic accumulation and oxidative stress. Cisplatin was identified as a RecA inhibitor, enhanced multiple antibiotic efficacies against MDR pathogens both in vitro and in vivo.Conclusions: Our work provides novel insights into the development of broad-spectrum antibiotic adjuvants to restore the efficacy of existing antibiotics, and offers an effective solution to combat the growing threat of drug-resistant bacterial infections.引言：抗生素的滥用与过度使用已催生多重耐药及泛耐药细菌的出现，构成严峻的全球公共卫生威胁。能够增强现有抗生素疗效的抗生素佐剂，是应对这一挑战的极具前景的研究方向之一。 方法：本研究通过全基因组CRISPR干扰（CRISPRi）筛选，将同源重组通路鉴定为广谱抗生素佐剂的潜在靶点。此外，我们采用三种不同策略抑制同源重组通路关键蛋白RecA的表达与功能：包括通过接合方式递送的dCas9-sgRNA系统、融合细胞穿膜结构域的RecX-20肽，以及经表面等离子体共振（SPR）验证的抑制剂顺铂。 结果：抑制同源重组通路可显著提升细菌对多类抗生素（喹诺酮类、β-内酰胺类、氨基糖苷类及呋喃妥因）的敏感性，同时降低抗生素耐药基因的水平转移。RecA缺陷会引发细胞膜损伤，并削弱外排泵活性，导致细胞内抗生素蓄积量升高与氧化应激加剧。顺铂被鉴定为RecA抑制剂，可在体外与体内环境中增强多种抗生素对多重耐药（MDR）病原菌的抗菌效果。 结论：本研究为开发广谱抗生素佐剂以恢复现有抗生素的临床疗效提供了全新视角，同时为应对日益严峻的耐药菌感染威胁提供了可行解决方案。