Laboratory for Applied Science and Technology in Health, Carlos Chagas Institute, Fiocruz, Curitiba 81350-010, PR, Brazil.
Laboratory of Cell Biology, Carlos Chagas Institute, Fiocruz, Curitiba 81350-010, PR, Brazil.
Int J Mol Sci. 2022 Aug 16;23(16):9175. doi: 10.3390/ijms23169175.
Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria.
抗微生物药物耐药性是一种具有全球影响的公共卫生负担，最近被确定为 2019 年主要死亡原因之一。磷霉素是一种常用于治疗尿路感染的抗生素，肠杆菌科对其的耐药性主要是由于fosA3 基因编码的金属酶 FosA3 所致。在这项工作中，我们改造了一种名为 pRE-FOSA3 的 CRISPR-Cas9 系统，以恢复 fosA3+大肠杆菌菌株的敏感性。fosA3+大肠杆菌菌株是通过将合成的 fosA3 转化为非致病性大肠杆菌 TOP10 而产生的。为了介导 fosA3 的破坏，选择了两个 gRNA（guide RNA），它们使用了超过 700 个 fosA3+大肠杆菌分离株中 fosA3 序列的保守区域，并且 resensitization 质粒 pRE-FOSA3 通过将 gRNA 克隆到 pCas9 中进行组装。gRNA_195 在使细菌对磷霉素重新敏感方面的效率达到了 100%。此外，尽管编辑菌株不包含 CRISPR-Cas9 的靶标，但仍失去了同一质粒中编码的氨苄青霉素抗性，这表明质粒已被清除。这里呈现的体外分析指出了一条可以探索的途径，以协助开发针对 fosA3+细菌的有效替代治疗方法。
