Resensitization of fosfomycin-resistant Escherichia coli using the CRISPR system

• Autor(a) principal: Walflor, Haniel Siqueira Mortagua
• Data de Publicação: 2022
• Outros Autores: Lucena, Aline Rodrigues Castro, Tuon, Felipe Francisco, Medeiros, Lia Carolina Soares, Faoro, Helisson
• Tipo de documento: Artigo
• Idioma: por
• Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
• Texto Completo: https://www.arca.fiocruz.br/handle/icict/55241
• topic: Metaloenzima FosA3; CRISPR–Cas9; Escherichia coli; Fosfomycin; Drug Resistance, Microbial; Enzymes; CRISPR-Associated Protein 9; Fosfomicina; Farmacorresistencia Microbiana; Proteína 9 Asociada a CRISPR; Fosfomycine; Résistance microbienne aux médicaments; Enzymes; Protéine-9 associée à CRISPR; Fosfomicina; Resistência Microbiana a Medicamentos; Enzimas; Proteína 9 Associada à CRISPR

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 pREFOSA3 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 CRISPRCas9 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.