Resensitization of Fosfomycin-Resistant Escherichia coli Using the CRISPR System.

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Antimicrobial resistance (AMR) poses a significant global health threat, identified as a leading cause of death in 2019.
• Fosfomycin is a key antibiotic for treating urinary tract infections, but resistance mediated by the FosA3 enzyme, encoded by the fosA3 gene in Enterobacteriaceae, is a growing concern.

Purpose of the Study:

• To develop and evaluate a CRISPR-Cas9 system for restoring fosfomycin sensitivity in fosA3-positive Escherichia coli.
• To investigate the potential of this system for combating AMR by targeting the fosA3 gene.

Main Methods:

• A CRISPR-Cas9 system, pRE-FOSA3, was engineered using two guide RNAs (gRNAs) targeting conserved regions of the fosA3 gene.
• A fosA3-positive E. coli strain was created by introducing a synthetic fosA3 gene into E. coli TOP10.
• The pRE-FOSA3 plasmid, containing the gRNA, was introduced to mediate fosA3 disruption.

Main Results:

• One selected gRNA (gRNA_195) demonstrated 100% efficiency in resensitizing the bacteria to fosfomycin.
• The CRISPR-Cas9 editing process resulted in the clearance of the ampicillin resistance gene present on the same plasmid, indicating successful plasmid elimination.
• In vitro analysis confirmed the potential of this method against fosA3-positive bacteria.

Conclusions:

• The adapted CRISPR-Cas9 system effectively restores antibiotic sensitivity in resistant bacteria.
• This strategy offers a promising avenue for developing novel therapeutic approaches against fosA3-mediated antimicrobial resistance in E. coli.
• CRISPR-Cas9 mediated plasmid clearance presents a dual benefit in combating antibiotic resistance.