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The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of  Enterobacteriaceae . One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of  Escherichia coli . Here we exploited this system further under challenging conditions: by targeting the  bla  TEM–  1  AMR gene located on a high-copy plasmid (i.e., 100–300 copies/cell) and by directly tackling  bla  TEM–  1 -positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of  E. coli  harboring  bla  TEM–  1  on the plasmid pSB1A2, the plasmid number and, accordingly, the  bla  TEM–  1  gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in  bla  TEM–  1  were observed, likely resulting in a dysfunction of the gene product. As a consequence, a full reversal to an antibiotic sensitive phenotype was achieved, despite plasmid maintenance. In a clinical isolate of  E. coli , plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of  Galleria mellonella  infected with CRISPR-Cas9-treated  E. coli , as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of  Enterobacter hormaechei  and to a lesser extent in  Klebsiella variicola , both of which harbored additional resistance genes affecting beta-lactams. The data show that targeting drug resistance genes is encouraging even when facing high-copy plasmids. In clinical isolates, the simultaneous interference with multiple genes mediating overlapping drug resistance might be the clue for successful phenotype reversal.

frontiers in microbiology

Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae