AUTHOR=Tagliaferri Thaysa Leite , Guimarães Natália Rocha , Pereira Marcella de Paula Martins , Vilela Liza Figueiredo Felicori , Horz Hans-Peter , dos Santos Simone Gonçalves , Mendes Tiago Antônio de Oliveira 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.00578

DOI=10.3389/fmicb.2020.00578

ISSN=1664-302X

ABSTRACT=<p>The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of <italic>Enterobacteriaceae</italic>. One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of <italic>Escherichia coli</italic>. Here we exploited this system further under challenging conditions: by targeting the <italic>bla</italic><sub>TEM–</sub><sub>1</sub> AMR gene located on a high-copy plasmid (i.e., 100–300 copies/cell) and by directly tackling <italic>bla</italic><sub>TEM–</sub><sub>1</sub>-positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of <italic>E. coli</italic> harboring <italic>bla</italic><sub>TEM–</sub><sub>1</sub> on the plasmid pSB1A2, the plasmid number and, accordingly, the <italic>bla</italic><sub>TEM–</sub><sub>1</sub> gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in <italic>bla</italic><sub>TEM–</sub><sub>1</sub> 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 <italic>E. coli</italic>, plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of <italic>Galleria mellonella</italic> infected with CRISPR-Cas9-treated <italic>E. coli</italic>, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of <italic>Enterobacter hormaechei</italic> and to a lesser extent in <italic>Klebsiella variicola</italic>, 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.</p>