{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown"],"volume":["11"],"submitter":["Tagliaferri TL"],"pubmed_abstract":["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."],"journal":["Frontiers in microbiology"],"pagination":["578"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC7203346"],"repository":["biostudies-literature"],"pubmed_title":["Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae."],"pmcid":["PMC7203346"],"pubmed_authors":["Horz HP","Tagliaferri TL","Pereira MPM","Dos Santos SG","Guimaraes NR","Mendes TAO","Vilela LFF"],"additional_accession":[]},"is_claimable":false,"name":"Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae.","description":"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.","dates":{"release":"2020-01-01T00:00:00Z","publication":"2020","modification":"2021-02-20T01:34:56Z","creation":"2020-05-23T07:11:30Z"},"accession":"S-EPMC7203346","cross_references":{"pubmed":["32425894"],"doi":["10.3389/fmicb.2020.00578"]}}