CRISPR-Cas9 targeting the blaKPC gene in a clinical isolate of Klebsiella michiganensis: Reduction of imipenem resistance and changes in genomic carbapenem resistance determinants

PLoS ONE, vol. 20, pp. e0328521

Abstract

The CRISPR-Cas technology can be used to disable drug resistance genes. Since carbapenem resistance can be mediated by multiple resistance determinants, we here investigated the extent of re-sensizitation when targeting the blaKPC carbapenemase gene and assessed possible effects on porins and efflux pumps. While full re-sensitization was achieved in a laboratory strain of Escherichia coli solely equipped with blaKPC, resistance reduction in a clinical isolate of Klebsiella michiganensis was achieved in 63% of analyzed transformants, which was a consequence of plasmid copy number reduction and decreased blaKPC gene expression. Damages in the Cas9, as well as alterations in carbapenem-resistance promoting genes including ompK36 downregulation and mutations in the acrB gene were found, likely preventing more efficient re-sensitization. Hence, interference with a single resistance gene promoted the emergence of clonal variants that exhibit alterations in outer membrane proteins. Those bacterial countermeasures, however, were not sufficient to restore the original carbapenem-resistant phenotype.

Authors 5

1. Thaysa Leite Tagliaferri corresponding Aachen Institute of Medical Microbiology

   RWTH Aachen University · Universitätsklinikum Aachen · Universidade Federal de Minas Gerais

   Affiliation as printed

   Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

   Institute of Medical Microbiology, RWTH Aachen University Hospital, Aachen, Germany

2. Alexander Krüttgen Aachen Laboratory Diagnostic Center

   RWTH Aachen University · Universitätsklinikum Aachen

   Affiliation as printed

   Laboratory Diagnostic Center, RWTH Aachen University Hospital, Aachen, Germany

3. Tiago Antônio de Oliveira Mendes

   Universidade Federal de Viçosa

   Affiliation as printed

   Department of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Viçosa, Brazil

4. Simone Gonçalves dos Santos

   Universidade Federal de Minas Gerais

   Affiliation as printed

   Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

5. Hans‐Peter Horz Aachen Institute of Medical Microbiology

   RWTH Aachen University · Universitätsklinikum Aachen

   Affiliation as printed

   Institute of Medical Microbiology, RWTH Aachen University Hospital, Aachen, Germany