Tagliaferri Thaysa Leite, Krüttgen Alex, Mendes Tiago Antônio de Oliveira, Gonçalves Dos Santos Simone, Horz Hans-Peter
Institute of Medical Microbiology, RWTH Aachen University Hospital, Aachen, Germany.
Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
PLoS One. 2025 Aug 12;20(8):e0328521. doi: 10.1371/journal.pone.0328521. eCollection 2025.
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.
CRISPR-Cas技术可用于使耐药基因失活。由于碳青霉烯耐药性可由多种耐药决定因素介导，我们在此研究了靶向blaKPC碳青霉烯酶基因时重新致敏的程度，并评估了对孔蛋白和外排泵的可能影响。虽然仅配备blaKPC的大肠杆菌实验室菌株实现了完全重新致敏，但在密歇根克雷伯菌的临床分离株中，63%的分析转化体实现了耐药性降低，这是质粒拷贝数减少和blaKPC基因表达降低的结果。发现Cas9受损，以及碳青霉烯耐药促进基因发生改变，包括ompK36下调和acrB基因突变，这可能阻止了更有效的重新致敏。因此，干扰单个耐药基因促进了在外膜蛋白中表现出改变的克隆变体的出现。然而，这些细菌对策不足以恢复原来的碳青霉烯耐药表型。
