International microbiology : the official journal of the Spanish Society for Microbiology · 2026
Sensitizing carbapenem-resistant Klebsiella pneumoniae using a compact Cascade-Cas3 system.
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Huang Jun, Chen Jiahui, Huang Luyao, Qiu Shaofu, Wang Ligui, Du Xinying, Wang Chao, Yuan Zhengquan, Li Ping, Song Hongbin, Liu Hongbo
Аннотация
BACKGROUND: Klebsiella pneumoniae is a 'superbug' characterized by high virulence and carbapenem resistance. CRISPR-Cas3 is capable of performing large-scale deletions in bacterial genomes. The purpose of this study is to sensitize Klebsiella pneumoniae A324 to carbapenem antibiotics by eliminating the multidrug resistance plasmid pA324-IMP using a compact Cascade-Cas3 system.
METHODS: Recombinant plasmids were constructed using molecular cloning techniques. Three sgRNAs targeting different positions within the blaIMP-38 gene were designed. Recombinant plasmids were separately transformed into host bacteria carrying blaIMP38 genes. The elimination efficiency of each CRISPR-Cas3 system target was determined by PCR and qPCR. Fluorescence confocal microscopy was used to observe the fluorescence intensity. Bacterial drug susceptibility phenotypes were determined using E-test drug sensitivity strips.
RESULTS: In this study, we successfully constructed the high copy number plasmid, the fluorescent plasmid, and the recombinant CRISPR plasmids. PCR and qPCR results demonstrated that the CRISPR-Cas3 system could efficiently eliminate blaIMP-38 genes, with pCas3-T3 showing the highest elimination efficiency. Fluorescence confocal microscopy indicated that the fluorescence intensity of the experimental group significantly decreased compared to the control group. Additionally, drug susceptibility tests revealed that the resistance of Klebsiella pneumoniae to imipenem and meropenem was significantly reduced. Furthermore, CRISPR-Cas3 could enhance the effectiveness of meropenem in killing Klebsiella pneumoniae A324.
CONCLUSION: In conclusion, this work demonstrates that the compact Cascade-Cas3 system can serve as a novel tool to eliminate the resistance gene blaIMP-38 and restore the sensitivity of host bacteria to antibiotics. This strategy shows great potential to combat the spread of the blaIMP-38 gene in Klebsiella pneumoniae and holds significant application value in the field of combating drug-resistant bacteria.
METHODS: Recombinant plasmids were constructed using molecular cloning techniques. Three sgRNAs targeting different positions within the blaIMP-38 gene were designed. Recombinant plasmids were separately transformed into host bacteria carrying blaIMP38 genes. The elimination efficiency of each CRISPR-Cas3 system target was determined by PCR and qPCR. Fluorescence confocal microscopy was used to observe the fluorescence intensity. Bacterial drug susceptibility phenotypes were determined using E-test drug sensitivity strips.
RESULTS: In this study, we successfully constructed the high copy number plasmid, the fluorescent plasmid, and the recombinant CRISPR plasmids. PCR and qPCR results demonstrated that the CRISPR-Cas3 system could efficiently eliminate blaIMP-38 genes, with pCas3-T3 showing the highest elimination efficiency. Fluorescence confocal microscopy indicated that the fluorescence intensity of the experimental group significantly decreased compared to the control group. Additionally, drug susceptibility tests revealed that the resistance of Klebsiella pneumoniae to imipenem and meropenem was significantly reduced. Furthermore, CRISPR-Cas3 could enhance the effectiveness of meropenem in killing Klebsiella pneumoniae A324.
CONCLUSION: In conclusion, this work demonstrates that the compact Cascade-Cas3 system can serve as a novel tool to eliminate the resistance gene blaIMP-38 and restore the sensitivity of host bacteria to antibiotics. This strategy shows great potential to combat the spread of the blaIMP-38 gene in Klebsiella pneumoniae and holds significant application value in the field of combating drug-resistant bacteria.