OBJECTIVE To restore the bactericidal efficacy of antimicrobial drugs by removing the multi-drug resistance genes IMP-4 and KPC-2 using the CRISPR/Cas9 system.

METHODS The CRISPR/Cas9 plasmid was constructed using molecular cloning method. The competent cells of drug-resistant bacteria were prepared, and the CRISPR/Cas9 system was delivered to the drug-resistant bacteria by plasmid transformation. Bacterial colony polymerase chain reaction (PCR) was used to detect the presence of drug resistance genes. The clearance efficiency of each CRISPR/Cas9 target for drug resistance genes was determined by real-time fluorescence quantitative PCR (RT-qPCR). Drug sensitive phenotypes of bacteria were determined by Sensititre drug sensitive plate and E-test drug sensitive strip.

RESULTS The CRISPR/Cas9 plasmids carrying the target spacer was constructed for IMP-4 and KPC-2. The results of colony counting showed that the drug-resistant genes were eradicated by CRISPR/Cas9 system, and the clearance rate was 100.00% (5/5). The relative quantitative analysis of bacterial drug resistance genes showed that the clearance efficiency of each target site reached 99.9% within 24 hours. Drug sensitivity test results showed that the bacteria in the experimental group restored their sensitivity to antibiotics, while the bacteria in the control group remained resistant to antibiotics. In the E-test strip experiment, the minimum inhibitory concentration (MIC) of piperacillin in the experimental group was reduced by about 204.8 times compared with the control group (P＜0.05), which restored the bacterial susceptibility to the antimicrobial drug. The CRISPR/Cas9 system was able to block bacterial acquisition of drug-resistant plasmids through the transformation pathway, and the efficiency of blockage was as high as 99.9% (P＜0.05).

CONCLUSION The rapid removal of bacterial drug-resistant genes IMP-4 and KPC-2 to restore the bacterial efficacy of antimicrobial drugs using the CRISPR/Cas9 system demonstrated great clinical application value.