Suppression of Antibiotic Resistance In Methicillin-Resistant Clinical Staphylococcus aureus (MRSA) Isolates Using CRISPR/dCas9 Based Genome Editing

Project no. 21898 · Status: Kapanmış (Yayın bekleniyor) · 2020

Summary

In recent years, the inadequacy of potential therapeutic agents in the treatment of infectious diseases and the fact that the pharmaceutical industry has not favoured the production of new antibiotics for many reasons over the years have initiated new approaches for antimicrobial effect in the fight against the increasing resistance problem and the search for new drugs suitable for these approaches. The CRISPR/Cas system is an RNA-based nuclease complex that bacteria and archaea develop against foreign DNA such as plasmids or phages and destroy the foreign genome. The search for new therapeutics in the fight against antibiotic resistance has accelerated the research on CRISPR/Cas antimicrobials. The aim of our project is to target methicillin, gentamicin and ciprofloxacin resistance genes in methicillin-resistant clinical Staphylococcus aureus (Methicillin Resistant S. aureus - MRSA) strains, which is a serious threat, by using CRISPR/Cas9 system and to change the resistance profile of the bacteria by suppressing these genes and to make the bacteria sensitive to antibiotics. Within the scope of the project, antibiotic susceptibilities of S. aureus strains isolated in the Bacteriology Laboratory of Ege University Faculty of Medicine, Department of Medical Microbiology were determined by VITEK-2, an automated system. According to the antibiotic susceptibility results, the presence of mecA, grlA, grlB, aacA resistance genes in all strains that were determined to be methicillin resistant was demonstrated by conventional PCR. The study was continued with a representative isolate selected from the strains that showed clonal relationship in REP-PCR with RW3A primer. Considering the sequence data in the NCBI database, sgRNAs were designed for the resistance genes mentioned above. Target region-specific sgRNAs were transferred to the competent MRSA strain by electroporation. Colony PCR was performed on selected representatives of colonies grown in TSA with chloramphenicol after receiving the plasmid. After total RNA isolation from these colonies, cDNA synthesis was performed and expression ratio changes of mecA, grlA, grlB, aacA genes were analysed by RT-PCR. Depending on the suppression of resistance genes, changes in resistance status were shown by disc diffusion and broth microdilution. Protein concentration was measured by protein isolation from knock-out (KO)-mecA and wild type (WT) strains and the change in PBP2a expression was analysed by Western blotting. Genomic DNA was isolated from KO strains and 1000-1200 bp areas were amplified by PCR to include target regions and confirmed by Sanger sequence analysis. According to VITEK-2 results, all 20 clinical S. aureus isolates were found to be methicillin resistant. mecA, grlA, grlB genes were detected in all isolates, while aacA gene was detected by conventional PCR in all but three isolates. According to RT-PCR results, it was determined that mecA gene expression decreased 1.5-fold in mecA-KO isolate, grlA gene decreased 5.5-fold in grlA-KO isolate, grlB gene decreased 6-fold in grlB-KO isolate and aacA gene decreased 4-fold in aacA-KO isolate. When the results of phenotypic antibiotic susceptibility tests were evaluated, the suppression of mecA, grlA, grlB and aacA genes in the bacteria led to the breakdown of methicillin, quinolone and aminoglycoside resistance. As a result, the strains switched from resistant to susceptible category to the related antibiotics. When the change in PBP2a was analysed by Western blot, a 70% decrease in PBP2a expression was detected in the KO strain compared to WT. According to Sanger sequence analysis, point mutations were detected in grlB and aacA genes, while 3 base substitutions were observed in grlA and mecA genes. Our study is important in terms of showing that CRISPR antimicrobials can be used to eliminate resistance to different antibiotics in many bacteria. Our study also provides data that will form the basis for future studies that will examine the use of CRISPR antimicrobials and traditional antibiotics that cannot be used due to the development of resistance in combined therapy, phage cocktails containing CRISPR-Cas and their combination with nanopharmaceuticals.

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