Study On The Activity And Mechanism Of Benzimidazole Derivative TFBZ Against MRSA Biofilm

Infection caused by drug-resistant bacteria is one of the main threats to public health security in the world.During the competition between bacteria and antibiotics,bacteria gradually develop into multiple drug-resistant pathogens that can resist various antibiotics,among which methicillin-resistant is known as superbacteria Staphylococcus aureus(MRSA)can not only produce a variety of toxins and invasive enzymes,but also easily adhere to solid surfaces to form Biofilm,which can easily cause repeated persistence Large-scale infection has brought unprecedented difficulties to clinical treatment,so it is extremely urgent to find and develop effective drugs for MRSA biofilm treatment.Benzimidazole derivatives can be used as potential sources of antibacterial drugs due to their diverse substituents,varied structures and broad spectrum biological activitiesIn this study,MRSA ATCC43300 was used as the experimental strain to comprehensively and systematically analyze the inhibitory effect and mechanism of benzimidazole derivative TFBZ on MRSA biofilms from three levels of cell level,molecular level and animal level.(1)The drug sensitivity test model of MRSA was established,and the antibacterial and anti-biofilm activity of benzimidazole derivative TFBZ was measured to optimize the optimal compound TFBZ,which had excellent inhibition and clearance effect on MRSA in the floating state and the biofilm form(MIC=4 g/m L and MBC=8 μg/m L,minimum biofilm clearance concentration MBEC=8 g/m L)In this study,the optimal compound TFBZ will be used as the target compound to carry out subsequent experimental exploration(2)Cell level: The antibacterial activity of compound TFBZ was evaluated by measuring the time-growth curve,cell activity,adhesion and invasion of MRSA to host cells.The results showed that the compound had bacteriostatic and bactericidal effects.Crystal violet staining was used to evaluate the scavenging effect of the compound on MRSA mature biofilm,and it was found that the compound had significant scavenging activity(clearance rate =84.41%).Optical microscope imaging technology was used to characterize the scavenging effect of the compound on MRSA biofilm,and obvious morphological changes were observed.The antibiofilm activity of the compound was confirmed.The above results all proved that compound TFBZ could inhibit and clear MRSA biofilms.(3)Molecular level: 1)RNA-sequencing technology was used to analyze the regulation of compound TFBZ on the spatio-temporal specific expression of related genes during biofilm formation.(1)Enrichment of differential genes: the expression levels of 140 genes were changed: 63 genes were up-regulated and 77 genes were down-regulated.(2)KEGG pathway enrichment results show that the main pathway is:Ribosome pathway,Histidine metabolism pathway,RNA degradation pathway,Messenger RNA biogenesis pathway.(3)Transcriptome analysis results were verified by q RT-PCR,and it was confirmed that the compound could significantly downregulate genes related to cell surface proteins and agglutination factors(sdr C,clf B),,related to coenzyme synthesis and transport(moe A),related to biologic membrane formation(ica D,ica A),related to ribosomal protein synthesis(rpl N),related to protein synthesis(inf C),and related to cell wall organisms(mur Q,dlt B).2)By analyzing the results of molecular docking experiment,the main target protein was predicted to be cythioether lyase(CSE).Therefore,The predictive compound TFBZ inhibited and cleared MRSA biofilms by affecting multiple pathways(4)Animal level: Compound TFBZ was confirmed to be able to clear MRSA biofilm in vivo by establishing mouse pyomyositis model(biofilm model),evaluating basic signs(body weight,limb activity score,wound ulceration trend,etc.),observing pathological tissues,and detecting bacterial load.The results of this study can provide theoretical support for the development of the compound as a new antibacterial drug and broaden the development of antibiotics.