The Effect And Mechanism Of Novel Coumarins Derivatives Against Persisters Of Staphylococcus Aureus

ObjectivePersisters are a special drug-resistant subgroup of bacteria,which is an important cause of infection recurrence,chronic refractory infection treatment failure and biofilm formation.Biofilm is a kind of special existence form with a strong resistance and immune escape.It was reported that bacteria in biofilm exhibited high persistent level and was not easy to be cleared by antimicrobial drugs.Currently,most of the antibiotics in clinic are effective to the proliferative bacteria,but ineffective to the persisters,which brings great difficulties and challenges to clinical treatment.In order to select potential therapeutic drugs and provide new strategies for bacterial persisters-and biofilm-associated infections,this study analyzed the effects of nine novel coumarins derivatives anti-bacterial persisters and anti-biofilm formation against S.aureus in vitro and in vivo,and explored the possible underlying mechanism as well.MethodIn this study,we evaluated the effect of 9 novel coumarins on the LAC(USA 300).First of all,we used antibiotic pressure test to evaluate the activity of the nine novel coumarins derivatives against LAC persisters.Next,we used antibiotic pressure test to evaluate the anti-bacterial persisters activity of the most potent compound in different growth stages and different media and also used crystal violet staining,scanning electron microscopy,fluorescence microscopy to evaluate its anti-biofilm activity.Then,the anti-bacterial persisters activity and anti-biofilm activity of it in vivo was evaluated by the neutropenic mouse thigh infection model and the subcutaneous tissue cage biofilm infection mouse model,respectively.Finally,Trascriptome sequencing(RNA-seq)was applied to analyze the difference in the gene expression of LAC persisters after the compound treatment,and real-time PCR was used to verify the transcriptome results.Result1.Among the nine novel coumarins derivatives,the compound HNMH exhibited the most potent activity against LAC persisters.2.In TSB medium,both the logarithmic and stable phase LAC persisters were sensitive to the compound HNMH,and in NB medium,the logarithmic phase LAC persisters were sensitive to compound HNMH,while the stable phase LAC persisters were resistant to compound HNMH.3.Compound HNMH inhibited the LAC biofilm formation with a dose-dependent manner in vitro.4.The compound HNMH significantly decreased LAC persisters in the neutropenic mouse thigh infection model,and inhibited the biofilm formation in LAC induced subcutaneous tissue cage biofilm infection mouse model in a dose-dependent manner.5.Trascriptome sequencing(RNA-seq)showed that 2629 genes were detected in LAC persisters,among which 1447 genes were expressed different significantly after compound HNMH treatment comparing to the control,including 713 up-regulated genes and 734 down-regulated genes.Using the Padj<0.05 as the threshold to screen the KEGG functional enrichment in LAC persisters,and there were three signaling pathways significantly down-regulated,including purine synthesis,peptidoglycan synthesis and protein efflux.6.The compound HNMH inhibited the expression of gmk,pyrH,pyrB,pyrG,cmk in LAC persisters,this result was consistent with RNA-seq.Conclusion1.Compound HNMH exhibited anti-LAC persisters activity in TSB medium.2.The compound HNMH inhibited the LAC biofilm formation in a dose-dependent manner.3.The compound HNMH had both anti-bacterial persisters activity and anti-biofilm activity on LAC in vivo.4.The compound HNMH maybe inhibite LAC persisters formation by blocking purine synthesis,peptidoglycan synthesis or protein efflux.