Study On The Mechanisms Of Cross-resistance To Antibiotics In Staphylococcus Aureus Mediated By Rhizoma Coptidis And The Drug-resistance Reverse

Objective:The purpose of this study was focused on the mechanisms of cross-resistance to antibiotics in Staphylococcus aureus（SA）mediated by the Rhizoma Coptidis（RC）and its resistance reverse.This results obtained in this research will contribute to broadening the knowledge of the bacterial drug-resistance and particularly providing the basic theory for the development of the new-style antibacterial agents.Methods:1.The selected strains were cultivated in medium with the corresponding drugs at half-the minimum inhibitory concentration（MIC）for12 days.The change in susceptibility of these treated bacteria was determined and the culture medium was replaced according to MIC value every day.After that,the drug-exposed strains were subjected to the drug-free culture for 10days.The susceptibility change of the drug-exposed strains was determined by MIC analysis.2.The 16S rDNA sequencing method was used to identify the RCE-exposed strains.The sequenced results were blasted on the NCBI website for homology analysis.3.The expression of the resistant genes in the variants was analyzed by transcriptome sequencing.Differential expression analysis was calculated by EBSeq,an R/Bioconductor package.A gene was considered to be differentially expressed when the false discovery rate（Fdr）was less than 0.05and the Log₂fold change was greater than 1 or less than-1（Log₂fold change of>1 or Log₂fold change of<-1）.The m RNA levels of the resistant genes determined by RT-q PCR were consistent with those from the transcriptome analysis.4.The drug-resistant strains obtained in the experiment were used as research objects,and five antimicrobial traditional Chinese medicines（Rhizoma Coptidis,Scutellaria Baicalensis,Radix Paeoniae Rubra,Mint and Forsythia）were used for the induction of resistance reversal,in order to screen antimicrobial traditional Chinese medicines（TCMs）with reversal effect of drug-resistance.Then RT-q PCR was used to detect the gene expression of the treated strains.Results:1.After Rhizoma Coptidis extract（RCE）exposure,the decreased susceptibility to RCE（≥4-fold MIC increase）was observed in almost all tests of the treated isolates,while there was no obvious change in the susceptibility to CHX in all treated isolates.Additionally,the treated ATCC 25923 showed notable cross-resistance to TET,GEN and PIP with not less than 8-fold increase in MIC.However,the increased susceptibility to TET with an 8-fold decrease in MIC was observed in all three parallel tests of the treated SA cp.Notably,none of these selected strains was cross-resistant to CIP,FEP and MEM after RCE exposure.Thus,it was suggested that the cross-resistance selected by RCE targeted differently for TET,GEN and PIP.2.After exposure of TET,both the reference and clinical strains showed high frequency of antibiotic cross-resistance.The exposed ATCC 25923showed cross-resistance to TET（MIC increase by 8-128 fold）,GEN（8-16fold increased MIC）and PIP（8-32 fold increased MIC）.However,there was an emergency of increased susceptibility to AMK（MIC decreased by 4-8fold）.SA czx showed cross-resistance to TET（MIC increase by 128 fold）and GEN（4-16 fold increased MIC）.SA lqq decreased the susceptibility from resistance to TET to sensitivity,while its sensitivity to AMK decreased 16times.SA cp was resistant to GEN,and the sensitivity to TET and AMK was decreased 4-16 fold.3.ATCC 25923 and its RCE-exposed isolate were selected for 16S r DNA sequencing.By BLAST algorithm of the results,it was determined that the isolates could be identified as SA.Moreover,after assaying by NCBI-Blast,it was indicated that the homology between the sequenced strains and SA CP042008.1 as well as SA MK809240.1 was no less than99.51%.4.It was found that there were 433 differentially expressed genes,among these 261 genes were up-regulated and 172 genes were down-regulated.There were 13 differentially expressed genes related to antibiotic resistance,including smr B、bmr3、clp L、ica R and nor A and so on.5.The results of TCMs reversal experiment showed that Radix Paeoniae Rubra（RPR）had a reversal effect on TET and PIP resistance caused by RCE,and had a reversal effect on GEN resistance of clinical multi-drug resistant SA.6.The gene expression level was detected by RT-qPCR.The expression levels of nor A and fmt A-1,related genes of TET and PIP resistance,were decreased after the reversal of RPR.In addition,the expression levels of vra G and HMPREF0776-1664 were also decreased.These results suggested that genes vra G and HMPREF0776-1664 were related to regulatory factors of TET and PIP resistance.Conclusion:1.Both the sub-lethal doses of antibiotics and antimicrobial TCMs can mediate the cross-resistance of SA,suggesting that the action mechanism of antimicrobial TCMs and antibiotics may be same or similar to some extent.2.The reference strain ATCC 25923 selected by RCE was fostered the preferential cross-resistance to the specific antibiotics,including TET,PIP and GEN.3.Sub-lethal doses of RCE could induce the increased expression of ica R、smr B、his G、nho A and fmt A-1 in SA.4.The cross-resistant mechanisms of SA to TET,PIP and GEN after RCE selection were mainly due to the gene over-expression associated with efflux pumps and some modifying enzymes.5.RPR can reverse the drug-resistance caused by antimicrobial TCMs and clinical bacterial resistance to some extent..6.It was suggested that fmtA-1 was a potential drug target for PIP resistance reverse.The genes vra G and HMPREF0776-1664 may be regulatory genes associated with TET and PIP resistance.