Exploring The Mechanisms Of Berberine Against Staphylococcus Aureus

Staphylococcus aureus is a kind of common pathogen that causes purulent infections and nosocomial infections.The drug resistance variation and persisters formation have brought great difficulties to clinical treatment and are the important causes of recurrent infections and chronic infections.Currently,there are few drugs available for treatment.In particular,there are lacks of drugs for the treatment of persisters.Therefore,it is of great significance to actively seek effective drugs against S.aureus,especially for its persisters.Berberine is an alkaloid extracted from plants such as coptis chinensis and cypress of traditional Chinese medicine.It has extensive pharmacological activities such as antibacterial,but its antibacterial mechanisms have not been fully understood.In this study,the effects of berberine against S.aureus and its persisters were studied and its mechanisms were discussed.Objective:1.To explore the effects and influencing factors of berberine against S.aureus and its persisters.2.To study the mechanisms of berberine against S.aureus and its persisters.Methods:1.Norfloxacin?20 ?g/ml?,rifampicin?0.2 ?g/ml?,gentamicin?40 ?g/ml?and vancomycin?40 ?g/ml?in Tryptone Soy Broth?TSB?and Nutrient Broth?NB?were chosen for exposure experiments to S.aureus to determine the persisters' formation situation of the different growing period of S.aureus.2.The minuscule broth dilution method was adopted to determine of the minimum inhibitory concentration?MIC?and minimum bactericidal concentration?MBC?of berberine against S.aureus in NB.Ampicillin?10 ?g/ml?and berberine?250 ?g/ml?were used to conduct exposure experiments in TSB and NB respectively to determine the effects of berberine against S.aureus and its persisters.3 Stable phase bacteria which grew in TSB or NB were resuspended with TSB culture filtrate,normal saline,and NB culture filtrate.Ampicillin?10 ?g/ml?and berberine?250 ?g/ml?were chosen to carry out exposure experiment to determine of medium factors on berberine against S.aureus persisters.4.The filtrate of the used TSB and NB and the filtrate which were adjusted the pH were used to resuspend S.aureus which had formed a large amount of persisters.Ampicillin?10 ?g/ml?and berberine?250 ?g/ml?were chosen to carry out exposure experiment to determine the pH of culture medium on berberine against S.aureus persisters.5.Total RNA of S.aureus of cultivation for 24 h and cultivation for 24 h and then using berberine?250 ?g/ml?treated for 3h were extracted.Adopt the transcriptome sequencing determination of berberine to deal with the change of S.aureus genome transcription.RT-qPCR was used to verify the results,and bioinformatics was used to analyze of the transcriptome sequencing results to discuss mechanisms for berberine against S.aureus and its persisters.6.The mutant strain of S.aureus which were resistant to high concentration of berberine was induced.The genomic DNA of the mutant were extracted and resequenced.The resequencing results were verified by PCR,and bioinformatics were used to analyze its mutation sites and related genes.Results:1.In the TSB and NB,stability culture of S.aureus to lethal concentration of norfloxacin,rifampicin,gentamicin and vancomycin could produce tolerance,which showed that there were still a large number of viable bacteria after 7 days for drug treated,and the logarithmic phase of bacteria were completely killed after 2 days of exposure.Gentamycin could completely kill the stable phase S.aureus after 4 days exposure in NB.2.The range of MIC and MBC of berberine in NB against S.aureus were 62.5 ?g/mL¹25?g/mL and 125?g/mL ²50?g/mL respectively.Two folds MIC of berberine could kill all stable phase S.aureus after be treated for 24 h.The drugs in the same conditions of TSB did not produce significant antimicrobial activity and there were still a large number of viable bacteria?8.19±0.13?after treated for 7 days.In TSB and NB,stability of S.aureus culture of S.aureus could produce tolerance to 10 folds MIC of ampicillin,and there were still a large number of viablebactera?3.82 ±0.24 and 7.12±0.16?after 7 days treatment..3.TSB and NB stable phase cultures which resuspended with NB filtrate were highly sensitive to berberine,and all bacteria were killed within 24 h.The precipitates of the above cultured substances which resuspended by TSB filtrate were resistant to ampicillin and berberine,and the viable cell numbers were 9.06±0.18 and 8.98±0.03 respectively when drugs treated for 7 days.With normal saline and NB filtrate resuspended deposits of the cultures in a slightly higher sensitivity to the role of ampicillin and the viable cells were 6.69±0.13 and 6.39±0.40 after 7 days exposure.The sensitivity of resuspension cultures of normal saline to berberine was higher than that of TSB filtrate,and all bacteria was killed within 72 h.4.At a pH of 6.50,berberine showed against stably phase S.aureus effect in TSB and NB,and all bacteria were killed within 24 h.Conversely,at a pH of 4.65,berberine lost its against stably phase S.aureus effect.Ampicillin had certain against stably phase S.aureus effect at pH 6.50,but its effect was significantly lower than that of berberine,and it lost its antibacterial effect at pH4.65.5.The sequencing results of the transcriptome showed that,comparing with the untreated group,25 genes of S.aureus in the berberine treatment group were significantly up-regulated?NWMN_tRNA26,NWMN₀907,NWMN_tRNA41,NWMN_tRNA32,NWMN_tRNA31,NWMN_tRNA53,NWMN₁874,NWMN_tRNA22,NWMN_tRNA33,NWMN₁861,NWMN_tRNA28,NWMN_tRNA29,araC,NWMN_tRNA27,NWMN_tRNA54,NWMN_tRNA45,NWMN₁068,NWMN_tRNA55,NWMN₁556,NWMN_tRNA24,NWMN₀869,NWMN_tRNA23,NWMN_tRNA18,NWMN_tRNA16,NWMN₀678?.There were 18 genes whose expression was significantly down-regulated?hisG,NWMN_rRNA13,NWMN_tRNA49,NWMN_rRNA01,NWMN_rRNA16,NWMN_rRNA05,NWMN_rRNA10,NWMN_rRNA15,NWMN_rRNA12,NWMN_rRNA02,NWMN_rRNA06,NWMN_rRNA09,hisZ,NWMN_tRNA11,NWMN_tRNA47,hisD,ilvB,NWMN₀387?.RT-qPCR was performed on 9 genes,and the results of transcriptome sequencing were completely correct.After bioinformatics analysis,differentially expressed genes were mainly focused on signaling pathways and pathways such as RNA metabolism and histidine biosynthesis.6.After drug induction,the mutant strain of S.aureus resistant to 5 folds MIC of berberine was obtained.The mutant strains of genomic DNA resequencing results showed that single nucleotide polymorphism?SNP?loci were 42,and meaningful small fragments sequence of insertions and deletions?insertion and deletion,InDel?loci were 11.These mutations genes were concentrated distribut in membrane proteins,ion transport,drug efflux pump,cell division and DNA damage repair,RNA metabolism,lipid metabolism,glucose metabolism,amino acid metabolism,and many other aspects.The sequencing results of the PCR amplification fragment were compared with the relevant genes of the mutant strain,and the results showed that the mutation sites information obtained by genome sequencing were accurate.Conclusions:S.aureus could form a large number of persisters at a stable phase after culture.Berberine had strong effects against S.aureus and its persisters in the neutral pH range.The signal pathways of inhibiting histidine biosynthesis,affecting DNA damage repair,and inhibiting RNA metabolism,interfering with the metabolism of bacteria,the transport of substances and the division of bacteria maybe include in the mechanisms of berberine against S.aureus.