| Staphylococcus aureus,a food borne pathogen,not only seriously endanger safety of food,but also has serious harm in animal production.Staphylococcus aureus often cause purulent infection in humans and animals in clinical medicine and veterinary medicine.Since methicillin-resistant Staphylococcus aureus been firstly isolated in 1961,the rate of clinical detection has rising.The infection caused by MRSA has become a global health problem.It is one of the important research directions to find effective antibacterial substances from natural drugs in recent years.Gueldenstaedtia delavayi Franch is special medicinal resources in the west of Sichuan,it is mainly used to cure exogenous disease.Gueldenstaedtia delavayi Franch has been used in folk for a long time,so far,there are few reports on the study of Gueldenstaedtia delavayi Franch at home and abroad.This study intended to extract the different fraction of Gueldenstaedtia delavayi Franch and screening the effective fraction inhibiting Staphylococcus aureus.On the basis of it,the antibacterial mechanism were researched.The results and final conclusions are as follows: 1.Study on the antibacterial activity of different fraction of Gueldenstaedtia delavayi Franch on Staphylococcus aureusThe petroleum ether,ethyl acetate,n-butanol were used to extract different fraction from Gueldenstaedtia delavayi Franch.The MIC and MBC of the different fraction from Gueldenstaedtia delavayi Franch against the Staphylococcus aureus ATCC25923 and the strain isolated from clinical RC-1?RC-3?RC-5?DZ-1were determined by broth microdilution method.The results showed that there was no significant effect on the growth of Staphylococcus aureus in n-butanol fraction and aqueous phase at the experimental concentration.The Petroleum ether extract and ethyl acetate extract can inhibit growth of Staphylococcus aureus ATCC25923 and the strain isolated from clinical.The minimum inhibitory concentration(MIC)were 15 mg·mL-1 and 7.5 mg·mL-1,respectively.The minimum bactericidal concentration(MBC)were 30 mg·mL-1 and 15 mg·mL-1,respectively.The result indicated the ethyl acetate extract from Gueldenstaedtia delavayi Franch showed better antibacterial activity.The growth curve of Staphylococcus aureus was determined by counting the colony count of Staphylococcus aureus at different time points under different concentrations of ethyl acetate extract from Gueldenstaedtia delavayi Franch.The results show that the bacteria concentration of 1 MIC group was significantly lower than that of control group and 1/4 MIC group at 2,4,6,10,24 h(P<0.01),also being significantly lower than the 1/2 MIC group at 4,6,10,24 h(P<0.01).The bacteria concentration of 1/2 MIC group was significantly lower than that of control group at 2,4,6,10 h(P<0.01)and was significantly lower than that of 1/4 MIC group at 2,4,6 h(P<0.01).The bacteria concentration of 1/4 MIC group was significantly lower than that of control group at 2,4,10 h(P<0.01).The results suggested that petroleum ether extract and ethyl acetate extract from Gueldenstaedtia delavayi Franch can inhibit the growth of MRSA and MSSA,and high concentration showed bactericidal effect.Compared with the petroleum ether extract,the ethyl acetate extract showed better antibacterial activity,and the inhibitory effect was positively related with the concentration of drug.2.Study on the antibacterial mechanism of ethyl acetate extract from Gueldenstaedtia delavayi Franch on Staphylococcus aureusThrough the determination of Staphylococcus aureus extracellular alkaline phosphatase(AKP)content,medium conductivity,glucose content changes,enzymes activity related sugar metabolism and soluble protein content,the mechanism of ethyl acetate extract from Gueldenstaedtia delavayi Franch against Staphylococcus aureus were studyed on cell wall and membrane permeability,energy metabolism and soluble protein synthesis.The results showed that the Staphylococcus aureus culture fluid conductivity didn't change under MBC concentration of ethyl acetate extract from Gueldenstaedtia delavayi Franch.The extracellular alkaline phosphatase activity was significantly higher than the control group at 1 h and 3 h(P<0.05),there were no significant difference in other time points.The results indicate the ethyl acetate extract from Gueldenstaedtia delavayi Franch didn't influence membrane permeability,but can increase cell wall permeability of Staphylococcus aureus.The determination results of glucose content showed that the glucose content of 1MIC,1/2 MIC and 1/4 MIC group were significantly higher than that of the control group at 4 h,6 h,8 h(P<0.01).The glucose of 0 MIC group were depleted by Staphylococcus aureus in 6 h,the glucose of 1/2 MIC and 1/4 MIC group were depleted by Staphylococcus aureus in 10 h,however,culture fluid of 1 MIC still contained glucose after 10 h.The concentration of 1 MIC,1/2 MIC and 1/4 MIC of the ethyl acetate extract from Gueldenstaedtia delavayi Franch can significantly inhibit the utilization of glucose by Staphylococcus aureus.The detection results of enzymes activity related sugar metabolism showed that the succinate dehydrogenase(SDH)and lactate dehydrogenase(LDH)activity of 1MIC group were generally lower than that of 0 MIC group.The LDH activity of 1 MIC group was significantly lower than that of 0 MIC group(P<0.01).The SDH activity was significantly lower than that of 0 MIC group at 1h and 3 h(P<0.01).The results indicated the ethyl acetate extract from Gueldenstaedtia delavayi Franch can significantly inhibit SDH and LDH activity.The results of detecting the content of soluble protein of Staphylococcus aureus showed that the content of soluble protein of 1 MIC group was significantly lower than the control group(P<0.01).There were extremely significant difference at 3 h and 5 h(p<0.01),significant difference at 7 h(p<0.05)between 1 MIC group and control group.The concentration of soluble protein of Staphylococcus aureus reduced by 42.54% at 3 h,reduced by 17.78% at 5 h,reduced by 15.87% at 7 h.The results suggested that the ethyl acetate extract from Gueldenstaedtia delavayi Franch can affect the cell wall permeability in early drug action and interference energy metabolism and protein synthesis process of Staphylococcus aureus.3.Global transcriptional profiles of Staphylococcus aureus treated with ethyl acetate extract from Gueldenstaedtia delavayi FranchIn this study,the RNA-sep high throughput sequencing technology was used to compare the transcripts of Staphylococcus aureus from the 1MIC of ethyl acetate extract from Gueldenstaedtia delavayi Franch group and control group.The sequencing results showed that,a total of 195 genes were identified to be differentially regulated by ethyl acetate extract from Gueldenstaedtia delavayi Franch(FC = 2,P<0.05),101 of which showed a significant increase in transcription and 94 of which showed a significant decrease in transcription.Compared with the control group,the genes related protein synthesis from 1MIC group differently expressed.The expression of genes of 50 S ribosomal protein L27,50 S ribosomal protein L25,50 S ribosomal protein L15,ribosomal subunit RNA methyltransferase,elongation factor G was down regulated(P<0.05).Many genes related amino acid biosynthesis differently expressed.The genes of branched chain amino acid aminotransferase,aspartate kinase and N-succinyltransferase were up regulated.The gene of diaminopimelate decarboxylase was down regulated.Additional,the genes of folylpolyglutamate synthase,glycine ligase,deoxy guanosine kinase,hypoxanthine phosphoribosyl transferase,pyrimidine nucleoside phosphorylase were down regulated.The genes of fructose-bisphosphate aldolase and L-iditol-2-dehydrogenase were down regulated.The genes encoding various ABC transporter permease were upregulated,including spermidine/putrescine ABC transport protein,oligopeptide ABC transport protein,iron complex transporter,iron/manganese/zinc/copper transport protein,nickel transport protein,phosphoric acid transport protein.Additional,the gene encoding ferritin was upregulated.The genes related peptidoglycan synthesis,such as penicillin binding protein 1A,UDP-N-acetylmuramate dehydrogenase,transglycosylase domain-containing protein were upregulated.However,the genes expression of multiple virulence factors were decreased,including exfoliative toxin A,clumping factor,pantonvalentine leucocidin and icaR?icaC.10 genes from the differentially expressed genes were verified by RT-PCR.The result suggested that there was a good correlation between RNA-Seq data and real time RT-PCR data.In short,the differentially expressed genes of Staphylococcus aureus treated by the ethyl acetate extract from Gueldenstaedtia delavayi Franch after 1h were mainly concentrated in the metabolic process,catalytic activity,cellular process,binding and virulence factor expression.The result suggested the ethyl acetate extract from Gueldenstaedtia delavayi Franch can down-regulate the genes related virulence factors,influence the expression of genes encoding enzymes in amino acid metabolism,glucose metabolism,purine metabolism and pyrimidine metabolism,up-regulate the genes related transporter,and may interfere with the synthesis of protein by down regulation of ribosomal genes and elongation factor G of Staphylococcus aureus.In conclusion,this study showed that the ethyl acetate extract from Gueldenstaedtia delavayi Franch had good antibacterial activity against Staphylococcus aureus.The ethyl acetate extract from Gueldenstaedtia delavayi Franch can interference energy metabolism and protein synthesis process of Staphylococcus aureus.The differentially expressed genes of Staphylococcus aureus treated by the ethyl acetate extract from Gueldenstaedtia delavayi Franch after 1h were mainly concentrated in the metabolic process,catalytic activity,cellular process,binding and virulence factor expression.The antibacterial mechanism of Gueldenstaedtia delavayi Franch against Staphylococcus aureus maybe be related to the genes differentially expressed. |
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