| Objective To investigate the variation of ?-lactam antibiotic sensitivity of clinical isolates of Escherichia coli,which are resistant to ?-lactam antibiotics,after the action of Iris Japonica aqueous extracts and ethanol extracts.And to explore the mechanism preliminary,to provide an experimental basis for the development of new anti-drug-resistant bacteria.Methods ? Boiling methods and Ethanol reflux extractions were used to extract tectoridin of Iris Japonica respectively.Macroporous resin technology were applied to purify crude Iris japonica extracts.and then to prepare Iris Japonica aqueous extracts and ethanol extracts samples.The color reaction,ammonia water reaction,aluminum trichloride reaction,hydrochloric acid-magnesium reaction were used by Iris japonica aqueous extracts or ethanol extracts to qualitative flavonoids,in tectoridin standard as a control,UV spectrophotometry methods were used to determine the content of tectoridin.?E-test methods were applied to measure minimal inhibitory concentration(MIC)of 183 Escherichia coli to penicillin G and cefotaxime sodium.PCR was used to detect the major ESBLs genes(TEM?CTX-M?SHV and KPC)of Escherichia coli.?Microtube dilution methods were applied to measure minimal inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of Iris japonica extracts to 183 Escherichia coli..Microtube dilution methods were applied to explore sensitive variation to penicilin G and cefotaxime sodium of Escherichia coli E001(TEM+),E002(CTX-M+),E003(KPC+),E004(SHV+),E005(TEM+),E006(CTX-M+),E007(CTX-M+),E008(TEM+),E009(SHV+)and E010(TEM+),which were resistant to ?-lactam antibiotics and containing only one single ?-lactamase gene,after the action 0.5 h of Iris Japonica aqueous extracts(final concentration were 250 mg/mL,125 mg/mL and 62.5 mg/mL)and ethanol extracts(final concentration were 100?g/mL,50 ?g/mL and 25 ?g/mL),which were in subinhibitory concentrations.?Real-time fluorescent quantitation RT-PCR were performed to determine the inducing effects of 1/4 MIC penicillin G and cefotaxime sodium to Escherichia coli TEM,SHV,CTX-M and KPC single gene expression,and the interrupting effect of Iris japonica extracts.Results ?Flavonoids qualitative analysis to Iris Japonica aqueous extracts and ethanol extracts samples shows,the results of color reaction,ammonia water reaction,aluminum trichloride reaction,hydrochloric acid-magnesium reaction were positive.Full wavelength scanning get a result that tectoridin had a maximum absorption peak at 266.5 nm wavelength.In tectoridin standard as a control,In 266.5 nm,UV spectrophotometry quantitatively measured that the content of tectoridin in aqueous extracts,ethanol extracts respectively was 11.12%and 36.75%.?E-test shows that the MIC of penicillin G and cefotaxime sodium to 183 clinical isolates of Escherichia coli resistant to ?-lactam antibiotics respectively were>32 and?16 ?g/mL.these results concord with the mensurations of hospitals which used by microtube dilution methods.PCR results showed that TEM,CTX-M and/or SHV genes all can be detected in 183 Escherichia coli to ?-lactam antibiotics.TEM gene had the highest detection rate,(83.1%,152/183),then CTX-M(77.1%,141/183),SHV(8.2%,15/183),KPC had the lowest detection rate(2.2%,4/183),as a result,TEM and CTX-M gene detection rate was significantly higher than SHV and KPC gene.(31.7%,58/183)strains carried KPC,TEM,CTX-M or SHV single gene,(68.3%,125/183)strains carried two or more ESBLs genes among the total,Therefore,the detection rate of strains who carried two or more ESBLs genes was significantly higher than the detection rate of strains who carried only one gene.(92.0%,115/125)strains carried TEM and CTX-M genes(TEM+CTX-M)among the total,was significantly higher than the number of strains who carried TEM+SHV(4.8%,6/125)or TEM+CTX-M+SHV(3.2%,4/125).Given the mechanism of gene expression of single-gene strains contrast to multi-gene strains is relatively simple,clear.Therefore,in this text,we selected E001(TEM+),E002(CTX-M+),E003(KPC+)……E010(TEM+),ten single-gene Escherichia coli strains to investigate the variation of ?-lactam antibiotic sensitivity of this ten strains,after the action of Iris Japonica aqueous extracts and ethanol extracts.At the same time,we selected E001(TEM+),E002(CTX-M+),E003(KPC+)and E004(SHV+),four strains to detecte the effect of Iris Japonica extracts to expression of ESBLs single resistance gene.?The bactericidal results of Iris japonica aqueous extracts,ethanol extracts to 183 Escherichia coli,respectively in Microtube dilution methods were MIC?500 mg/mL and?200 ?g/mL,MBC?1000 mg/mL and>400 ug/mL.To provide an experimental basis for determining the working concentration of Iris japonica aqueous extracts(250 mg/mL,125 mg/mL and 62.5 mg/mL)and ethanol extracts(100 ?/mL,50 ?g/mL and 25 ?g/mL),as well as exploring the variation of ?-lactam antibiotic sensitivity for Iris japonica extracts to Escherichia coli,and the drug resistance gene expression.?We can get that in E001(TEM4),E002(CTX-M+),E003(KPC+)......E010(TEM+),only E003(KPC+)invariant resistant to penicillin G and cefotaxime sodium,the remaining 9 isolates are resistant to sensitive.Such as the MICs of penicillin G to E001,E004,E006 and E009 from the original 32?g/mL to 8?g/mL.to E002 and E005 from the original 32 ?g/mL to 4 ?g/mL;to E007 and E010 from the original 64 ?g/mL to 16?g/mL,while to E008,the MIC from the original 128 ?g/mL changed to 16 ?g/mL.Demonstrating that Iris japonica aqueous extracts(250 mg/mL),ethanol extracts(100 ?g/mL)can enhance the susceptibility of the resistant Escherichia coli to penicillin G and cefotaxime sodium.while,Iris japonica aqueous extracts(125 mg/mL)and Iris japonica ethanol extracts(50 ?g/mL)can only enhance the susceptibility of one or two resistant Escherichia coli to penicillin G but they can enhance the susceptibility of five resistant Escherichia coli to cefotaxime sodium.Nevertheless,Iris japonica aqueous extracts(62.5 mg/mL),ethanol extracts(25 ?g/mL)had hardly any effect on resistant Escherichia coli to penicillin G or cefotaxime sodium.?With 16S RNA as a reference,compared with the blank control group,1/4 MIC penicillin or cefotaxime could induce more than 10 times elevation of TEM-,SHV-and CTX-M-mRNAs,showing that 1/4 MIC antibiotics could induce elevation of the gene expression.While,Iris japonica aqueous extracts(250 mg/mL)and ethanol extracts(100 ?g/mL or 50 ?g/mL)could decreased more than 40%of gene expression,demonstrating that Iris japonica extracts in these concentrations could interrupt the antibiotics-induced elevation of ESBLs-mRNAs.Conclusion The MICs of Iris japonica aqueous extracts and ethanol extracts to E.coli were?500 mg/mL and ?200 pg/mL respectly.We choose 250 mg/mL?125 mg/mL ? 62.5 mg/mL as the working concentration of Iris japonica aqueous extracts,100 ?g/mL,50 ?g/mL and 25?g/mL as the working concentration of Iris japonica ethanol extracts,they were lower than bacteriostasis concentration.In the reseach of susceptibility enhancement of the resistant Escherichia coli to penicillin G and cefotaxime sodium,The results showed that Iris japonica aqueous extracts(250 mg/mL)and ethanol extracts(100 ?g/mL or 50 ?g/mL)can enhance the susceptibility of the resistant Escherichia coli to penicillin G or cefotaxime sodium.In the reseach of measureing the single gene expression,We found that these samples can interrupt 1/4 MIC penicillin G or cefotaxime sodium induce elevation of TEM-,SHV-and CTX-M-mRNAs.Therefore,we get a conclusion that Iris japonica extracts can enhance the susceptibility of the resistant Escherichia coli to penicillin G and cefotaxime sodium,may by interrupting the expression of TEM,SHV and CTX-M single gene in E.coli isolates,which induced by penicillin G or cefotaxime sodium. |
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