| Objective: In this study,E.coli CP9,which is resistant to traditional Chinese medicine,was used as the donor bacteria and S.aureus RN450 RF was used as the recipient bacteria.The aim was to target the Mpf system that plays a decisive role in plasmid replication,conjugative transfer,and regulation(Research on junction-matching pair formation system)and Dtr system(DNA transfer and replication system).To explore the mechanism of E.coli resistance to traditional Chinese medicine,the mechanism of the effect of low-concentration Chinese medicine on the conjugation and transfer of plasmids under the condition of liquid phase conjugation,and to analyze the properties of the plasmids of resistance to traditional Chinese medicine in E.coli.Methods: This research is divided into four parts.1.Induction experiment of Chinese medicine resistance of Escherichia coli causing pyelonephritis: The molecular distillation technology was used to obtain small molecular substances in the water-soluble extracts of Chinese herbal medicines as experimental drugs.The clinically isolated E.coli CP9 was used as the experimental strain.Carry out in vitro antibacterial test to determine the minimum inhibitory concentration of E.coli CP9,and it was cultured and subcultured in M-H broth medium containing 1/2MIC drug to induce drug resistance.Pick a single colony to determine the MIC after drug induction.If the MIC after drug induction is more than 4times the MIC before induction,it will be regarded as drug resistance.2.Identification experiment of the properties of Chinese medicine resistant plasmids: E.coli CP9(after the induction of experiment one)was used as the donor bacteria,and the plasmid was extracted by the Alkaline lysis method.S.aureus RN450 RF and competent cell E.coli DH5α are used as recipient bacteria.Perform plasmid transformation competent cell experiments and plasmid conjugation and transfer experiments in vitro.The cultured transformant plasmids and transconjugant plasmids were extracted by alkaline lysis method,and the plasmids of drug-resistant bacteria were detected by agarose gel electrophoresis.3.Western blot method to determine the expression of the replication initiation protein encoded by the Trf A gene and the immunological activity experiment: The E.coli CP9 with resistance plasmid were used as the experimental strains,and the non-resistant E.coli CP9 was used as the control strain.The total bacterial proteome samples were prepared by the urea method,and the samples were denatured and then subjected to SDS-PAGE electrophoresis experiments.After the electrophoresis,transfer the protein bands on the separation gel to the NC membrane.Rabbit antiserum anti-Trf A was used as the primary antibody,goat anti-rabbit Ig G/HRP was used as the secondary antibody,and HRP-ECL chemiluminescence was used for immunoassay.4.PCR method to detect the expression of tra gene in Chinese medicine resistant plasmid: E.coli CP9,which is resistant to traditional Chinese medicine,was the standard for the experimental strain.E.coli J96 was used as the control strain,and the bacterial plasmid was extracted by alkaline lysis method.Using traditional Chinese medicine resistant plasmid DNA as a template,using Oligo software,10 pairs of primers were designed according to the sequence of the conserved region of the zinc finger protein of the tra gene.Prepare 50μL PCR reaction system and amplify on the set PCR machine.After the PCR is over,the PCR products are taken for electrophoresis detection.Results: 1.Using molecular distillation technology to obtain small molecular substances in water-soluble extracts of Chinese herbal medicines,as experimental drugs,equivalent to 0.5g crude drugs/ml.The physical and chemical properties of the experimental drug are: light brown,viscosity 505 c P,relative density 0.9886-0.9890,absorbance0.351-0.355,optical rotation +10.90,refraction 1.2066-1.2068,p H value 5.6-5.7.(Figure1-2/1-3/1-4/1-5).2.E.coli CP9 and S.aureus RN450 RF,which cause pyelonephritis,were isolated from clinical specimens of inpatients in the First Affiliated Hospital of Changchun University of Traditional Chinese Medicine,and were identified by an automatic bacterial identification instrument.Its biological characteristics: ⑴ E.coli CP9: β-hemolysis,O4/K54/H5 serotype,P pilus(class I Pap Gadhesin),aerobactin minus genotype;⑵ S.aureus RN450RF: RN450 derivation,phenotype α Phage-free,Rifr Fusr.3.Observe the in vitro antibacterial activity of "Wenyang Tonglin Decoction" against Pyelonephritis-causing E.coli in multiple groups,and compare the in vitro antibacterial activity of different extracts of "Wenyang Tonglin Decoction" against E.coli to determine the main effect of "Wenyang Tonglin Decoction" The ingredients are small molecules in the water-soluble extracts of Chinese herbal medicines(Figure 1-6).4.The minimum inhibitory concentration(MIC)experiment of the drug showed that the experiment can see obvious inhibition zone(Figure 1-7),the minimum inhibitory concentration of E.coli CP9 drug MIC1 is 50g/L,the zone of inhibition The diameter is2mm(Table 1-2).The minimum inhibitory concentration MIC2 of S.aureus RN450 RF is25g/L,and the diameter of the inhibition zone is 11 mm.The growth of clinical test bacteria and quality control bacteria,as well as the antibacterial status of E.coli and S.aureus,are consistent,and clinical strains do not show resistance to Chinese medicine preparations.5.Sub-inhibitory concentration(1/2 MIC)drug-induced drug resistance experiment showed that before the drug induction experiment,the MIC of E.coli CP9 drug was 50g/L,and the diameter of the inhibition zone was 2mm.After the drug induction experiment,the MIC of E.coli CP9 was 250g/L,and the area diameter was 4mm.After drug induction,MIC≥ 4 times the pre-MIC,so the secondary inhibitory concentration(1/2 MIC)can be determined.The drug can induce drug resistance in E.coli CP9,and E.coli CP9 is also determined to be resistant to traditional Chinese medicine.6.The experiment of plasmid transformation into competent cells showed that E.coli CP9(Experiment 1),which is resistant to Chinese medicine,was the donor bacteria,and the competent cells of E.coli DH5α were the recipient bacteria.The plasmid in E.coli CP9 was extracted by alkaline lysis.After the plasmid and competent cell E.coli DH5α were mated in the broth,the transformant was detected on the plate containing the Chinese herbal medicine mixture.All transformed zygotes were tested against serial dilutions of Chinese herbal medicine mixtures,and were found to be resistant to Chinese herbal medicine mixtures,with an MIC of 250g/L and an area diameter of 4mm.Agarose gel electrophoresis detection revealed that a plasmid DNA fragment of 45 kb in size was detected in E.coli CP9 resistant to Chinese medicine,but it did not exist in E.coli DH5α before mating.After mating,the transformed zygote of E.coli DH5α was observed to contain this fragment(Figure 2-1)and showed resistance to Chinese herbal medicine mixtures.Plasmid transformation experiments proved that plasmid fragments can be transformed from E.coli CP9 into E.coli DH5α competent cells.The presence of this plasmid increases the MIC of the herbal mixture of E.coli DH5α strain from 12.5g/L to 250 g /L.7.The results of plasmid conjugation and transfer experiments showed that E.coli CP9(Experiment 1)was the donor bacteria,and S.aureus RN450 RF was the recipient bacteria,which were cultured in L-B broth medium.After the ex vivo match,the transzygote was detected on the plate containing the Chinese herbal medicine mixture.In vitro antibacterial experiments show that the transzygote is resistant to Chinese herbal medicine mixtures,with an MIC of 125g/L and an area diameter of 3mm.The presence of this plasmid increased the MIC of S.aureus RN450 RF Chinese herbal medicine mixture from 25g/L to 125g/L.The transzygote was subcultured,and it was found that the plasmid persisted in the next generation of S.aureus,which still showed resistance to the mixture of Chinese medicines.In addition,after comparing the mass spectra of plasmids,it was found that the Chinese medicine-resistant E.coli and the transconjugants contained plasmids of the same size(45kb),which did not exist in S.aureus RN450 RF before mating(Figure 2-2).8.The results of western blot experiments showed that E.coli CP9(with resistant plasmid)was used as the experimental strain,and E.coli CP9(without resistance)was used as the control strain.Rabbit antiserum anti-Trf A was used as the primary antibody,and goat anti-rabbit Ig G/HRP antibody was used as the secondary antibody.Bright fluorescent bands(33k Da and 44 k Da specific protein bands)were observed in the E.coli CP9 samples with resistant plasmids,but not in the control samples(Figure 3-1).Western blot experiments showed that the Trf A-33 protein(33k Da)and Trf A-44 protein(44k Da)encoded by the trf-A gene were successfully expressed in E.coli CP9(with a resistant plasmid).9.The PCR results showed that E.coli CP9(with resistant plasmid)was used as the experimental strain,and E.coli J96 was used as the control strain.Eight clear bands were visible in the E.coli CP9 plasmid DNA sample extracted by alkaline lysis,but no amplified band was detected in the control sample(Figure 4-1).The size,fragment length and fluorescence intensity of the amplified products can be distinguished,including orf1(406bp),orf2(1035bp),orf4(433bp),orf5(359bp),orf6(322bp),orf7(326bp),orf8(932bp)and orf10(690bp),a total of 8 open reading frames.The results of PCR experiments show that the Chinese medicine resistant plasmid contains the tra gene for the formation of regulatory fimbriae and the formation of conjugative mating pairs.This not only provides the necessary prerequisites for the conjugative transfer of the plasmid,but is also a characteristic marker of conjugative plasmids.Conclusion: 1.Sub-inhibitory concentration drugs(1/2MIC)can induce drug resistance in Escherichia coli;2.The plasmid of E.coli CP9,which is resistant to Chinese medicine,carries the resistance gene,and the plasmid can be transferred between different species and strains through conjugative transfer and/or transformation;3.Chinese medicine drug-resistant plasmids contain Trf A genes encoding regulatory replication initiation proteins;4.Chinese medicine drug-resistant plasmids contain regulatory bacterial conjugative transfer and the transformed tra gene;5.The nature of the Chinese medicine-resistant E.coli plasmid is an autonomous conjugative plasmid.Summary:Plasmids extracted from E.coli CP9 resistant to Chinese medicine: carrying resistance genes,containing the necessary nucleotide sequences required for DNA replication(or origin of replication),and tra genes that control bacterial conjugation and transfer.Therefore,it can be concluded that the Chinese medicine resistant plasmid is a conjugative plasmid. |
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