Study On Hazard Factors Confirmation Of Streptomycin Dregs And Resistance Mechanisms Induced By The Factors In Bacteria

Streptomycin dregs is a kind of industrial waste produced in the process of biological fermentation.Streptomycin dregs contains a certain concentration of crude protein and residual streptomycin.However,the streptomycin dregs contain some unknown secondary metabolites.So there are food safety risks and risks of bacterial resistance to feed animals by streptomycin dregs.Now,there is no analysis of hazard factors of Streptomycin dregs and risk assessments of drug dregs in China and abroad.And there is no confirmatory analysis methods of streptomycin dregs in feed.In order to contribute to the realization of an effective"post-mortem supervision"and"early-warning",In the research,it is including identification of hazard factors of Streptomycin dregs,analytic procedures for the determination of streptomycin dregs in feeds,analysis of antibiotic resistance induced by harmful factors and induced resistance mechanism.Followings are specific research contents and results.1.Identification of hazard factors of Streptomycin dregs.The aims of the study are to screen the streptomycin and its bio-synthetic intermediates as hazard factors?25?,using three kinds of solvents?high water phase,high organic phase and low pH?to extract the metabolites from drug dregs,two chromatographic patterns?RPLC,HILIC?retention and separation,positive and negative?ESI±?ionization,and high resolution mass spectrum detected in the m/z:50-1200Da range.It is found that 2kinds of compounds contained in the dregs including streptidine and streptomycin.According to hazard analysis,there are cumulative toxicities of animal foods and risks of bacterial resistance in streptidine and streptomycin.So it is identified as the hazard factors of Streptomycin dregs.2.Analytical approaches for the determination of streptomycin residues in feeds are established by using the characteristic markers of streptidine and streptomycin.The 20%TCA solution is used as the extraction solvent.Na₂EDTA is used to eliminate the metal ion interference in matrix.Purification and enrichment of target compounds by weak cation exchange solid phase extraction?WCX?.Two featured markers are detected by ultra-high performance liquid chromatography tandem mass spectrometry?UPLC-MS/MS?positive ion?ESI+?multiple reaction monitoring?MRM?mode.The method is accurate?Streptomycin:71%-78%,Streptidine:75%-84%?,Precision?5.4%-9.6%?,linear range?in the range of 50-1000 g/kg R²?0.99?,LODs?20?g/kg?,LOQs?50?g/kg?and other performance indicators meet the requirements of streptomycin dregs confirmation analysis in the feeds.3.Analysis of bacterial drug resistance induced by streptomycin dregs hazards factors.Including that E.coli and Salmonella bacteria is as the tested bacteria,and streptidine and streptomycin is as induced factor,the subject has been carried out comparative analysis in vitro induced resistance.The results show that the streptomycin can induce two kinds of tested bacteria resistance;Streptidine can't induce two kinds of tested bacteria producing streptomycin resistance.But with streptomycin existing,streptidine has synergistic inducing the streptomycin resistant action to E.coli.The results from the experiments in vitro prove that the hazard factors of streptomycin dregs can induce the risk of bacterial resistance,and greater than that of the same concentration streptomycin induced risks.4.Objective to study the mechanism of streptidine's synergistic induction of drug resistance by using non targeted metabolomics.Establish the pretreatment methods of E.coli metabonomics samples,namely use low temperature saline-quenching,freeze grinding-ultrasonic lyses cells and cold methanol-water?50/50,V/V?to extract the metabolites.This method is applied to the pretreatment of E.coli with streptomycin resistant,and we can separate and determinate metabolites by UPLC-QTOF,At the same time,we can screen and identify 22 kinds of differential metabolites induced by different induction methods by PCA?OPLS-DA and online search in chemspider database.Through the KEGG metabolic pathway analysis and metabolic pathways of MetaboAnalyst topological analysis,we find that glutathione metabolism,valine,leucine,isoleucine degradation,pyruvate metabolism,butanoate metabolism,fatty acid metabolism,propanoate metabolism,pantothenate and CoA biosynthesis,glycerophospholipid metabolism are tied directly or indirectly to E.coli streptomycin resistance.In the meanwhile,we find that glycerophospholipid metabolism have connection with the effects of synergistic inducing resistance mechanisms of streptidine and streptomycin.