| Objective:Antibiotic misuse has created a global dangers of bacterial resistance,resulting in disease nonadherence.Metallic products are beginning to be commonly used as novel antibacterial drugs.Integrons have the function of capturing and excising drug resistance gene cassettes,which is a key reason for bacterial drug resistance.This study mainly investigated the effects of copper and silver ions on class 1 integron trapping resistance gene cassettes and the related mechanismsMethods:An in vivo model of a class 1 integron capture resistance gene cassette was constructed in Escherichia coli.q PCR was used to determine the frequency of integron integration in E.coli after intervention with different concentrations of silver（0.3,0.9,and 1.5μg/m L of Ag+）and copper ions(5,50,100,150,and 220μg/m L of Cu2+)and control group without metal ion intervention and validated by phenotypic screening.Determination of the concentration of metal ions absorbed by bacteria by mass spectrometry.Furthermore,the molecular mechanism of silver ion inhibiting bacterial integron capturing drug resistance gene cassette was analyzed by transcriptome sequencing.Result:1.The results of fluorescence quantitative PCR showed that the integration frequencies were 9.42×10-5(7.91×10-5)in the 0.9μg/m L Ag+group and 7.29×10-5(4.58×10-5)in the 1.5μg/m L Ag+group compared to 2.59×10-4(1.09×10-4)in the control group,with a significantly lower integration frequency,with statistical significance（P<0.001）.However,there was no statistically significant difference between the 0.9μg/ml Ag+and 1.5μg/m L Ag+groups（P>0.05）.There was no statistically significant difference in the frequency of integration between the control group and the group with different concentrations of copper ions（P>0.05）,nor between the groups with different concentrations of copper ions（P>0.05）.2.The results of the phenotypic screening method showed that the 0.9μg/m LAg+and 1.5μg/m LAg+groups had few colonies on LB plates containing streptomycin compared to the control group,and the control group,0.3μg/m LAg+group and different concentrations of copper ions groups had more colonies on LB plates containing streptomycin,with no significant change in the frequency of integration.The phenotypic screening method was consistent with the q PCR results.3.Inductively coupled plasma mass spectrometry（ICP-MS）showed that the metal ions absorbed by Escherichia coli increased with the increase of bacterial exposure to silver and copper ions.4.Transcriptional sequencing method was used to analyze the gene expression level of Escherichia coli before and after silver ion treatment.Compared with the control group,there were 241 genes significantly different in 1.5μg/m L Ag+group,among which 139 genes were up-regulated and 102 genes were down-regulated.5.Through GO function and KEGG pathway enrichment,it was found that differentially expressed gene was associated with methyl galactoside transport,maltose transport,phosphoenolpyruvate-glycerol phosphotransferase activity and glycerone kinase activity,as well as amino sugar and nucleotide sugar metabolism,flagellum assembly,CAMP resistance,fructose and mannose metabolism and PTS metabolic pathways.6.The top 12 hub genes（pts G,mal E,lam B,lac Z,mal K,bas R,ais,ugd,nag E,met N,mal Q,and mal F）were screened by protein interaction network.Conclusion:A certain concentration of silver ions can inhibit integron integration of drug-resistant gene cassettes,while not all metal ions with bactericidal properties can have an effect on integration frequency.It is speculated that the silver ion inhibition of bacterial integron captured drug resistance gene cassette may be regulated by maltose transport and carbon catabolism.However,carbon catabolism is very complex and needs further study.At present,there is no related study on bacterial integron transcriptome,which provides a new way to solve the problem of bacterial drug resistance. |
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