| The dairy cow mastitis is one of the most common and multiple disease in cows,and it has caused enormous economic losses on cow culture.At present,most of antibiotics is used to prevent and treat the dairy cow mastitis.However,due to the long-term use and abuse of antibiotics,it has resulted in resistance of cow mastitis pathogens being more and more increasing,and it has become an important problem for the dairy cow farms.The main pathogenic bacteria of causing cow mastitis are Staphylococcus aureus,Escherichia coli(E.coli)and Streptococcus.In recent years,more researches on the pathogenic mechanisms of Staphylococcus aureus and Streptococcus causing the dairy cow mastitis were reported,but the reports about the clinical isolates pathogenicity and resistance mechanisms of E.coli mastitis were relatively less.Quorum sensing is a process of adapting to some special environment where pathogens use their own chemical molecules as a signal to change their physiological behavior.Quorum sensing signaling molecule Autoinducer 2(AI-2)is considered as a kind of interspecies communication signal in Gram-positive and Gram-negative bacteria.In Escherichia coli model strains,the functional mechanism of AI-2 has been well studied,however,in clinical pathogenic E.coli strains,whether AI-2 affects the expression of antibiotic resistance-associated and AI-2-associated genes have not been reported.In this study,we showed that how AI-2 affected the regulatory mechanism of the antibiotic resistance of a clinical E.coli strain isolated from a dairy cow with mastitis,which will provide new clues for control the dairy cow mastitis.1.To detect the antibiotic resistance of the clinical E.coli strain isolated from a dairy cow with mastitis,this study used double dilution method and gradient dilution method to detect various antibiotic resistance of the clinical E.coli DCM1 mastitis.We found that minimum inhibitory concentrations of ampicillin,oxacillin,penicillinG and chloramphenicol were 4.5 mg/mL,6.5 mg/mL,3.5 mg/mL and 2.0 ?g/mL,respectively.This results showed that the clinical mastitis of E.coli DCM1 was a typical multi-drug resistance strain.2.PCR amplification technique and restriction enzymes were used to detect E.coli DCM1 antibiotic resistance gene and the location of antibiotic resistance gene and plasmid with transfer element.The results showed that plasmid-mediated antibiotic resistance tem gene encode extended spectrum ?-lactamases(ESBLs),and the extended spectrum?-lactamases(ESBLs)positive E.coli was a major etiological organism responsible forbovine mastitis.3.This study used colony-counting method and turbidimetric method to detect if the addition of exogenous AI-2 effect the antibiotic resistance phenotype of E.coli DCM1.The results showed that exogenous additional AI-2 enhance the antibiotic resistance of E.coli DCM1.4.This study performed real-time reverse transcription PCR(RT-qPCR)experiment to detect the transcription level of the tem gene.The results showed that addition of AI-2resulted in 2.1-fold(ampicillin),2.4-fold(oxacillin),and 2.4-fold(penicillinG)increase in the tem transcription under the presence of antibiotics,respectively.5.The intracellular receptor protein LsrR of the quorum sensing AI-2 was over-expressed and purification,then the DIG-gel shift assays were performed to make various amounts of LsrR binding to the promoter region of tem.These results confirmsd that exogenous AI-2 through the receptor protein LsrR directly up-regulated tem transcription So,this study showed that exogenous additional AI-2 increased the antibiotic resistance of a clinical E.coli strain isolated from a dairy cow with mastitis by up-regulating the expression of tem in an lsrR-dependent manner.In conclusion,PCR amplication methods,RT-qPCR methods and DIG-gel shift assays were used to prove that quorum sensing signaling molecule AI-2 could up-regulate the transcription level of tem,and the regulation mechanism is directly related to the intracellular receptor protein LsrR of the quorum sensing AI-2.Therefore,this study provides an effective experimental method for the further study of the mechanism of Escherichia coli multi-antibiotic resistance. |
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