Regulatory Mechanism Of AI-2 Quorum Sensing On Antibiotic Resistance In Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)is one of the common pathogenic bacteria in poultry breeding industry,is one of the reasons to restrict the sustainable development of poultry breeding industry in our country,and lead to huge losses of the farm and other relative industry about meat and eggs.Long-term overuse of antibiotics in poultry breeding industry resulted in decreasing susceptibility of the avian pathogenic Escherichia coli to antibiotics and brought a great challenge for the routine antimicrobial therapy.Therefore,it is important to investigate the mechanism of the occurrence and transfer of the antibiotic resistance of E.coli.Searching of the new target for treatment of antibiotic-resistant E.coli has a great meaning for medical control of the APEC strain.Quorum sensing(QS)is a process of cell-cell communication widely present in bacteria.In E.coli,autoinducer2(AI-2)is the main QS signal molecule which has been produced.whether AI-2 is related to the pathogenesis and antibiotic resistant gene expression in multi antibiotic-resistant E.coli,has seldom been reported.We explored the effect of AI-2 on avian pathogenic E.coli kanamycin resistance,then studied the regulatory mechanism of AI-2 worked on kanamycin resistant gene aadA,found that the AI-2 quorum-sensing system could make avian pathogenic E.coli more sensitivec to kanamycin by reduced the expression level of aadA.1.In order to detect the drug resistance of avian pathogenic E.coli strain isolated,this test used double dilution method to detect kanamycin resistance of the avian pathogenic E.coli strains isolated: APEC 28,APEC 32 and APEC 56.It was found that the minimum inhibitory concentrations of kanamycin were 2.5 mg/mL,2.5 mg/mL and 5 mg/mL.The results showed that this three avian pathogenic E.coli strains isolated is kanamycin resistant bacterias.2.PCR amplification technique and BLAST software analyze were used to detect the drug resistance gene of avian pathogenic E.coli strain isolated.It was found that the adenyltransferase were encoded by aadA drug-resistant gene is the main cause of antibiotic resistance of avian pathogenic E.coli strain.3.In this test,growth curve method and colony counting method(CFU)were used to detect the influence on avian pathogenic E.coli growth via adding AI-2 in vitro.The results showed that add AI-2 in vitro can down-regulate the drug resistance of APEC 28,APEC 32 and APEC 56.To prove that avian pathogenic E.coli strains isolated can adjust the drug resistance through the AI-2.4.This test used real time fluorescence quantitative PCR(RT-qPCR)technology to detect the transcription level of kanamycin resistance gene aadA.Results show that,under the pressure of kanamycin,add AI-2 in vitro will lead to 1.75-fold,2.6-fold and 1.2-fold decrease of kanamycin resistance gene aadA transcription level,respectively,in APEC 28 at different times;1.2-fold,2.7-fold and 1.5-fold decrease in APEC 32 at different time srespectively;1.5-fold,2-fold and 1.2-fold decrease in APEC 56 at different times respectively.Based on the previous work,this study will further investigate how the AI-2 QS system affects the aadA expression and regulation mechanism of kanamycin resistance gene of the APEC strain with the effcet of AI-2.The further study could provide new drug targets and treatment methods for control the infection of resistant avian pathogenic E.coli.