Study On The Resistance Of Erysipelothrix Rhusiopathiae Based On Transcriptome And Proteomics

Swine erysipelas is a kind of acute and hot zoonotic infectious disease caused by erysipelas suis(E.rhusiopathiae),which belongs to the second class of animal diseases published by the Ministry of agriculture and rural areas of China.It mainly damages pigs to develop septicemia,endocarditis and polyarthritis.Since 2010,there has been an outbreak of erysipelas in many provinces of China,causing serious economic losses.Vaccination and antibiotic treatment are effective methods to prevent erysipelas.?-lactams(?-lactams)antibiotics are commonly used in the prevention and treatment of erysipelas,of which penicillin(PG)is always the first and most effective.However,due to the unreasonable use of antibiotics in veterinary clinic for a long time,bacterial resistance has been produced and become increasingly serious.Based on the epidemiological investigation of swine bacterial diseases in Anhui,Jiangsu,Shandong,Hebei,Guangxi and other five provinces,we found and obtained a strain of e.rhusiopatiae resistant to PG.Based on the understanding of the reasons for the occurrence of tolerance to PG in e.rhusiopatiae,this study made a preliminary exploration by using transcriptome and proteomics in order to lay a foundation for further research on the mechanism of e.rhusiopatiae resistance.In this study,E.rhusiopathiae PG resistant strain(code AEr51),E.rhusiopathiae standard strain ATCC19414(code AEr S)and E.rhusiopathiae PG sensitive strain(code AEr31)were used as the test strains for the following tests:(1)the paper agar diffusion method(K-B method)was used to detect three test strains for 27 ?-species-The sensitivity of lactams was determined to determine whether there was multiple drug resistance(MDR).The minimal inhibitory concentration(MIC)of three strains to PG,Amoxicillin(AMX)and Ampicillin(AMP)was determined by microdilution method.(2)The resistance related differentially expressed genes of three strains were screened by RNA-seq technology,and the changes of gene expression were compared.The reliability of RNA-seq results was verified by real-time fluorescent quantitative PCR(q RT-PCR).(3)TMT quantitative proteomics was used to screen the different expression proteins related to drug resistance of three tested strains and compare the changes of protein expression.The results showed that:(1)AEr51 was resistant to PG,sensitive to AMX and AMP,and sensitive to all ?-lactams.AEr51,AEr S and AEr31 were sensitive to lincomycin and tetracycline antibiotics,and resistant to gentamicin,streptomycin and tilmicosin.The results showed that the three strains had no MDR to 27 antibiotics,except that the mic value of AEr51 to PG was 32?g/m L,which was highly resistant(MIC ?16-20?g/m L),AEr S and AEr31 were highly sensitive to PG,AMX and AMP(MICs ?0.025-0.78?g/m L).The MIC values of AEr51 to AMX and AMP were 4 ?g/m L and2?g/m L,respectively.The sensitivity of AEr51 to AMX and AMP was slightly lower than that of AEr31 and AEr31,and it was moderately sensitive(mics,0.1-25?g/m L).(2)The differentially expressed genes were screened under the condition of | log2(foldchange)| > 1 and Qvalue < 0.005,AEr51/AEr S a total of 668 differential genes were screened in the comparison group,including 434 up-regulated genes and 234 down regulated genes;a total of 403 differential genes were screened in the AEr51/AEr31 comparison group,including 275 up regulated genes and 128 down regulated genes;a total of 316 differential genes were screened in the AEr31/AEr S comparison group,including 175 up regulated genes and 141 down regulated genes.Six differentially expressed genes were selected for q RT-PCR verification,and the data results were consistent with the gene expression trend of transcriptome analysis,which confirmed the reliability of RNA-seq and data analysis.(3)Using 1.2 times as the change threshold(fold change ? 1.2 or ? 0.83),P ? 0.05 as the standard to screen differential expression protein,AEr51/AEr S a total of 167 differential proteins were screened in the comparison group,of which 86 were up-regulated and 81 were down regulated;159 differential proteins were screened in the AEr51/AEr31 comparison group,of which 80 were up regulated and 79 were down regulated;177 differential proteins were screened in the AEr31/AEr S comparison group,of which 80 were up regulated and 97 were down regulated.It was found that the metabolic pathways related to microbial,amino acid,carbon,sulfur,pyrimidine metabolism were significantly enriched.According to KEGG annotation information,a total of 18 proteins related to drug resistance were screened for differential expression,including ABC transporters,efflux pump related proteins,bicomponent related proteins and pathways,bacterial chemotaxis related proteins and pathways,etc.The results showed that AEr51 and AEr31 were resistant to PG and sensitive to PG respectively.Twenty-one differentially expressed genes related to drug resistance were screened by RNA-seq,including efflux pump,chemotaxis,two-component system,etc.A total of 18 differentially expressed proteins related to drug resistancewere screened by quantitative proteomics of TMT,including ABC transporter and PBP.