| China is the largest producer and consumer of pork all over the world.The extensive use of feed antibiotics has promoted the growth of livestock products,while brought serious hidden dangers.The spread of drug resistance microorganisms and their resistant genes through the pork industry chain has brought harm to public health.Sulfonamides which are widely used in the prevention and treatment of livestock and poultry bacterial infectious diseases,are one of the most widely used veterinary antibacterial drugs.Their drug-resistant microorganisms and drug-resistant genes(sull,sul2 and sul3)are one of the main sources of drug-resistant genes in the pork industry chain.Escherichia coli,as the donor,acceptor and intermediate vector of drug resistant genes,is an important carrier and potential source base for the spread of resistant genes.Pig slaughterhouses are one of the most important intermediate links in the pork industry chain.Therefore,the study set the E.coli in the slaughterhouse as the research object,which explored the distribution characteristics of sulfonamide resistance genes in the pork production and environments,and their dissemination and fitness cost in the E.coli and possible fitness mechanism.It was aimed to lay the foundation for control the spread of bacterial drug resistance genes.The main contents are as follows:1.The distribution characteristics of E.coli sulfonamide resistance genes in the pig slaughterhouse and the genetic relationship between the sulfonamide resistance strains:Through RPA-LFD rapid detection technology,a visual and efficient simultaneous detection method for sulfonamide resistance genes sull,sul2 and sul3 was established.Through this method,different sources of E.coli sulfonamide resistance genes were detected,and the results showed the distributions of sulfonamide resistance genes sull,sul2 and sul3.The total detection rate of sulfonamide resistance genes was 70.29%,of which there was 18.86%of sull,22.29%of sul2 and 3.43%of sul3.The carrying rates of sull and sul2 genes were various in each sample sites,but were significantly higher than that of sul3(p<0.05).The carrying rate of composite sulfonamide resistance genes was 25.71%,in which there was 20.00%of sull+sul2,1.71%of sull+sul3,3.14%of sul2+sul3,0.86%of sull+sul2+sul3,and the carrying rate of sull+sul2 combination was significantly higher than that of other combinations at all sampling points(p<0.05).After using pulsed field gel electrophoresis(PFGE)and multiple loci sequence analysis(MLST)molecular subtyping technology,the genetic relationships among 34 representative E.coli strains containing all 7 kinds of arrays of sulfonamide resistance genes from different sources were analyzed,and they were successfully divided into 29 PFGE types and 25 ST types.The two methods were basically consistent in the differentiation of strains.34 strains of E.coli were dispersed,the number of strains of the same clone was less,and the strains of the same clone earrjied different types of sulfonamide resistance genes,which indicated that there were a small number of genes with vertical spread of the bacteria,and the horizontal dissemination was probably the main spread way mediated by horizontal gene transbacterial fer(HGT).2.Horizontal dissemination of E.coli sulfonamide resistance genes mediated by HGT in the pig slaughterhouse:In this study,17 strains of E.coli carrying composite sulfonamide resistance genes were used to locate sull,sul2 and sul3 through southern hybridization.Among them,9 strains of sulfonamide resistance genes were all located in plasmids,4 were all located in genomes,and another 4 were both located in plasmids and genomes.The transconjugants or transformants were obtained of all the 13 isolates which plasmids carrying sulfonamide resistance genes through conjugation or plasmid transformation.The results of the plasmid replicons of the transconjugants and transformants were consistent,and the 11 plasmids were IncF/IncK multi-replicon plasmids.The PCR walking method was used to analyze the flanking sequence and the genetic environment of sulfonamide resistance genes located in the 13 plasmids.Among them,7 of 9 sull genes were located between two IS6 insertion sequences,11 of 11 sul2 genes were adjacent to streptomycin resistance gene strA and strB,and 7 sul2 genes were located between transposon sequences tnpA and tnpB or insertion sequences IS6 and IS5.6 of 9 sul3 genes were located between the insertion sequences IS6 and IS256.In addition,the flanking sequence and the genetic environment analysis were carried out on the sulfonamide resistance genes of 8 E.coli strains located on the genome.Among them,5 sull genes were all located on the class 1 integron,all 7 sull genes were located between IS6 and IS91 insertion sequences,and neither of the 2 sul3 genes was adjacent to a mobile element.The above results indicated that the horizontal dissemination mechanisms mediated by HGT of E.coli sulfonamide resistance genes in the pig slaughterhouse were as follows:(1)The horizontal dissemination was mediated by IncF/IncK multi-replicon plasmids.(2)The horizontal dissemination of sull,sul2 and sul3,which located on the plasmind,is mainly mediated by mobile genetic elements such as insertion sequences(IS6,IS5,IS256 and IS26),transposons(tnpA and tnpB)and class 1 integron.The sul2 gene can also be spread horizontally under the co-selection of streptomycin.(3)In the genome,the sull gene was mainly spread horizontally mediated by the class 1 integron.The sul2 gene was spread horizontally mediated by insertion sequences IS6 and IS91.The flanking sequence of sul3 has no mobile genetic element and has little horizontal dissemination potential.3.The differences of fitness between sul3 gene and sull and sul2 genes in E.coli:In order to study the fitness of sull,sul2 and sul3 genes in E.coli,engineering strains with a consistent genetic background,carrying sull,sul2 or sul3 genes respectively were constructed.Pairwise competitive tests were carried out respectively,and it was found that the selection coefficient(S)of E.coli BL21:pET23a-sul3 was-0.087±0.020,S<0,which indicated that it has fitness cost.The selection coefficient S of E.coli BL21:pET23a-sul1 and E.coli BL21:pET23a-sul2 were 0.021±0.012 and 0.019±0.016,S>0,respectively,which indicated a good fitness in the competition test.In addition,E.coli BL21:pET23a-sul3 had significantly lower growth capacity,plasmid stability and movement capacity than the other 2 strains(p<0.05).Then the mechanism of changes in the fitness of E.coli under the expression of different sulfonamide resistance genes was explored at the protein level through Label-free proteomics technology,and functional verification was carried out at the transcription level by Real-time PCR.The reasons why the fitness cost of E.coli BL21:pET23a-sul3 was significantly higher than that of the other 2 strains were as follows:1)The differential expression of bacterial motility related proteins FliZ,FliA,FliC and LrhA might affected the motility of E.coli BL21:pET23a-sul3.2)The differential expression of outer membrane pore protein OmpD and the ATP-binding proteins UgpC,RbsA,GsiA in ABC transport systems affected the energy metabolism of E.coli BL21:pET23a-sul3.3)The expression of SUL3 dihydropteroate synthase was significantly lower than that of SUL1 and SUL2(p<0.05),which resulted in a decline of ability in the regulation of folate synthesis,and affected the reproduction capacity of E.coli BL21:pET23a-sul3.In conclusion,the carrying rates of E.coli sull and sul2 genes from different sources in the pig slaughterhouse were significantly higher than that of sul3 genes(p<0.05).The horizontal dissemination of sull,sul2 and sul3 on plasmids is mainly mediated by HGT through IncF/IncK multi-replicon plasmids as well as insertion sequences(IS6,IS5,IS256 and IS26),transposon sequences(tnpA and tnpB)and class 1 integron sequences.The horizontal dissemination of sull and sul2 genes in the genome is mainly mediated by HGT through the class 1 integron and insertion sequences IS6 and IS91.Flanking sequences of sul3 gene located in the genome lacks mobile genetic elements,so its horizontal dissemination potential is low.The differences in genetic environment and fitness between sul3 gene and sull and sul2 genes are one of the main reasons why the carrying rate of sul3 gene is significantly lower than that of sull and sul2 genes.The results of this study have deepened the understanding of the dissemination of sulfonamide resistance genes in pig slaughterhouses,and laid a theoretical foundation for the search of methods to control the dissemination of sulfonamide resistance genes along with the pork industry chain. |
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