| Riemerella anatipestifer(R.anatipestifer,RA)is an important waterfowl pathogen.In farming,the extensive resistance of R.anatipestifer has brought great difficulties to the effective prevention and treatment of this disease.In order to understand the mechanism of R.anatipestifer resistance,we first investigated the resistant phenotypes of R.anatipestifer isolates,and then detected their corresponding resistance genes.Finally,the functions of multi-resistance gene clusters were studied in R.anatipestifer.The main results were as follows:?.Detection of resistant phenotypes and genes in R.anatipestifer isolates and functional verification of novel resistance genesFirst,108 R.anatipestifer filed isolates from different regions were performed the antimicrobial susceptibility test and resistance genes were detected by PCR in this thesis.Meanwhile,the functions of tet(A),tet(M),tet(O)and tet(Q)gens newly detected in R.anatipestifer isolates were verified by transferring into ATCC 11845 strain.The result of antimicrobial susceptibility test showed that the resistance of R.anatipestifer to six kinds of antibiotics,i.e.tetracycline,erythromycin,lincomycin,ceftiofur,aztreonam and sulfamethoxazole,were serious,and their resistance rates were 94.4%,90.7%,92.6%,43.5%,81.5%and 100%,respectively.The detected frequency of resistance gene was quite high,especially tet(X),erm(F)and bla-1 gene,their detected frequency were90.7%,77.8%and 83.3%,respectively.Except for tet(B),tet(C),tet(E),tet(G),tet(K),tet(W)and tet(O/W/32/O),the other remaining resistance genes were detected in varying degrees,including tetracycline resistance genes,such as tet(A),tet(M),tet(O)and tet(Q),which have not been reported in R.anatipestifer.The transconjugants carrying tet(A),tet(M),tet(O)and tet(Q)genes exhibited tetracycline resistance with MIC values ranging from 4 to 64?g/m L.?.Analysis and functional activity verification of multi-resistance gene cluster in R.anatipestiferIn this chapter,the resistant phenotypes were tested in RA-CH-1,RA-CH-2 and ATCC 11845 strains and their genomes were compared.Simultaneously,the bioinformatics analysis,functional activity and transferability of multi-resistance gene cluster in RA-CH-2 were studied.Compared with ATCC 11845,RA-CH-1 and RA-CH-2exhibited more serious resistance to multi-class antibiotics.The results of comparative genomic analysis showed that there were some multi-resistance gene clusters in the genome of RA-CH-1 and RA-CH-2,which was presumed to be related to their resistant phenotypes.According to the bioinformatic analysis of the multi-resistance gene cluster of the RA-CH-2,the resistance gene cluster was predicted as tet(X)-lnu(H)-bla-1-aads-cat-ere(D)-dhfr-cat-bla-2-tet(X).Through the deletion of the cluster and antimicrobial susceptibility test,it was preliminarily confirmed that the multi-resistance gene cluster could confer the corresponding resistance phenotype.Natural transformation experiments confirmed that,except for bla-1 and bla-2,all the other resistance genes in multi-resistance gene cluster could be transferred by nature transformation,including the whole gene cluster.?.Study on the function of incosamide nucleotidylyltransferases gene lnu(H)In this chapter,bioinformatics analysis,gene deletion,protein expression and purification,enzyme activity assay,mass spectrometry analysis of enzyme inactivated products,and epidemiological and transferable investigations of the G148?1775 gene in RA-CH-2 strain were performed.The results showed that the G148?1775 protein,with?41%similarity to other reported lincosamide nucleotidylyltransferases,was a new lincosamide-resistant gene lnu(H).RA-CH-2,the expression and complementation strains of lnu(H)gene showed high-level lincosamide resistance.While,the lnu(H)gene deletion strains exhibited 512-and 32-fold decreases in lincomycin and clindamycin MICs.Enzyme activity assay and mass spectrometry analysis demonstrated that Lnu(H)protein catalysed adenylylation of lincosamides lincomycin and clindamycin.The natural transformation frequency of lnu(H)gene was about 10-7,and the prevalence was 38%in R.anatipestifer field isolates.?.Study on the function of the erythromycin ester gene ere(D)This chapter was peformed bioinformatics analysis,gene deletion,protein expression,enzyme activity assay,epidemiological and transferable investigations of G148?1771 gene in RA-CH-2 strain.The results showed that G148?1771 belonged to the type D erythromycin esterase gene,and was designated as ere(D).RA-CH-2 and the expression strain of ere(D)gene showed erythromycin resistance,while the ere(D)gene deletion strain showed sensitivity(MIC value was 0.25?g/m L).The enzymatic decomposition of 4-nitrophenylbutyrate showed that Ere(D)had an inefficient hydrolysis,and the catalytic efficiency was only 34.0 L mol-1 s-1.The natural transformation rate of ere(D)gene was about 10-7,and the prevalence was 28.7%in R.anatipestifer field isolates.?.Study on the function of the tetracycline inactivated enzyme gene tet(X)In this chapter,bioinformatics analysis,gene deletion,protein expression,enzyme activity assay,real-time fluorescence quantitative PCR(q RT-PCR),epidemiological and transferable investigations were performed to study the G148?1767 and G148?1777genes in RA-CH-2 strain.The results showed that the G148?1767 and G148?1777 genes were two-copies tet(X)gene,and 100%similarity with each other.RA-CH-2,G148?1767gene deletion strain and complementation strains showed resistance to tetracycline antibiotics,whereas the G148?1777 gene deletion and the double deletion strains exhibted sensitivity.Enzyme activity assays showed that Tet(X)protein could inactivate tetracycline antibiotics.q RT-PCR analysis revealed that the expression of tet(X)gene was affected by tetracycline concentration,and the expression of tet(X)gene was the highest when the concentration was 8mg/m L.Compared with the expression of tet(X)gene in parent strain,there was no change in G148?1767 gene deletion strain,while the deletion of G148?1777 gene results in a significant decrease(at least 10 times).The natural transformation frequency of tet(X)gene was about 10-6,and the prevalence was 90.7%in R.anatipestifer field isolates.?.Study on the function of?-lactamase geneIn this chapter,the G148?1768 and G148?1774 genes in RA-CH-2 strains,and the AWB56?10175 and AWB56?01405 genes in RCAD0122 strain were studied by bioinformatics analysis,gene deletion,protein expression,enzyme activity assay,epidemiological and transferable investigations.The results showed that gene G148?1774and AWB56?10175 were 100%similarity.The G148?1774 and AWB56?01405 were new class A?-lactamase genes,tentatively designated as bla-1 and bla RAA-1.The G148?1768was a class D?-lactamase gene,named as bla-2.Compared with the?-lactam antibiotic resistance phenotype of RA-CH-2 strain,there was no change in bla-1 or bla-2 gene deletion strains,while the bla-1 and bla-2 double gene deletion strain showed sensitivity.When the bla-1 gene was deleted,the?-lactam antibiotic resistance of the RCAD0122strain did not change,while the bla RAA-1gene deletion strain exhibited?-lactam antibiotic sensitivity.At the same time,the cloning and expression strains of bla-1 and bla RAA-1also showed high-level?-lactam antibiotics resistance,and all tested strains showed typical synergy effects between antibiotics and inhibitors.The enzyme activity assay results showed that the G148?1774 and RAA-1 proteins had inactivated?-lactam antibiotic activity.Both bla-1 and bla-2 genes could not be transferred by natural transformation,while the natural transformation rate of bla RAA-1gene was about 10-7.In conclusion,this study found that the multi-resistence phenomenon was serious and multiresistence clusters were widely existence in R.anatipestifer.At the same time,the functions of lnu(H),ere(D),tet(X),bla-1,bla-2,bla RAA-1,tet(A),tet(M),tet(O)and tet(Q)genes were confirmed.These results contributed to the systematic elucidation of the molecular mechanism of R.anatipestifer resistance and provided some scientific guidance for clinical drug use rationally.In particular,the discovery of the novel resistance genes lnu(H),bla-1 and bla RAA-1has important scientific significance. |
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