Resistance Characterizations Of β-lactam Antibiotics And Prevalence And Variability Of CTX-M β-lactamase In Salmonella

Objectives:1.We study the phenotypic traits of p-lactam antibiotics and the corresponding 6 kinds of genes(blaTEM,blaCTXX-M,blaOAX,blaCMX,blaPSE and blaSHV),and analyze the correlation ofβ-lactam antibiotic resistance with Salmonella from different sources.We aim to offer the reference data for clinical and poultry animal husbandry to reduce the resistance to β-lactam antibiotics with Salmonella,and to guide the treatment of salmonellosis.2.We also study the distribution and prevalence of cefotaxime extended-spectrumβ-lactamase(CTX-M)family in Jiangsu.We make the molecular traceability to isolates harbored different CTX-Ms by pulsed-field gel electrophoresis(PFGE)to learn the relationship between different genotypes of CTX-M and PFGE molecular types.We aim to find the regional differences,the homology and epidemiological characteristics of CTX-M genotypes.3.The whole gene sequencing method was used to sequence some strains carrying different CTX-Ms.We aim to find the amino acid variation,and the mobile genetic sequences located in the upstream and downstream with the different CTX-M genotypes.We try to learn what caused the variation of CTX-Ms,and what were the communication mechanism of difterent CTX-M.Methods:1.Two hundred and seventy-one Salmonella isolates from different sources were typed with serotypes.The resistant tests were going on with 7 kinds of β-lactam antibiotics by paper diffusion method.Six kinds of β-lactam genes were detected by polymerase chain reaction(PCR).2.Sixty-one harbored CTX-M Salmonella isolates were classified to different CTX-M genotypes with PCR.These isolates were also made the PFGE with Xbal for the restriction endonuclease digestion to carry out molecular typing.3.The DNAs of 12 isolates carrying CTX-M gene were extracted by TAKARA DNA extraction kit to make whole genome sequencing.The whole genome sequences were annotated using the Comprehensive Antibiotic Resistance Database(CARD)database.The varies of the amino acid sequences of different CTX-M types were found by nucleic acid BLAST alignment,comparing with the published sequences of CTX-M-1,CTX-M-9 and CTX-M-64.Results:1.S.Enteritidis,S.Indiana,S.Derby and S.Typhimurium were the major seroytpes among 271 isolates.S.Enteritidis and S.Typhimurium were the major seroytpes in human-origin isolates from while S.Enteritidis and S.Indiana were the major in animal-origin isolates.The human-origin isolates showed higher resistance to ampicillin(43.16%),followed by cefoperazone(18.95%)and amoxicillin(9.47%),while the animal-origin isolates showed higher resistance to ampicillin(69.32%),followed by cefoperazone(57.39%)and cefotaxime(33.52%).The Pearson correlation coefficient,which shows the resistance relationship with the isolates from human and animal,was 0.821.Approximately 36.99%isolates showed resistance to five antibiotics,and 27.4%to six.Approximately 37.89%human-origin isolates harbored blaTEM,and 5.26%harbored blaOXA,while 60.23%animal-origin isolates had blaTEM,and 32.39%had blaCTX-M.2.There were 3 CTX-M group and 6 CTX-M genotypes in 61 isolates harbored CTX-M.Two genotypes,CTX-M-55 and CTX-M-79,belong to CTX-M-1 group;three genotypes,CTX-M-65,CTX-M-14 and CTX-M-27,belong to CTX-M-9 group;one genotype,CTX-M-123,belongs to CTX-M-64 group.CTX-M-65 was prevalent in S.Indiana,S.Thompson and S.Typhimurium,while CTX-M-55 in S.Enteritidis.Four groups,A,B,C,D were found by PFGE molecular typing.The isolates in group A and group B,harboring CTX-M-55 majority,were from different sources,respectively.The isolates in group C was from the same source,harboring CTX-M-65 majority;the isolates in group D was from different areas,harboring CTX-M-65 majority.3.According to the whole gene sequencing,there are 4 labile amino acid sites in CTX-M-1 group and CTX-M-9 group,respectively.The labile amino acid sites in CTX-M-1 group located in 1st,117th,143th and 289th,while in CTX-M-9 group were 80th,234th,242th and 289th.There were sequence(IS)located on the upstream and downstream of CTX-Ms.ISEcpl was on upstream,and IS903 on downstream of CTX-M-65 and CTX-M-27,while ISEcpl was on upstream of CTX-M-55,and IS903 was on downstream of CTX-M-14.Conclusions:1.In this study,Salmonella was more resistance to β-lactam antibiotics,especially in animal-origin Salmonella.Pearson correlation analysis showed that the resistant animal-derived Salmonella could affect the resistance of human-derived Salmonella.It is the great potential harm for human that animal-derived Salmonella carries a large number of drug resistance genes,2 times higher than that of human-origin.2.CTX-M genes are variable,which is the most prevalent extended-spectrum β-lactamase.The major genotypes in Jiangsu were CTX-M-55 and CTX-M-65.The avian-derived S.Indiana carried a variety of CTX-M types,which was the major origin of the CTX-M.Results of PFGE molecular traceability indicates that the serotypes,the PFGE genotypes,the origin and areas could result in the difference of CTX-M.3.The labile amino acid sites were the main reason that caused the variability of CTX-M genotype.The CTX-M genotypes in the same group had the fixed mutation sites,which caused the changes of CTX-M genotype.On the other hand,the labile amino acid sites with different CTX-M group were different and not fixed.The upstream and downstream of CTX-M had the insertion sequences,ISEcp1 and IS903,respectively,which are the most common ISs in CTX-Ms.It is the two ISs,ISEcp1 and IS903,that caused CTX-M mobile among Salmonella,Salmonella and other Enterobacteriaceae,and also accelerate the spread and variation of CTX-M genotype.The mutability and the spreadability easily of CTX-M extended-spectram β-lactamase were the major factors for Salmonellae that caused the multi-drug resistance,even extensive drug resistance.