IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的结构基因组学分析
|
摘要
【背景】IncFII-FIA-FIB型质粒广泛存在于肠杆菌科细菌中,介导了许多耐药基因的水平转移,并导致细菌多重耐药问题日益严重。【目的】分析IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的基因组结构,并研究其介导大肠杆菌BTR株的耐药基因水平转移机制。【方法】利用PCR进行耐药基因筛查;接合转移和电转化实验验证质粒pBTR-CTXM是否具备自主接合转移的特性;VITEK 2 Compact全自动细菌鉴定及药敏分析仪测定相关菌株对抗生素的药物敏感性;构建MatePair文库并进行细菌全基因组高通量测序和质粒结构基因组学分析。【结果】菌株BTR是携带blaNDM-1、blaCTX-M-15、blaTEM、qnrD、qnrS1、mph(A)、erm(B)和tetA(B)等耐药基因的多重耐药大肠杆菌,其中blaCTX-M-15、mph(A)、erm(B)和tet A(B)等耐药基因均位于大小为144 939 bp的质粒p BTR-CTXM (GenBank登录号MF156697)上,该质粒可与菌株BTR内质粒pNDM-BTR接合共转移到受体菌大肠杆菌EC600中。pBTR-CTXM具备IncFII-FIA-FIB型质粒典型的骨架区结构,其多重耐药(Multidrug-resistant,MDR)区由新的复合型转座子Tn6492、Tn2残余、Tn10残余、ISEcp1-blaCTX-M-15-Δorf477转座单元和一些插入序列组成。【结论】pBTR-CTXM中新复合型转座子Tn6492与Tn10残余和ISEcp1-blaCTX-M-15-Δorf477转座单元共同介导大肠杆菌BTR株的多重耐药与耐药基因的水平传播。
[Background] The IncFII-FIA-FIB incompatibility group plasmids are widely encountered in Enterobacteriaceae species. They mediate the horizontal transfer of many resistance genes and lead to the upsurge of multidrug-resistant strains. [Objective] To investigate the genomic characterization of the multidrug-resistant plasmid pBTR-CTXM assigned into IncFII-FIA-FIB incompatibility group and the plasmid-mediated horizontal transfer mechanism of resistance genes of Escherichia coli BTR. [Methods] The screening of antibiotic resistance genes was determined using PCR. The transferability of plasmid pBTR-CTXM was confirmed by conjugation experiments and electroporation experiments. The minimal inhibitory concentration(MIC) values were tested by VITEK 2 Compact system. The complete nucleotide sequence of pBTR-CTXM was determined using next-generation sequencing technology from a mate pair library. Structural genomics of pBTR-CTXM was analyzed subsequently. [Results] The multidrug-resistant E. coli BTR isolate harbored the blaNDM-1, blaCTX-M-15, blaTEM, qnrD, qnrS1, mph(A), erm(B), and tetA(B) genes. The blaCTX-M-15, mph(A), erm(B), and tetA(B) genes were located on pBTR-CTXM(Gen Bank accession number MF156697) with 144 939 bp in length. The pBTR-CTXM could be conjugatively mobilized to the recipient strain E. coli EC600 by p NDM-BTR, a conjugative plasmid existed in the E. coli BTR. p BTR-CTXM possessed typical backbones of Inc FII-FIA-FIB plasmids and a multidrug-resistant(MDR) region, which was comprised of a novel composite transposon Tn6492, the Tn2 remnant, the Tn10 remnant, the ISEcp1-blaCTX-M-15-Δorf477 unit and some insertion sequences(IS) elements. [Conclusion] The novel composite transposon Tn6492, the Tn10 remnant, and the ISEcp1-blaCTX-M-15-Δorf477 unit mediated the horizontal transfer of resistance genes and the antibiotic resistance of E. coli BTR.
[Background] The IncFII-FIA-FIB incompatibility group plasmids are widely encountered in Enterobacteriaceae species. They mediate the horizontal transfer of many resistance genes and lead to the upsurge of multidrug-resistant strains. [Objective] To investigate the genomic characterization of the multidrug-resistant plasmid pBTR-CTXM assigned into IncFII-FIA-FIB incompatibility group and the plasmid-mediated horizontal transfer mechanism of resistance genes of Escherichia coli BTR. [Methods] The screening of antibiotic resistance genes was determined using PCR. The transferability of plasmid pBTR-CTXM was confirmed by conjugation experiments and electroporation experiments. The minimal inhibitory concentration(MIC) values were tested by VITEK 2 Compact system. The complete nucleotide sequence of pBTR-CTXM was determined using next-generation sequencing technology from a mate pair library. Structural genomics of pBTR-CTXM was analyzed subsequently. [Results] The multidrug-resistant E. coli BTR isolate harbored the blaNDM-1, blaCTX-M-15, blaTEM, qnrD, qnrS1, mph(A), erm(B), and tetA(B) genes. The blaCTX-M-15, mph(A), erm(B), and tetA(B) genes were located on pBTR-CTXM(Gen Bank accession number MF156697) with 144 939 bp in length. The pBTR-CTXM could be conjugatively mobilized to the recipient strain E. coli EC600 by p NDM-BTR, a conjugative plasmid existed in the E. coli BTR. p BTR-CTXM possessed typical backbones of Inc FII-FIA-FIB plasmids and a multidrug-resistant(MDR) region, which was comprised of a novel composite transposon Tn6492, the Tn2 remnant, the Tn10 remnant, the ISEcp1-blaCTX-M-15-Δorf477 unit and some insertion sequences(IS) elements. [Conclusion] The novel composite transposon Tn6492, the Tn10 remnant, and the ISEcp1-blaCTX-M-15-Δorf477 unit mediated the horizontal transfer of resistance genes and the antibiotic resistance of E. coli BTR.
引文
[1]Mellata M.Human and avian extraintestinal pathogenic Escherichia coli:infections,zoonotic risks,and antibiotic resistance trends[J].Foodborne Pathogens and Disease,2013,10(11):916-932
[2]Liu CY.Comparative genomics analysis and study on subunit vaccine of porcine extraintestinal pathogenic Escherichia coli[D].Wuhan:Doctoral Dissertation of Huazhong Agricultural University,2014(in Chinese)刘璨颖.猪源肠外致病性大肠杆菌比较基因组学和亚单位疫苗的研究[D].武汉:华中农业大学博士学位论文,2014
[3]Bengtsson S,Naseer U,Sundsfjord A,et al.Sequence types and plasmid carriage of uropathogenic Escherichia coli devoid of phenotypically detectable resistance[J].Journal of Antimicrobial Chemotherapy,2012,67(1):69-73
[4]Hammerum AM,Larsen J,Andersen VD,et al.Characterization of extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli obtained from danish pigs,pig farmers and their families from farms with high or no consumption of third-or fourth-generation cephalosporins[J].Journal of Antimicrobial Chemotherapy,2014,69(10):2650-2657
[5]Duan SS.The dissemination mechanism and genetic characteristics of CTX-M clusters among Avian Escherichia coli[D].Zhengzhou:Master’s Thesis of Henan Agricultural University,2015(in Chinese)段沙沙.禽大肠埃希菌不同CTX-M亚群的散播机制及遗传特性[D].郑州:河南农业大学硕士学位论文,2015
[6]MarcadéG,Deschamps C,Boyd A,et al.Replicon typing of plasmids in Escherichia coli producing extended-spectrumβ-lactamases[J].Journal of Antimicrobial Chemotherapy,2009,63(1):67-71
[7]Villa L,García-Fernández A,Fortini D,et al.Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants[J].Journal of Antimicrobial Chemotherapy,2010,65(12):2518-2529
[8]Osborn AM,da Silva Tatley FM,Steyn LM,et al.Mosaic plasmids and mosaic replicons:evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons[J].Microbiology,2000,146(9):2267-2275
[9]Szczepanowski R,Braun S,Riedel V,et al.The 120 592 bp IncFplasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants,two iron-acquisition systems and other putative virulence-associated functions[J].Microbiology,2005,151(4):1095-1111
[10]Womble DD,Rownd RH.Genetic and physical map of plasmid NR1:comparison with other IncFII antibiotic resistance plasmids[J].Microbiological Reviews,1988,52(4):433-451
[11]Cavalli LL,Lederberg J,Lederberg EM.An infective factor controlling sex compatibility in Bacterium coli[J].Microbiology,1953,8(1):89-103
[12]Zhan Z,Feng J,Yin Z,et al.Antibiotic-resistant mechanisms of plasmid pA1137 carrying aminoglycoside resistance gene aac C2[J].Military Medical Sciences,2017,41(12):973-977(in Chinese)占喆,冯娇,殷喆,等.携带氨基糖苷类耐药基因aacC2的质粒pA1137耐药机制研究[J].军事医学,2017,41(12):973-977
[13]Zhao YC,Wang LJ,Zhang ZY,et al.Structural genomics of pNDM-BTR harboring In191 and Tn6360,and other blaNDM-carrying IncN1 plasmids[J].Future Microbiology,2017,12(14):1271-1281
[14]Frank JA,Reich CI,Sharma S,et al.Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNAgenes[J].Applied and Environmental Microbiology,2008,74(8):2461-2470
[15]Poirel L,Walsh TR,Cuvillier V,et al.Multiplex PCR for detection of acquired carbapenemase genes[J].Diagnostic Microbiology and Infectious Disease,2011,70(1):119-123
[16]Dallenne C,Da Costa A,DecréD,et al.Development of a set of multiplex PCR assays for the detection of genes encoding importantβ-lactamases in Enterobacteriaceae[J].Journal of Antimicrobial Chemotherapy,2010,65(3):490-495
[17]Ciesielczuk H,Hornsey M,Choi V,et al.Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants[J].Journal of Medical Microbiology,2013,62(12):1823-1827
[18]Phuc Nguyen MC,Woerther PL,Bouvet M,et al.Escherichia coli as reservoir for macrolide resistance genes[J].Emerging Infectious Diseases,2009,15(10):1648-1650
[19]Maynard C,Fairbrother JM,Bekal S,et al.Antimicrobial resistance genes in enterotoxigenic Escherichia coli O149:K91isolates obtained over a 23-year period from pigs[J].Antimicrobial Agents and Chemotherapy,2003,47(10):3214-3221
[20]Clinical and Laboratory Standards Institute.M100-S27 Performance standards for antimicrobial susceptibility testing:twenty-seventh informational supplement[Z].Wayne,PA,USA:CLSI,2017
[21]Margulies M,Egholm M,Altman WE,et al.Genome sequencing in microfabricated high-density picolitre reactors[J].Nature,2005,437(7057):376-380
[22]Aziz RK,Bartels D,Best AA,et al.The RAST server:rapid annotations using subsystems technology[J].BMC Genomics,2008,9(1):75
[23]Boratyn GM,Camacho C,Cooper PS,et al.BLAST:a more efficient report with usability improvements[J].Nucleic Acids Research,2013,41:W29-W33
[24]O’Leary NA,Wright MW,Brister JR,et al.Reference sequence(RefSeq)database at NCBI:current status,taxonomic expansion,and functional annotation[J].Nucleic Acids Research,2016,44(D1):D733-D745
[25]Boutet E,Lieberherr D,Tognolli M,et al.UniProtKB/Swiss-Prot,the manually annotated section of the UniProt KnowledgeBase:how to use the entry view[A]//Plant Bioinformatics.Methods in Molecular Biology[M].New York:Humana Press,2016:23-54
[26]Siguier P,Perochon J,Lestrade L,et al.ISfinder:the reference centre for bacterial insertion sequences[J].Nucleic Acids Research,2006,34(suppl 1):D32-D36
[27]Moura A,Soares M,Pereira C,et al.INTEGRALL:a database and search engine for integrons,integrases and gene cassettes[J].Bioinformatics,2009,25(8):1096-1098
[28]Roberts AP,Chandler M,Courvalin P,et al.Revised nomenclature for transposable genetic elements[J].Plasmid,2008,60(3):167-173
[29]Wibberg D,Szczepanowski R,Eikmeyer F,et al.The IncFplasmid pRSB225 isolated from a municipal wastewater treatment plant’s on-site preflooder combining antibiotic resistance and putative virulence functions is highly related to virulence plasmids identified in pathogenic E.coli isolates[J].Plasmid,2013,69(2):127-137
[30]Partridge SR,Hall RM.Complex multiple antibiotic and mercury resistance region derived from the r-det of NR1(R100)[J].Antimicrobial Agents and Chemotherapy,2004,48(11):4250-4255
[31]Cain AK,Liu XL,Djordjevic SP,et al.Transposons related to Tn1696 in IncHI2 plasmids in multiply antibiotic resistant Salmonella enterica serovar typhimurium from Australian animals[J].Microbial Drug Resistance,2010,16(3):197-202
[32]Chalmers R,Sewitz S,Lipkow K,et al.Complete nucleotide sequence of Tn10[J].Journal of Bacteriology,2000,182(10):2970-2972
[33]Partridge SR.Analysis of antibiotic resistance regions in Gram-negative bacteria[J].FEMS Microbiology Reviews,2011,35(5):820-855
[34]Zong ZY,Partridge SR,Iredell JR.ISEcp1-mediated transposition and homologous recombination can explain the context of blaCTX-M-62 linked to qnrB2[J].Antimicrobial Agents and Chemotherapy,2010,54(7):3039-3042
[35]Bailey JK,Pinyon JL,Anantham S,et al.Distribution of the blaTEM gene and bla TEM-containing transposons in commensal Escherichia coli[J].Journal of Antimicrobial Chemotherapy,2011,66(4):745-751
[2]Liu CY.Comparative genomics analysis and study on subunit vaccine of porcine extraintestinal pathogenic Escherichia coli[D].Wuhan:Doctoral Dissertation of Huazhong Agricultural University,2014(in Chinese)刘璨颖.猪源肠外致病性大肠杆菌比较基因组学和亚单位疫苗的研究[D].武汉:华中农业大学博士学位论文,2014
[3]Bengtsson S,Naseer U,Sundsfjord A,et al.Sequence types and plasmid carriage of uropathogenic Escherichia coli devoid of phenotypically detectable resistance[J].Journal of Antimicrobial Chemotherapy,2012,67(1):69-73
[4]Hammerum AM,Larsen J,Andersen VD,et al.Characterization of extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli obtained from danish pigs,pig farmers and their families from farms with high or no consumption of third-or fourth-generation cephalosporins[J].Journal of Antimicrobial Chemotherapy,2014,69(10):2650-2657
[5]Duan SS.The dissemination mechanism and genetic characteristics of CTX-M clusters among Avian Escherichia coli[D].Zhengzhou:Master’s Thesis of Henan Agricultural University,2015(in Chinese)段沙沙.禽大肠埃希菌不同CTX-M亚群的散播机制及遗传特性[D].郑州:河南农业大学硕士学位论文,2015
[6]MarcadéG,Deschamps C,Boyd A,et al.Replicon typing of plasmids in Escherichia coli producing extended-spectrumβ-lactamases[J].Journal of Antimicrobial Chemotherapy,2009,63(1):67-71
[7]Villa L,García-Fernández A,Fortini D,et al.Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants[J].Journal of Antimicrobial Chemotherapy,2010,65(12):2518-2529
[8]Osborn AM,da Silva Tatley FM,Steyn LM,et al.Mosaic plasmids and mosaic replicons:evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons[J].Microbiology,2000,146(9):2267-2275
[9]Szczepanowski R,Braun S,Riedel V,et al.The 120 592 bp IncFplasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants,two iron-acquisition systems and other putative virulence-associated functions[J].Microbiology,2005,151(4):1095-1111
[10]Womble DD,Rownd RH.Genetic and physical map of plasmid NR1:comparison with other IncFII antibiotic resistance plasmids[J].Microbiological Reviews,1988,52(4):433-451
[11]Cavalli LL,Lederberg J,Lederberg EM.An infective factor controlling sex compatibility in Bacterium coli[J].Microbiology,1953,8(1):89-103
[12]Zhan Z,Feng J,Yin Z,et al.Antibiotic-resistant mechanisms of plasmid pA1137 carrying aminoglycoside resistance gene aac C2[J].Military Medical Sciences,2017,41(12):973-977(in Chinese)占喆,冯娇,殷喆,等.携带氨基糖苷类耐药基因aacC2的质粒pA1137耐药机制研究[J].军事医学,2017,41(12):973-977
[13]Zhao YC,Wang LJ,Zhang ZY,et al.Structural genomics of pNDM-BTR harboring In191 and Tn6360,and other blaNDM-carrying IncN1 plasmids[J].Future Microbiology,2017,12(14):1271-1281
[14]Frank JA,Reich CI,Sharma S,et al.Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNAgenes[J].Applied and Environmental Microbiology,2008,74(8):2461-2470
[15]Poirel L,Walsh TR,Cuvillier V,et al.Multiplex PCR for detection of acquired carbapenemase genes[J].Diagnostic Microbiology and Infectious Disease,2011,70(1):119-123
[16]Dallenne C,Da Costa A,DecréD,et al.Development of a set of multiplex PCR assays for the detection of genes encoding importantβ-lactamases in Enterobacteriaceae[J].Journal of Antimicrobial Chemotherapy,2010,65(3):490-495
[17]Ciesielczuk H,Hornsey M,Choi V,et al.Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants[J].Journal of Medical Microbiology,2013,62(12):1823-1827
[18]Phuc Nguyen MC,Woerther PL,Bouvet M,et al.Escherichia coli as reservoir for macrolide resistance genes[J].Emerging Infectious Diseases,2009,15(10):1648-1650
[19]Maynard C,Fairbrother JM,Bekal S,et al.Antimicrobial resistance genes in enterotoxigenic Escherichia coli O149:K91isolates obtained over a 23-year period from pigs[J].Antimicrobial Agents and Chemotherapy,2003,47(10):3214-3221
[20]Clinical and Laboratory Standards Institute.M100-S27 Performance standards for antimicrobial susceptibility testing:twenty-seventh informational supplement[Z].Wayne,PA,USA:CLSI,2017
[21]Margulies M,Egholm M,Altman WE,et al.Genome sequencing in microfabricated high-density picolitre reactors[J].Nature,2005,437(7057):376-380
[22]Aziz RK,Bartels D,Best AA,et al.The RAST server:rapid annotations using subsystems technology[J].BMC Genomics,2008,9(1):75
[23]Boratyn GM,Camacho C,Cooper PS,et al.BLAST:a more efficient report with usability improvements[J].Nucleic Acids Research,2013,41:W29-W33
[24]O’Leary NA,Wright MW,Brister JR,et al.Reference sequence(RefSeq)database at NCBI:current status,taxonomic expansion,and functional annotation[J].Nucleic Acids Research,2016,44(D1):D733-D745
[25]Boutet E,Lieberherr D,Tognolli M,et al.UniProtKB/Swiss-Prot,the manually annotated section of the UniProt KnowledgeBase:how to use the entry view[A]//Plant Bioinformatics.Methods in Molecular Biology[M].New York:Humana Press,2016:23-54
[26]Siguier P,Perochon J,Lestrade L,et al.ISfinder:the reference centre for bacterial insertion sequences[J].Nucleic Acids Research,2006,34(suppl 1):D32-D36
[27]Moura A,Soares M,Pereira C,et al.INTEGRALL:a database and search engine for integrons,integrases and gene cassettes[J].Bioinformatics,2009,25(8):1096-1098
[28]Roberts AP,Chandler M,Courvalin P,et al.Revised nomenclature for transposable genetic elements[J].Plasmid,2008,60(3):167-173
[29]Wibberg D,Szczepanowski R,Eikmeyer F,et al.The IncFplasmid pRSB225 isolated from a municipal wastewater treatment plant’s on-site preflooder combining antibiotic resistance and putative virulence functions is highly related to virulence plasmids identified in pathogenic E.coli isolates[J].Plasmid,2013,69(2):127-137
[30]Partridge SR,Hall RM.Complex multiple antibiotic and mercury resistance region derived from the r-det of NR1(R100)[J].Antimicrobial Agents and Chemotherapy,2004,48(11):4250-4255
[31]Cain AK,Liu XL,Djordjevic SP,et al.Transposons related to Tn1696 in IncHI2 plasmids in multiply antibiotic resistant Salmonella enterica serovar typhimurium from Australian animals[J].Microbial Drug Resistance,2010,16(3):197-202
[32]Chalmers R,Sewitz S,Lipkow K,et al.Complete nucleotide sequence of Tn10[J].Journal of Bacteriology,2000,182(10):2970-2972
[33]Partridge SR.Analysis of antibiotic resistance regions in Gram-negative bacteria[J].FEMS Microbiology Reviews,2011,35(5):820-855
[34]Zong ZY,Partridge SR,Iredell JR.ISEcp1-mediated transposition and homologous recombination can explain the context of blaCTX-M-62 linked to qnrB2[J].Antimicrobial Agents and Chemotherapy,2010,54(7):3039-3042
[35]Bailey JK,Pinyon JL,Anantham S,et al.Distribution of the blaTEM gene and bla TEM-containing transposons in commensal Escherichia coli[J].Journal of Antimicrobial Chemotherapy,2011,66(4):745-751