禽源奇异变形杆菌质粒介导AmpC酶基因型检测与质粒分析  

Analysis of Plasmid-Mediated AmpCβ-lactamases Gene and Plasmid in Poultry Proteus mirabilis Strains

作　　者：赵世玉 焦嘉杰 董宁宁 潘圆月 崔孟梅 潘玉善[1] ZHAO ShiYu;JIAO JiaJie;DONG NingNing;PAN YuanYue;CUI MengMei;PAN YuShan(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002;College of Life Sciences,Henan Agricultural University,Zhengzhou 450002)

机构地区：[1]河南农业大学牧医工程学院,郑州450002 [2]河南农业大学生命科学学院,郑州450002

出　　处：《中国农业科学》2021年第17期3780-3788,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金-河南人才培养联合基金(U1504326);河南农业大学本科教学实验室开放创新训练项目。

摘　　要：【目的】研究禽源奇异变形杆菌携带ampC基因的分型和bla_(CMY-2)阳性接合质粒pC12的序列结构,为防控多重耐药禽源奇异变形杆菌的传播提供理论基础。【方法】利用头孢西丁三维试验和PCR方法对21株奇异变形杆菌进行AmpC酶的检测和基因分型研究;对bla_(CMY-2)阳性菌株进行脉冲场凝胶电泳(PFGE)分型和接合试验;利用高通量测序技术获得pC12的核苷酸序列并进行比较分析。【结果】头孢西丁三维试验表明21株禽源奇异变形杆菌中有6株产AmpC酶。6株产AmpC酶奇异变形杆菌对氨苄西林、头孢西丁、多西环素、氟苯尼考、粘菌素全部耐药,而对头孢他啶、阿米卡星全部敏感。耐药基因PCR扩增和测序结果表明,6株菌均携带bla_(CMY-2),检出率为28.6%。接合试验表明,1株奇异变形杆菌C12接合成功并获得接合子,其余5株接合不成功。PFGE分型结果表明,酶切图谱分为3个型别,bla_(CMY-2)在禽源变形杆菌中存在垂直和水平传播。测序结果表明菌株C12含有一个1b型IncC质粒,其全长161319 bp,GC含量为52.45%,预测有161个开放阅读框,提交NCBI并获得序列号MT320534。该质粒包含3个耐药区:第一个抗生素耐药区(antibiotic resistance islands,ARI-B)携带floR、tet(A)、strA、strB和sul2;第二个耐药区是一个典型的ISEcp1-bla_(CMY-2)-blc-sugE结构,其中的ISEcp1被一个插入的IS10R截断;第三个耐药区(ARI-A)是一个杂合的Tn1696tnp-pDUmer转座子,包含一个1类整合子基因盒(aac(6')-Ib-cr|arr3|dfrA27|aadA16)和汞抗性基因簇merEDBAPTR,其插入到质粒骨架产生两个重复序列TTGTA,该耐药区也是1型IncC耐药区变化最大的区域。【结论】6株产AmpC酶禽源奇异变形杆菌均携带bla_(CMY-2),其酶切图谱分为3个PFGE型别,其中一株菌携带了一个流行的bla_(CMY-2)阳性1型IncC可接合质粒。广宿主的IncC质粒是bla_(CMY-2)、tet(A)、floR等多个耐药基因及整合子的重要载体之一,该质粒�【Objective】The aim of this study was to probe the genotype of AmpCβ-lactamases gene and the complete nucleotide sequence of the conjugative plasmid carrying bla_(CMY-2) in poultry P.mirabilis strains,so as to provide a theoretical basis for the prevent spreading of multidrug-resistant poultry P.mirabilis strains.【Method】Twenty-one P.mirabilis strains were characterized for the confirmation of AmpCβ-lactamases genes by using three-dimensional test,polymerase chain reaction(PCR)amplification and sequencing.The bla_(CMY-2)-carrying P.mirabilis strains were further evaluated using pulse field gel electrophoresis(PFGE)and conjugation experiments.The complete nucleotide sequence of conjugative plasmid pC12 was determined by using high-throughput sequencing platform and compared with closely related plasmids.【Result】Six of twenty-one P.mirabilis strains produced AmpC enzymes,all of which carried the bla_(CMY-2) gene and the detection rate was 28.6%.Antimicrobial susceptibility testing showed that six P.mirabilis strains exhibited high resistance to ampicillin,cefoxitin,doxycycline,florfenicol and colistin,but were susceptible to ceftazidime and amikacin.Conjugation assay revealed the bla_(CMY-2) gene was successfully transferred from P.mirabilis C12 to E.coli C600 recipient strain,however,conjugation experiments failed to obtain transconjugants for other bla_(CMY-2)-bearing strains,despite repeated attempts.Three PFGE patterns of six P.mirabilis strains were determined.The findings demonstrated the vertical and horizontal dissemination of bla_(CMY-2) gene in poultry P.mirabilis isolates.Sequence analysis revealed the P.mirabilis C12 harbored a conjugative plasmid,designated as pC12.pC12 was found to be a multi-drug resistant type 1b IncC plasmid with 161319-bp size and an average GC content of 52.45%,and had at least 161 predicted open reading frames.The complete sequence of pC12 has been submitted to GenBank with the accession number MT320534.The pC12 harbored three antibiotic resistance regions:the first

关 键 词：奇异变形杆菌 IncC质粒 AMPCΒ-内酰胺酶 bla_(CMY-2) Tn1696转座子 

分 类 号：S852.6[农业科学—基础兽医学]