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

【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,该质粒在动物源奇异变形杆菌的传播扩散进一步增加了治疗该菌感染的难度,应引起足够的重视。

【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 region,antibiotic resistance island ARI-B,carried floR,tet(A),strA,strB,and sul2 genes;the second region,ISEcp1-bla_(CMY-2)-blc-sugE,was a typical structure,and the ISEcp1 was truncated by IS10R;the third region,ARI-A,was a hybrid Tn1696tnp-pDUmer module.The ARI-A contained a sul1-containing class 1 integron with cassette array(aac(6')-Ib-cr|arr3|dfrA27|aadA16),and a mercury resistance cluster merEDBAPTR,and inserted into the plasmid backbone generating 5-bp direct repeats(TTGTA).【Conclusion】All the AmpC-producing P.mirabilis strains carried the bla_(CMY-2) gene,and one of them harbored an epidemic type 1 IncC conjugative plasmid.Three PFGE patterns were identified.The findings demonstrated the vertical and horizontal dissemination of bla_(CMY-2) gene in poultry Proteus mirabilis isolates.IncC plasmid was one of the predominant vehicles for the dissemination of multiple resistance genes,such as bla_(CMY-2),tet(A),floR or class 1 integron cassette,which further increased the difficulty for the treatment of the infection caused by P.mirabilis.More attention should be paid on the epidemiology of IncC plasmid in pathogenic bacteria.