肺炎克雷伯菌耐药基因和毒力基因传播机制

Study on transmission mechanisms of resistance gene and virulence gene of Klebsiella pneumoniae

    摘要
  • 摘要:
    目的 研究高毒力肺炎克雷伯菌(HvKP)的毒力基因与耐碳青霉烯肺炎克雷伯菌(CRKP)耐药基因的横向转移机制及其遗传演化特征。
    方法 供体菌为1株同时含VIM、KPC耐药基因的CRKP, 阳性对照菌为1株含blaIMP-4CRKP, 受体菌为大肠埃希菌J53, 通过质粒接合实验验证耐药基因的水平转移能力。收集暨南大学附属第一医院2017-2021年50株HvKP菌株, 将50株HvKP菌株与具有介导耐药基因传播能力的5株CRKP菌株进行质粒接合试验, 通过含有美罗培南抗菌药物的选择性培养基筛选质粒接合成功的接合菌, 采用聚合酶链式反应(PCR)检测接合菌的耐药基因和毒力基因, 通过大蜡螟幼虫实验验证接合菌的致死性。
    结果 供体菌CRKP具有介导耐药基因传播的能力, 其携带的blaKPC基因成功转移至大肠埃希菌J53(但VIM基因转移失败), 即共筛选到5株具有介导耐药基因传播能力的CRKP菌, 其中1株携带VIM、KPC耐药基因, 另外4株均携带IMP-4耐药基因；只有1株含blaIMP-4耐药基因的CRKP与HvKP菌接合成功, 接合菌同时含有毒力基因(aerobactin和RmpA)和耐药基因blaIMP-4, 且拉丝实验阳性。大蜡螟幼虫实验显示接合菌在24 h内导致全部幼虫死亡。
    结论 医院环境中, CRKP与HvKP可通过质粒接合形成兼具耐药性和高毒力的“超级细菌”, 对临床抗感染治疗构成严峻挑战, 需加强院感防控。

     

    Abstract:
    OBJECTIVE To investigate the horizontal transfer mechanisms and genetic evolutionary characteristics of virulence genes in hypervirulent Klebsiella pneumoniae (HvKP) and resistance genes in carbapenem-resistant K. pneumoniae (CRKP).
    METHODS The donor strain was a CRKP strain harboring both VIM and KPC resistance genes. The positive control was a CRKP strain containing blaIMP-4. The recipient strain was Escherichia coli J53. The horizontal transfer capability of the resistance genes was verified by plasmid conjugation assay. Fifty HvKP strains were collected from the First Affiliated Hospital of Jinan University from 2017 to 2021 and conjugated with the plasmids of five CRKP strains which could mediate the transmission of drug resistance genes. The conjugants were screened by selective medium containing meropenem, and polymerase chain reaction (PCR) was used to detect the resistance and virulence genes in the conjugation-successful strains. The lethality of conjugants was verified by Galleria mellonella larval assays.
    RESULTS The donor CRKP strain had the ability to mediate resistance gene transmission. Its blaKPC gene was successfully transferred to E. coli J53, but the transference of the VIM gene failed. Five CRKP strains with the ability to mediate resistance gene transmission were screened, of which one strain carried both VIM and KPC resistance genes, and the other four strains carried the IMP-4 resistance gene. Only one CRKP strain with the blaIMP-4 resistance gene was successfully conjugated with HvKP, carrying both virulence genes (aerobactin and RmpA) and the blaIMP-4 resistance gene, with positive wire drawing test result. The Galleria mellonella larvae test showed that the conjugation-successful strain caused the death of all larvae within 24 hours.
    CONCLUSIONS In the hospital environment, CRKP and HvKP can form "superbugs" with both drug resistance and high virulence through plasmid conjugation, posing a severe challenge to clinical anti-infection treatment. It is necessary to strengthen the prevention and control of hospital-acquired infections.

     

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