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Abstract：
This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp).
Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted.
Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105.
In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.
Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted.
Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105.
In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.
摘要：
■这项研究旨在揭示非碳青霉烯酶产生的高毒力肺炎克雷伯菌（hvKp）中头孢他啶-阿维巴坦敏感性损失和获得的潜在机制。
■在这里，我们以一个月的间隔从一名老年男性中纵向回收了3种不产生碳青霉烯酶的K1-ST23hvKp菌株（KP29105，KP29499和KP30086）。抗菌药物敏感性试验,全基因组测序，转录组测序，基因克隆,质粒接合，实时定量PCR(qRT-PCR),进行SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)。
■在3个hvKp菌株中，KP29105对第三代和第四代头孢菌素耐药，KP29499获得了对头孢他啶-阿维巴坦和碳青霉烯类的抗性，而KP30086恢复了对头孢他啶-阿维巴坦的敏感性，亚胺培南和美罗培南，但保留了对厄他培南的低水平抗性。KP29105和KP29499分别携带质粒编码基因blaCTX-M-15和blaCTX-M-71,但是KP30086两个都输了.基因blaCTX-M-71的克隆和携带blaCTX-M-71的质粒的接合实验表明，转化体和转接合体对头孢他啶-阿维巴坦敏感，但MIC增加了8倍以上。补充外膜渗透剂可使头孢他啶-阿维巴坦的MIC降低32倍，表明孔蛋白在头孢他啶-阿维巴坦耐药性中起关键作用。3个分离株的OmpK35不表达，KP29499和KP30086的OmpK36具有新的氨基酸取代(L359R)。SDS-PAGE和qRT-PCR显示KP29499和KP30086的孔蛋白OmpK36的表达较KP29105显著下调。
■总之，我们报道了在不产生碳青霉烯酶的hvKp菌株中罕见的头孢他啶-阿维巴坦抗性。携带blaCTX-M-71和突变的OmpK36的抗性质粒对抗性具有协同作用。
■在这里，我们以一个月的间隔从一名老年男性中纵向回收了3种不产生碳青霉烯酶的K1-ST23hvKp菌株（KP29105，KP29499和KP30086）。抗菌药物敏感性试验,全基因组测序，转录组测序，基因克隆,质粒接合，实时定量PCR(qRT-PCR),进行SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)。
■在3个hvKp菌株中，KP29105对第三代和第四代头孢菌素耐药，KP29499获得了对头孢他啶-阿维巴坦和碳青霉烯类的抗性，而KP30086恢复了对头孢他啶-阿维巴坦的敏感性，亚胺培南和美罗培南，但保留了对厄他培南的低水平抗性。KP29105和KP29499分别携带质粒编码基因blaCTX-M-15和blaCTX-M-71,但是KP30086两个都输了.基因blaCTX-M-71的克隆和携带blaCTX-M-71的质粒的接合实验表明，转化体和转接合体对头孢他啶-阿维巴坦敏感，但MIC增加了8倍以上。补充外膜渗透剂可使头孢他啶-阿维巴坦的MIC降低32倍，表明孔蛋白在头孢他啶-阿维巴坦耐药性中起关键作用。3个分离株的OmpK35不表达，KP29499和KP30086的OmpK36具有新的氨基酸取代(L359R)。SDS-PAGE和qRT-PCR显示KP29499和KP30086的孔蛋白OmpK36的表达较KP29105显著下调。
■总之，我们报道了在不产生碳青霉烯酶的hvKp菌株中罕见的头孢他啶-阿维巴坦抗性。携带blaCTX-M-71和突变的OmpK36的抗性质粒对抗性具有协同作用。
