Investigation on the mechanisms of porins OmpK35 and OmpK36 impacting antimicrobial susceptibility of hypervirulent Klebsiella pneumoniae

Objective To investigate the gene background of porin OmpK35/36 in Klebsiella pneumoniae type K1 (KP), construct the deletion mutant of OmpK35/36 and analyze the effect of OmpK35/36 on the antimicrobial sensitivity of Klebsiella pneumoniae type K1.Methods The whole KP genome in GenBanrk was collected, then the distribution of porins and the polymorphism of gene and protein sequences in Klebsiella pneumoniae were analyzed through BLAST. Single and double knocking of ompK35/36 was performed on K1 strain NTUH-K2044 by CRISPR-Cas9 knockout method, and the changes of antibiotic sensitivity before and after knockout were measured.Results ompK35/36 was found to be common in KP genome with homology of more than 90%. After OmpK36 deletion, the minimum inhibitory concentration (MIC) of NTUH-K2044 against piperacillin, cefoxitin, cefazolin, cefuroxime and imipenem increased by 4, 4, 4, 8 and 4 times respectively. After OmpK35/36 double deletion, the MIC of piperacillin, cefoxitin, cefazolin, cefuroxime, imipenem and meropenem increased by 8, 32, 32, 16, 8 and 8 times respectively.Conclusions OmpK35/36 is common in Klebsiella pneumoniae with conserved sequence. Loss of OmpK35/36 leads to increased resistance of strains to certain beta-lactam antimicrobials, and when additional OmpK35 is further lost on basis of the loss of OmpK36, MIC will be further increased.

目的 探究K1型肺炎克雷伯菌（Klebsiella pneumoniae type K1, KP）中孔蛋白OmpK35/36的基因背景，构建OmpK35/36缺失突变株，并分析OmpK35/36对K1型肺炎克雷伯菌抗菌药物敏感性的影响。
方法 收集GenBanrk中的所有KP基因组序列，通过BLAST分析肺炎克雷伯菌中孔蛋白的分布特征及基因与蛋白序列的多态性；采用CRISPR-Cas9敲除技术对K1型菌株NTUH-K2044进行ompK35/36单基因及双基因敲除，并测定敲除前后菌株对抗菌药物敏感性的变化。
结果 发现ompK35/36在KP基因组中广泛存在，同源性超过90%；OmpK36缺失后，NTUH-K2044对哌拉西林、头孢西丁、头孢唑林、头孢呋辛及亚胺培南的最低抑菌浓度（minimum inhibitory concentration, MIC）分别升高4、4、4、8及4倍；OmpK35/36双缺失后，菌株对哌拉西林、头孢西丁、头孢唑林、头孢呋辛、亚胺培南及美罗培南的MIC分别升高8、32、32、16、8及8倍。
结论 OmpK35/36在肺炎克雷伯菌中广泛存在且序列保守；OmpK35/36缺失会导致菌株对部分β-内酰胺类抗菌药物的耐药性增强，且在OmpK36缺失的基础上进一步缺失OmpK35时，MIC会进一步升高。