Li Guiling, Wang Li, Zhang Heguang, Luan Ying, Sun Qi, Duo Libo
Department of Clinical Laboratory, the Second Hospital Affiliated to Harbin Medical University, Harbin, Heilongjiang, 150086, People's Republic of China.
Department of Clinical Laboratory, School of Medicine, Chengdu Women's and Children's Central Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan, 611731, People's Republic of China.
Infect Drug Resist. 2023 Aug 24;16:5587-5598. doi: 10.2147/IDR.S421598. eCollection 2023.
In this study, we constructed knock-out and knock-in strains from a clinically isolated Kp1strain carrying in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between and -induced expression.
We created the gene deletion mutant strains Kp1-Δ and Kp NTUH-K2044-Δ with pKO3-km plasmid using homologous recombination technology. We constructed the Kp NTUH-K2044-RC and Kp NTUH-K2044-Δ-RC drug resistance model strains with plasmid pACYC184. We constructed the knock-in strains by introducing the genes of Kp1, 029M, PAO1, ATCC25922, and LT2 into the gene-deleted strains with carrier pet-30a. Real-time polymerase chain reaction (real-time PCR) was used to detect the relative expressions of and mRNAs.
Compared with Kp1, the induction phenotype of the of Kp1-Δ strain disappeared, the expression was reduced, and the minimal inhibitory concentration (MIC) values of cefoxitin and ceftazidime significant decrease from 128 μg/mL to 1 μg/mL. Based on Kp1, five strain were successfully constructed to complement the genes from five knock-in strain, and all of the above complemented strains showed inducible expression of and restored the expression of to varying degrees, as well as restored resistance to the antimicrobial drugs cefoxitin and ceftazidime ( < 0.05). The and genes were barely expressed in Kp NTUH-K2044-ΔG-RC when compared with Kp NTUH-K2044-RC. The expressions of and in each knock-in strain were recovered, the induction phenotype of was restored, and the MIC values of cefoxitin and ceftazidime were increased. ( < 0.05).
In this study, we found that was an essential regulator for the plasmid-mediated induced expression in .
在本研究中，我们从一株临床分离的携带质粒的肺炎克雷伯菌1型菌株构建了基因敲除和基因敲入菌株，并将它们与肺炎克雷伯菌NTUH-K2044菌株进行比较，以研究与诱导表达之间的关系。
我们使用同源重组技术，用pKO3-km质粒创建了基因缺失突变株Kp1-Δ和Kp NTUH-K2044-Δ。我们用质粒pACYC184构建了Kp NTUH-K2044-RC和Kp NTUH-K2044-Δ-RC耐药模型菌株。我们通过将肺炎克雷伯菌1型、029M、铜绿假单胞菌PAO1、大肠埃希菌ATCC25922和鼠伤寒沙门菌LT2的基因导入带有载体pet-30a的基因缺失菌株中，构建了基因敲入菌株。采用实时聚合酶链反应（实时PCR）检测和mRNA的相对表达。
与肺炎克雷伯菌1型相比，Kp1-Δ菌株的诱导表型消失，表达降低，头孢西丁和头孢他啶的最低抑菌浓度（MIC）值从128μg/mL显著降至1μg/mL。基于肺炎克雷伯菌1型，成功构建了5株菌株以补充来自5株基因敲入菌株的基因，所有上述互补菌株均表现出的诱导表达，并不同程度地恢复了的表达，以及恢复了对头孢西丁和头孢他啶的抗菌药物耐药性（<0.05）。与Kp NTUH-K2044-RC相比，Kp NTUH-K2044-ΔG-RC中的和基因几乎不表达。每个基因敲入菌株中的和表达均得以恢复，的诱导表型得以恢复，头孢西丁和头孢他啶的MIC值升高。（<0.05）。
在本研究中，我们发现是质粒介导的肺炎克雷伯菌诱导表达的关键调节因子。
