Luan Ying, Li Gui-Ling, Duo Li-Bo, Wang Wei-Ping, Wang Cheng-Ying, Zhang He-Guang, He Fei, He Xin, Chen Shu-Juan, Luo Dan-Ting
Department of Medicine Laboratory, Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086, P.R. China.
Medicine Laboratory, Department of Urology Surgery, Daqing Oilfield General Hospital, Daqing, Heilongjiang 163001, P.R. China.
Mol Med Rep. 2015 Apr;11(4):3069-77. doi: 10.3892/mmr.2014.3054. Epub 2014 Dec 5.
The present study aimed to investigate the regulatory mechanism of the AmpC enzyme by analyzing the construction and function of AmpCR, AmpE and AmpG genes in the Dhahran (DHA)‑1 plasmid of Klebsiella pneumoniae (K. pneumoniae). The production of AmpC and extended‑spectrum β‑lactamase (ESBL) were determined following the cefoxitin (FOX) inducing test for AmpC, preliminary screening and confirmation tests for ESBL in 10 DHA‑1 plasmid AmpC enzymes of K. pneumoniae strains. AmpCR, AmpD, AmpE and AmpG sequences were analyzed by polymerase chain reaction. The pACYC184‑X plasmid analysis system was established and examined by regulating the pAmpC enzyme expression. The electrophoretic bands of AmpCR, AmpD, AmpE and AmpG were expressed. Numerous mutations in AmpC + AmpR (AmpCR) and in the intergenic region cistron of AmpC‑AmpR, AmpD, AmpE and AmpG were observed. The homology of AmpC and AmpR, in relation to the Morganella morganii strain, was 99%, which was determined by comparing the gene sequences of Kp1 with those of Kp17 AmpCR. The specific combination of AmpR and labeled probe demonstrated a band retarded phenomenon and established a spatial model of AmpR. All the enzyme production strains demonstrated Val93→Ala in AmpG; six transmembrane domains were found in AmpE in all strains, with the exception of Kp1 and Kp4, which had only three transmembrane segments that were caused by mutation. The DHA‑1 plasmid AmpC enzymes encoded by plasmid are similar to the inducible chromosomal AmpC enzymes, which are also regulated by AmpD, AmpE, AmpR and AmpG.
本研究旨在通过分析肺炎克雷伯菌(K. pneumoniae)达兰(DHA)-1质粒中AmpCR、AmpE和AmpG基因的结构与功能,探讨AmpC酶的调控机制。对10株肺炎克雷伯菌菌株的DHA-1质粒AmpC酶进行头孢西丁(FOX)诱导试验以检测AmpC的产生,并进行超广谱β-内酰胺酶(ESBL)的初步筛选和确认试验。通过聚合酶链反应分析AmpCR、AmpD、AmpE和AmpG序列。建立pACYC184-X质粒分析系统,并通过调节pAmpC酶表达进行检测。表达AmpCR、AmpD、AmpE和AmpG的电泳条带。观察到AmpC + AmpR(AmpCR)以及AmpC-AmpR、AmpD、AmpE和AmpG基因间区域顺反子存在大量突变。通过比较Kp1与Kp17 AmpCR的基因序列,确定AmpC和AmpR与摩根摩根菌菌株的同源性为99%。AmpR与标记探针的特异性结合表现出条带阻滞现象,并建立了AmpR的空间模型。所有产酶菌株在AmpG中均表现出Val93→Ala;除Kp1和Kp4仅有三个跨膜区段是由突变导致外,所有菌株的AmpE中均发现有六个跨膜结构域。质粒编码的DHA-1质粒AmpC酶与诱导型染色体AmpC酶相似,其也受AmpD、AmpE、AmpR和AmpG调控。