Podbielski A, Schönling J, Melzer B, Warnatz K, Leusch H G
Institute of Medical Microbiology, University of Aachen, FRG.
J Gen Microbiol. 1991 Mar;137(3):569-78. doi: 10.1099/00221287-137-3-569.
Between 1986 and 1988, multiresistant Klebsiella pneumoniae strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMP1. The MIC values for cefotaxime of the original isolates and the transconjugants were greater than 128 mg l-1 and 64 mg l-1, respectively. Isoelectric focusing of protein preparations from the transconjugants showed a beta-lactamase with a pI of 7.6. A 3.6 kb BamHI fragment containing the beta-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and PstI subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3' noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5' upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the beta-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available beta-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant beta-lactamase of the SHV-type and name it SHV-2a.
1986年至1988年间，从亚琛工业大学医院重症监护病房患者标本中分离出对头孢噻肟呈现高水平耐药的多重耐药肺炎克雷伯菌菌株。研究表明，负责耐药性的基因编码在一个名为pZMP1的66 kb接合性质粒上。原始分离株和转接合子对头孢噻肟的MIC值分别大于128 mg/L和64 mg/L。对转接合子蛋白质制剂进行等电聚焦分析，显示出一种pI为7.6的β-内酰胺酶。将一个包含β-内酰胺酶基因的3.6 kb BamHI片段克隆到pLG339中，得到重组质粒pZMP1-1。构建了克隆插入片段的限制性图谱，并将插入片段的PstI亚片段进一步亚克隆到pBGS18中。测定了完整3.6 kb片段的核苷酸序列。在3663 bp内发现一个858 kb的开放阅读框，与SHV-2和-3核苷酸序列具有99%的同源性。推导的氨基酸序列与这些已确定的SHV酶在一个和两个位置上分别不同。3'非编码序列与SHV-2的非编码序列几乎完全同源，但5'上游序列与相应的SHV-2序列的同源性小于50%，表明该变异SHV酶的启动子区域发生了改变。对β-内酰胺酶的动力学分析表明，与其他SHV酶相比，其对头孢噻肟的水解效率提高了50-100%。该酶不水解头孢西丁、头孢他啶、氨曲南和亚胺培南。该变异酶被常用的β-内酰胺酶抑制剂所抑制。克拉维酸对该酶具有最高的亲和力，在阻断其作用方面效果最佳。基于遗传和动力学数据，我们建议将该酶归类为SHV型新的变异β-内酰胺酶，并将其命名为SHV-2a。
