SHV-16, a β-lactamase with a pentapeptide duplication in the omega loop
SHV-16, a β-lactamase with a pentapeptide duplication in the omega loop
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DOI：
10.1128/aac.45.9.2480-2485.2001
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发表时间：
2001-09-01
影响因子：
4.9
通讯作者：
Quentin, C
中科院分区：
文献类型：
--
作者：
Arpin, C;Labia, R;Quentin, C
临床分离的肺炎克雷伯菌对氨苄西林(MIC)、替卡西林(MIC512)和头孢他啶(MIC128)耐药，对所有其他β-内酰胺类药物敏感；克拉维酸和头孢他啶的协同作用提示存在超广谱β-内酰胺酶(ESBL)。在大肠杆菌中获得了低水平的转接子(每个供体细胞10(-7)个)，并表现出类似的β-内酰胺耐药模式(对氨苄西林、替卡西林和头孢他啶的耐药率为µg/ml)。超广谱β-内酰胺酶(ESBL)的pI为7.6，由一个大的、不携带任何其他耐药决定簇的质粒(~gt；100kb)编码。利用BLA(SHV)特异性引物，通过聚合酶链式反应扩增出ESBL编码基因，并进行了序列测定。推导的氨基酸序列表明，SHV-16ESBL与SHV-1只有一个五肽插入(163DRWET167)，对应于omega环中的串联复制。将163a-DRWET163b-DRWET序列克隆到同一载体中，并导入同一菌株中，证实了163a-DRWET163b-DRWET序列在头孢他啶耐药中的意义。在此等基因条件下，SHV-16的头孢他啶MIC是SHV-1的32倍。此外，E166aA或E166bA的定点突变实验表明，功能性谷氨酸残基位于复制序列的第一个拷贝中。但令人惊讶的是，第二个E166b也对头孢他啶产生了低水平的耐药性。这项工作是首次在临床分离株中描述一种A类酶，由于插入而不是核苷酸替代(S)而表现出延长的底物专一性。
A clinical isolate of Klebsiella pneumoniae was found to be resistant to ampicillin (MIC of 128 mug/ml), ticarcillin (MIC of 512 mug/ml), and ceftazidime (MIC of 128 mug/ml) and susceptible to all other P-lactams; a synergistic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum P-lactamase (ESBL). Transconjugants in Escherichia coli were obtained at low levels (10(-7) per donor cell) and exhibited a similar beta -lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 mug/ml). The ESBL, pI 7.6, was encoded by a large plasmid (> 100 kb) which did not carry any other resistance determinant. The ESBL-encoding gene was amplified by PCR using bla(SHV)-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that it differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corresponding to a tandem duplication in the omega loop. The implication of the 163a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by cloning either bla(SHV-1) or bla(SHV-16) in the same vector, subsequently introduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1. Furthermore, site-directed mutagenesis experiments modifying either E166aA or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is the first description of a class A enzyme exhibiting an extended substrate specificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.