肠杆菌科细菌去阻遏持续高产Amp C酶的检测与amp D基因突变的研究

Detection of AmpC-derepressed enzyme-stable hyperproducer and analysis of ampD gene mutation of Enterobacteriaceae

目的 了解ampD基因突变与持续高产AmpC酶的关系。方法 对 5 3株细菌用三维试验方法检测AmpC酶 ,进行等电点测定和ampD基因扩增 ,2株测定核苷酸序列。结果 32株阴沟肠杆菌中 9株检出AmpC酶 ,检出率为 2 8% ,1 2株枸橼酸杆菌有 3株检出 ,检出率为 2 5 %。其余细菌没有检出。能被邻氯西林抑制的条带主要有 7 96、8 7、8 8和 8 9。有 2株三维试验阳性而ampD基因扩增阴性。对 2株亚胺培南中介其余抗生素耐药的细菌ampD基因测序 ,结果突变主要发生在表型的C′ 末端。阴沟肠杆菌主要在 1 0 1、1 4 9、1 5 5、1 6 6位氨基酸发生改变 ,枸橼酸杆菌主要在 95、1 5 8、1 6 4位氨基酸发生改变。结论 ampD基因突变可导致AmpC酶C′ 端氨基酸改变 ,从而使AmpC酶去阻遏持续高产 ,并导致细菌对 β 内酰胺类抗生素耐药。

Objective To detect AmpC derepressed enzyme in Enterobacter cloacae, Citrobacter freundii , Serratia and Morganella morganii by three dimensional extract test and analyze the variant of phenotypes due to the mutations within amp D gene resulting in derepressed AmpC enzyme stable hyperproducer.Methods derepressed AmpC enzyme in 53 strains of Enterobacteriaceae was detected by three dimensional test and the isoelectric points of enzyme were measured.The amp D gene was detected in the 12 positive strains by PCR and the nucleotides in 2 of them were sequenced.Results Positive derepressed AmpC enzyme was found in 9 of 32 strains of Enterobacter cloacae (28%) and in 3 of 12 strains of Citrobacter freundii (25%),but undetectable in the other strains of Enterobacteriaceaes .The determination of isoelectric points showed that 7.96, 8.7, 8.8 and 8.9 of strips were inhibited by cloxacillin.The sequencing of PCR products of amp D gene from 2 strains which was intermediate by imipenem (IPM) but resistant to other β Lactam antibiotics showed the mutation predominantly happened at the C terminal of phenotypes.The variation of amino acid sequence was found at 101,149,155 and 166 residue in Enterobacter cloacae and at 95,158 and 164 residue in Citrobacter freundii .Conclusion The mutant of amp D gene may result in variation of amino acid sequence at the C terminal,thus expression of AmpC β Lactamase was derepressed and stably hyperproduced.It leads Enterobacteriaceae to be highly resistant to β lactams antibiotics.Therefore three dimensional extract test is necessary for detection of AmpC enzyme in clinical laboratories.