Schmidtke Amber J, Hanson Nancy D
Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE 68178, USA.
Antimicrob Agents Chemother. 2006 Jun;50(6):2030-7. doi: 10.1128/AAC.01458-05.
Mutations within the structural gene of ampD can lead to AmpC overproduction and increases in beta-lactam MICs in organisms with an inducible ampC. However, identification of mutations alone cannot predict the impact that those mutations have on AmpD function. Therefore, a model system was designed to determine the effect of ampD mutations on ceftazidime MICs using an AmpD(-) mutant Escherichia coli strain which produced an inducible plasmid-encoded AmpC. ampD genes were amplified by PCR from strains of E. coli, Citrobacter freundii, and Pseudomonas aeruginosa. Also, carboxy-terminal truncations of C. freundii ampD genes were constructed representing deletions of 10, 21, or 25 codons. Amplified ampD products were cloned into pACYC184 containing inducible bla(ACT-1)-ampR. Plasmids were transformed into E. coli strains JRG582 (AmpD(-)) and K-12 259 (AmpD(+)). The strains were evaluated for a derepressed phenotype using ceftazidime MICs. Some mutated ampD genes, including the ampD gene of a derepressed C. freundii isolate, resulted in substantial decreases in ceftazidime MICs (from >256 microg/ml to 12 to 24 microg/ml) for the AmpD(-) strain, indicating no role for these mutations in derepressed phenotypes. However, ampD truncation products and ampD from a partially derepressed P. aeruginosa strain resulted in ceftazidime MICs of >256 microg/ml, indicating a role for these gene modifications in derepressed phenotypes. The use of this model system indicated that alternative mechanisms were involved in the derepressed phenotype observed in strains of C. freundii and P. aeruginosa. The alternative mechanism involved in the derepressed phenotype of the C. freundii isolate was downregulation of ampD transcription.
ampD结构基因内的突变可导致AmpC过量产生，并使具有可诱导ampC的生物体中的β-内酰胺最低抑菌浓度（MIC）升高。然而，仅通过鉴定突变无法预测这些突变对AmpD功能的影响。因此，设计了一个模型系统，利用产生可诱导的质粒编码AmpC的AmpD（-）突变型大肠杆菌菌株来确定ampD突变对头孢他啶MIC的影响。通过聚合酶链反应（PCR）从大肠杆菌、弗氏柠檬酸杆菌和铜绿假单胞菌菌株中扩增ampD基因。此外，构建了弗氏柠檬酸杆菌ampD基因的羧基末端截短体，分别缺失10、21或25个密码子。将扩增的ampD产物克隆到含有可诱导的bla（ACT-1）-ampR的pACYC184中。将质粒转化到大肠杆菌菌株JRG582（AmpD（-））和K-12 259（AmpD（+））中。使用头孢他啶MIC评估菌株的去阻遏表型。一些突变的ampD基因，包括一株去阻遏的弗氏柠檬酸杆菌分离株的ampD基因，导致AmpD（-）菌株的头孢他啶MIC大幅降低（从>256μg/ml降至12至24μg/ml），表明这些突变在去阻遏表型中不起作用。然而，ampD截短产物和来自部分去阻遏的铜绿假单胞菌菌株的ampD导致头孢他啶MIC>256μg/ml，表明这些基因修饰在去阻遏表型中起作用。该模型系统的使用表明，弗氏柠檬酸杆菌和铜绿假单胞菌菌株中观察到的去阻遏表型涉及其他机制。弗氏柠檬酸杆菌分离株去阻遏表型所涉及的替代机制是ampD转录的下调。
