血流感染病原菌AmpC酶及相关耐药机制的初步研究

Preliminary study on AmpC β-lactamase and related drug-resistant mechanism of pathogenic bacteria in blood stream

目的了解血流感染病原菌中产AmpCβ-内酰胺酶(简称AmpC酶)菌株的分布情况和临床病例特点,并对相关基因ampC和ampD进行初步研究。方法收集2006年1月—2008年9月血流感染革兰阴性菌181株,分别采用头孢西丁初筛试验和三维试验筛选产AmpC酶菌株和高产AmpC酶菌株;对初筛试验阳性相关临床病例进行回顾性分析。同时对初筛试验阳性菌株ampC、ampD基因PCR扩增、测序和比对,Rep-PCR分析ampC阳性菌株基因型。结果181株病原菌中头孢西丁初筛试验法检出产AmpC酶39株,概率为21.5%(39/181);三维试验法检出高产AmpC酶菌株的概率为43.6%(17/39)。PCR结果显示ampC和ampD基因阳性率分别为41%(16/39)和56.4%(22/39)。对16株ampC阳性菌进行基因测序,与GenBank相应ampC核酸序列比对发现同源性达98%～100%;2株阴沟肠杆菌ampD基因的测序发现在ampD基因羧基端存在可疑的突变位点。结论本研究相关败血症患者血流耐药病原菌感染主要源自院内感染。在产AmpC酶的血流病原菌中,ampC基因突变比较少见,而阴沟肠杆菌ampD基因突变发生率比较高,且ampD蛋白羧基末端氨基酸的缺失或替代可能与AmpC酶去阻遏表达高度相关。

Objective To investigate the characteristics of strains of AmpC β-lactamase （AmpC enzyme） production in pathogenic bacteria in blood stream and clinical presentations of the cases, and study the related ampC and ampD genes. Methods One hundred and eighty-one strains of gram negative bacillus in blood stream were collected, Cefoxitin screening test and three-dimensional test were performed for screening of strains of AmpC enzyme, production and those of AmpC enzyme hyperproduction retrospective analysis was condected in the strains with positive results, ampC and ampD gene PCR amplification, sequencing and sequence analysis of positive strains were performed, and gene homology of ampC positive strains was analysed by Rep-PCR. Results Among 181 strains in blood stream, strains of AmpC enzyme production were detected in 39 isolates by Cefoxitin screening test, with the detection rate of 21.5% （39/181）. The detection rate of strains of AmpC enzyme hyperproduction by three-dimensional test was 43.6% （17/39）. PCR revealed that the positive rates for ampC and ampD genes were 41% （16/39） and 56.4% （22/39）, respectively. The ampC gene sequencing of 16 positive strains indicated that the homology was 98% to 100% by comparison with the GenBank, while the ampD gene sequencing of 2 strains of Enterobacter cloacae demonstrated that the suspected gent mutations existed in the carboxy-tcrminal of ampD gene. Conclusion The prevalence of drug-resistant pathogenic bacteria in blood stream in this study is due to nosocomial infection. The mutation of ampC gene is rare in the pathogenic bacteria in blood stream with production of AmpC enzyme, while the rate of gene mutation in Enterobacter cloacae is higher, and the deletion and amino acid substitutions in the carboxy-terminal of ampD is highly relevant to the depressed expression of AmpC enzyme.