不动杆菌属临床分离株Amp C酶及其相关基因研究

Analysis of the Amp C β-lactamase produced by Acinetobacter

目的了解不动杆菌产Amp C酶的情况,研究不动杆菌属临床分离株Amp C酶及调控基因amp D、amp R的变化。方法 Nitrocefin显色法、头孢西丁纸片筛选法和三维试验测定临床分离的126株鲍曼不动杆菌产Amp C酶的情况。用amp C 基因引物对提取的质粒DNA和染色体DNA进行PCR扩增。对amp C基因扩增阳性的菌株进行amp R与amp D基因的扩增。结果 126株鲍曼不动杆菌总β-内酰胺酶的检出率为78.6%(99/126)。119株菌头孢西丁纸片法筛选阳性,62株三维试验阳性。三维试验和amp C基因扩增均阳性的有38株,三维试验阴性、amp C基因扩增阳性的有8株。对三维试验阳性与 amp C基因扩增阴性的24株菌进行等电聚焦显示,未发现pI】8.0的条带,但发现有pI为5.2左右的β-内酰胺酶。用amp C 基因引物对质粒DNA和染色体DNA进行PCR扩增并测序显示,在染色体中存在amp C基因,在质粒DNA中未检测出amp C 基因。三维试验与PCR法扩增amp C基因相比,假阳性率为54.7%,假阴性率为17.4%;阳性率为82.6%。三维试验与染色体amp C基因扩增同时为阳性的菌株中,amp D(-)19株;amp R(-)5株;amp D(+)与amp R(+)16株,x2=11.94,P【 0.01。三维试验阴性与染色体amp C基因扩增阳性的菌株中,amp D(+)、amp R(+):8株。结论本组不动杆菌普遍产生β-内酰胺酶。amp C基因位于染色体上,在质粒上未发现。临床分离的产生Amp C酶的不动杆菌株中amp D、amp R基因有变化,amp D的改变比amp R改变更多见。三维试验与PCR法检测Amp C酶方法相比,假阳性较高。

Objective To analyse the resistance β-lactamase in Acinetobacter and investigate the Amp C β-lactamase produced by Acinetobacter. To research amp R and amp D gene. Methods Cefoxitin disc was used to preliminarily detect Amp C β-lactamase. Three-dimension test、soelectric focus and PCR amplification were used to detect the Amp C β-lactamase by 126 strains of Acinetobacter. Results The β-lactamase ratio was 78.6%. The positive isolates of cefoxitin disc detect were 119 strains, and three-dimension test were 62 strains. The positive isolates of three-dimension test and PCR amplification of amp C gene were 8 strains. The Isoelectric Focusing Electrophoresis of the 24 isolated strains that were positive of three-dimension test but negative of PCR amplification of amp C gene , it was not found the specified strap(PI > 8. 0) , but found another strap(PI =5.2). The amp C gene consisted in the chromosome and not in the plasmids. In the positive isolate strains of three-dimension test and PCR amplification of amp C gene, amp D(-) were 19 strains; amp R(-) were 5 strains; both amp D(+) and amp R(+) were 16,by the x2 test,P<0.01. In the isolate strains that were negative of three-dimension test but positive of amp C gene amplification, both amp D(+) and amp R(+) were 8strains. Conclusion the β-lactamases was produced by Acinetobacter widespread. The amp C gene consisted in the chromosome. There was no the amp C gene in the plasmid. The amp D and amp R gene mutants maybe result in hyperexpression of Amp C β-lactamases. The three-dimensional extract test to detect the Amp C B-lactamase had highly false-positive and lowly false-negative ratio.