临床分离变形杆菌不典型第1类整合子结构分析

Structural analysis of atypical class 1 integrons in clinical Proteus isolates

目的确定临床分离变形杆菌中不典型第1类整合子的结构。方法在26株第1类整合酶基因阳性可变区扩增失败的变形杆菌临床分离株中，选取6株通过反向PCR扩增第1类整合酶基因周围DNA片段，PCR产物测序结果通过BLAST进行比对分析，确定同源目标序列后，查看GenBank该提取码对应序列文件靶序列周围序列，再根据周围序列设计合成引物进行重叠PCR并测序验证。结果通过反向PCR及重叠PCR测序分析，26株变形杆菌临床分离株中，25株细菌第1类整合子可变区结构全部或部分确定，其中22株细菌第1类整合子可变区结构为estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3，另3株第1类整合子可变区分别为estX-psp-aadA2-cmlA1、dfrA14、IS26，均缺失3′保守区。结论反向PCR可用于临床菌株中不典型第1类整合子结构分析，在临床分离变形杆菌中首次检出基因盒psp及基因盒组合estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3、estX-psp-aadA2-cmlA1。

ObjectiveTo analyze the structures of atypical class 1 integrons in clinical Proteus isolates.MethodsThis study included 26 class 1 integron integrase gene-positive clinical Proteus isolates, from which the variable regions of class 1 integrons could not be amplified.Six isolates were chosen from them to amplify the flanking DNA segments of class 1 integron integrase gene using inverse PCR.The sequences of PCR products were analyzed with BLAST to identify the target homologous sequences as well as their accession numbers in GenBank.Primers for overlap PCR were designed according to the flanking sequences.Then the 26 clinical Proteus isolates were analyzed with overlap PCR and sequencing analysis.ResultsThe variable regions of class 1 integrons in 25 out of the 26 clinical Proteus isolates were completely or partly identified by using inverse PCR, overlap PCR and sequencing analysis.A gene cassette array of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 was detected in 22 isolates.The variable regions of class 1 integrons in the other three isolates were identified to be estX-psp-aadA2-cmlA1, dfrA14 and IS26, respectively.All of the 25 isolates lacked the 3′ conserved segements in class 1 integron.ConclusionInverse PCR can be used to analyze the structures of atypical class 1 integrons.Gene cassette psp and gene cassette arrays of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 and estX-psp-aadA2-cmlA1 in clinical Proteus isolates are reported for the first time.