伤寒沙门菌主动外排多重耐药基因acrB与表达水平研究

目的调查临床分离伤寒沙门菌对常用抗生素的耐药情况与多重耐药主动外排基因acrB的检测、序列分析及其表达水平。方法根据NCCLS推荐的琼脂二倍稀释法测定7种抗生素对伤寒沙门菌的抗菌活性，以基因库序列为参考设计引物PCR扩增acrB、测序并用RT—PCR方法检测其表达水平。结果32株伤寒沙门菌对氧氟沙星、环丙沙星、头孢噻肟、哌拉西林、氯霉素、四环素、庆大霉素的耐药率分别为0、0、0、28．13％、43．75％、40．63％和12．5％，其中多重耐药菌10株。所有细菌均检测到多重耐药外排基因acrB．测序显示与参考沙门菌序列（No．AL627267）比较，有2处碱基变异。与大肠埃希菌（No．ECU00734）比较，碱基同源性为85．51％（61／421），提示其碱基序列同源性极高。对不同种类抗生素耐药及不耐药部分伤寒沙门菌共16株进行一步法RT—PCR检测acrB表达水平，结果所测伤寒沙门菌均检测到多重耐药外排基因acrB的表达，多重耐药株主动外排的表达较其它菌株明显增强。结论伤寒沙门菌对喹诺酮类、第三代头孢菌素类耐药率低，对哌拉西林、氯霉素、四环素耐药率相对较高，且有多重耐药。伤寒沙门菌中均存在acrAB主动外排系统，acrB与大肠埃希菌同源性高，对不同结构抗生素耐药的种类越多，表达水平越高。主动外排机制可能是伤寒沙门菌多重耐药的主要原因之一。

Multiple-antibiotic-resistant active efflux gene acrB and its expression in Salmonella typhi

Objective To investigate the antibiotic resistance of Salmonella typhi clinical isolates and(identify) the active efflux gene acrB and its expression.Methods The antimicrobial susceptibilities of 32 strains of S.typhi were tested with agar dilution method according to NCCLS and the acrB gene was identified and sequenced by PCR DNA-sequencing technique,the expression of acrB gene was measured by RT-PCR.(Results) The antibiotic resistant rates of S.typhi to ofloxacin,ciprofloxacin,cefotaxime,piperacillin,chloramphenicol,tetracycline and gentamicin were 0,0,0,28.13%,43.75%,40.63% and 12.5%,respectively.PCR detection result showed all of the tested strains having acrB gene.Compared with the GenBank sequence(No.AL627267),acrB of S.typhi 275 has two nucleotide bases different,and 85.51% identity with that of(Escherichia coli)(GenBank No.ECU00734).All the nucleic acid base mutations did not lead to the alteration in the amino acids of AcrB.There was significant statistical different of the expression level of acrB gene between the multiple-antibiotic-resistant strains and others,which meant that the broader the antibiotic resistant spectrum was,the higher the expression level of acrB gene.Conclusions The resistant rates of S.typhi to fluoroquinolones and the third generation of cephalosporins were low in Chongqing area.The acrB gene of S.typhi exists in all of the clinical strains and has high homology with that of Escherichia coli,the expression of the gene is responsible for the multiple-antibiotic-resistance.