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为探讨反义寡核苷酸(ASODN)沉默acrA、marA和acrD基因前后,黄芩苷对临床耐药大肠埃希菌(E.coli)环丙沙星敏感性的影响,设计并合成3个基因的ASODN引物,设置空白对照组、黄芩苷组、ASODN沉默组、ASODN沉默+黄芩苷组,分别转染到临床耐药菌感受态细胞中,涂布于一定浓度环丙沙星平板上,培养观察菌落生长情况并计数。结果发现,相对于空白对照组,黄芩苷组菌落数显著降低;acrA、acrD和marA基因沉默组与空白对照组相比,除了acrD基因沉默1号菌株和marA基因沉默的Y17菌株外,其余菌株在环丙沙星平板上的菌落数也出现不同程度地下降,具有显著性差异;acrA、acrD和marA基因沉默+黄芩苷组与黄芩苷组相比,多数菌株在环丙沙星平板上的菌落数均出现不同程度地下降;acrA基因沉默+黄芩苷组与acrA基因沉默组相比,4株菌株变化差异不显著,Y17号菌株菌落数有所上升,J45号菌株在该基因沉默后,环丙沙星平板上的菌落数出现下降的趋势,差异显著;acrD基因沉默+黄芩苷组与acrD基因沉默组相比,除了1号菌株变化差异不显著外,其余5株菌株变化差异显著,但Y17号菌株菌落数上升;marA基因沉默+黄芩苷组与marA基因沉默组相比,除了Y17、Y35这2株菌株变化差异显著外,其余4株菌株变化差异不显著。以上结果表明,黄芩苷主要通过抑制acrA和marA发挥外排抑制作用,acrD不是其增强环丙沙星敏感性的主要靶点。
To investigate the effects of antisense oligonucleotide (ASODN) silencing acrA, marA and acrD genes on the sensitivity of ciprofloxacin to clinical drug-resistant Escherichia coli before and after acrA, three genes were designed and synthesized ASODN primer set, blank control group, baicalin group, ASODN silencing group, ASODN silencing + baicalin group were transfected into clinical drug-resistant competent cells, coated on a certain concentration of ciprofloxacin plate, culture Observed colony growth and counting. The results showed that compared with the blank control group, the number of colonies in the baicalin group was significantly decreased. Compared with the blank control group, the acrA, acrD and marA gene silencing groups, except for the acrD gene silencing strain No.1 and the Y17 strain in which the marA gene was silenced, The number of colonies on ciprofloxacin plate also decreased to some extent, with significant differences; acrA, acrD and marA gene silencing + baicalin group compared with the baicalin group, the majority of strains in the ciprofloxacin plate The number of colonies decreased to varying degrees; acrA gene silencing + baicalin group compared with the acrA gene silencing group, the four strains showed no significant difference, the number of strains Y17 colonies increased, J45 strains in the gene silencing, The number of colonies on ciprofloxacin plate tended to decrease, and there was significant difference between the two groups. In acrD gene silencing + baicalin group and acrD gene silencing group, except for the no significant difference between strains 1 and 5, However, the number of colonies of strain Y17 increased. Compared with marA gene silencing + baicalein group and marA gene silencing group, the four strains except Y17 and Y35 showed significant difference. The above results show that baicalin mainly inhibits the efflux inhibition of acrA and marA, and acrD is not the main target of enhancing the sensitivity of ciprofloxacin.