摘要
为了研究槲皮素对多重耐药沙门菌耐药性的消减作用,试验采用微量肉汤稀释法测定槲皮素对多重耐药肠沙门菌肠亚种0044(Salmonella enterica subsp. Enterica 0044,SENT0044)的最小抑菌浓度(MIC)、最小杀菌浓度(MBC);不同质量浓度槲皮素作用SENT0044后,检测生长曲线、细菌胞外碱性磷酸酶(AKP)活性及通过扫描电子显微镜和透射电子显微镜观察细菌形态结构;通过槲皮素刺激SENT0044,检测槲皮素刺激后SENT0044的MIC,并与刺激前的MIC相比较;采用反转录实时荧光定量PCR(RT-q PCR)方法检测槲皮素对SENT0044耐药基因qnr S、floR表达的影响。结果表明:槲皮素对SENT0044的MIC和MBC分别为0.640 mg/mL和0.512 mg/mL,阿莫西林、硫酸新霉素、恩诺沙星等10种抗生素对SENT0044的MIC和MBC分别为0.002~0.256 mg/mL和0.004~0.640 mg/mL。0.16,0.32 mg/mL槲皮素对SENT0044抑菌强度不断增加,0.64,1.28 mg/mL槲皮素对SENT0044有良好的抑制效果且完全抑制细菌生长。不同质量浓度(0.16,0.32,0.64,1.28 mg/mL)槲皮素作用下的细菌胞外AKP活性分别为1.8,2.3,2.4,2.8 Kings/100 mL,均极显著高于菌液对照(P<0.01),且在一定程度上呈剂量依赖性。槲皮素能够破坏受试菌的细胞壁和细胞膜结构,对菌体正常生长造成影响。SENT0044耐药消除前后阿莫西林、盐酸环丙沙星、庆大霉素的MIC未变;氧氟沙星、恩诺沙星、多西环素、硫酸新霉素、泰妙菌素及氟苯尼考的MIC均有不同程度的下降。SENT0044携带喹诺酮类耐药基因qnr S和氯霉素类耐药基因floR,且槲皮素可极显著或显著下调qnr S和floR mRNA的相对表达量(P<0.01或P<0.05)。说明槲皮素通过破坏多重耐药沙门菌SENT0044的细胞壁和细胞膜,使其通透性增加导致细菌死亡,并下调耐药基因相对表达量,提高细菌对抗生素的敏感性。
In order to investigate the effect of quercetin on reducing the drug resistance of multidrug-resistant Salmonella,the experiment used the microbroth dilution method to determine the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of quercetin against multidrug-resistant Salmonella enterica subsp.Enterica 0044(SENT0044).After treating SENT0044 with different mass concentrations of quercetin,the growth curve and extracellular alkaline phosphatase(AKP)activity of the bacteria were detected,and the ultrastructure of the bacteria was observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).By stimulating SENT0044 with quercetin,the MIC of SENT0044 after quercetin stimulation was detected and compared with the MIC before stimulation.The reverse transcription real-time fluorescent quantitative PCR(RT-qPCR)method was used to detect the effect of quercetin on the expression of drug resistance genes qnrS and floR in SENTOO44.The results showed that the MIC and MBC of quercetin against SENTO044were 0.640 mg/mL and 0.512 mg/mL,respectively,while the MIC and MBC of 10 antibiotics including amoxicillin,neomycin,and enrofloxacin against SENT0044 were 0.0020.256 mg/mL and 0.004-0.640 mg/mL,respectively.The antibacterial strength of quercetin at 0.16,0.32 mg/mL against SENT0044 increased continuously,and quercetin at 0.64,1.28 mg/mL had a good inhibitory effect on SENT0044,which completely inhibited bacterial growth.The extracellular AKP activities of bacteria treated with quercetin at different mass concentrations(0.16,0.32,0.64,1.28 mg/mL)were 1.8,2.3,2.4,and 2.8 Kings/100 mL,respectively,which were all extremely significantly higher than that of the bacterial suspension control(P<0.01)and showed a dose-dependent manner to a certain extent.Quercetin could damage the integrity and morphological structure of the cell walls and cell membranes of the tested bacteria,affecting the normal growth of the bacterial cells.The MIC values of amoxicillin,ciprofloxacin,and gentamicin remained unchanged before and after the elimination of SENT0044resistance;the MIC values of ofloxacin,enrofloxacin,doxycycline,neomycin,tiamulin,and florfenicol all decreased to various degrees.SENT0044 carried the fluoroquinolone resistance gene qnrS and the chloramphenicol resistance gene floR,and quercetin could basically and extremely significantly or significantly down-regulate the mRNA relative expression levels of qnrS and floR(P<0.01 or P<0.05).This indicated that quercetin destroied the cell wall and cell membrane of multidrug-resistant Salmonella SENT0044,increased their permeability leading to bacterial death,and down-regulated the relative expression levels of resistance genes,thereby improving the sensitivity of bacteria to antibiotics.
作者
周昱含
吕世明
杨露情
余永梅
何江
程振涛
杨剑
宋旭琴
ZHOU Yuhan;LYU Shiming;YANG Luqing;YU Yongmei;HE Jiang;CHENG Zhentao;YANG Jian;SONG Xuqin(College of Animal Science,Guizhou University,Guiyang 550025,China;Guiyang Agricultural Products Quality and Safety Supervision,Inspection and Testing Center,Guiyang 550081,China)
出处
《黑龙江畜牧兽医》
北大核心
2026年第1期1-7,共7页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(32202854)
贵州省基础研究计划(自然科学)面上项目(黔科合基础MS[2025]586)。
关键词
槲皮素
沙门菌
多重耐药
耐药性
消减
quercetin
Salmonella
multidrug-resistance
drug resistance
attenuating
