木香、黄精提取液在小鼠体内抑制R质粒接合传递的实验研究

目的观察中药木香、黄精提取液在小鼠体内抑制S.flexneriD15 R-plasmid CmR和E.coli1485 RifR之间R质粒的接合传递作用。方法木香、黄精提取液分别与S.flexneriD15 R-plasmid CmR和E.coli1485 RifR混合后注入昆明小鼠腹腔;取作用24和48 h的腹腔液接种含氯霉素和不含氯霉素麦康凯琼脂平板,37℃培养。实验设溴化乙锭组和未加药对照组。根据培养24 h后的菌落数计算各组的R质粒转移率及R质粒转移抑制率。结果木香组、黄精组及未加药对照组24 h R质粒转移率分别为(4.17±0.59)%、(11.83±2.37)%和(58.67±5.80)%,差异有统计学意义(P0.01);48 h R质粒转移率分别为(1.94±0.62)%、(15.61±0.12)%和(57.61±3.32)%,差异有统计学意义(P0.01)。木香组和黄精组24 h的R质粒转移抑制率分别为(92.9±7.81)%和(79.82±6.94)%;48 h的R质粒转移抑制率分别为(96.63±8.52)%和(72.91±7.42)%。结论木香、黄精提取液在体内能有效抑制S.flexneriD15 R-plas-mid CmR和E.coli1485 RifR之间R质粒的接合传递。     

福氏志贺菌主动外排基因acrA的克隆及原核表达

目的对福氏志贺菌外排基因acrA进行克隆和原核表达,为进一步研究其在志贺菌外排机制中的作用奠定基础。方法参考福氏志贺菌2aacrA基因序列设计一对特异引物,在引物的5′和3′端分别加入含有BamHⅠ和SalⅠ限制性酶切位点序列。以福氏志贺菌2a菌株基因组DNA为模板,通过PCR扩增acrA基因并与pMD18-T载体连接,然后转化DH5α。提取重组质粒pMD18-acrA,经BamHI/SalI双酶切并与载体pET30a连接后转化宿主菌BL21(DE3)pLys,通过IPTG诱导表达目的蛋白。结果克隆的acrA基因长度为1122bp,核苷酸序列与GenBank上公布的序列完全相同。原核表达经SDS-PAGE及WesternBlotting检测和鉴定,结果表明重组载体pET-30a-acrA可成功地在大肠杆菌中表达AcrA蛋白。结论成功构建了福氏志贺菌acrA基因的原核表达质粒,并在大肠杆菌中得到有效表达,该研究为了解AcrA蛋白的特性、功能以及对福氏志贺菌多药耐药机理的深入研究奠定了基础。     

Transfer patterns of integron-associated and antibiotic resistance genes in S. flexneri during different time intervals in Tianjin,China

Background: Shigella is one of the common genera of pathogens responsible for bacterial diarrhoea in humans. According to World Health Organisation (WHO), 800,000–1,700,000 patients in China were infected with Shigella spp. in 2000, and Shigella flexneri is the most common serotype (86%). Objectives: We investigated the transfer patterns of integron-associated and antibiotic resistance genes in S. flexneri during different time intervals in the city of Tianjin in the People’s Republic of China. Materials and Methods: The integrase-encoding and variable regions of the integrons of the bacterial strains were amplified by polymerase chain reaction (PCR), followed by gene sequencing. Fifty-six S. flexneri strains, 32 of which were stored in our laboratory and the other 24 were isolated from tertiary hospitals in Tianjin during different time intervals, were tested for their sensitivity to 12 antibiotics by using the Kirby–Bauer antibiotic testing method (K-B method). Results and Conclusion: Of the 32 strains of S. flexneri isolated from 1981 to 1983 and stored in our laboratory, class 1 integron was detected in 28 strains (87.50%), while 27 strains (84.37%) harboured an aminoglycoside resistance gene, aadA, in the variable region of their integrons. Class 1 integron was identified in 22 (91.67%) of the 24 S. flexneri strains isolated from 2009 to 2010, whereas the variable region and 3′-end amplification were not present in any of the strains. Class 2 integron was not found in the 1981–1983 group (group A) of strains; although 19 (79.17%) of the 24 strains in the 2009–2010 group (group B) possessed class 2 integron, and the variable region of the integron harboured dfrA1 + sat1 + aadA1 genes, which, respectively, mediate antibiotic resistance to trimethoprim, streptothricin and streptomycin. Seventeen strains of the total 56 possessed both class 1 and 2 integrons. Strains belonging to group A were highly resistant to tetracycline, chloramphenicol and a combination of trimethoprim-sulfamethoxazole; 65.63% of the strains were multi-resistant to three or more antibiotics. In group B, the strains showed high resistance to ampicillin, trimethoprim-sulfamethoxazole, piperacillin and tetracycline; 83.33% of the strains were multi-resistant to three or more antibiotics. Class 1 and 2 integrons exist extensively in S. flexneri, and the 3′-conserved segments of class 1 integron may have deletion or other types of mutations. Comparing the antibiotic and multi-drug resistance of group A with that of group B, it is apparent that the antibiotic resistance and the incidence of genes that confer multi-drug resistance have increased over the years in S. flexneri.     

痢疾菌体内诱导基因表达文库的构建

目的 构建筛选痢疾菌福氏2a体内诱导基因的融合基因表达文库。方法 以自杀载体pGP704为骨架,用氯霉素乙酰转移酶基因作为报告基因,构建了用于筛选体内诱导基因的载体pGP-cat。把痢疾菌福氏2a基因组DNA的随机酶切片段（0．6－1kb)亚克隆到pGPcat载体报告基因上游的BglⅡ位点,构建了融合基因文库。结果 与痢疾菌福氏2a2457T结合转移后,获得融合基因表达文库。以氯霉素为选择标记,经过体外初步筛选,得到3．2％的具有氯霉素抗性的菌株。结论 构建的融合基因表达文库适用于筛选福氏2a痢疾菌的体内诱导基因。     

一株福氏志贺菌中同时检出CTX-M-3型超广谱β-内酰胺酶和质粒介导的喹诺酮耐药基因qnrS

目的明确1株福氏志贺菌RJ506对第3代头孢菌素和氟喹诺酮同时耐药的分子机制。方法通过E-test检测RJ506对各抗菌药物的最低抑菌浓度（MIC）,接合试验了解耐药性是否由质粒介导;用聚合酶链反应（PCR）分别扩增染色体和质粒介导的喹诺酮耐药基因并进行序列分析,检测染色体介导耐药的突变位点和质粒介导耐药的基因型;用PCR扩增CTX-M各组超广谱β-内酰胺酶（ESBEs）基因并进行序列分析,检测ESBLs的基因型。结果RJ506对头孢噻肟和环丙沙星同时耐药,其染色体DNA旋转酶gyrA亚基的83位发现Ser→Leu突变,而gyrB和parC未见突变;该菌株同时含有ESBLs基因blaCTX-M-3和喹诺酮耐药基因qnrS,且分别位于不同的可接合质粒上。结论本实验在国内的志贺菌中检出质粒介导的喹诺酮耐药基因qnrS和CTX-M-3型ESBLs。     

福氏志贺菌O-抗原磷酸乙醇胺修饰特异抗血清的制备

目的 制备针对福氏志贺菌O-抗原磷酸乙醇胺（PEtN）修饰的抗血清。方法 用血清型Yv菌株036_Yv（O-抗原PEtN修饰）免疫兔子制备抗血清,用036_Yv 的宿主菌036（O-抗原无PEtN修饰）吸附后,制备针对O-抗原PEtN修饰的特异抗血清,并用135株福氏志贺菌检测血清的特异性。结果 制备的血清能够特异地与O-抗原存在PEtN修饰的福氏志贺菌发生凝集,效价为1:32;除了O-抗原携带PEtN修饰的福氏志贺菌血清型Xv,Yv和4av外,制备的血清不能与其他血清型发生交叉凝集,具有很好的特异性。结论 成功制备了针对福氏志贺菌O-抗原PEtN修饰的特异抗血清,可应用于痢疾检测和监测。     

Multidrug-Resistant Atypical Variants of Shigella flexneri in China

We identified 3 atypical Shigella flexneri varieties in China, including 92 strains with multidrug resistance, distinct pulse types, and a novel sequence type. Atypical varieties were prevalent mainly in developed regions, and 1 variant has become the dominant Shigella spp. serotype in China. Improved surveillance will help guide the prevention and control of shigellosis.     

北京地区福氏志贺菌4c亚型毒力基因分析

目的 了解北京地区福氏志贺菌4 c亚型生化特征和毒力基因携带情况.方法 对分离自北京地区的44株福氏4 c亚型志贺菌采用Api生化板条进行鉴定,玻片凝集法进行血清分型,应用PCR技术对其侵袭性质检抗原(ipaH)、志贺肠毒素(setlA、setlB)、侵袭相关位点(sen、ial)和毒力表达调控基因(virF)6种毒力基因进行检测.结果 全部福氏4 c志贺菌只发酵葡萄糖、甘露醇、蜜二糖和阿拉伯糖;血清凝集抗原结构式为(Ⅳ:7,8);ipaH、setlA、setlB、sen、ial和virF6毒力基因携带率分别为100%、97.7%、100%、77.2%、72.7%和77.2%;54.5%的菌株携带全部6种毒力基因.结论 福氏4 c志贺菌毒力基因携带率高,具有较强致病性.     

从白面包中检出一株与福氏志贺氏菌6型交叉凝集的阴沟肠杆菌的报告

目的 分析研究阴沟肠杆菌和福氏志贺氏菌6型的生物学关系,以防志贺氏菌的误诊.方法 分离培养后,结合形态学、血清学和全面生化确定检验结果.结果 该菌株与福氏志贺氏菌6型发生程度深的交叉凝集,结合形态学和全面生化确定为阴沟肠杆菌.结论 阴沟肠杆菌与福氏志贺氏菌6型存在共同抗原.     

福氏志贺氏菌3a型污染水源水引起的细菌性痢疾

对2005年9月2-13日,广西隆安县某中学和某幼儿园出现的发热、呕吐及粘液脓血便症状患者的肛拭308份及饮用水7份进行致病性微生物检测,经血清学及细菌生化学鉴定,从1份水样及16份肛拭中检出3a型福氏志贺氏菌。证实本次腹泻爆发流行为饮用3a型福氏志贺氏菌污染的水源水引起。