Cernat Ramona, Lazăr Veronica, Balotescu Carmen, Cotar Ani, Coipan Elena, Cojocaru Cristina
Facultatea de Biologie, Dep. Microbiologie-Imunologie Universitatea din Bucureşti.
Bacteriol Virusol Parazitol Epidemiol. 2002 Jul-Dec;47(3-4):179-84.
Several multiple antibiotic resistant E. coli strains isolated from river and polluted waters were compared for their genetic relatedness. Antibiotic susceptibility testing was performed for gentamycin, kanamycin, ampicillin, tetracycline, chloramphenicol, ceftazidime and cefotaxime, as described by Kirby-Bauer disk diffusion method following NCCLS recommendations. Minimum inhibitory concentrations (MICs) were performed using dilution method in Mueller-Hinton broth with a 0.06-64 micrograms/ml concentration range for all antimicrobials and bacterial inoculum of about 1-2 x 10(8) CFU/ml. For the data analysis NCCLS breakpoints for resistance and sensitivity were used. Genomic DNA was isolated from E. coli strains by CTAB method and digested to completion with HindIII enzyme. Genetic characterization was performed by agarose gel electrophoresis and spectrophotometric analysis. Genetic similarity and clustering were calculated using NISIS program. All E. coli strains isolated from river and polluted waters show a high incidence of multiple antibiotic resistance phenotype, 16% of them being resistant to 7, 6 and 4 antibiotics, 40% to 5 and 8% to 2 antibiotics, respectively. A moderate resistance was observed to kanamycin (higher than 30%) and cefotaxime (68%). The percentage of resistant E. coli strains ranged from 76% (to ampicillin, gentamicin and chloramphenicol) to 85% (to ceftazidime). The best results (resistance about 99%) were obtained with tetracycline. Screening for plasmids relieved the presence into 4 E. coli strains of several plasmids ranging from 3.8 kpb to more than 50 kpb. The number of fragments produced by HindIII digestion of genomic DNA ranged from 11 to 25, with sizes of approximately 22 to more than 750 kb. The phenotypic data shows the dynamic flow of multiple antibioresistant E. coli strains in aquatic media (river and polluted waters). Electrophoretic patterns analysis reflects the incidence and diversity of analyzed plasmids. DNA fingerprinting with genomic DNA RE suggested that, depending of the isolation source, E. coli strains could be grouped in two distinct populations with a different plasmid diversity.
对从河流和污水中分离出的几种多重耐药大肠杆菌菌株的遗传相关性进行了比较。按照美国国家临床实验室标准化委员会（NCCLS）的建议，采用 Kirby-Bauer 纸片扩散法对庆大霉素、卡那霉素、氨苄青霉素、四环素、氯霉素、头孢他啶和头孢噻肟进行药敏试验。使用稀释法在 Mueller-Hinton 肉汤中测定最低抑菌浓度（MIC），所有抗菌药物的浓度范围为 0.06 - 64 微克/毫升，细菌接种量约为 1 - 2×10⁸ CFU/毫升。数据分析采用 NCCLS 耐药和敏感断点。通过 CTAB 法从大肠杆菌菌株中分离基因组 DNA，并用 HindIII 酶完全消化。通过琼脂糖凝胶电泳和分光光度分析进行遗传特征分析。使用 NISIS 程序计算遗传相似性和聚类。从河流和污水中分离出的所有大肠杆菌菌株均表现出多重耐药表型的高发生率，其中 16%对 7 种、6 种和 4 种抗生素耐药，40%对 5 种抗生素耐药，8%对 2 种抗生素耐药。观察到对卡那霉素（高于 30%）和头孢噻肟（68%）有中度耐药。耐药大肠杆菌菌株的百分比范围从 76%（对氨苄青霉素、庆大霉素和氯霉素）到 85%（对头孢他啶）。四环素的耐药率最高（约 99%）。质粒筛选显示 4 株大肠杆菌中存在几种大小从 3.8 kpb 到超过 50 kpb 的质粒。基因组 DNA 经 HindIII 消化产生的片段数量从 11 到 25 不等，大小约为 22 到超过 750 kb。表型数据显示了多重耐药大肠杆菌菌株在水生介质（河流和污水）中的动态传播。电泳图谱分析反映了所分析质粒的发生率和多样性。用基因组 DNA 限制性内切酶进行的 DNA 指纹分析表明，根据分离来源，大肠杆菌菌株可分为两个具有不同质粒多样性的不同群体。
