海洋病原细菌的氯霉素耐药菌株筛选鉴定及质粒消除  

Screening,identification of marine pathogenic bacteria with chloramphenicol resistance and elimination of their plasmids

作　　者：孟繁梅[1] 汪凯[1] 潘子强[1] 尹德胜[1] 艾云灿[1] 

机构地区：[1]中山大学生命科学院有害生物控制与资源利用国家重点实验室,广州510275

出　　处：《中国抗生素杂志》2006年第7期437-441,共5页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(No.39870579;No.30270064;No.40376046);广东省自然科学基金(No.031624);教育部回国留学人员科研启动基金;教育部高等学校骨干教师资助计划;广东省科技计划重点项目(No.2005B20501001);中山大学测试科学基金;985-II期专项资助项目

摘　　要：目的从污染海域筛选非临床氯霉素耐药株,研究耐药基因定位及耐药机制多样性。方法TCBS和EMB选择平板筛选,结合16SrDNA序列分析鉴定耐药株。MIC测定、基因组DNA-RAPD分型和质粒消除评估多样性。结果评估187株氯霉素耐药株的MIC值分布、属种分布、耐药基因定位及耐药机制。发现80株耐药MIC25～128μg/ml是CLSI/NCCLS(1995年)定义临床标准(MIC12.5μg/ml)的2～10倍,分布在弧菌科和肠杆菌科主要属种;58株MIC>25μg/ml基因组DNA-RAPD分型与菌落表型分组结果吻合,显示多样性丰富。采用多轮高温(～43℃)和高浓度(～1%)SDS双重处理和交替培养,77株MIC>25μg/ml分离株的质粒消除效率为28.6%;55株不能消除耐药表型,暗示染色体编码耐药基因;22株可消除氯霉素耐药表型,其中16株完全消除,暗示质粒独立编码耐药基因,6株部分消除,暗示质粒和染色体分别编码耐药基因。结论来自污染海域环境的非临床氯霉素耐药株可作为新模型,提供耐药基因定位及耐药机制多样性的新视野。Objective To screen out non-clinical isolates with chloramphenicol resistance from polluted marine environment, and study on the diversities of resistance gene locations and resistance mechanisms. Methods The isolates were screened on TCBS and EMB plates containing 25 μg/ml chloramphenicol, and co-identified by 16S rDNA sequencing and analysis. The diversity of isolates for resistance to chloramphenicol was assayed by determination of MIC values, genomic DNA-RAPD typing, and plasmid elimination. Results The distributions of MIC values, genus and species, locations of chloramphenicol resistance gene and resistance mechanisms of 187 isolates were determined. 80 isolates of them possessed MIC 25 - 128μg/ml up to 2 - 10 times of the standard for clinical isolates of MIC 12.5μg/ml recommended by the CLSI/NCCLS（1995）. These isolates were belonged to typical genera within Vibrionaceae and Enterobacteriaceae. The 58 isolates of them possessing MIC〉25μg/ml had well consistence of genomic DNA-RAPD types with their colony phenotypes showing abundant diversities. By multiple round treatments with high temperature （ -43℃ ） and high concentration - 1% ） of SDS coupled with alternative culture procedures, the rate of plasmid elimination for 77 isolates of MIC〉25μg/ml was only 28.6%. The resistance phenotype of 55 isolates were not eliminated suggesting chromosomal locations of resistance genes. The resistance phenotype of 22 isolates were eliminated, among them, the resistance of 16 isolates were entirely eliminated, suggesting plasmid encoding resistance genes, whereas the other resistance eliminating partially 6 isolates indicated both plasmid and chromosome encoded resistance genes. Conclusion The non-clinical isolates from polluted marine environment with chloramphenicol resistance were unique models to provide new insights into the diversities of resistance gene locations and their resistance mechanisms.

关 键 词：海洋环境 病原细菌 氯霉素 耐药性 基因定位 质粒 消除 

分 类 号：R378.99[医药卫生—病原生物学]