制药用水及受控环境中寡营养微生物的生物负载监测  

Monitoring the bioburden of oligotrophs in pharmaceutical water systems and controlled environments

作　　者：范一灵 李琼琼 王培恩 杨美成 FAN Yi-ling;LI Qiong-qiong;WANG Pei-en;YANG Mei-cheng(China State Institute of Pharmaceutical Industry,Shanghai 201203,China;NMPA Key Laboratory for Testing Technology of Pharmaceutical Microbiology,Shanghai Quality Inspection and Testing Center for Innovative Biological Products,Shanghai Institute for Food and Drug Control,Shanghai 201203,China;Shanghai Food and Drug Packaging Material Control Center,Shanghai 201203,China)

机构地区：[1]中国医药工业研究总院,上海201203 [2]国家药品监督管理局药品微生物检测技术重点实验室,上海市创新生物制品质量检验检测中心,上海市食品药品检验研究院,上海201203 [3]上海市食品药品包装材料测试所,上海201203

出　　处：《药物分析杂志》2024年第11期1899-1908,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：上海市药品监督管理局监管科学研究项目(ZD-2023-06);中国医药工业研究总院研究生创新基金项目(YJS2022009)。

摘　　要：目的:筛选适用于制药环境中寡营养微生物的监测方法,了解制药用水及洁净环境等寡营养条件下的生物负载及微生物特征。方法:通过比较R2A和TSA培养基对实验室纯水中微生物在不同温度、培养基及培养时间的计数结果比较,优化寡营养条件下微生物的培养条件和检测方法。以传统方法与寡营养检测方法同时开展制药生产环境中寡营养微生物的监测,结合16s rDNA测序和MALDI-TOF MS等技术,对分离的微生物开展多相微生物鉴定及分析。结果:菌落计数方法、培养基种类和培养时间在微生物计数中有显著性影响,采用寡营养R2A培养基分离环境微生物的效果优于TSA培养基。上述2种培养基的微生物分离谱差异较大,在R2A培养基中革兰阴性菌的分离率可达37.0%,在TSA培养基中革兰阴性菌的分离率仅为14.0%。此外,初次分离于R2A培养基的多株条件致病微生物在TSA培养基中生长缓慢,在传统监测方案中易被漏检。结论:随着制药工艺的发展,以革兰阳性球菌为主的人源性微生物污染比例将逐步下降,适时选用寡营养微生物监测方法作为传统技术的补充,有助于在早期发现生产环境中潜在的不可接受微生物污染风险。Control;Shanghai Food and Drug Packaging Material Control Center;Objective:To screen the suitable monitoring method applicable to oligotrophs and review the bioburden and microbial species of oligotrophic environments in pharmaceutical water systems and cleanrooms.Methods:The culture conditions and detection methods suitable for microorganisms under oligotrophic conditions were optimized by comparing the counting results of microorganisms in laboratory pure water with R_2A and TSA media at different temperatures,media,and incubation times.In addition,monitoring of oligotrophs in pharmaceutical environments using both the traditional and oligotrophic assays were conducted,combining with the techniques of 16s rDNA sequencing and MALDI-TOF MS for multiphase microbial identification and analysis of the isolated microorganisms under oligotrophic conditions.Results:The colony counting methods,the type of culture media,and the incubation intervals significantly affected microbial enumeration.The isolation method using oligotrophic medium R_2A was more effective than the TSA medium in this study.The profiles of microorganisms isolated from the R_2A and TSA media were unique.The isolation rate of Gram-negative bacteria in the R_2A medium reached 37.0%,however,that of Gram-negative bacteria in the TSA medium was only 14.0%.In addition,several strains of potential pathogens initially isolated from the R_2A medium grew slowly in the TSA medium and were easily missed by the traditional monitoring method.Conclusion:With the development of the pharmaceutical industry,the contamination proportion of human-associated Gram-positive cocci might gradually decrease,the oligotrophic culture method as a powerful supplement to traditional monitoring techniques can help to detect potential objectionable microorganism contamination risks at an early stage.

关 键 词：寡营养微生物 制药用水 受控环境 生物负载 不可接受微生物 

分 类 号：R917[医药卫生—药物分析学]