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Article(id=1240945596094272210, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1240945593548337937, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2024-0357, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1716912000000, receivedDateStr=2024-05-29, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773794085762, onlineDateStr=2026-03-18, pubDate=1732982400000, pubDateStr=2024-12-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773794085762, onlineIssueDateStr=2026-03-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773794085762, creator=13701087609, updateTime=1773794085762, updator=13701087609, issue=Issue{id=1240945593548337937, tenantId=1146029695717560320, journalId=1205117023404326918, year='2024', volume='44', issue='11', pageStart='1827', pageEnd='2010', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773794085156, creator=13701087609, updateTime=1773796488495, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1240955673937236736, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1240945593548337937, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1240955673937236737, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1240945593548337937, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1899, endPage=1908, ext={EN=ArticleExt(id=1240945596794721014, articleId=1240945596094272210, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=Monitoring the bioburden of oligotrophs in pharmaceutical water systems and controlled environments*, columnId=1240945594596913943, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Bioassay • Metabolism Analysis, runingTitle=null, highlight=null, articleAbstract=
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 R2A 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 R2A was more effective than the TSA medium in this study. The profiles of microorganisms isolated from the R2A and TSA media were unique. The isolation rate of Gram-negative bacteria in the R2A 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 R2A 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.

, correspAuthors=Mei-cheng YANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Yi-ling FAN, Qiong-qiong LI, Pei-en WANG, Mei-cheng YANG), CN=ArticleExt(id=1240945597998486329, articleId=1240945596094272210, tenantId=1146029695717560320, journalId=1205117023404326918, language=CN, title=制药用水及受控环境中寡营养微生物的生物负载监测*, columnId=1240945594722743066, journalTitle=药物分析杂志, columnName=生物检定+代谢分析, runingTitle=null, highlight=null, articleAbstract=
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。

, correspAuthors=杨美成, authorNote=null, correspAuthorsNote=
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Airborne microbiological sampling and future trends [J]. Chin J Health Lab Technol，2003，13(5)：538, articleTitle=Airborne microbiological sampling and future trends, refAbstract=null)], funds=[Fund(id=1240954727328969025, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, awardId=ZD-2023-06, language=CN, fundingSource=*上海市药品监督管理局监管科学研究项目(ZD-2023-06), fundOrder=null, country=null), Fund(id=1240954727417049413, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, awardId=YJS2022009, language=CN, fundingSource=中国医药工业研究总院研究生创新基金项目(YJS2022009), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1240954722694262817, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, xref=1., ext=[AuthorCompanyExt(id=1240954722702651427, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, companyId=1240954722694262817, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.China State Institute of Pharmaceutical Industry，Shanghai 201203，China), AuthorCompanyExt(id=1240954722711040037, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, companyId=1240954722694262817, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.中国医药工业研究总院，上海 201203)]), AuthorCompany(id=1240954722807509031, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, xref=2., ext=[AuthorCompanyExt(id=1240954722824286249, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, companyId=1240954722807509031, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.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), AuthorCompanyExt(id=1240954722836869162, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, companyId=1240954722807509031, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=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)]), AuthorCompany(id=1240954722933338159, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, xref=3., ext=[AuthorCompanyExt(id=1240954722941726768, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, companyId=1240954722933338159, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.Shanghai Food and Drug Packaging Material Control Center，Shanghai 201203，China), AuthorCompanyExt(id=1240954722950115377, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, companyId=1240954722933338159, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3.上海市食品药品包装材料测试所，上海 201203)])], figs=[ArticleFig(id=1240954725760299227, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Fig.1, caption=Schematic diagram of microbial sampling points in production cleanrooms, figureFileSmall=bmi4KJPzfqf7Bwexw2CThw==, figureFileBig=HjtSyNDtYxQ54E/JXjXgBw==, tableContent=null), ArticleFig(id=1240954725852573921, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=图1　, caption=生产洁净车间微生物采样布点示意图, figureFileSmall=bmi4KJPzfqf7Bwexw2CThw==, figureFileBig=HjtSyNDtYxQ54E/JXjXgBw==, tableContent=null), ArticleFig(id=1240954725957431529, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Fig.2, caption=Bioburden results for purified water systems under different influencing factors, figureFileSmall=s3TiNxihkVtr2DshcWbZ9g==, figureFileBig=ICBrMtdbzU/v26DtFUsHqA==, tableContent=null), ArticleFig(id=1240954726037123313, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=图2　, caption=不同影响因素下纯化水系统生物负载计数结果

A.以直接接种法与薄膜过滤法为分类因素(direct inoculation and membrane filtration as categorizing factors)　B.以R2A培养基和TSA培养基为分类因素(R2A medium and TSA medium as categorizing factors)　C.以培养时间分别为3、5和7 d为分类因素(incubation times of 3，5 and 7 d as categorizing factors)　D.以培养温度分别为20~25 ℃和30~35 ℃为分类因素(incubation temperatures of 20-25 ℃ and 30-35 ℃ as categorizing factors)

, figureFileSmall=s3TiNxihkVtr2DshcWbZ9g==, figureFileBig=ICBrMtdbzU/v26DtFUsHqA==, tableContent=null), ArticleFig(id=1240954726116815092, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Fig.3, caption=Isolation rates of microorganisms in R2A and TSA media in pharmaceutical environment, figureFileSmall=DAX9t6bpnRvJzJPS5RDQDw==, figureFileBig=4geHAu3J/n4pe9/zNiOiUg==, tableContent=null), ArticleFig(id=1240954726213284091, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=图3　, caption=制药环境中R2A和TSA培养基中微生物的分离率

A.R2A培养基(R2A medium)　B.TSA培养基(TSA medium)

, figureFileSmall=DAX9t6bpnRvJzJPS5RDQDw==, figureFileBig=4geHAu3J/n4pe9/zNiOiUg==, tableContent=null), ArticleFig(id=1240954726334918911, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Tab.1, caption=

Summary of microbiological sampling points in production cleanrooms

, figureFileSmall=null, figureFileBig=null, tableContent=
房间
(room)		功能
(function)		洁净度
(cleanliness)		TSA培养基采样点数
(number of sampling points using TSA media)		R2A培养基采样点数
(number of sampling points using R2A media)
ABaSTSFABSTSF
206一更(1st changing room)D22-1--
207缓冲间(buffer room)C22门把手(door handle)：211内侧把手(inside door handle)：1
210C级走道(class C aisle)C22墙面(wall)：211墙面(wall)：1
219缓冲间(buffer room)C-b-门把手(door handle)：2--内侧把手(inside door handle)：1
220二更(2 nd changing room)B--门把手(door handle)：2
墙面(wall)：2
台面(desktop)：1		--内侧把手(inside door handle)：1
墙面(wall)：1
226准备间(preparatory room)B--门把手(door handle)：1
墙面(wall)：2
冰箱(refrigerator)：1
台面(desktop)：1		--冰箱(refrigerator)：1
台面(desktop)：1
221B级走道(class B aisle)B-1门把手(door handle)：2
墙面(wall)：2		-1内侧把手(inside door handle)：1
墙面(wall)：1
223缓冲(buffer room)B--门把手(door handle)：2
墙面(wall)：2		---
225缓冲(buffer room)B--门把手(door handle)：2
墙面(wall)：2		---
235细胞制备间(cell preparation room)B-1门把手(door handle)：2
墙面(wall)：2
台面(desktop)：1
设备表面(equipment surface)：7		-1内侧把手(inside door handle)：2
墙面(wall)：2
台面(desktop)：1
设备表面(equipment surface)：2
235生物安全柜(biological safety cabinet)A22设备表面(equipment surface)：422设备表面(equipment surface)：2
237缓冲间(buffer room)B--门把手(door handle)：2
墙面(wall)：2		---
239污物走道(waste path)C1-墙面(wall)：21-墙面(wall)：1
采样人员(sampler)--手套(gloves)：2--手套(gloves)：2
总计(total)910526621
), ArticleFig(id=1240954726422999301, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=表1, caption=

生产洁净车间微生物采样点信息汇总

, figureFileSmall=null, figureFileBig=null, tableContent=
房间
(room)		功能
(function)		洁净度
(cleanliness)		TSA培养基采样点数
(number of sampling points using TSA media)		R2A培养基采样点数
(number of sampling points using R2A media)
ABaSTSFABSTSF
206一更(1st changing room)D22-1--
207缓冲间(buffer room)C22门把手(door handle)：211内侧把手(inside door handle)：1
210C级走道(class C aisle)C22墙面(wall)：211墙面(wall)：1
219缓冲间(buffer room)C-b-门把手(door handle)：2--内侧把手(inside door handle)：1
220二更(2 nd changing room)B--门把手(door handle)：2
墙面(wall)：2
台面(desktop)：1		--内侧把手(inside door handle)：1
墙面(wall)：1
226准备间(preparatory room)B--门把手(door handle)：1
墙面(wall)：2
冰箱(refrigerator)：1
台面(desktop)：1		--冰箱(refrigerator)：1
台面(desktop)：1
221B级走道(class B aisle)B-1门把手(door handle)：2
墙面(wall)：2		-1内侧把手(inside door handle)：1
墙面(wall)：1
223缓冲(buffer room)B--门把手(door handle)：2
墙面(wall)：2		---
225缓冲(buffer room)B--门把手(door handle)：2
墙面(wall)：2		---
235细胞制备间(cell preparation room)B-1门把手(door handle)：2
墙面(wall)：2
台面(desktop)：1
设备表面(equipment surface)：7		-1内侧把手(inside door handle)：2
墙面(wall)：2
台面(desktop)：1
设备表面(equipment surface)：2
235生物安全柜(biological safety cabinet)A22设备表面(equipment surface)：422设备表面(equipment surface)：2
237缓冲间(buffer room)B--门把手(door handle)：2
墙面(wall)：2		---
239污物走道(waste path)C1-墙面(wall)：21-墙面(wall)：1
采样人员(sampler)--手套(gloves)：2--手套(gloves)：2
总计(total)910526621
), ArticleFig(id=1240954726523662602, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Tab.2, caption=

One-way ANOVA results of oligotrophic microbial monitoring data in purified water systems

, figureFileSmall=null, figureFileBig=null, tableContent=
因素
(factor)		自由度
（degree of freedom）		平方和
（sum of squares）		均方
（mean variance）		 F P
计数方法（counting method）14 983.3254 983.32595.1920.000
培养基种类（media）113 450.75613 450.756256.9370.000
培养时间（time）25 383.5592 691.78051.4190.000
计数方法×培养基种类（counting method×media）16 463.2116 463.211123.4610.000
计数方法×培养时间（counting method×time）23 245.3771 622.68930.9970.000
培养基种类×培养时间（media×time）24 592.6072 296.30443.8640.000
计数方法×培养基种类×培养时间（counting method×media×time）23 274.2511 637.12531.2730.000
模型（modelling）1132 915.2692 992.29757.1590.000
误差（error）34818 217.90952.3500.0000.000
修正整体（statistical modification）35951 133.1780.0000.0000.000
), ArticleFig(id=1240954726624325903, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=表2, caption=

纯化水系统寡营养微生物监测数据的单因素方差分析

, figureFileSmall=null, figureFileBig=null, tableContent=
因素
(factor)		自由度
（degree of freedom）		平方和
（sum of squares）		均方
（mean variance）		 F P
计数方法（counting method）14 983.3254 983.32595.1920.000
培养基种类（media）113 450.75613 450.756256.9370.000
培养时间（time）25 383.5592 691.78051.4190.000
计数方法×培养基种类（counting method×media）16 463.2116 463.211123.4610.000
计数方法×培养时间（counting method×time）23 245.3771 622.68930.9970.000
培养基种类×培养时间（media×time）24 592.6072 296.30443.8640.000
计数方法×培养基种类×培养时间（counting method×media×time）23 274.2511 637.12531.2730.000
模型（modelling）1132 915.2692 992.29757.1590.000
误差（error）34818 217.90952.3500.0000.000
修正整体（statistical modification）35951 133.1780.0000.0000.000
), ArticleFig(id=1240954726691434773, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Tab.3, caption=

Summary of microorganisms isolated by TSA and R2A media

, figureFileSmall=null, figureFileBig=null, tableContent=
初次分离的培养基(medium for primary isolation)菌株(species)特性(character)
初次仅分离于TSA培养基(separated from the TSA medium)米兰农霉菌(Agromyces mediolanus)真菌(fungi)
巨大芽孢杆菌(Bacillus meqaterium)产芽孢(spore producing)
壁芽孢杆菌(Bacillus muralis)产芽孢(spore producing)
短短芽孢杆菌(Brevibacillus brevis)产芽孢(spore producing)
滕黄短小杆菌(Curtobacterium luteum)条件致病菌(opportunistic pathogen)
埃吉类芽孢杆菌(Paenibacillus elqii)/
解葡聚糖类芽胞杆菌(Paenibacillus glycanilyticus)/
山羊葡萄球菌(Staphylococcus caprae)条件致病菌(opportunistic pathogen)
嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)条件致病菌(opportunistic pathogen)
初次仅分离于R2A培养基(separated from the R2A medium)高地芽孢杆菌(Bacillus altitudinis)产芽孢(spore producing)
土壤短芽孢杆菌(Brevibacillus agri)产芽孢(spore producing)
桥石短芽孢杆菌(Brevibacillus choshinensis)产芽孢(spore producing)
在TSA和R2A共同分离的菌株(separated from the TSA and R2A media)蜡样芽孢杆菌(Bacillus cereus)产芽孢(spore producing)
海泥芽孢杆菌(Bacillus oceanisediminis)产芽孢(spore producing)
简单芽孢杆菌(Bacillus simplex)产芽孢(spore producing)
类短短芽孢杆菌(Brevibacillus parabrevis)产芽孢(spore producing)
罗伊氏短芽孢杆菌(Brevibacillus reuszeri)产芽孢(spore producing)
腐殖质类芽孢杆菌(Paenibacillus humicus)/
皮氏罗尔斯顿菌(Ralstonia pickettii)条件致病菌(opportunistic pathogen)
), ArticleFig(id=1240954726787903775, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=表3, caption=

TSA与R2A培养基分离微生物汇总表

, figureFileSmall=null, figureFileBig=null, tableContent=
初次分离的培养基(medium for primary isolation)菌株(species)特性(character)
初次仅分离于TSA培养基(separated from the TSA medium)米兰农霉菌(Agromyces mediolanus)真菌(fungi)
巨大芽孢杆菌(Bacillus meqaterium)产芽孢(spore producing)
壁芽孢杆菌(Bacillus muralis)产芽孢(spore producing)
短短芽孢杆菌(Brevibacillus brevis)产芽孢(spore producing)
滕黄短小杆菌(Curtobacterium luteum)条件致病菌(opportunistic pathogen)
埃吉类芽孢杆菌(Paenibacillus elqii)/
解葡聚糖类芽胞杆菌(Paenibacillus glycanilyticus)/
山羊葡萄球菌(Staphylococcus caprae)条件致病菌(opportunistic pathogen)
嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)条件致病菌(opportunistic pathogen)
初次仅分离于R2A培养基(separated from the R2A medium)高地芽孢杆菌(Bacillus altitudinis)产芽孢(spore producing)
土壤短芽孢杆菌(Brevibacillus agri)产芽孢(spore producing)
桥石短芽孢杆菌(Brevibacillus choshinensis)产芽孢(spore producing)
在TSA和R2A共同分离的菌株(separated from the TSA and R2A media)蜡样芽孢杆菌(Bacillus cereus)产芽孢(spore producing)
海泥芽孢杆菌(Bacillus oceanisediminis)产芽孢(spore producing)
简单芽孢杆菌(Bacillus simplex)产芽孢(spore producing)
类短短芽孢杆菌(Brevibacillus parabrevis)产芽孢(spore producing)
罗伊氏短芽孢杆菌(Brevibacillus reuszeri)产芽孢(spore producing)
腐殖质类芽孢杆菌(Paenibacillus humicus)/
皮氏罗尔斯顿菌(Ralstonia pickettii)条件致病菌(opportunistic pathogen)
), ArticleFig(id=1240954726863401251, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Tab.4, caption=

Colony counts of TSA and R2A media at pharmaceutical environmental monitoring sites

, figureFileSmall=null, figureFileBig=null, tableContent=
房间号
(room)		监测项目
(monitoring item)		菌落计数(colony count)/（CFU·皿-1）(CFU per dish)
R2A培养基(R2A medium)TSA培养基*(TSA medium)
206沉降菌（settling microbe）2822
207浮游菌（airborne microbe）175
207把手表面菌（surface microbe on door handles）21
210沉降菌（settling microbe）62
210浮游菌（airborne microbe）88
219把手表面菌（surface microbe on door handles）<1#1
220墙面表面菌（surface microbe on walls）<10.5
220把手表面菌（surface microbe on door handles）2<1
221沉降菌（settling microbe）<10.5
221浮游菌（airborne microbe）10.5
224沉降菌（settling microbe）3814.5
235把手表面菌（surface microbe on door handles）20.5
239浮游菌（settling microbe）11.5
239墙面表面菌（surface microbe on walls）38<1
235把手表面菌（surface microbe on door handles）1<1
), ArticleFig(id=1240954726959870251, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=表4, caption=

制药环境监测点TSA与R2A培养基的计数结果

, figureFileSmall=null, figureFileBig=null, tableContent=
房间号
(room)		监测项目
(monitoring item)		菌落计数(colony count)/（CFU·皿-1）(CFU per dish)
R2A培养基(R2A medium)TSA培养基*(TSA medium)
206沉降菌（settling microbe）2822
207浮游菌（airborne microbe）175
207把手表面菌（surface microbe on door handles）21
210沉降菌（settling microbe）62
210浮游菌（airborne microbe）88
219把手表面菌（surface microbe on door handles）<1#1
220墙面表面菌（surface microbe on walls）<10.5
220把手表面菌（surface microbe on door handles）2<1
221沉降菌（settling microbe）<10.5
221浮游菌（airborne microbe）10.5
224沉降菌（settling microbe）3814.5
235把手表面菌（surface microbe on door handles）20.5
239浮游菌（settling microbe）11.5
239墙面表面菌（surface microbe on walls）38<1
235把手表面菌（surface microbe on door handles）1<1
), ArticleFig(id=1240954727043756334, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=EN, label=Tab.5, caption=

Isolation rates of typical microorganisms in R2A and TSA media in pharmaceutical environment

, figureFileSmall=null, figureFileBig=null, tableContent=
序号
(No.)		微生物种类
(type of microbes)		革兰染色
(Gram stain)		分离率(isolation rate)/%
R2A培养基(R2A medium)TSA培养基(TSA medium)
1微球菌属(Micrococcus spp.)G+22.519.6
2芽孢杆菌属(Bacillus spp.)G+14.38.9
3寡养单胞菌(Stenotrophomonas spp.)G-14.31.8
4葡萄球菌属(Staphylococcus spp.)G+12.239.3
5微小杆菌属某些种(Microbacterium spp.)G+8.27.1
6莫拉菌属某些种(Moraxella spp.)G-6.11.8
7假单胞菌属(Pseudomonas spp.)G-4.11.8
8分枝盐场单胞菌属(Salinarimonas spp.)G-4.10.0
9短杆菌属(Brachybacterium spp.)G+2.00.0
10短杆菌属某些种(Brevibacterium spp.)G+2.00.0
11戈登氏菌属(Gordonia spp.)G+2.01.8
12甲基杆菌属（Methylobacterium spp.)G-2.00.0
13副球菌属(Paracoccus spp.)G-2.00.0
14鞘氨醇杆菌属（Sphingobacterium spp.)G-0.03.6
15不动杆菌属（Acinetobacter spp.）G-0.01.8
16嗜碱杆菌属（Alkalihalobacillus spp.)G+0.03.6
17布鲁氏菌属（Brevundimonas spp.)G-0.01.8
18皮肤球菌属（Dermacoccus spp.)G+0.01.8
19库克氏菌属（Kocuria spp.)G+0.01.8
20瑙曼氏菌属（Naumannella spp.)G+0.01.8
21泛菌属某些种（Pantoea spp.)G-0.01.8
), ArticleFig(id=1240954727115059506, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1240945596094272210, language=CN, label=表5, caption=

制药环境R2A和TSA培养基中典型微生物的分离率

, figureFileSmall=null, figureFileBig=null, tableContent=
序号
(No.)		微生物种类
(type of microbes)		革兰染色
(Gram stain)		分离率(isolation rate)/%
R2A培养基(R2A medium)TSA培养基(TSA medium)
1微球菌属(Micrococcus spp.)G+22.519.6
2芽孢杆菌属(Bacillus spp.)G+14.38.9
3寡养单胞菌(Stenotrophomonas spp.)G-14.31.8
4葡萄球菌属(Staphylococcus spp.)G+12.239.3
5微小杆菌属某些种(Microbacterium spp.)G+8.27.1
6莫拉菌属某些种(Moraxella spp.)G-6.11.8
7假单胞菌属(Pseudomonas spp.)G-4.11.8
8分枝盐场单胞菌属(Salinarimonas spp.)G-4.10.0
9短杆菌属(Brachybacterium spp.)G+2.00.0
10短杆菌属某些种(Brevibacterium spp.)G+2.00.0
11戈登氏菌属(Gordonia spp.)G+2.01.8
12甲基杆菌属（Methylobacterium spp.)G-2.00.0
13副球菌属(Paracoccus spp.)G-2.00.0
14鞘氨醇杆菌属（Sphingobacterium spp.)G-0.03.6
15不动杆菌属（Acinetobacter spp.）G-0.01.8
16嗜碱杆菌属（Alkalihalobacillus spp.)G+0.03.6
17布鲁氏菌属（Brevundimonas spp.)G-0.01.8
18皮肤球菌属（Dermacoccus spp.)G+0.01.8
19库克氏菌属（Kocuria spp.)G+0.01.8
20瑙曼氏菌属（Naumannella spp.)G+0.01.8
21泛菌属某些种（Pantoea spp.)G-0.01.8
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制药用水及受控环境中寡营养微生物的生物负载监测*
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范一灵 1, 2 , 李琼琼 2 , 王培恩 2 , 杨美成 1, 3, **
药物分析杂志 | 生物检定+代谢分析 2024,44(11): 1899-1908
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药物分析杂志 | 生物检定+代谢分析 2024, 44(11): 1899-1908
制药用水及受控环境中寡营养微生物的生物负载监测*
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范一灵1, 2 , 李琼琼2, 王培恩2, 杨美成1, 3, **
作者信息
  • 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

    • Tel：（021）38839900；E-mail：

通讯作者:

**Tel：（021）38839900；E-mail：
Monitoring the bioburden of oligotrophs in pharmaceutical water systems and controlled environments*
Yi-ling FAN1, 2 , Qiong-qiong LI2, Pei-en WANG2, Mei-cheng YANG1, 3, **
Affiliations
  • 1.China State Institute of Pharmaceutical Industry，Shanghai 201203，China
  • 2.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
  • 3.Shanghai Food and Drug Packaging Material Control Center，Shanghai 201203，China
出版时间: 2024-12-01 doi: 10.16155/j.0254-1793.2024-0357
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摘要
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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。

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
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 R2A 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 R2A was more effective than the TSA medium in this study. The profiles of microorganisms isolated from the R2A and TSA media were unique. The isolation rate of Gram-negative bacteria in the R2A 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 R2A 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.

oligotrophs  /  pharmaceutical water  /  controlled environment  /  bioburden  /  objectionable microorganism
范一灵, 李琼琼, 王培恩, 杨美成. 制药用水及受控环境中寡营养微生物的生物负载监测*. 药物分析杂志, 2024 , 44 (11) : 1899 -1908 . DOI: 10.16155/j.0254-1793.2024-0357
Yi-ling FAN, Qiong-qiong LI, Pei-en WANG, Mei-cheng YANG. Monitoring the bioburden of oligotrophs in pharmaceutical water systems and controlled environments*[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (11) : 1899 -1908 . DOI: 10.16155/j.0254-1793.2024-0357
正文
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寡营养微生物（oligotrophs）是一类可存活于有机物含量极低环境中（含碳量1~15 mg·L-1），并保持一定生长速率的微生物[1]，主要由非发酵革兰阴性菌组成，广泛存在于天然水体、空气和土壤等自然环境样本中[2]。寡营养微生物自身耗氧量低、营养需求少，极易在药品生产环境及其水系统中长期存活和生长[3]。寡营养微生物一般都具有较强的环境耐受性，可产生生物被膜、生物毒素和耐受性等自我保护机制，同时携带多种抗生素耐药性[4-5]，抵抗一定浓度的消毒剂[6-7]。此外，该类微生物可共享环境种群的生物学特性，通过质粒在微生物群落间传播耐药性和抗逆性[8-9]。在营养条件的选择压力下，寡营养微生物往往处于活的不可培养状态，很难通过常规手段培养分离[10]。已知的寡营养微生物主要为不动杆菌属（Acinetobacter spp.）、甲基杆菌属（Methylobacterium spp.）、假单胞菌属（Pseudomonas spp.）、伯克霍尔德菌属（Burkholderia spp.）、寡养单胞菌属(Stenotrophomonas spp.)、气单胞菌属（Aeromonas spp.）、军团菌属（Legionella spp.）和分枝杆菌属（Mycobacterium spp.）等微生物[11]。其中，不少寡养微生物属于条件致病菌，每年可引起上万人感染[12-13]。寡营养微生物可随药品原辅料和生产工艺最终进入终产品中，导致药品和医院制剂的污染[14-15]，是医药产品中内毒素的主要来源之一[9]，易感人群、免疫抑制和免疫缺陷患者的安全带来巨大影响。
制药生产过程中生物负载的监控方式主要以胰酪大豆胨液体培养基（Soybean-Casein Digest Agar，SCDA）或沙氏葡萄糖琼脂（Sabouraud’s Dextrose Agar，SDA）等富营养培养基为主，适合培养嗜中温微生物[10，16-17]。该类微生物以人源革兰阳性球菌为主要污染源[18-19]。因在自然环境中寡营养微生物生可能处于活的不可培养状态（Viable but non-culturable，VBNC），且生长缓慢，在首次复苏培养时无法迅速适应富营养的培养条件，容易被其他微生物群落掩盖[20-21]，难以在制药行业日常监测中快速获得可培养和可观察的菌落[22]。因此，传统制药环境监测中使用的富营养培养方法可能并不适用于寡营养微生物种群的监测。
随着技术进步，以基因治疗产品等为代表的新兴生物制品已全面步入了全封闭生产（隔离系统）和自动化生产（连续制造）的新阶段。在核心生产工艺中，操作人员直接接触产品的概率持续降低，预期将显著减少人源性微生物的污染。药品生产链中污染微生物的种群必然随着生产工艺进步而变化。已有研究显示，在临床样本和无菌药物生产车间中大量分离到了寡营养微生物种群[15-23]。制药洁净环境中由物料、空气和制药用水等为主要污染源的寡营养微生物极有可能成为未来药品污染微生物的重要因素之一。探索寡营养微生物的有效监测方法已迫在眉睫。常见的寡营养培养基主要有R2A培养基和1/10或其他低浓度的SCDA等培养基[24-25]。为有效分离水系统寡营养环境引入的微生物污染，提高微生物检出率，国际主流药典已普遍选用R2A培养基作为制药用水微生物监测的首选培养基[26-27]。
本研究通过筛选适宜的寡营养微生物培养方法，监测制药企业及药品检验实验室典型环境（制药用水、洁净空气和洁净表面等），解决传统生物负载监测技术可能漏检寡营养微生物的问题。依托微生物多相鉴定技术，分析真实制药环境中微生物的种类与群落分布，丰富我们对制药行业潜在的风险微生物种类的认知，为企业建立更为全面的环境微生物监测方案，满足企业对不可接受微生物的风险评估需求。
1　实验材料、仪器与试剂
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1.1　实验材料
实验中选用的R2A和TSA培养基均采购自北京陆桥技术有限责任公司，按厂家说明书制备。表面菌采样平皿规格为55 mm，其余平皿规格为90 mm。试验用纯化水样本来自上海市食品药品检验研究院微生物实验室管道纯化水。环境样本来自国内某生物制药公司细胞生产车间。
1.2　实验仪器
试验所用主要仪器为Biotyper基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS，布鲁克公司）；Veritii PCR扩增仪（赛默飞公司）；QuantityOne紫外凝胶成像仪（伯乐公司）。数据统计软件选用OriginPro 2018。
1.3　实验试剂
细菌基因组DNA提取试剂盒采购自北京天根生化科技公司。细菌核酸扩增及测序上游引物27F：5’-AGAGTTTGATCCTGGCTCAG-3’，下游引物1492R：5’-ACGGCTACCTTGTTACGACTT-3’。DNA引物合成与测序服务由生工生物工程（上海）股份有限公司提供。
2　实验方法
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2.1　药品检测实验室纯水系统微生物监测
以直接接种法和薄膜过滤法分别监测药品检测实验室内10个管道纯水使用点。每个使用点的纯化水采样量不少于100 mL。取4份纯化水各1 mL直接接种于TSA培养基中，于20~25 ℃和30~35 ℃各培养2个平皿；另取4份纯化水各1 mL接种于R2A培养基中，于20~25 ℃和30~35 ℃培养2个平皿。同法以薄膜过滤法替代直接接种法接种2种培养基，薄膜过滤法（滤膜孔径≥0.45 μm，直径约为50 mm）过滤纯化水量为10 mL，并置2个温度培养。记录上述培养物在培养第3、5和7 d时的需氧菌总数计数结果，分别计算不同条件下的菌落数平均值（CFU·mL-1）。分析2种培养基在不同培养时间、培养温度和接种方法对纯化水中需氧菌总数计数结果的影响。
2.2　制药企业环境微生物监测
对制药业洁净室的采样按2020年版《中华人民共和国药典》通则9205要求执行，生产洁净车间采样布点示意图见图1。
采用R2A和TSA培养基分别收集生产车间洁净室的沉降菌、浮游菌、设备与操作人员的表面菌，采样信息见表1。其中，以TSA培养基监测的采样点取2个样品，以R2A培养基监测的采样点取1个样品，墙面均仅取1个样品。采样后置30~35 ℃培养7 d，统计需氧菌总数计数结果。
2.3　微生物的鉴定
挑取每个监测平板中表形形态不同的微生物，分别划线接种至R2A和TSA培养基，置30~35 ℃培养纯化。采用MALDI-TOF MS技术对分离的微生物进行鉴定，对无法获得准确鉴定结果的微生物（置信度小于2.0），采用16S rDNA测序技术鉴定。
MALDI-TOF MS鉴定方法如下[28]：采用原位甲酸提取法，将待测微生物均匀涂抹于检测靶板上，自然干燥后加入70%甲酸溶液1 μL，干燥后加入α-氰基-4-羟基肉桂酸（HCCA）基质溶液1 μL，充分晾干后上机检测，确定微生物鉴定结果。16S rDNA测序鉴定方法如下[29]：将待测微生物用500 μL生理盐水分散，按细菌基因组DNA提取试剂盒说明书提取核酸，置100 μL TE缓冲液-20 ℃保存。PCR反应体系：DNA模板2 μL，rTag DNA聚合酶1U，1×PCR缓冲液，4种dNTP各200 μmol·L-1，上下游引物各200 pmol·L-1，加纯化水至50 μL。PCR反应条件：95 ℃变性1 min；95 ℃变性30 s，56 ℃退火30 s，72 ℃延伸90 s，共30个循环；72 ℃延伸10 min，产物置-20 ℃保存、测序。测序数据拼接后与GenBank数据库(www.ncbi.nlm.nih.gov)的核酸信息比对，确定菌株鉴定结果。
3　试验结果
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3.1　纯化水系统微生物监测
3.1.1　纯化水系统的微生物计数
采用单因素方差分析方法统计药品检测实验室10个纯化水点位的需氧菌总数计数结果（表2）。试验选取的3个主要参数（计数方法、培养基种类和培养时间）显著影响水系统需氧菌总数计数结果（P<0.01）。将每个监测点的菌落计数结果按计数方法（直接接种法与薄膜过滤法）、培养基种类（R2A培养基和TSA培养基）、培养时间（3、5和7 d）和培养温度（20~25 ℃和30~35 ℃）进行分类汇总，采用Origin 3D条状图功能绘图（图2），可更直观地确定开展纯化水寡营养微生物检查的关键条件，即采用R2A培养基直接倾注法处理，置30~35 ℃培养不少于7 d。
3.1.2　纯化水系统的微生物鉴定
在10个纯化水采样点中，从R2A和TSA培养基共分离并鉴定出68株典型微生物。其中，R2A培养基分离得到菌种数量占比为42.6%（29/68），TSA培养基分离得到菌株数量占比为57.4%（39/68）。按菌株初次分离的培养基分类鉴定结果汇总见表3，不同培养基监测到的微生物种群有较大差异。本次监测中在TSA和R2A培养基中共同分离出了7种微生物，其中Ralstonia pickettii是报道对水基质产品可能具备潜在危害的不可接受微生物。在R2A培养基中分离出3种在TSA培养基上不易分离或不易生长的条件致病菌株，即Bacillus altitudinis、Brevibacillus agri和Brevibacillus choshinensis。这3种微生物均可产生芽孢，能抵抗部分高温、消毒等不利条件。当然，在TSA培养基中也分离出独特的微生物种群，包括葡萄球菌属微生物。TSA作为常用监测培养基可检测到更多的人源性污染微生物，R2A培养基作为监测寡营养体系的培养基，可有效分离部分生长较慢的微生物。因此，在水系统监测过程中，可根据实际情况联合选用寡营养和富营养培养基，以期更全面、有效的监控水系统污染微生物种类和数量，制定有针对性的消杀策略。
3.2　制药环境寡营养微生物的监测
3.2.1　制药环境寡营养微生物的计数
对制药生产车间13个采样点位的104个监测数据进行统计分析。排除同一采样点2种培养基上均为零值的结果，将非零值的监测数据列于表4。其中，R2A培养基的结果为平皿计数值，TSA培养基的结果为2个重复测试的平均值。采用R2A和TSA培养基对企业洁净环境监测的结果均满足2020年版《中华人民共和国药典》四部通则9205对洁净环境的要求。以双样本T检验方法分析，R2A和TSA培养基的计数结果虽然在统计上无显著性差异（P>0.05），但R2A培养基的计数总平均值（9.6 CFU·皿-1）大于TSA培养基的计数结果（3.8 CFU·皿-1）。R2A培养基菌落计数的结果略高于TSA培养基的计数结果。
3.2.2　制药环境寡营养微生物的鉴定
在制药环境各采样点中，共分离出105种典型微生物。其中，从R2A培养基中共分离微生物49株（占比46.7%），从TSA培养基共分离微生物56株（占比53.3%）。对上述2种培养基分离的微生物进行鉴定（表5），按革兰染色结果分类，R2A培养基中革兰阴性菌的分离率为37.0%，革兰阳性菌的分离率为63.0%。而TSA培养基中革兰阴性菌的分离率仅为14.0%，革兰阳性菌的分离率高达86.0%。以“属”分类，从R2A培养基中分离率最高的前3种微生物为Micrococcus spp.、Bacillus spp.和Stenotrophomonas spp.，占比分别为22.5%、14.3%和14.3%。从TSA培养基中分离率最高的前3种微生物为Staphylococcus spp.、Micrococcus spp.和Bacillus spp.，占比分别为39.3%、19.6%和8.9%。其中，Stenotrophomonas spp.在TSA培养基中分离率占比仅为1.8%。相反的，在TSA培养基中Staphylococcus spp.的分离率占比超过39.3%，而该微生物在R2A培养基中的分离率仅为12.2%。
R2A和TSA培养基在同一环境中对微生物种群的分离率存在明显差异，在R2A培养基上单一微生物种群占比明显低于TSA培养基（图3）。同时，微生物在R2A培养基的菌落明显小于TSA培养基，即R2A培养基中微生物的生长速度相对较慢，符合寡营养微生物的生长特点。在充分的培养时间下（7 d），R2A培养基上的微生物在形成独立菌落前，不易互相掩盖，有利于非优势种群的发现和分离。
TSA培养基适合于监测常见人源性污染微生物，主要为革兰阳性菌；R2A培养基更适合监测和分离环境中革兰阴性微生物，能提高洁净环境中寡养微生物的检出概率。寡养培养方法可作为常规环境监测方法的补充，更全面的揭示环境污染风险。
4　讨论
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生物负载监控是制药生产过程中的一项重要工作，其目的是确保在药品全生命周期中微生物污染保持在可接受水平，以满足法规和产品质量标准的要求。对具体产品及相应工艺的风险评估要求而言，工艺中的微生物种类和污染量同样重要。传统制药工艺中无法有效隔离人员操作的影响，人源性微生物是制药工业过程中引入外源性污染的主要来源。因此，采用TSA培养基为主的监控技术可有效反映传统工艺中微生物的总体污染情况[29-30]。R2A属于寡营养培养基，有助于抑制常见微生物，尤其是革兰阳性球菌的生长速度，使得其他寡营养微生物不易被掩盖。且在R2A培养基上微生物更容易产生色素，便于在菌落形成初期的识别。各国药典已规定在制药用水的监测中使用寡营养培养基[26]。尝试使用TSB和R2B液体培养基分析寡营养微生物（Ralstonia pickettii、Paenibacillus lautus等）的生长情况，初步结果显示，寡营养液体培养基R2B中微生物的生长速率显著低于富营养培养基TSB，培养60 h后R2B培养基中微生物的终浓度约为TSB培养基的30%~70%。
随着制药工艺的不断进步，尤其在细胞治疗产品和基因治疗产品等新兴生物制药工艺中已广泛应用隔离系统和密闭式生产工艺，极大的避免了人员与生产过程的直接接触。可以预测随着自动化与隔离程度的不断提高，人源性微生物污染比例将显著下降。而自然界中广泛存在的寡营养微生物，如洋葱伯克霍尔德菌群（Burkholderia cepacia complex）、罗尔斯通菌属（Ralstonia spp.）、寡氧单胞菌属（Stenotrophomonas spp.）、鞘氨醇单胞菌属（Sphingomonas spp.）、粘质沙雷菌（Serratia marcescens）、肺炎克雷伯菌（Klebsiella pneumonia）、阴沟肠杆菌（Enterobacter cloacae）、不动杆菌属（Acinetobacter spp.）等微生物，可能会成为药品工艺污染监控的重点。由于寡营养微生物的生长特性和培养条件与传统方法监测的人源性微生物有较大差异，适时引入专属的寡营养微生物监测技术可以有助于发现生产过程中的潜在风险，为基于风险的质量管理提供数据。
本研究结果显示平皿倾注法的菌落计数结果优于薄膜过滤。可能是因为薄膜的有效过滤面积较小，菌落易重叠，同时在薄膜上不易观察微小菌落，更易被掩盖。本研究中发现，在R2A中分离的3种微生物，即Bacillus altitudinis、Brevibacillus agri和Brevibacillus choshinensis，虽也可在TSA培养基中生长，但其生长较为缓慢，培养超过5 d后仍较难识别。与水系统微生物存活状态不同，空气中微生物分布具有更大随机性。使用空气采样器的采样效率和重复性较水样采集方法差[31]。虽然，在洁净环境监测结果中R2A和TSA培养基的菌落计数结果在统计上不显著，可能与洁净环境的“零”值结果较多有关，但R2A培养基可复苏更为丰富的微生物种群。另一方面，TSA培养基的检测时间较短（2~3 d），有利于对企业对时间成本的控制。企业在选择监控方法时，可以考虑以传统环境监控技术为基础，辅以寡营养检测技术，基于风险评估原则建立质量风险管理原则下的环境微生物质量控制技术体系，分析微生物污染控制中的风险，有效发现生产过程中的潜在不可接受微生物，有助于指导环境消毒、方法验证等过程控制方式，消除潜在隐患。
本研究比较了R2A和TSA培养基在制药用纯化水系统和常见洁净环境生物负载监测的微生物计数和鉴定结果，初步形成了较为完善的寡养环境微生物过程监控模式，为制定微生物污染控制策略提供有效信息。提高实验室对环境微生物种群多样性的监测能力，完善制药环境微生物的质量监控体系，为企业质量风险管理提供案例参考。
基金
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  • *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)
文献
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2024年第44卷第11期
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doi: 10.16155/j.0254-1793.2024-0357
  • 接收时间：2024-05-29
  • 首发时间：2026-03-18
  • 出版时间：2024-12-01
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  • 收稿日期：2024-05-29
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)
作者信息
    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

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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