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Article(id=1239231267858870564, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239231265254207730, articleNumber=null, orderNo=null, doi=10.16155/j.0254-1793.2024-0072, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1706630400000, receivedDateStr=2024-01-31, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773385358061, onlineDateStr=2026-03-13, pubDate=1725033600000, pubDateStr=2024-08-31, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773385358061, onlineIssueDateStr=2026-03-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773385358061, creator=13701087609, updateTime=1773385358061, updator=13701087609, issue=Issue{id=1239231265254207730, tenantId=1146029695717560320, journalId=1205117023404326918, year='2024', volume='44', issue='8', pageStart='1285', pageEnd='1462', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=0, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773385357440, creator=13701087609, updateTime=1773385579800, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1239232197937393856, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239231265254207730, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1239232197937393857, tenantId=1146029695717560320, journalId=1205117023404326918, issueId=1239231265254207730, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1407, endPage=1414, ext={EN=ArticleExt(id=1239231268118917428, articleId=1239231267858870564, tenantId=1146029695717560320, journalId=1205117023404326918, language=EN, title=Using metagenomic sequencing to identify and trace drug-contaminating bacteria in lincomycin hydrochloride and lidocaine hydrochloride gel, columnId=1206272757852074373, journalTitle=Chinese Journal of Pharmaceutical Analysis, columnName=Safety Monitoring, runingTitle=null, highlight=null, articleAbstract=
Objective:

To evaluate the application of metagenomic sequencing in identification and tracking of drug-contaminating bacteria.

Methods:

Both metagenomic sequencing and isolation culturing method were employed to identify and genotype bacteria in two batches of possibly contaminated gel medicines as well as the environmental samples from testing laboratory. Bacteria isolated by culturing method were identified through Gram’s stain，biochemical reaction，MALIDI-TOF-MS，16srDNA sequencing and Bacillus specific PCR. The detected Bacillus cereus were typed by multilocus sequence typing (MLST) .The metagenomic sequencing results were analyzed at the strain level using MetaPhlan software.

Results:

Both MLST and MetaPhlan analysis revealed a substantial contamination of Bacillus cereus in gel medicines as well as environmental samples，with the presence of the same Bacillus cereus group. Both metagenomic sequencing and culturing methods detected Paenibacillus，Brevibacillus and Neobacillus in gel medicines，but amplicon sequencing also detected these bacteria in environment which might be the source of contamination.

Conclusion:

These findings indicate that metagenomic sequencing technology is reliable and has the advantage over isolation culturing in comprehensively detecting potentially hazardous bacterial species and accurately performing strain-level traceability analysis. It holds significant application value in pharmaceutical contamination identification and traceability.

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目的：

评价宏基因组测序法在药品污染细菌鉴定和溯源中的应用。

方法：

分别通过培养法、扩增子测序法和宏基因组测序法，对2批微生物限度控制菌检查中检出未知微生物的林可霉素利多卡因凝胶药剂进行菌种鉴定和溯源。培养法分离得到的细菌，通过革兰氏染色、生化反应、生物质谱法、16srDNA测序及芽孢杆菌特异性PCR方法进行菌种鉴定，并通过多位点序列分型（MLST）法对药品和环境中均检出的蜡样芽孢杆菌进行菌株分型。宏基因组测序结果通过MetaPhlan软件进行菌株水平的分析。

结果：

MLST与MetaPhlan结果均显示药品与检测环境中存在同一组蜡样芽孢杆菌，显示药品中的蜡样芽孢杆菌极有可能来自检测环境的污染。培养法与宏基因组测序法均在药品中检出污染微生物土壤短芽孢杆菌、类芽孢杆菌及新芽孢杆菌，但扩增子测序还在环境样本中检出上述细菌，因此不能排除药品中此类污染菌来自检测环境的可能性。

结论：

宏基因组测序技术相比培养法，能够更全面发现具有潜在风险的微生物，同时也能准确地进行菌株水平的溯源分析，在药品污染菌鉴定与溯源中具有重要的应用价值。

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Tel：13957203861；E-mail：

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Vegetatio，1987，69：57, articleTitle=Compositional dissimilarity as a robust measure of ecological distance, refAbstract=null)], funds=[Fund(id=1239231272644571649, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, awardId=2023010, language=CN, fundingSource=*浙江省药监局科技计划项目(2023010), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1239231269897302357, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, xref=1., ext=[AuthorCompanyExt(id=1239231269901496662, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, companyId=1239231269897302357, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.Zhoushan Institute for Food and Drug Control，National Ocean Food Quality Inspection Center，Zhoushan 316012，China), AuthorCompanyExt(id=1239231269909885271, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, companyId=1239231269897302357, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.舟山市食品药品检验检测研究院 国家海洋食品质量检验检测中心，舟山 316012)]), AuthorCompany(id=1239231269972799840, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, xref=2., ext=[AuthorCompanyExt(id=1239231269981188448, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, companyId=1239231269972799840, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.Shenzhen Institutes of Advanced Technology，Chinese Academy of Sciences，Shenzhen Institute of Synthetic Biology，CAS Key Laboratory of Quantitative Engineering Biology，Shenzhen 518055，China), AuthorCompanyExt(id=1239231269985382753, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, companyId=1239231269972799840, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.中国科学院深圳先进技术研究院 深圳合成生物学创新研究院 中国科学院定量工程生物学重点实验室，深圳 518055)])], figs=[ArticleFig(id=1239231271860236756, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=EN, label=Tab.1, caption=

Specific PCR primer pairs for Bacillus

, figureFileSmall=null, figureFileBig=null, tableContent=
细菌种类
（bacteria species)		PCR引物
（PCR primer）		基因
（gene）		PCR产物
（PCR product）/bp		参考文献
（reference）
枯草芽孢杆菌（Bacillus subtilis）F：AAGCCGTGAAATTATCGCAGACTCilvD640自行设计（designed by this research）
R：CGCCGCCCGCTTCGTGA
42F：CAGTCAGGAAATGCGTACGTCCTTgyrA1 000[10]
1066R：CAAGGTAATGCTCCAGGCATTGCT
解淀粉芽孢杆菌（Bacillus amyloliquefaciens）42F：CAGTCAGGAAATGCGTACGTCCTTgyrA1 000[10]
1066R：CAAGGTAATGCTCCAGGCATTGCT
蜡样芽孢杆菌（Bacillus cereus）F：GCGTCTGCAACGTTTAACTGGgyrA1 084[13]
R：TGTCGCTACCTCTTGCTCATC
F：TGGCCCAAATGAAGATpanC600自行设计（designed by this research）
R：TAACTGCAATAGCTAAAATGAT
F：CGG GGC AAA CAT TAA GAG AAilvD600[14]
R：GGT TCT GGT CGT TTC CAT TC
苏云金芽孢杆菌（Bacillus thuringiensis）F：GCTGTGACACGAAGGATATAGCCACcry1 600[15]
R：AGGACCAGGATTTACAGGAGG
), ArticleFig(id=1239231271952511450, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=CN, label=表1, caption=

芽孢杆菌特异性PCR引物

, figureFileSmall=null, figureFileBig=null, tableContent=
细菌种类
（bacteria species)		PCR引物
（PCR primer）		基因
（gene）		PCR产物
（PCR product）/bp		参考文献
（reference）
枯草芽孢杆菌（Bacillus subtilis）F：AAGCCGTGAAATTATCGCAGACTCilvD640自行设计（designed by this research）
R：CGCCGCCCGCTTCGTGA
42F：CAGTCAGGAAATGCGTACGTCCTTgyrA1 000[10]
1066R：CAAGGTAATGCTCCAGGCATTGCT
解淀粉芽孢杆菌（Bacillus amyloliquefaciens）42F：CAGTCAGGAAATGCGTACGTCCTTgyrA1 000[10]
1066R：CAAGGTAATGCTCCAGGCATTGCT
蜡样芽孢杆菌（Bacillus cereus）F：GCGTCTGCAACGTTTAACTGGgyrA1 084[13]
R：TGTCGCTACCTCTTGCTCATC
F：TGGCCCAAATGAAGATpanC600自行设计（designed by this research）
R：TAACTGCAATAGCTAAAATGAT
F：CGG GGC AAA CAT TAA GAG AAilvD600[14]
R：GGT TCT GGT CGT TTC CAT TC
苏云金芽孢杆菌（Bacillus thuringiensis）F：GCTGTGACACGAAGGATATAGCCACcry1 600[15]
R：AGGACCAGGATTTACAGGAGG
), ArticleFig(id=1239231272040591842, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=EN, label=Tab.2, caption=

Identification results of 8 bacteria strains isolated from gel medicines and environment

, figureFileSmall=null, figureFileBig=null, tableContent=
批号
（batch No.）		菌株
（strain）		比对结果（百分比）[results (per. identity)]
质谱结果
（MALIDI-TOF-MS）		16srDNA比对结果
（The BLAST result of 16srDNA）		特异性PCR产物比对结果（the BLAST result of specific PCR product）
gyrApanCilvDCRY
D2300391-1蜡样芽孢杆菌群
（Bacillus cereus group）(99.9%)		蜡样芽孢杆菌（Bacillus cereus）(99.79%)蜡样芽孢杆菌/苏云金芽孢杆菌（Bacillus cereus/thuringiensis ）(98.86%)蜡样芽孢杆菌/苏云金芽孢杆菌（Bacillus cereus/thuringiensis ）(99.18%)蜡样芽孢杆菌
（Bacillus cereus）
(99.27%)		无PCR产物（no PCR product）
91-2短芽孢杆菌属
（Brevibacillus spp）
(99.9%)		土壤短芽孢杆菌（Brevibacillus agri）(99.58%)////
40无对应数据（no record in database)类芽孢杆菌（Paenibacillus albicereus）(99.03%)////
D2201332无对应数据（no record in database)巴达维亚新芽孢杆菌（Neobacillus bataviensis）(99.53%)////
HJ4HJ-1蜡样芽孢杆菌群
（Bacillus cereus group）		热带/副覃状/蜡样/淤泥/白色芽孢杆菌
（Bacillus tropics/paramycoides/cereus/luti/albus）(99.58%)		副炭疽/蜡样芽孢杆菌（Bacillus paranthracis/cereus）
(99.52%)		副炭疽/蜡样芽孢杆菌（Bacillus paranthracis/cereus）
(99.34%)		副炭疽芽孢杆菌
（Bacillus paranthracis）(99.80%)		无PCR产物（no PCR product）
HJ-2枯草/解淀粉/死谷芽孢杆菌（Bacillus subtiliis/amyloliquefaciens/vallismortis）卢戈斯芽孢杆菌（Bacillus rugosus）(99.53%)斯皮宰曾氏芽孢杆菌（Bacillus spizizenii） (99.28%)/枯草芽孢杆菌
（Bacillus subtilis）
(99.85%)		/
HJ5HJ-3蜡样芽孢杆菌群
（Bacillus cereus group）		蜡样芽孢杆菌（Bacillus cereus）(99.93%)/蜡样芽孢杆菌
（Bacillus cereus）
(99.17%)		蜡样芽孢杆菌/苏云金芽孢杆菌
（Bacillus cereus/thuringiensis）
(99.45%)		无PCR产物（no PCR product）
HJ-4枯草/解淀粉/死谷芽孢杆菌（Bacillus subtiliis/amyloliquefaciens/vallismortis）死谷芽孢杆菌（Bacillus vallismortis） (99.71%)解淀粉芽孢杆菌（Bacillus amyloliquefaciens）(99.89%)///
), ArticleFig(id=1239231272124477925, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=CN, label=表2, caption=

药品及检测环境中分离得到8株细菌的鉴定结果

, figureFileSmall=null, figureFileBig=null, tableContent=
批号
（batch No.）		菌株
（strain）		比对结果（百分比）[results (per. identity)]
质谱结果
（MALIDI-TOF-MS）		16srDNA比对结果
（The BLAST result of 16srDNA）		特异性PCR产物比对结果（the BLAST result of specific PCR product）
gyrApanCilvDCRY
D2300391-1蜡样芽孢杆菌群
（Bacillus cereus group）(99.9%)		蜡样芽孢杆菌（Bacillus cereus）(99.79%)蜡样芽孢杆菌/苏云金芽孢杆菌（Bacillus cereus/thuringiensis ）(98.86%)蜡样芽孢杆菌/苏云金芽孢杆菌（Bacillus cereus/thuringiensis ）(99.18%)蜡样芽孢杆菌
（Bacillus cereus）
(99.27%)		无PCR产物（no PCR product）
91-2短芽孢杆菌属
（Brevibacillus spp）
(99.9%)		土壤短芽孢杆菌（Brevibacillus agri）(99.58%)////
40无对应数据（no record in database)类芽孢杆菌（Paenibacillus albicereus）(99.03%)////
D2201332无对应数据（no record in database)巴达维亚新芽孢杆菌（Neobacillus bataviensis）(99.53%)////
HJ4HJ-1蜡样芽孢杆菌群
（Bacillus cereus group）		热带/副覃状/蜡样/淤泥/白色芽孢杆菌
（Bacillus tropics/paramycoides/cereus/luti/albus）(99.58%)		副炭疽/蜡样芽孢杆菌（Bacillus paranthracis/cereus）
(99.52%)		副炭疽/蜡样芽孢杆菌（Bacillus paranthracis/cereus）
(99.34%)		副炭疽芽孢杆菌
（Bacillus paranthracis）(99.80%)		无PCR产物（no PCR product）
HJ-2枯草/解淀粉/死谷芽孢杆菌（Bacillus subtiliis/amyloliquefaciens/vallismortis）卢戈斯芽孢杆菌（Bacillus rugosus）(99.53%)斯皮宰曾氏芽孢杆菌（Bacillus spizizenii） (99.28%)/枯草芽孢杆菌
（Bacillus subtilis）
(99.85%)		/
HJ5HJ-3蜡样芽孢杆菌群
（Bacillus cereus group）		蜡样芽孢杆菌（Bacillus cereus）(99.93%)/蜡样芽孢杆菌
（Bacillus cereus）
(99.17%)		蜡样芽孢杆菌/苏云金芽孢杆菌
（Bacillus cereus/thuringiensis）
(99.45%)		无PCR产物（no PCR product）
HJ-4枯草/解淀粉/死谷芽孢杆菌（Bacillus subtiliis/amyloliquefaciens/vallismortis）死谷芽孢杆菌（Bacillus vallismortis） (99.71%)解淀粉芽孢杆菌（Bacillus amyloliquefaciens）(99.89%)///
), ArticleFig(id=1239231272237724141, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=EN, label=Tab.3, caption=

MLST results of 4 Bacillus cereus strains

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株
（strain）		等位基因（allele）等位基因编号
（allelic profile）		BURST聚类结果
(cluster result of BURST)
glpFgmkilvDptapycApurHtpiA
91-1138911124293 257Group1
HJ-132315316426singleton
HJ-31228858129103 258Group1
HJ138911129532 573Group1
), ArticleFig(id=1239231272329998836, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=CN, label=表3, caption=

蜡样芽孢杆菌菌株的MLST分型结果

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株
（strain）		等位基因（allele）等位基因编号
（allelic profile）		BURST聚类结果
(cluster result of BURST)
glpFgmkilvDptapycApurHtpiA
91-1138911124293 257Group1
HJ-132315316426singleton
HJ-31228858129103 258Group1
HJ138911129532 573Group1
), ArticleFig(id=1239231272409690616, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=EN, label=Tab.4, caption=

Bactria detected by culturing method and sequencing methods

, figureFileSmall=null, figureFileBig=null, tableContent=
样本号
（sample No.）		培养法分离得到细菌
（bactria isolated by culturing method）		扩增子测序优势细菌
（dominant bactria in Amplicon sequencing result）		宏基因组测序结果MetaPhlan分析
（MetaPhlan analysis of metagenomics sequencing result）
32巴达维亚新芽孢杆菌（Neobacillus bataviensis）内生新芽孢杆菌（Neobacillus endophyticus）(99.99%)吉达新芽孢杆菌（Neobacillus jeddahensis SGB30241）（99.64%）
魏德曼氏芽孢杆菌（Bacillus wiedmannii）(0.007%)蜡样芽孢杆菌（Bacillus cereus SGB7697）（0.36%）
40类芽孢杆菌（Paenibacillus albicereus）类芽孢杆菌（Paenibacillus sp. UniB2）(89.6%)类芽孢杆菌（Paenibacillus albicereus SGB82357）（100%）
魏德曼氏芽孢杆菌（Bacillus wiedmannii）(2.0%)
玛斯格雷芽孢杆菌（Bacillus massiliglaciei）(1.9%)
内生新芽孢杆菌（Neobacillus endophyticus）（0.03%）
91-1蜡样芽孢杆菌（Bacillus cereus）魏德曼氏芽孢杆菌（Bacillus wiedmannii）(99.99%)蜡样芽孢杆菌（Bacillus cereus SGB7697）（100%）
内生新芽孢杆菌（Neobacillus endophyticus）(0.003%)
土壤短芽孢杆菌（Brevibacillus agri）（0.002%）
91-2土壤短芽孢杆菌（Brevibacillus agri）土壤短芽孢杆菌（Brevibacillus agri）(99.97%)土壤短芽孢杆菌（Brevibacillus agri SGB7330）（99.99%）
魏德曼氏芽孢杆菌（Bacillus wiedmannii）（0.03%）蜡样芽孢杆菌（Bacillus cereus SGB7697）（0.01%）
HJ4蜡样芽孢杆菌（Bacillus cerus）魏德曼氏芽孢杆菌（Bacillus wiedmannii）(95.2%) 蜡样芽孢杆菌（Bacillus cereus SGB7697）(98.34%)
类芽孢杆菌（Paenibacillus sp. UniB2）(2.1%)蜡样芽孢杆菌（Bacillus cereus SGB7703）(1.26%)
玛斯格雷芽孢杆菌（Bacillus massiliglaciei）（0.03%）地衣芽孢杆菌（Bacillus licheniformis SGB7780）(0.34%)
枯草芽孢杆菌（Bacillus subtilis）内生新芽孢杆菌（Neobacillus endophyticus）(0.03%)溶血葡萄球菌（Staphylococcus haemolyticus SGB7861）(0.06%)
枯草芽孢杆菌（Bacillus subtilis SGB7788）(0.0006%)
HJ5蜡样芽孢杆菌（Bacillus cereus）魏德曼氏芽孢杆菌（Bacillus wiedmannii）(99.998%)蜡样芽孢杆菌（Bacillus cereus SGB7697）(99.98%)
解淀粉芽孢杆菌（Bacillus amyloliquefaciens） 土壤短芽孢杆菌（Brevibacillus agri）（0.002%）解淀粉芽孢杆菌（Bacillus velezensis SGB7785）(0.02%)
), ArticleFig(id=1239231272480993785, tenantId=1146029695717560320, journalId=1205117023404326918, articleId=1239231267858870564, language=CN, label=表4, caption=

扩增子测序、宏全基因组测序优势细菌与培养法得到菌种比较

, figureFileSmall=null, figureFileBig=null, tableContent=
样本号
（sample No.）		培养法分离得到细菌
（bactria isolated by culturing method）		扩增子测序优势细菌
（dominant bactria in Amplicon sequencing result）		宏基因组测序结果MetaPhlan分析
（MetaPhlan analysis of metagenomics sequencing result）
32巴达维亚新芽孢杆菌（Neobacillus bataviensis）内生新芽孢杆菌（Neobacillus endophyticus）(99.99%)吉达新芽孢杆菌（Neobacillus jeddahensis SGB30241）（99.64%）
魏德曼氏芽孢杆菌（Bacillus wiedmannii）(0.007%)蜡样芽孢杆菌（Bacillus cereus SGB7697）（0.36%）
40类芽孢杆菌（Paenibacillus albicereus）类芽孢杆菌（Paenibacillus sp. UniB2）(89.6%)类芽孢杆菌（Paenibacillus albicereus SGB82357）（100%）
魏德曼氏芽孢杆菌（Bacillus wiedmannii）(2.0%)
玛斯格雷芽孢杆菌（Bacillus massiliglaciei）(1.9%)
内生新芽孢杆菌（Neobacillus endophyticus）（0.03%）
91-1蜡样芽孢杆菌（Bacillus cereus）魏德曼氏芽孢杆菌（Bacillus wiedmannii）(99.99%)蜡样芽孢杆菌（Bacillus cereus SGB7697）（100%）
内生新芽孢杆菌（Neobacillus endophyticus）(0.003%)
土壤短芽孢杆菌（Brevibacillus agri）（0.002%）
91-2土壤短芽孢杆菌（Brevibacillus agri）土壤短芽孢杆菌（Brevibacillus agri）(99.97%)土壤短芽孢杆菌（Brevibacillus agri SGB7330）（99.99%）
魏德曼氏芽孢杆菌（Bacillus wiedmannii）（0.03%）蜡样芽孢杆菌（Bacillus cereus SGB7697）（0.01%）
HJ4蜡样芽孢杆菌（Bacillus cerus）魏德曼氏芽孢杆菌（Bacillus wiedmannii）(95.2%) 蜡样芽孢杆菌（Bacillus cereus SGB7697）(98.34%)
类芽孢杆菌（Paenibacillus sp. UniB2）(2.1%)蜡样芽孢杆菌（Bacillus cereus SGB7703）(1.26%)
玛斯格雷芽孢杆菌（Bacillus massiliglaciei）（0.03%）地衣芽孢杆菌（Bacillus licheniformis SGB7780）(0.34%)
枯草芽孢杆菌（Bacillus subtilis）内生新芽孢杆菌（Neobacillus endophyticus）(0.03%)溶血葡萄球菌（Staphylococcus haemolyticus SGB7861）(0.06%)
枯草芽孢杆菌（Bacillus subtilis SGB7788）(0.0006%)
HJ5蜡样芽孢杆菌（Bacillus cereus）魏德曼氏芽孢杆菌（Bacillus wiedmannii）(99.998%)蜡样芽孢杆菌（Bacillus cereus SGB7697）(99.98%)
解淀粉芽孢杆菌（Bacillus amyloliquefaciens） 土壤短芽孢杆菌（Brevibacillus agri）（0.002%）解淀粉芽孢杆菌（Bacillus velezensis SGB7785）(0.02%)
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宏基因组测序法对林可霉素利多卡因凝胶中污染细菌的鉴定与溯源研究*
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严卓彦 1 , 谭宇翔 2 , 夏瑛瑛 1
药物分析杂志 | 安全监测 2024,44(8): 1407-1414
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药物分析杂志 | 安全监测 2024, 44(8): 1407-1414
宏基因组测序法对林可霉素利多卡因凝胶中污染细菌的鉴定与溯源研究*
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严卓彦1 , 谭宇翔2, 夏瑛瑛1
作者信息
  • 1.舟山市食品药品检验检测研究院 国家海洋食品质量检验检测中心，舟山 316012
  • 2.中国科学院深圳先进技术研究院 深圳合成生物学创新研究院 中国科学院定量工程生物学重点实验室，深圳 518055

    • Tel：13957203861；E-mail：

Using metagenomic sequencing to identify and trace drug-contaminating bacteria in lincomycin hydrochloride and lidocaine hydrochloride gel
Zhuo-yan YAN1 , Yu-xiang TAN2, Ying-ying XIA1
Affiliations
  • 1.Zhoushan Institute for Food and Drug Control，National Ocean Food Quality Inspection Center，Zhoushan 316012，China
  • 2.Shenzhen Institutes of Advanced Technology，Chinese Academy of Sciences，Shenzhen Institute of Synthetic Biology，CAS Key Laboratory of Quantitative Engineering Biology，Shenzhen 518055，China
出版时间: 2024-08-31 doi: 10.16155/j.0254-1793.2024-0072
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摘要
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目的：

评价宏基因组测序法在药品污染细菌鉴定和溯源中的应用。

方法：

分别通过培养法、扩增子测序法和宏基因组测序法，对2批微生物限度控制菌检查中检出未知微生物的林可霉素利多卡因凝胶药剂进行菌种鉴定和溯源。培养法分离得到的细菌，通过革兰氏染色、生化反应、生物质谱法、16srDNA测序及芽孢杆菌特异性PCR方法进行菌种鉴定，并通过多位点序列分型（MLST）法对药品和环境中均检出的蜡样芽孢杆菌进行菌株分型。宏基因组测序结果通过MetaPhlan软件进行菌株水平的分析。

结果：

MLST与MetaPhlan结果均显示药品与检测环境中存在同一组蜡样芽孢杆菌，显示药品中的蜡样芽孢杆菌极有可能来自检测环境的污染。培养法与宏基因组测序法均在药品中检出污染微生物土壤短芽孢杆菌、类芽孢杆菌及新芽孢杆菌，但扩增子测序还在环境样本中检出上述细菌，因此不能排除药品中此类污染菌来自检测环境的可能性。

结论：

宏基因组测序技术相比培养法，能够更全面发现具有潜在风险的微生物，同时也能准确地进行菌株水平的溯源分析，在药品污染菌鉴定与溯源中具有重要的应用价值。

宏基因组测序  /  扩增子测序  /  多位点序列分型  /  MetaPhlan分析  /  药品污染菌的鉴定与溯源
Objective:

To evaluate the application of metagenomic sequencing in identification and tracking of drug-contaminating bacteria.

Methods:

Both metagenomic sequencing and isolation culturing method were employed to identify and genotype bacteria in two batches of possibly contaminated gel medicines as well as the environmental samples from testing laboratory. Bacteria isolated by culturing method were identified through Gram’s stain，biochemical reaction，MALIDI-TOF-MS，16srDNA sequencing and Bacillus specific PCR. The detected Bacillus cereus were typed by multilocus sequence typing (MLST) .The metagenomic sequencing results were analyzed at the strain level using MetaPhlan software.

Results:

Both MLST and MetaPhlan analysis revealed a substantial contamination of Bacillus cereus in gel medicines as well as environmental samples，with the presence of the same Bacillus cereus group. Both metagenomic sequencing and culturing methods detected Paenibacillus，Brevibacillus and Neobacillus in gel medicines，but amplicon sequencing also detected these bacteria in environment which might be the source of contamination.

Conclusion:

These findings indicate that metagenomic sequencing technology is reliable and has the advantage over isolation culturing in comprehensively detecting potentially hazardous bacterial species and accurately performing strain-level traceability analysis. It holds significant application value in pharmaceutical contamination identification and traceability.

metagenomic sequencing  /  amplicon sequencing  /  MLST  /  MetaPhlan analysis  /  bacteria identification and tracking in contaminated drugs
严卓彦, 谭宇翔, 夏瑛瑛. 宏基因组测序法对林可霉素利多卡因凝胶中污染细菌的鉴定与溯源研究*. 药物分析杂志, 2024 , 44 (8) : 1407 -1414 . DOI: 10.16155/j.0254-1793.2024-0072
Zhuo-yan YAN, Yu-xiang TAN, Ying-ying XIA. Using metagenomic sequencing to identify and trace drug-contaminating bacteria in lincomycin hydrochloride and lidocaine hydrochloride gel[J]. Chinese Journal of Pharmaceutical Analysis, 2024 , 44 (8) : 1407 -1414 . DOI: 10.16155/j.0254-1793.2024-0072
正文
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林可霉素利多卡因凝胶为非处方类皮肤科用药，是非无菌外用制剂，用于轻度烧伤、创伤及蚊虫叮咬引起的各种皮肤感染。马帅等[1]对全国5个生产企业11批次的林可霉素利多卡因凝胶进行质量考察和评价，按现行标准检验微生物限度控制菌，结果均符合规定；按非标准检验，有1批检出洋葱伯克霍尔德菌，3批检出其他微生物，说明该产品在微生物方面尚存在质量风险。舟山市食品药品检验检测研究院对全国10个生产企业40批次的林可霉素利多卡因凝胶进行质量考核，虽未检出控制菌金黄色葡萄球菌和铜绿假单胞菌，也未检出不可接受微生物洋葱伯克霍尔德菌，但在一家生产企业2个批号的药品培养液中均发现有未知微生物生长。通过传统培养法在1批药品中分离得到蜡样芽孢杆菌，另1批药品中分离得到伪芽孢杆菌。考虑到药品中可能存在其他未分离得到的污染微生物，因此，采用宏基因组测序法结合传统培养法对这2批药品进行菌种鉴定和菌株分型，旨在全面分析该批药品的污染情况，并尝试对污染微生物进行溯源分析。
宏基因组学(或元基因组学，metagenomics)是一种以环境样品中的微生物群体基因组为研究对象，以功能基因筛选和/或测序分析为研究手段，以微生物多样性、种群结构、进化关系、功能活性、相互协作关系及与环境之间的关系为研究目的的新的微生物研究方法[2]。目前，宏基因组研究紧密依赖高通量测序技术，包括扩增子测序（amplicon sequencing）与宏基因组测序（metagenome sequencing）。扩增子测序主要针对核糖体RNA基因和功能基因，比如细菌16srDNA、真菌ITS可变区等分子标记进行PCR扩增后再测序；宏基因组测序指对环境样品中所有微生物基因组DNA进行高通量测序，其优点在于能够得到微生物的全部基因组信息，进行菌株水平的溯源分析[4]。目前已有宏基因组测序法用于中药饮片表面污染微生物的研究报道，如李琼琼等[5]利用16srDNA扩增子测序技术研究中药饮片中污染微生物的群落特征，车阳等[6]利用宏基因组测序技术检测胎菊饮片表面微生物污染。宏基因组菌株水平分析已经应用到食品污染[7]和院内感染爆发[8]的溯源分析中，但是尚未有药品中污染菌溯源分析的相关研究报道。
本研究对有污染菌检出的2批林可霉素利多卡因凝胶及检测环境进行扩增子测序和宏全基因组测序分析，并基于宏全基因组测序数据，通过MetaPhlan算法[9]比较各样本间的菌株相似度，与培养法分离得到的污染菌进行鉴定和溯源分析的比较。
1　实验材料、仪器试药与标准菌株
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1.1　实验材料
实验材料为来自同一生产厂家的2批林可霉素利多卡因凝胶，分别为批号D23003的3个样品（编号91-1、91-2、40）和批号D22013的1个样品（编号32）。环境样本2次取样（分别编号HJ4和HJ5）均采集自舟山市食品药品检验检测研究院药品微生物限度检查室，包括洁净工作台面、集菌仪、操作人员的无菌手套和无菌服、实验用胰酪胨大豆液体培养基（TSB）和pH 7.0氯化钠蛋白胨缓冲液。
1.2　仪器和试药
HTY-601集菌仪，杭州泰林生物技术设备有限公司；SPX-250B-Z生化培养箱，上海博讯实业有限公司医疗设备厂；MLS-3780高压蒸汽灭菌器，三洋工业株式会社；DK-S24电热恒温水浴锅，上海精宏实验设备有限公司；KB240恒温培养箱，BRAND公司；My Cycler PCR扩增仪、Mini-PROTEAN Tetra水平电泳仪、Gel Doc XR凝胶成像系统，BIO-RAD公司；VITEK MS全自动快速微生物质谱系统，梅里埃生物公司；Illumina Miseq测序仪，Illumina公司；DNBSEQ-T7测序仪，深圳华大智造科技股份有限公司。
胰酪大豆胨琼脂培养基(TSA) （批号20220505）、pH 7.0氯化钠蛋白胨缓冲液（批号20220909）、胰酪大豆胨液体培养基(TSB）（批号20210608）、沙氏葡萄糖琼脂培养基（SDA）（批号20220424）、甘露醇氯化钠琼脂培养基（MSA）（批号20221213）、溴化十六烷基三甲铵琼脂（CTAB）（批号20230307）、洋葱伯克霍尔德菌群选择性琼脂培养基（BCCSA）（批号20230525），均购自青岛高科园海博生物技术有限公司。氯化镁（批号20220105），国药集团化学试剂有限公司生产。革兰氏染色液试剂盒（批号230308）、蜡样芽孢杆菌干制生化鉴定试剂盒（DBI-07，批号231121），均购自北京陆桥技术股份有限公司。
1.3　标准菌株
金黄色葡萄球菌（Staphylococcus aureus）[CMCC(B)26003] 、铜绿假单胞菌(Pseudomonas aeruginosa) [CMCC(B)10104]，中国食品药品检验研究院；洋葱伯克霍尔德菌（Burkholderia cepacia）[ATCC BAA-245]，美国菌种保藏管理中心。
2　实验方法
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2.1　控制菌检查和未知污染微生物分离培养
2.1.1　供试液制备及检查方法
取供试品10 g，加入中和剂10%氯化镁溶液20 mL，摇匀后，加入pH 7.0无菌氯化钠蛋白胨缓冲液至100 mL，充分振摇使混匀，作为1:10的供试液。取10 mL供试液进行薄膜过滤，用pH 7.0无菌氯化钠蛋白胨缓冲液进行冲洗，冲洗量为每膜500 mL，冲洗后将薄膜无菌转移至100 mL TSB中。同时按照以上方法制备3份阳性对照，分别加入不大于100 cfu的金黄色葡萄球菌、铜绿假单胞菌和洋葱伯克霍尔德菌，混匀，30~35 ℃培养24~48 h，然后划线接种于MSA、CTAB、BCCSA等选择性培养琼脂和TSA平板上，30~35 ℃培养24~72 h，观察平板上菌落生长情况。TSB培养液划线后立即寄往上海生工生物工程有限公司（简称上海生工）进行基因组提取和高通量测序。
2.1.2　环境微生物采集
在样品处理完毕后立即对实验室环境进行取样，用灭菌后的棉签分别收集操作者无菌手套及无菌服、集菌仪表面、TSB培养液、pH 7.0蛋白胨缓冲液等可能污染源，于装有100 mL TSB的无菌袋中32.5 ℃培养18~24 h，用接种环取培养液划线于TSA平板上，32.5 ℃培养5 d，划线于SDA平板上，23 ℃培养7 d。划线后立即将TSB培养液寄往上海生工进行宏基因组提取和测序。另外，检测环境的常规监控（浮游菌/沉降菌)在每次实验过程中都会进行，以排除可能的空气污染。
2.1.3　菌种鉴定方法
将选择性平板和TSA平板上的单菌落划线到营养琼脂上进行纯化培养，纯化的菌落通过革兰氏染色、生物质谱法及16srDNA基因测序法进行初步鉴定，对前几种方法无法确认的芽孢杆菌采用生化反应及芽孢杆菌特异性PCR方法（具体条件见表1)进行鉴定。针对某种芽孢杆菌的特异性PCR方法能较好地鉴别枯草芽孢杆菌群的某些近缘菌种与蜡样芽孢杆菌群，比如基于gyrA基因[10]、rpoB基因和panC基因[11]的特异性PCR能较好地鉴别枯草芽孢杆菌、解淀粉芽孢杆菌和蜡样芽孢杆菌群，但蜡样芽孢杆菌和苏云金芽孢杆菌基因组几乎完全相同，唯一的区别是苏云金芽孢杆菌在质粒上有编码蛋白毒素结晶体的cry基因[12]，因此，还需要增加基于cry基因的特异性PCR反应。
2.1.4　菌株分型方法
应用国际通用的多位点序列分型（MLST）法，对样品和环境中均检出的蜡样芽孢杆菌进行菌株分型。蜡样芽孢杆菌采用pubMLST网站(http：//pubmlst.org)上公布的PCR引物。PCR扩增产物送往上海生工进行双向测序，将7个管家基因的正反双向引物扩增产物的测序结果进行拼接，并在PubMLST数据库进行序列比对，得到相应的序列号，将等位基因序列号在数据库上传后得到相应的序列型（ST）。对于有基因变异和多态性位点变异的序列，向数据库提交新序列的信息并申请新的分离株号（isolate）和ST，将得到的分离株ST采用在线BURST软件进行聚类分析。
2.2　宏基因组测序法
2.2.1　扩增子测序法
委托上海生工进行基因组提取、文库构建和测序，测序平台为Miseq。16srRNA测序引物为341F(CCTACGGGNGGCWGCAG)和805R(GACTACHVGGGTATCTAATCC)，ITS测序引物为ITS1F(CTTGGTCATTTAGAGGAAGTAA)和ITS2R（GCTGCGTTCTTCATCGATGC)，测序平台为Illumina MiSeq。二代测序得到的reads首先根据重叠关系进行拼接，区分样本后用Cutadapt和PRINSEQ软件对序列质量进行质控和过滤，然后用Usearch软件进行OTU聚类（97%以上的序列相似度），用BLAST软件参考NCBI数据库进行物种分类学分析（大于97%相似率，大于90%覆盖率）。
2.2.2　宏基因组测序及MetaPhlan分析
宏基因组测序法采用二代高通量测序法，测序平台为DNBSEQ-T7。具体步骤包括基因组DNA片段化、文库构建、文库质控和测序。测序数据分析包括数据评估及质控、拼接组装、基因集构建和功能注释、基因集丰度及丰度分析。首先对原始数据通过Fastp进行质量评估和数据过滤，测序数据分析应用MetaPhlan（4.0.2），使用默认参数利用快速比对工具bowtie2与参比基因集（marker)数据库（mpa_vOct22_CHOCOPhlAnSGB_202212）进行比对，对宏基因组测序数据进行每个样品菌群定性和定量分析，具体步骤为参比基因组的确定、reads与marker的比对和含量的计算。
3　实验结果
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3.1　传统培养法分离得到的菌株鉴定及溯源
经过培养，阳性对照样品均在选择性平板上分离得到目的菌金黄色葡萄球菌、铜绿假单胞菌及洋葱伯克霍尔德菌，证明该药品微生物检查方法的可行性。2批样品均未在选择性平板上分离得到目的控制菌，而在TSA平板上分离得到4株细菌，SDA平板上无真菌生长。环境样本在TSA平板上分离得到4株细菌，在SDA平板上得到1株霉菌。
8株细菌染色结果除了菌株32为革兰氏阳性长杆菌外，其余均为革兰氏阳性有芽孢短杆菌。其中，编号为91-1、HJ-1、HJ-3的菌株符合蜡样芽孢杆菌生化特征。通过生物质谱法、16srDNA测序法对检出细菌进行鉴定，并通过芽孢杆菌特异性PCR方法对检出芽孢杆菌进行鉴别（见表2）。批号为D23003的3个样品中分别检出蜡样芽孢杆菌、土壤短芽孢杆菌（Brevibacillus agri）和类芽孢杆菌（Paenibacillus albicereus），批号为D22013的1个样品（编号32）中检出巴达维亚新芽孢杆菌（Neobacillus bataviensis）。检测环境中分离得到4株菌，经鉴定2株为蜡样芽孢杆菌，1株为枯草芽孢杆菌，1株为解淀粉芽孢杆菌。
对药品与环境中都检出的3株蜡样芽孢杆菌（91-1、HJ-1、HJ-3）及1株曾经在实验室环境中检出并保存的蜡样芽孢杆菌（编号HJ）进行MLST分型。参照pubMLST网站上蜡样芽孢杆菌的PCR引物序列对蜡样芽孢杆菌的7个管家基因进行PCR扩增，扩增产物测序结果在pubMLST网站上搜索得到特异性等位基因编号，根据等位基因编号组合得到每个菌株的ST。其中2株蜡样芽孢杆菌无对应的ST，将相关序列上传至pubMLST网站，得到新的分离株号和ST。将得到的分离株ST采用在线BURST算法（参数n-4）进行聚类分析，结果显示药品中的蜡样芽孢杆菌与环境中的2株蜡样芽孢杆菌聚为同一组（Group 1），且与保存的原实验室环境菌HJ最接近（7个位点中有5个位点相同），说明药品中的蜡样芽孢杆菌极有可能来源于检测环境的污染。见表3。
3.2　宏基因组测序结果分析
对培养法分离得到的细菌菌种与扩增子测序、宏基因组测序得到的细菌菌种进行比较，结果见表4。培养法得到的细菌基本上都能体现在扩增子测序和宏基因组测序结果中，且宏基因组测序比扩增子测序所得细菌在种水平上与培养法更一致。比如，扩增子测序将样本中普遍存在的蜡样芽孢杆菌鉴定为魏德曼芽孢杆菌（Bacillus wiedmannii），其他细菌也基本只鉴定到属水平；宏基因组测序Metaphlan分析虽然在新芽孢杆菌（Neobacillus）种的鉴定上与培养法不一致，但在其他菌种上完全一致，尤其是在环境样本HJ4和HJ5中检出含量极低的枯草芽孢杆菌Bacillus subtilis SGB7788（0.006%）和解淀粉芽孢杆菌Bacillus velezensis SGB7785（0.02%），与培养法在环境中分离得到的2株细菌HJ-2和HJ-4完全一致。
扩增子测序和宏基因组测序得到菌种信息都多于培养法，且扩增子测序菌种信息多于宏基因组测序的MetaPhlan分析结果。比如扩增子测序在药品40和91-1及环境样本HJ4中检出其他方法都未检出的新芽孢杆菌，在药品91-1和环境样本HJ5中检出其他方法都未检出的土壤短芽孢杆菌（Brevibacillus agri），在环境样本HJ4中检出其他方法都未检出的类芽孢杆菌。原因可能是环境中这些细菌的含量极低，培养法和宏基因组测序法都未能检测到，但是扩增子测序通过PCR扩增得到这几种细菌的信息，说明扩增子测序在检测痕量菌种时优势。
扩增子测序与宏基因组测序结果均显示药品和环境样本中普遍存在蜡样芽孢杆菌菌群，采用培养法只在1个样品中分离得到蜡样芽孢杆菌，MetaPhlan分析结果显示药品32、91-1、91-2中的蜡样芽孢杆菌与环境样本HJ4和HJ5中的蜡样芽孢杆菌属于同一组Bacillus cereus SGB7697，与培养法所得菌株MLST分型结果一致，显示药品中的蜡样芽孢杆菌极有可能来自检测环境的污染。Metaphlan分析结果还发现，环境样本HJ4中含有另外1组蜡样芽孢杆菌Bacillus cereus SGB7703，在其他样本中均未发现该菌组，该结果也与培养法得到环境菌株HJ-1在MLST分型中单独归为一类相符合。
4　讨论
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本研究采用传统培养法、扩增子测序法和宏全基因组测序法，对2批药品及检测环境中的污染微生物进行鉴定，3种方法均在药品样本中检出土壤短芽孢杆菌、类芽孢杆菌和新芽孢杆菌，并在药品和环境样本中都检出蜡样芽孢杆菌。宏基因组测序MetaPhlan分析结果显示环境样本HJ4中含有2组不同的蜡样芽孢杆菌菌株，其中含量较高的1组（SGB7697)在药品样本及环境样本HJ5中均有检出，该结果与培养法的溯源结果一致，都显示药品中的蜡样芽孢杆菌来源于检测环境；另外1组含量较低的蜡样芽孢杆菌(SGB7703)与培养法得到菌株HJ-1在MLST分型中单独归为一类相符合。与培养法和宏基因组测序结果不同，扩增子测序还在环境样本中检出少量的土壤短芽孢杆菌、类芽孢杆菌和新芽孢杆菌，因此不能排除药品中的这3种污染菌也可能来源于检测环境的假设。另外，鉴于蜡样芽孢杆菌等芽孢杆菌属为环境中普遍存在的污染菌，并不能完全排除药品在生产环境中就已经被污染这一可能性，建议生产厂家进一步分析生产环境。
3种检测方法各有优缺点：传统培养法可以在菌株水平上分析微生物的生理代谢，但这种方法无法掌握样品中绝大部分菌种信息[16]；基于二代测序的扩增子测序技术可免培养且进行群落分析，具有通量高、准确率高、可定性及价格低廉等优点[17-18]，可以全面、快速地对污染微生物多样性进行分析，但由于只可识别部分高变区扩增和测序靶点，导致对微生物的鉴定仅精确到属水平[3]，且无法从全基因组水平上进行菌株水平的溯源；宏全基因组测序对环境样品中所有微生物基因组DNA进行高通量测序，然后进行序列组装和基因注释，获得可培养和不可纯培养微生物的基因组序列，分析该环境下所有微生物的物种分类、基因功能等信息，但也存在成本较高、检测时间较长、对痕量菌种无法鉴别等劣势。基于宏全基因组测序的菌株水平分析分为无参考基因组(无参)的组装分析和有参考基因组(有参)的比对分析两大类。有参比对的菌株分析方法又分为基于SNP进行识别和基于基因组成(genetic constitution)差异进行识别。基于SNP的分析方法是菌株分析算法的主流，利用的是宏基因组数据中单碱基水平的信息，通过使用宏基因组片段序列和参考序列(全基因组或标记基因)的比对结果进行SNP位点的等位基因相对比例分析，最后根据这些比例信息，推算菌株的组成[19]。其中，MetaPhlan可以标记基因定位到亚种级别，进一步实现菌株级别溯源。基于株水平的宏基因组学方法不仅能准确识别菌株间亲缘关系，还能节省样本分离纯培养时间，同时有助于更全面发现具有潜在风险的细菌物种，在药品微生物鉴定与溯源分析中具有重要的应用价值。但无论是扩增子测序还是宏全基因组测序，都存在一定的测序偏差，参比数据库不够全面，无法区分死活菌等缺陷，因此，还需与培养法相结合，才能更加全面准确地进行药品污染菌的分析及溯源。
基金
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  • *浙江省药监局科技计划项目(2023010)
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2024年第44卷第8期
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doi: 10.16155/j.0254-1793.2024-0072
  • 接收时间：2024-01-31
  • 首发时间：2026-03-13
  • 出版时间：2024-08-31
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  • 收稿日期：2024-01-31
基金
*浙江省药监局科技计划项目(2023010)
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    1.舟山市食品药品检验检测研究院 国家海洋食品质量检验检测中心，舟山 316012
    2.中国科学院深圳先进技术研究院 深圳合成生物学创新研究院 中国科学院定量工程生物学重点实验室，深圳 518055
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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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