目的：应用现有微生物分类鉴定技术手段，对《中国药典》拟收载洋葱伯克霍尔德菌群(Bcc) 检查法中所选用的标准菌株进行冻存及传代稳定性考察。方法：选取美国药典USP43-NF38 Bcc检查法中的一株Bcc标准菌株ATCC25416为对照菌株。以3株拟收载标准菌株和1株对照标准菌株为试验菌株，分别对这些试验菌株冻存0.5年、1年、2年的样本及连续6代的传代样本进行菌落表型观察、Biolog生化鉴定分析、16S rRNA基因测序及RiboPrinter全自动基因指纹图谱分析，并使用BioNumerics 7.6对核糖体图谱数据进行聚类分析。结果：试验菌株在经冻存及传代后，各菌株表型无明显差异；菌株的Biolog生化鉴定结果虽略有差异，但菌株关键生化代谢属性稳定，其鉴定结果均属Bcc；同时，各菌株的16S rRNA测序结果一致；核糖体带型相似值均大于0.95。结论：在本研究考察的冻存时间及传代次数内，3株《中国药典》洋葱伯克霍尔德菌群检查法拟收载的标准菌株的表型、基因型相对稳定。
Objective: Using the existing methods of microbial classifi cation and identifi cation techniques to investigate the cryopreservation and passage stability of the standard strains selected in the test of Burkholderia cepacia Complex in the Chinese Pharmacopoeia. Methods: A Bcc standard strain ATCC25416 in the USP43- NF38 Bcc inspection method was selected as the control strain. Three candidate standard strains and one control strain were selected as the test strains, then the colony phenotype, Biolog biochemical identifi cation system, 16S rRNA gene sequencing and RiboPrinter automatic genetic fi ngerprinting analysis were performed on samples of these experimental strains cryopreserved for 0.5, 1 and 2 years and passage samples for 6 consecutive generations. The BioNumerics 7.6 software was used to perform the cluster analysis of the fingerprint data. Results: Subsequent to the cryopreservation or subculture of test strains of Burkholderia cepacia Complex, the phenotypes of the strains had no signifi cant diff erence. The Biolog biochemical identifi cation results of the strains were all Bcc. Although there were slight diff erences, the key biochemical metabolic properties of the strains were stable; at the same time, the 16S rRNA sequencing results were consistent and the ribotyping bands similarity values of each strain were greater than 0.95. Conclusion: Within the freezing storage time and passage times investigated in this study, the phenotypes and genotypes of the these three standard strains included in the test of Burkholderia cepacia Complex in Chinese Pharmacopoeia are relatively stable.
[1] Correa-Ruiz A,Girón R,Buendía B,et al.Burkholderia Cepacia Complex Infection in an Adult Cystic Fibrosis Unit in Madrid[J].Enfermedades Infecciosas y Microbiología Clínica,2013,31(10):649-654.
[2] Garcia B A,Carden J L,Goodwin D L,et al.Implementation of a Successful Eradication Protocol for Burkholderia Cepacia Complex in Cystic Fibrosis Patients [J].BMC Pulmonary Medicine,2018,18(1):35.
[3] Zurita J,Mejia L,Zapata S,et al.HealthcareAssociated Respiratory Tract Infection and Colonization in an Intensive Care Unit Caused by Burkholderia Cepacia Isolated in Mouthwash[J].International Journal of Infectious Diseases,2014,29:96-99.
[4] Mahenthiralingam E,Urban T A,Goldberg J B.The Multifarious,Multireplicon Burkholderia Cepacia Complex [J].Nature Reviews Microbiology,2005,3(2):144-156.
[5] Torbeck L,Raccasi D,Guilfoyle DE,et al.Burkholderia Cepacia:This Decision is Overdue[J].PDA Journal of Pharmaceutical Science and Technology,2011,65(5):535-543.
[6] Sutton S,Jimenez L.A Review of Reported Recalls Involving Microbiological Control 2004-2011 with Emphasis on FDA Considerations of Objectionable Organisms[J].American Pharmaceutical Review,2012,15(1):42-57.
[7] FDA.FDA Advises Drug Manufacturers that Burkholderia Cepacia Complex Poses a Contamination Risk in Nonsterile，Water-based Drug Products[EB/OL].[2021-12-07].https://www.fda.gov/drugs/drug-safety-andavailability.
[8] USP43-NF38[S].2019：6454-6457，7845-7850.
[9] 国家药典委员会.中华人民共和国药典：四部 [S].北京：中国医药科技出版社，2020：499-500.
[10] European Pharmacopoeia Commission.EP 10.0：VolⅠ [S].Strasbourg：EDQM，2019：201-205.
[11] 范一灵，蒋波，房蕊，等.药品无菌检查中微生物污染的鉴定和污染溯源分析[J].药物分析杂志，2011，31（6）：1067-1072.
[12] 王似锦，余萌，胡昌勤，等.14株伯克霍尔德菌的鉴定分析[J].中国抗生素杂志，2019，44（10）：1214-1219.
[13] 周江林，孔娜，张琪，等.洋葱伯克霍尔德菌复合群部分菌株基于基因组的物种水平重鉴定[J].生物技术通讯，2019，30（6）：733-739.
[14] 余萌，王似锦，戴翚，等.recA基因及核糖体分型技术在洋葱伯克霍尔德菌复合体鉴别与溯源分析中的应用 [J].药物分析杂志，2019，39（8）：1521-1526.
[15] 黄新新，何苗，韩伟，等.迟缓爱德华氏菌EcoRⅠ 核糖体自动化分型与聚类分析[J].微生物学通报，2016，43（10）：2249-2253.
[16] Matloob Mazin，Griffiths Mansel.Ribotyping and Automated Ribotypingof ListeriaMonocytogenes[J].Methods in Molecular Biology，2014，1157：85-93.
