制药企业生产环境中污染葡萄球菌菌种鉴定方法的比较评价及毒素基因调查分析
|
摘要
比较评价16S rRNA序列比对、VITEK 2 Compact微生物鉴定系统和RiboPrinter鉴定系统对制药企业生产环境中分离的92株葡萄球菌菌种鉴定的准确性,研究12种毒素编码基因在制药企业生产环境污染葡萄球菌中的分布规律。结果表明92株葡萄球菌均为凝固酶阴性菌,可分为8个葡萄球菌菌种,其中表皮葡萄球菌污染率最高(27.2%)。16S rRNA序列比对分析不能有效区分头状葡萄球菌和山羊葡萄球菌,RiboPrinter鉴定系统易把假中间葡萄球菌错误鉴定为中间葡萄球菌,而VITEK 2 Compact鉴定系统对头状葡萄球菌、人葡萄球菌、巴氏葡萄球菌和科氏葡萄球菌中的部分菌株存在不能有效鉴定或鉴定错误。在92株凝固酶阴性菌中共检出7种毒素基因(pvl,sea,seb,sec,sed,egc和eta),其中杀白细胞毒素基因pvl的检出率最高(8.70%),其次为肠毒素基因sec(6.52%)。综上所述,16S rRNA序列比对分析和RiboPrinter鉴定系统对不同种葡萄球菌的鉴定准确性优于VITEK2Compact鉴定系统。制药企业生产环境中污染的凝固酶阴性葡萄球菌具有一定潜在致病性,今后在药品微生物检验与生产过程中应加强对凝固酶阴性葡萄球菌的监控。
The species identification accuracy of 16 S rRNA sequence alignment, VITEK 2 Compact microbial identification system and RiboPrinter bacterial identification system in 92 Staphylococcus strains isolated from production environment of drug manufacturing enterprises were compared and evaluated. The distribution of 12 toxin genes in 92 Staphylococcus strains was also investigated. It revealed that all the 92 Staphylococcus strains were coagulase-negative bacteria, and could be divided into 8 Staphylococcus species. Among the 8 species, S. epidermidis had the highest isolation rate(27.2%). The 16 S rRNA sequence alignment analysis could not distinguish S. capitis and S. caprae. The RiboPrinter bacterial identification system misidentified all S. pseudintermedius strains as S. intermedius. The VITEK 2 Compact microbial identification system displayed false or invalid identification results of some strains including S. capitis, S.hominis, S. pasteuri, and S. cohnii. 7 toxin genes(pvl, sea, seb, sec, sed, egc and eta) were detected in the 92 coagulasenegative strains, in which the leukotoxin gene pvl had the highest detection rate(8.70%), followed by enterotoxin gene sec(6.52%). In summary, 16 S rRNA sequence alignment analysis and RiboPrinter bacterial identification system are superior to the VITEK 2 Compact microbial identification system for the identification of different Staphylococci species.Contaminated coagulase-negative Staphylococci in the production environment of drug manufacturing enterprises have a certain potential pathogenicity, so the monitoring of coagulase-negative Staphylococci should be strengthened in the future.
The species identification accuracy of 16 S rRNA sequence alignment, VITEK 2 Compact microbial identification system and RiboPrinter bacterial identification system in 92 Staphylococcus strains isolated from production environment of drug manufacturing enterprises were compared and evaluated. The distribution of 12 toxin genes in 92 Staphylococcus strains was also investigated. It revealed that all the 92 Staphylococcus strains were coagulase-negative bacteria, and could be divided into 8 Staphylococcus species. Among the 8 species, S. epidermidis had the highest isolation rate(27.2%). The 16 S rRNA sequence alignment analysis could not distinguish S. capitis and S. caprae. The RiboPrinter bacterial identification system misidentified all S. pseudintermedius strains as S. intermedius. The VITEK 2 Compact microbial identification system displayed false or invalid identification results of some strains including S. capitis, S.hominis, S. pasteuri, and S. cohnii. 7 toxin genes(pvl, sea, seb, sec, sed, egc and eta) were detected in the 92 coagulasenegative strains, in which the leukotoxin gene pvl had the highest detection rate(8.70%), followed by enterotoxin gene sec(6.52%). In summary, 16 S rRNA sequence alignment analysis and RiboPrinter bacterial identification system are superior to the VITEK 2 Compact microbial identification system for the identification of different Staphylococci species.Contaminated coagulase-negative Staphylococci in the production environment of drug manufacturing enterprises have a certain potential pathogenicity, so the monitoring of coagulase-negative Staphylococci should be strengthened in the future.
引文
[1] EISSA M E. Quantitative microbial risk assessment of pharmaceutical products[J]. PDA J Pharm Sci Tech, 2017,71(3):245-251.
[2]胡昌勤.药品微生物控制现状与展望[J].中国药学杂志,2015,50(20):1747-1751.
[3]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.
[4]郑小玲,王征南,王知坚,等.药品微生物检测实验室环境菌库的建立[J].中国现代应用药学,2015, 32(7):847-850.
[5]范一灵,冯震,钟玮,等.无菌药品生产企业核心区微生物污染调查与分析[J].中国药事,2014, 28(6):586-590.
[6] ASAAD A M, ANSAR Q M, MUJEEB H S. Clinicalsignificance of coagulase-negative staphylococci isolates from nosocomial bloodstream infections[J]. Infect Dis,2016, 48(5):356-360.
[7]王锦萍,蔡常辉,梁连辉,等.肺结核并发肺部感染凝固酶阴性葡萄球菌的耐药性及耐药基因mecA分析[J].国际检验医学杂志,2018, 39(5):623-625.
[8]朱诗应,戚中田.16S rDNA扩增及测序在细菌鉴定与分类中的应用[J].微生物与感染,2013, 8(2):104-109.
[9]杨燕,冯震,鲍英,等.全自动基因指纹分析仪Riboprinter在药品中大肠埃希菌的鉴定分析中的应用(英文)[J].中国抗生素杂志,2013,38(7):536-539.
[10]叶乃芳,王中新.16S rRNA及相关技术用于临床细菌鉴定的研究进展[J].国际检验医学杂志,2015, 36(4):520-522.
[11]刘继超,姜铁民,姜阿赤,等.应用全自动核糖体RNA基因指纹技术快速鉴定细菌菌种[J].食品工业科技,2013,34(13):170-172.
[12]杨晶,王伟欢. VITEK 2 Compact系统的应用及鉴定结果分析[J].中国卫生检验杂志,2016, 26(18):2643-2645.
[13] VANDAMME P,POT B, GILLIS M, et al.Polyphasic taxonomy, a consensus approach to bacterial systematics[J]. Microbiol Rev. 1996, 60(2):407-438.
[14] KIM M, OH H S, PARK S C, et al. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[J]. Int J Syst Evol Microbiol, 2014, 64(2):346-351.
[15] HEIKENS E, FLEER A, PAAUW A, et al. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci[J]. J Clin Microbiol, 2005, 43(5):2286-2290.
[16]陈西勇,于淑渤,梁宏.无菌药品生产发生微生物污染的因素分析(上)[J].首都食品与医药,2009,16(6):23-25.
[17]冯言言,田伟.金黄色葡萄球菌杀白细胞毒素的研究[J].农家之友,2010,10:122-123.
[18]李琼琼,范一灵,宋明辉,等.食源性金黄色葡萄球菌肠毒素及其检测方法[J].食品安全质量检测学报,2016, 7(2):555-560.
[2]胡昌勤.药品微生物控制现状与展望[J].中国药学杂志,2015,50(20):1747-1751.
[3]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.
[4]郑小玲,王征南,王知坚,等.药品微生物检测实验室环境菌库的建立[J].中国现代应用药学,2015, 32(7):847-850.
[5]范一灵,冯震,钟玮,等.无菌药品生产企业核心区微生物污染调查与分析[J].中国药事,2014, 28(6):586-590.
[6] ASAAD A M, ANSAR Q M, MUJEEB H S. Clinicalsignificance of coagulase-negative staphylococci isolates from nosocomial bloodstream infections[J]. Infect Dis,2016, 48(5):356-360.
[7]王锦萍,蔡常辉,梁连辉,等.肺结核并发肺部感染凝固酶阴性葡萄球菌的耐药性及耐药基因mecA分析[J].国际检验医学杂志,2018, 39(5):623-625.
[8]朱诗应,戚中田.16S rDNA扩增及测序在细菌鉴定与分类中的应用[J].微生物与感染,2013, 8(2):104-109.
[9]杨燕,冯震,鲍英,等.全自动基因指纹分析仪Riboprinter在药品中大肠埃希菌的鉴定分析中的应用(英文)[J].中国抗生素杂志,2013,38(7):536-539.
[10]叶乃芳,王中新.16S rRNA及相关技术用于临床细菌鉴定的研究进展[J].国际检验医学杂志,2015, 36(4):520-522.
[11]刘继超,姜铁民,姜阿赤,等.应用全自动核糖体RNA基因指纹技术快速鉴定细菌菌种[J].食品工业科技,2013,34(13):170-172.
[12]杨晶,王伟欢. VITEK 2 Compact系统的应用及鉴定结果分析[J].中国卫生检验杂志,2016, 26(18):2643-2645.
[13] VANDAMME P,POT B, GILLIS M, et al.Polyphasic taxonomy, a consensus approach to bacterial systematics[J]. Microbiol Rev. 1996, 60(2):407-438.
[14] KIM M, OH H S, PARK S C, et al. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[J]. Int J Syst Evol Microbiol, 2014, 64(2):346-351.
[15] HEIKENS E, FLEER A, PAAUW A, et al. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci[J]. J Clin Microbiol, 2005, 43(5):2286-2290.
[16]陈西勇,于淑渤,梁宏.无菌药品生产发生微生物污染的因素分析(上)[J].首都食品与医药,2009,16(6):23-25.
[17]冯言言,田伟.金黄色葡萄球菌杀白细胞毒素的研究[J].农家之友,2010,10:122-123.
[18]李琼琼,范一灵,宋明辉,等.食源性金黄色葡萄球菌肠毒素及其检测方法[J].食品安全质量检测学报,2016, 7(2):555-560.