食源性金黄色葡萄球菌肠毒素基因的分布与时序性表达

doi:10.3864/j.issn.0578-1752.2012.19.018

摘要/Abstract

摘要： 【目的】研究食源性金黄色葡萄球菌各血清型肠毒素基因的分布，并进一步分析其时序性表达规律。【方法】针对51株各类食品中分离的金黄色葡萄球菌菌株，应用PCR技术对11种葡萄球菌肠毒素进行基因分型；提取细菌总RNA，以ftsZ和ropB作为内标基因，使用反转录荧光定量PCR研究各肠毒素基因在细菌生长周期中的表达水平变化。【结果】除see、ses和set外，其余8种肠毒素基因在51株食源性金黄色葡萄球菌菌株中均有检出，且sei和seg的检出率最高（27.45%）。各肠毒素基因在mRNA水平的时序性表达规律基本一致，均在对数后期达到峰值，随后快速下降。以内标基因为参照，同一菌株中不同肠毒素基因的相对表达量以及不同菌株中同一肠毒素基因的相对表达量均差异较大。【结论】本文系统研究了肠毒素基因在食源性金黄色葡萄球菌中的分布，并探究了肠毒素基因的时序性表达规律，对金黄色葡萄球菌毒力机制研究与食品质量安全控制具有参考意义。

关键词: 食源性金黄色葡萄球菌, 肠毒素, 基因分型, 时序性表达, 荧光定量实时PCR

Abstract: 【Objective】 The objective of this study is to investigate the distribution of different serum enterotoxins in food-borne Staphylococcus aureus and to further analyze their temporal expression of mRNA.【Method】Fifty-one foodborne S. aureus isolates were separated, and the presence of 11 staphylococcal enterotoxins was identified by PCR technology. After the total RNA was extracted, the expression of enterotoxin mRNAs during the cell cycle were assayed in selected isolates by reverse transcriptional real-time PCR with internal control genes ftsZ and ropB. 【Result】 Eight SE genes were detected in the 51 food-borne S. aureus isolates. The most prevalent SE genes were seg and sei. During the growth cycle, the patterns of SE mRNA gene expression were similar in all of the selected isolates. The enterotoxin mRNAs were peaked in the post-exponential growth phase and then rapidly decreased. However, the relative expression level of different enterotoxin genes in the same isolate was different, and the same SE gene's expression level also varied among strains.【Conclusion】The distribution and temporal expression of foodborne enterotoxin genes were studied, which would be valuable to the study of staphylococcal food poisoning (SPF) mechanism and the control of food safety.

Key words: foodborne Staphylococcus aureus, enterotoxin, gene typing, temporal expression, real-time PCR

杨静, 杨军, 黄继超, 何玮玲, 张驰, 黄明. 食源性金黄色葡萄球菌肠毒素基因的分布与时序性表达[J]. 中国农业科学, 2012, 45(19): 4057-4066.

YANG Jing, YANG Jun, HUANG Ji-Chao, HE Wei-Ling, ZHANG Chi, HUANG Ming. Distribution and Temporal Expression of Staphylococcus aureus Enterotoxin Genes in Foodborne Staphylococcus aureus Strains[J]. Scientia Agricultura Sinica, 2012, 45(19): 4057-4066.

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参考文献

[1]Arvidson S, Tegamrk K. Regulation of virulence determinants in Staphylococcus aureus. International Journal of Medical Microbiology, 2001, 291(2): 159-170.

[2]Marrack P, Kappler J. The Staphylococcal enterotoxins and their relatives. Science, 1990, 248(1): 705-711.

[3]Buzby J C, Roberts T. Economic costs and trade impacts of microbid food-borne illness. World Health Statistics Quarterly, 1997, 50(1/2): 57-66.

[4]Omoe K, Imanishi K, Hu D L, Kato H, Fugane Y, Abe Y, Hamaoka S, Watanabe Y, Nakane A, Uchiyama T, Shinagawa K. Characterization of novel Staphylococcal enterotoxin-like toxin type P. Infection and Immunity, 2005, 73(9): 5540-5546.

[5]Ono H K, Omoe K, Imanishi K, Iwakabe Y, Hu D L, Kato H, Saito N, Nakane A, Uchiyama T, Shinagawa K. Identification and characterization of two novel Staphylococcal enterotoxins types S and T. Infection and Immunity, 2008, 76 (11): 4999-5005.

[6]Thomas D Y, Jarraud S, Lemercier B, Cozon G, Echasserieau K, Etienne J, Gougeon M L, Lina G, Vandenesch F. Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster. Infection and Immunology, 2006, 74(8): 4724-4734.

[7]Zhang S P, Iandolo J J, Stewart G C. The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiology Letters, 1998, 168(2): 227-233.

[8]Lina G, Bohach G A, Nair S P, Hiramatsu K, Jouvin-Marche E, Mariuzza R. Standard nomenclature for the superantigens expressed by Staphylococcus. Journal of Infectious Diseases, 2004, 189(12): 2334-2336.

[9]Pereira V, Lopes C, Castro A, Silva J, Gibbs R, Teixeira P. Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal. Food Microbiology, 2009, 26(3): 278-282.

[10]Letertre C, Perelle S, Dilasser F, Fach P. A strategy based on 5’ nuclease multiplex PCR to detect enterotoxin genes sea to sej of Staphylococcus aureus. Molecular and Cellular Probes, 2003, 17(5): 227-235.

[11]Grumann D, Scharf S S, Holtfreter S, Kohler C, Steil L, Engelmann S, Hecker M, Volker U, Broker B M. Immune cell activation by enterotoxin gene cluster (egc)-encoded and non-egc superantigens from Staphylococcus aureus. Journal of Immunology, 2008, 181(7): 5054-5061.

[12]Dauwalder O, Pachot A, Cazalis M A, Paye M, Faudot C, Badiou C, Mougin B, Vandenesch F, Etienne J, Lina G, Monneret G. Early kinetics of the transcriptional response of human leukocytes to staphylococcal superantigenic enterotoxins A and G. Microbial Pathogenesis, 2009, 47(3): 171-176.

[13]Rosec J P, Gigaud O. Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. International Journal of Food Microbiology, 2002, 77(1-2): 61-70.

[14]李琳, 黄金海, 赵耘, 王聪明, 董艳娇, 王静思, 刘莹. 葡萄球菌肠毒素基因分型PCR检测技术的研究. 食品科学, 2008, 29(7): 340-344.

Li L, Huang J H, Zhao Y, Wang C M, Dong Y J, Wang J S, Liu Y. Study on PCR method for differentiating genetype of entertoxin of S.aureus. Food Science, 2008, 29(7): 340-344. (in Chinese)

[15]Tseng C W, Stewart G C. Rot repression of enterotoxin B expression in Staphylococcus aureus. Journal of Bacteria, 2005, 187: 5301-5309.

[16]中华人民共和国卫生部. 食品安全国家标准: 食品微生物学实验金黄色葡萄球菌检验GB 4789.10—2010 中国标准书号[S]. 北京: 中国标准出版社, 2010.

Ministry of Health of the People’s Republic of China. National food safety standard: food microbiological examination: Staphylococcus aureus.GB 4789.10—2010 China standard book number[S]. Beijing: China Standard Press, 2010. (in Chinese)

[17]Collery M M, Smyth C J. Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes ψent1 and ψent2 and the selu or seluv gene using PCR-RFLP. Journal of Medical Microbiology, 2007, 56(2): 208-216.

[18]Vandesompele J, De P, Pattyn P, Poppe B, Van-Roy N, De-Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 2002, 3(7): 0034.1–0034.11.

[19]Derzelle S, DIilasser F, Duquenne M, Deperrois V. Differential temporal expression of the Staphylococcal enterotoxins genes during cell growth. Food Microbiology, 2009, 26(8): 896-904.

[20]Klein D, Janda P, Steinborn R, Muller M, Salmons B, Gunzburg W H. Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: Influence of mismatches on quantification. Electrophoresis, 1999, 20(2): 291-299.

[21]张驰宇, 徐顺高, 黄新祥. 一种新颖简便的荧光实时RT-PCR相对定量方法的建立. 生物化学与生物物理进展, 2005, 32(9): 883-888. Zhang C Y, Xu S G, Huang X X. A novel and convenient relative quantitative method of fluorescence real time RT-PCR assay based on slope of standard curve. Progress In Biochemistry and Biophysics, 2005, 32(9): 883-888. (in Chinese)

[22]刘冬香, 刘书亮, 王印, 张晓利, 居华, 张兴. 四川省动物性食品中金黄色葡萄球菌的分离鉴定及污染分析. 食品科学, 2009, 30(8): 251-254.

Liu D X, Liu S L, Wang Y, Zhang X L, Ju H, Zhang X. Isolation, identification and contamination analysis of Staphylococcus aureus in animal food in Sichuan. Food Science, 2009, 30(8): 251-254. (in Chinese)

[23]Wallin-Carlquist N, Marta D, Borch E, Radstrom P. Prolonged expression and production of Staphylococcus aureus enterotoxin A in processed pork meat. International Journal of Food Microbiology, 2010, 141: S69-S74.

[24]Le L Y, Baron F, Gautier M. Staphylococcus aureus and food poisoning. Genetics and Molecular Research, 2003, 2(1): 63-76.

[25]Blaiotta G, Fusco V, Von-Eiff C, Becker K. Biotyping of enterotoxigenic Staphylococcus aureus by enterotoxin gene cluster (egc) polymorphism and spa typing analyses. Applied and Environmental Microbiology, 2006, 72(9): 6117-6123.

[26]Ferry T, Thomas D, Genestier A L, Bes M, Lina G, Vandenesch F. Comparative prevalence of superantigen genes in Staphylococcus aureus isolates causing sepsis with and without septic shock. Clinical Infectious Diseases, 2005, 41(6): 771-777.

[27]Peacock S J, Moore C E, Justice A, Kantzanou M, Story Y, Mackie K, O'Neil G, Day NP L. Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infection and Immunity, 2002, 70(9): 4987-4996.

[28]Proft T, Fraser J D. Bacterial superantigens. Clinical and Experimental Immunology, 2003, 133(3): 299-306.

[29]Su Y C, Wong C L. Detection of staphylococcal enterotoxin H by an enzyme-linked immunosorbent assay. Journal of Food Protection, 1996, 59(3): 327-330.

[30]Omoe K, Ishikawa M, Shimoda Y. Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh, or sei genes. Journal of Clinical Microbiology, 2002, 40(3): 857-862.

[31]赵建, 丁水军, 陆扁, 傅丹青. 48株金黄色葡萄球菌的肠毒素分布及其耐药性研究. 中国卫生检验杂志, 2005, 15(7): 841-842.

Zhao J, Ding S J, Lu B, Fu D Q. Study on enterotoxin distribution and drug resistance of 48 Staphylococcus aureus isolates. Chinese Journal of Health Laboratory Technology, 2005, 15(7): 841-842. (in Chinese)

[32]Chan P F, Foster S J. Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. Journal of Bacteriology, 1998, 180(23): 6232-6241.