金黄色葡萄球菌新型肠毒素sek基因在3 株食品分离菌株中的表达

doi:10.7506/spkx1002-6630-201613025

摘要/Abstract

摘要：

目的：肠毒素sek基因在临床分离的耐甲氧西林金黄色葡萄球菌菌株中较为流行，研究新型肠毒素sek基因的时序性表达可为食物中毒预防和疾病控制补充新的基础数据。方法：本研究针对实验室保存的食源性金黄色葡萄球菌菌株进行21 种肠毒素基因检测，筛选出3 株含sek基因的菌株SA005、SA008和SAB0，针对3 株菌株进行生长曲线测定，随后在培养不同时间段收集菌体提取RNA，利用实时荧光定量聚合酶链式反应（real time-polymerasechain reaction，real time-PCR）检测肠毒素sek基因在12～120 h的相对表达水平。结果：基因检测结果显示，菌株SA005含有16S、nuc、mecA、seb、sec、sek、sex基因，SA008含有16S、nuc、mecA、sea、sek、sex基因，SAB0含有16S、nuc、sek、sex基因。3 株菌株在胰酪胨大豆肉汤培养基中的生长曲线趋势较相似，18 h左右出现折点，之后细菌进入生长稳定期。3 株菌株的sek基因在12～120 h全程表达，SA005的sek基因在48 h时相对表达量最高，在84 h相对表达量最低；SA008在24 h时的相对表达量最高，在48 h相对表达量最低，在60～120 h表达量相对比较稳定；SAB0在12～120 h之间相对表达量呈现抛物线趋势，在60 h和72 h的表达量相对较高且差异不显著（P＞0.05）。结论：3 株菌株sek表达水平存在明显差异，相同菌株sek基因不同时间点表达也存在差异性，菌株基因背景的影响可能是造成sek基因表达差异性的关键。

关键词: 金黄色葡萄球菌, 新型肠毒素, sek基因, 实时荧光定量聚合酶链式反应, 时序性表达

Abstract:

Objective: The newly identified staphylococcal enterotoxin sek gene is found to be very popular in clinical
methicillin-resistant S. aureus isolates. The study of sek gene temporal expression would provide some valuable information
and new data for food poisoning prevention and disease control linked to this bacterium. Methods: Three strains (SA005,
SA008 and SAB0) with sek gene from foodborne isolates in our lab were selected by PCR detection of 21 different SEs
genes. The growth curves of three strains were monitored during the growth phase in TSB. Total RNAs from three strains
were extracted at different growth periods, and a quantitative real time-PCR was developed to monitor the relative expression
of sek gene. Results: The results of virulence gene detection showed that SA005 harbored 16S, nuc, mecA, seb, sec, sek and
sex genes, SA008 harbored 16S, nuc, mecA, sea, sek and sex genes, and SAB0 harbored 16S, nuc, sek and sex genes. The
characteristics of growth curves for three strains had similarity, which exhibited a turning point around 18 h, and then entered
the stationary growth phase. mRNA expression levels were shown during all the growth phases (12–120 h); however, there
were very obvious differences among three strains and among different sampling points. The relative expression of sek gene
was very high at 48 h and very low at 84 h for SA005. For SA008, it was very high at 24 h, very low at 48 h, and was stable
between 60–120 h. The relative expression of sek gene in SAB0 presented a parabolic trend, with a high level at 60 h and
72 h (P > 0.05). Conclusion: The relative expression of sek gene among three strains had a significant difference. Even in the
same strain, the relative expression of sek gene was different at different time points. Strain-to-strain variations with different
background genes may be the key points for different expression patterns of sek gene.

Key words: Staphylococcus aureus, newly identified enterotoxins, sek gene, real time-polymerase chain reaction (real time- PCR), temporal expression

中图分类号:

TS207.4

王琼，唐俊妮，汤承，陈娟，刘骥，蔡自建. 金黄色葡萄球菌新型肠毒素sek基因在3 株食品分离菌株中的表达[J]. 食品科学, doi: 10.7506/spkx1002-6630-201613025.

WANG Qiong, TANG Junni, TANG Cheng, CHEN Juan, LIU Ji, CAI Zijian. Temporal Expression of Staphylococcal Enterotoxin K (sek) Gene in Three Isolates from Food Samples[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201613025.

参考文献

[1] BALABAN N, RASOOLY A. Staphylococcal enterotoxins[J].

International Journal of Food Microbiology, 2000, 61(1): 1-10.

DOI:10.1016/s0168-1605(00)00377-9.

[2] ARGUDIN M A, MENDOZA M C, RODICIO M R. Food poisoning and

Staphylococcus aureus enterotoxins[J]. Toxins, 2010, 2(7): 1751-1773.

DOI:10.3390/toxins2071751.

[3] WILSON G J, SEO K S, CARTWRIGHT R A, et al. A novel core

genome-encoded superantigen contributes to lethality of communityassociated

MRSA necrotizing pneumonia[J]. PLoS Pathogens, 2011,

7(10): e1002271. DOI:10.1371/journal.ppat.1002271.

[4] ORWIN P M, LEUNG D Y, DONAHUE H L, et al. Biochemical and

biological properties of staphylococcal enterotoxin K[J]. Infection

and Immunity, 2001, 69(1): 360-366. DOI:10.1128/IAI.69.1.360-

366.2001.

[5] SUMBY P, WALDOR M K. Transcription of the toxin genes present

within the Staphylococcal phage phiSa3ms is intimately linked with the

phage’s life cycle[J]. Journal of Bacteriology, 2003, 185(23): 6841-6851.

DOI:10.1128/JB.185.23.6841-6851.2003.

[6] NOVICK R. Mobile genetic elements and bacterial toxinoses: the

superantigen-encoding pathogenicity islands of Staphylococcus

aureus[J]. Plasmid, 2003, 49(2): 93-105. DOI:10.1016/S0147-

619X(02)00157-9.

[7] OMOE K, HU D L, ONO H K, et al. Emetic potentials of newly

identified staphylococcal enterotoxin-like toxins[J]. Infection and

Immunity, 2013, 81(10): 3627-3631. DOI:10.1128/IAI.00550-13.

[8] 卫沛楠, 吕国平, 徐保红. 食源性金黄色葡萄球菌9种肠毒素基因的

多重PCR检测[J]. 现代预防医学, 2013, 40(17): 3269-3272.

[9] 岳丽琴, 王俊怡, 徐小静. 金黄色葡萄球菌分离株致病基因

研究[J]. 临床儿科杂志, 2013, 31(4): 347-350. DOI:10.3969/

j.issn.1000-3606.2013.04.013.

[10] AGUILAR J L, VARSHNEY A K, WANG X, et al. Detection and

measurement of staphylococcal enterotoxin-like K (Sel-K) secretion

by Staphylococcus aureus clinical isolates[J]. Journal of Clinical

Microbiology, 2014, 52(7): 2536-2543. DOI:10.1128/JCM.00387-14.

[11] WU D J, LI X M, YANG Y H, et al. Superantigen gene profiles and

presence of exfoliative toxin genes in community-acquired meticillinresistant

Staphylococcus aureus isolated from Chinese children[J].

Journal of Medical Microbiology, 2011, 60(1): 35-45. DOI:10.1099/

jmm.0.023465-0.

[12] VARSHNEY A K, MEDIAVILLA J R, ROBIOU N, et al. Diverse

enterotoxin gene profiles among clonal complexes of Staphylococcus

aureus isolates from the Bronx, New York[J]. Applied and

Environmental Microbiology, 2009, 75(21): 6839-6849. DOI:10.1128/

AEM.00272-09.

[13] DERZELLE S, DILASSER F, DUQUENNE M, et al. Differential

temporal expression of the staphylococcal enterotoxins genes

during cell growth[J]. Food Microbiology, 2009, 26(8): 896-904.

DOI:10.1016/j.fm.2009.06.007.

[14] 杨静, 杨军, 黄继超, 等. 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.

DOI:10.3864/j.issn.0578-1752.2012.19.018.

[15] 王琼, 唐俊妮, 汤承, 等. 一种采用微波炉加热快速提取细菌DNA

用于PCR扩增的方法[J]. 西南民族大学学报(自然科学版), 2015,

41(2): 150-155. DOI:10.11920/xnmdzk.2015.02.004.

[16] TANG J N, SHI X M, SHI C L, et al. Characterization of a duplex PCR

assay for the detection of enterotoxigenic strains of Staphylococcus

aureus[J]. Journal of Rapid Methods and Automation in Microbiology,

2006, 14(3): 201-217. DOI:10.1111/j.1745-4581.2006.00047.x.

[17] XU J, SHI C L, SONG M H, et al. Phenotypic and genotypic

antimicrobial resistance traits of foodborne Staphylococcus aureus

isolates from Shanghai[J]. Journal of Food Science, 2014, 79(4):

M635-M642. DOI:10.1111/1750-3841.12405.

[18] WONGBOOT W, CHOMVARIN C, ENGCHANIL C, et al. Multiplex

PCR for detection of superantigenic toxin genes in methicillin-sensitive

and methicillin-resistant Staphylococcus aureus isolated from patients

and carriers of a hospital in northeast Thailand[J]. The Southeast Asian

Journal of Tropical Medicine and Public Health, 2013, 44(4): 660-671.

[19] JARRAUD S, MOUGEL C, THIOULOUSE J, et al. Relationships

between Staphylococcus aureus genetic background, virulence factors,

agr groups (alleles), and human disease[J]. Infection and Immunity,

2002, 70(2): 631-641. DOI:10.1128/IAI.70.2.631-641.2002.

[20] SRINIVASAN V, SAWANT A A, GILLESPIE B E, et al.

Prevalence of enterotoxin and toxic shock syndrome toxin genes in

Staphylococcus aureus isolated from milk of cows with mastitis[J].

Foodborne Pathogens and Disease, 2006, 3(3): 274-283. DOI:10.1089/

fpd.2006.3.274.

[21] PFAFFL M W. A new mathematical model for relative quantification

in real-time RT-PCR[J]. Nucleic Acids Research, 2001, 29(9): e45.

DOI:10.1093/nar/29.9.e45.

[22] CZOP J K, BERGDOLL M S. Staphylococcal enterotoxin synthesis

during the exponential, transitional, and stationary growth phases[J].

Infection and Immunity, 1974, 9(2): 229-235.

[23] TSENG C W, ZHANG S, STEWART G C. Accessory gene regulator

control of staphyloccoccal enterotoxin d gene expression[J]. Journal of

Bacteriology, 2004, 186(6): 1793-1801. DOI:10.1128/JB.186.6.1793-

1801.2004.

[24] OTERO A, GARCÍA M C, GARCÍA M L, et al. Production of

staphylococcal enterotoxins C1 and C2 and thermonuclease in ewe’s

milk[J]. Food Microbiology, 1987, 4(4): 339-345. DOI:10.1016/

S0740-0020(87)80008-4.