茶多酚诱导铜绿假单胞菌交叉耐受性研究

doi:10.13305/j.cnki.jts.2015.06.004

摘要/Abstract

摘要： 检测了亚致死浓度的茶多酚（Tea Polyphenols,TP）处理对铜绿假单胞菌交叉耐受性的诱导作用。铜绿假单胞菌暴露于1βmg·mL^-1茶多酚1βh后能够显著增强细菌对多种环境条件的耐受性,包括氧化剂（1βmmol·L^-1 H₂O₂）、高温（47℃）,及酸性溶液[磷酸缓冲液（pH4.0）、含有有机酸（60βmmol·L^-1柠檬酸、60βmmol·L^-1乳酸、80βmmol·L^-1乙酸）的磷酸缓冲液（pH4.0）]。另外,通过荧光定量RT-PCR技术分析了茶多酚诱导下铜绿假单胞菌胁迫相关基因的表达情况。研究发现,茶多酚能够显著诱导铜绿假单胞菌氧化胁迫相关基因katB、sodM、ohr、lexA和recN的表达,以及热激蛋白基因dnaK、groEL、htpG、grpE和groES的表达。这些胁迫相关基因的表达很可能在细菌交叉耐受性形成过程中起到重要作用。以上研究结果表明,虽然茶多酚作为天然食品添加剂具有较高的安全性,但在实际应用过程中应充分考虑茶多酚诱导细菌交叉耐受性所导致的潜在风险,以优化食品保鲜策略。

关键词: 茶多酚, 铜绿假单胞菌, 交叉耐受性, 氧化胁迫相关基因, 热激蛋白基因

Abstract: Cross-resistance induced by sublethal concentration of tea polyphenols (TP) to Pseudomonas aeruginosa was investigated. One-hour exposure of Pseudomonas aeruginosa to 1βmg·mL^-1 TP significantly increased the tolerance to multiple environmental stresses, including oxidant (1βmmol·L^-1 H₂O₂), high temperature (47℃) and acid solutions [ phosphate buffer (pH4.0), phosphate buffer (pH4.0) containing 60βmmol·L^-1 citric acid, 60βmmol·L^-1 lactic acid and 80βmmol·L^-1 acetic acid]. In addition, the effects of TP on expression levels of a few stress-related genes were studied by quantitative real-time RT-PCR analysis. It was observed that the expression levels of a few oxidative stress-related genes katB, sodM, ohr, lexA and recN were up-regulated by TP, as well as some heat shock protein genes, including dnaK, groEL, htpG, grpE and groES. The expression of these genes might play important roles in the development of stress cross-resistance. Although TP was considered to be safe as food preservatives, the underlying hazards associated with cross-resistance induced by TP should be taken into account in food technology.

Key words: tea polyphenols, Pseudomonas aeruginosa, cross-resistance oxidative stress-related genes, heat shock protein genes

中图分类号:

TS272
Q946.8

刘小香, 孙爱华, 杜蓬, 陈文虎, 朱军莉. 茶多酚诱导铜绿假单胞菌交叉耐受性研究[J]. 茶叶科学, 2015, 35(6): 534-542. doi: 10.13305/j.cnki.jts.2015.06.004.

LIU Xiaoxiang, SUN Aihua, DU Peng, CHEN Wenhu, ZHU Junli. Cross-resistance Induced by Tea Polyphenols in Pseudomonas aeruginosa[J]. Journal of Tea Science, 2015, 35(6): 534-542. doi: 10.13305/j.cnki.jts.2015.06.004.

参考文献 33

[1]	Khan N, Mukhtar H.Tea polyphenols for health promotion[J]. Life Science, 2007, 81(7): 519-533.
[2]	Almajano MP, Carbó R, Jiménez JAL, et al. Antioxidant and antimicrobial activities of tea infusions[J]. Food Chemistry, 2008, 108(1): 55-63.
[3]	Friedman M.Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas[J]. Molecular Nutrition & Food Research, 2007, 51(1): 116-134.
[4]	Reygaert WC.The antimicrobial possibilities of green tea[J]. Front Microbiology, 2014, 5: 434.
[5]	Cui Y, Oh YJ, Lim J, et al. AFM study of the differential inhibitory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negative bacteria[J]. Food Microbiology, 2012, 29(1): 80-87.
[6]	Yi S, Li J, Zhu J, et al. Effect of tea polyphenols on microbiological and biochemical quality of Collichthys fish ball[J]. Journal of the Science of Food and Agriculture, 2011, 91(9): 1591-1597.
[7]	Sirk TW, Brown EF, Friedman M, et al. Molecular binding of catechins to biomembranes: relationship to biological activity[J]. Journal of Agricultural and Food Chemistry, 2009, 57(15): 6720-6728.
[8]	Navarro-Martínez MD, Navarro-Perán E, Cabezas-Herrera J, et al. Antifolate activity of epigallocatechin against Stenotrophomonas maltophilia[J]. Antimicrobial Agents and Chemotherapy, 2005, 49(7): 2914-2920.
[9]	Zhang YM, Rock CO.Evaluation of epigallocatechin gallate and related plant polyphenols as inhibitors of the FabG and FabI reductases of bacterial type II fatty-acid synthase[J]. The Journal of Biological Chemistry, 2004, 279(30): 30994-31001.
[10]	Arakawa H, Maeda M, Okubo S, et al. Role of hydrogen peroxide in bactericidal action of catechin[J]. Biological & Pharmaceutical Bulletin, 2004, 27(3): 277-281.
[11]	Maeta K, Nomura W, Takatsume Y, et al. Green tea polyphenols function as prooxidants to activate oxidativestress-responsive transcription factors in yeasts[J]. Applied and Environmental Microbiology, 2007, 73(2): 572-580.
[12]	Lin MH, Tsai TY, Hsieh SC, et al. Susceptibility of Vibrio parahaemolyticus to disinfectants after prior exposure to sublethal stress[J]. Food Microbiology, 2013, 34(1): 202-206.
[13]	Hsiao WL, Ho WL, Chou CC.Sub-lethal heat treatment affects the tolerance of Cronobacter sakazakii BCRC 13988 to various organic acids, simulated gastric juice and bile solution[J]. International Journal of Food Microbiology, 2010, 144(2): 280-284.
[14]	Gennari M, Dragotto F.A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water[J]. the Journal of Applied Bacteriology, 1992, 72(4): 281-288.
[15]	Hamanaka D, Onishi M, Genkawa T, et al. Effects of temperature and nutrient concentration on the structural characteristics and removal of vegetable-associated Pseudomonas biofilm[J]. Food Control, 2012, 24(1/2): 165-170.
[16]	Dumas JL, van Delden C, Perron K, et al. Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR [J]. FEMS Microbiology Letters, 2006, 254(2): 217-225.
[17]	Palma M, DeLuca D, Worgall S, et al. Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide[J]. Journal of Bacteriology, 2004, 186(1): 248-252.
[18]	Burt SA, van der Zee R, Koets AP, et al. Carvacrol induces heat shock protein 60 and inhibits synthesis of flagellin in Escherichia coli O157: H7 [J]. Applied and Environmental Microbiology, 2007, 73(14): 4484-4490.
[19]	Chang W, Small DA, Toghrol F, et al. Microarray analysis of Pseudomonas aeruginosa reveals induction of pyocin genes in response to hydrogen peroxide[J]. BMC Genomics, 2005, 6: 115-128.
[20]	Yi SM, Zhu JL, Fu LL, et al. Tea polyphenols inhibit Pseudomonas aeruginosa through damage to the cell membrane[J]. International Journal of Food Microbiology, 2010, 144(1): 111-117.
[21]	Rangel DE.Stress induced cross-protection against environmental challenges on prokaryotic and eukaryotic microbes[J]. World Journal of Microbiology & Biotechnology, 2011, 27(6): 1281-1296.
[22]	Arroyo C, Cebrián G, Condón S, et al. Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions[J]. Journal of Applied Microbiology, 2012, 112(3): 561-570.
[23]	Bikels-Goshen T, Landau E, Saguy S, et al. Staphylococcal strains adapted to epigallocathechin gallate (EGCG) show reduced susceptibility to vancomycin, oxacillin and ampicillin, increased heat tolerance, and altered cell morphology[J]. International Journal of Food Microbiology, 2010, 138(1/2): 26-31.
[24]	Cho YS, Schiller NL, Kahng HY, et al. Cellular responses and proteomic analysis of Escherichia coli exposed to green tea polyphenols[J]. Current Microbiology, 2007, 55(6): 501-506.
[25]	Lee PC, Bochner BR, Ames BN.AppppA, heat-shock stress, and cell oxidation[J]. Proceedings of the National Academy of Sciences of the United States of America, 1983, 80(24): 7496-7500.
[26]	Torres MA, Dangl JL.Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development[J]. Current Opinion in Plant Biology, 2005, 8(4): 397-403.
[27]	Aertsen A, De Spiegeleer P, Vanoirbeek K, et al. Induction of oxidative stress by high hydrostatic pressure in Escherichia coli[J]. Applied and Environmental Microbiology, 2005, 71(5): 2226-2231.
[28]	Kaku N, Hibino T, Tanaka Y, et al. Effects of overexpression of Escherichia coli katE and bet genes on the tolerance for salt stress in a freshwater cyanobacterium Synechococcus sp. PCC 7942[J]. Plant Science, 2000, 159(2): 281-288.
[29]	Yu P.Enhancing survival of Escherichia coli by increasing the periplasmic expression of Cu, Zn superoxide dismutase from Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology, 2007, 76(4): 867-871.
[30]	Van Bogelen RA, Kelley PM, Neidhardt FC.Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli [J]. Journal of Bacteriology, 1987, 169(1): 26-32.
[31]	Silva A, Sampaio-Marques B, Fernandes A, et al. Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid[J]. PLoS One, 2013, 8(8): e71294.
[32]	Singh VK, Utaida S, Jackson LS, et al. Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus [J]. Microbiology, 2007, 153(Pt 9): 3162-3173.
[33]	Lee S, Ahn S, Lee H, et al. Gene-related strain variation of Staphylococcus aureus for homologous resistance response to acid stress[J]. Journal of Food Protection, 2014, 77(10): 1794-1798.