溴化呋喃酮对单增李斯特菌生物膜形成及黏附作用的影响
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摘要
目的:为了预防控制单增李斯特菌的污染,研究群体感应抑制剂3,4-二溴-2(5H)-呋喃酮在亚抑菌浓度下对单增李斯特菌生物膜形成的调控及黏附作用。方法:采用倍比稀释法确定抑制剂的最小抑菌浓度(MIC)和最低致死剂量(MBC),研究在亚抑菌浓度MIC,5MIC,10MIC时,单增李斯特菌生长曲线、细菌总蛋白含量、胞外多糖含量变化规律。通过结晶紫染色法观察生物膜形成情况,计算抑制率;通过细胞黏附试验测定对黏附率的影响。结果:3,4-二溴-2(5H)-呋喃酮对单增李斯特菌的MIC为25μg/mL,MBC为400μg/mL;在工作质量浓度分别为25,125,250μg/mL时,对单增李斯特菌生物膜形成的抑制率分别为18.83%,39.98%和83.27%,细菌总蛋白和胞外多糖明显减少;对单增李斯特菌侵染Caco-2细胞黏附率抑制率分别为44.41%,67.68%和85.28%。结论:3,4-二溴-2(5H)-呋喃酮在亚抑菌浓度下,对单增李斯特菌生物膜的形成以及黏附细胞均存在一定的调控作用,且随着抑制剂浓度的增加抑制效果更明显。
Objective: To prevent and control the contamination of L. monocytogenes, effects of quorum sensing inhibitors on biofilm formation and the adhesion to Caco-2 cells were examined for L. monocytogenes. Methods: Different concentrations of brominated 2(5 H)-furanones were added into the culture medium during the experiment to determine the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC), then the colony growth curve, the content variations of extracellular proteins and polysaccharides of L. monocytogenes were measured, the formation of L. monocytogenes biofilms was observed by using crystal violet staining, and the effects of brominated 2(5 H)-furanones on the adhesion of L. monocytogenes to Caco-2 cells were studied at the concentrations of MIC, 5 MIC and10 MIC, respectively. Results: The results showed that the MIC and MBC of brominated 2(5 H)-furanones to L. monocytogenes were 25 μg/mL and 400 μg/mL, respectively. The inhibitory rate of brominated 2(5 H)-furanones on L. monocytogenes biofilm formation were 18.83%, 39.98% and 83.27%, extracellular proteins and polysaccharides of L. monocytogenes had a decreasing direction, and the inhibition of L. monocytogenes adhesion to Caco-2 cells were 44.41%, 67.68%and 85.28% at the brominated 2(5 H)-furanones working concentrations of MIC, 5 MIC and 10 MIC, respectively. Conclusion: This study confimed brominated 2(5 H)-furanones could play an important inhibitting role in L. monocytogenes biofilm formation and the adhesion of L. monocytogenes to Caco-2 cells, and the effects could be strengthened with the increase of its concentration.
Objective: To prevent and control the contamination of L. monocytogenes, effects of quorum sensing inhibitors on biofilm formation and the adhesion to Caco-2 cells were examined for L. monocytogenes. Methods: Different concentrations of brominated 2(5 H)-furanones were added into the culture medium during the experiment to determine the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC), then the colony growth curve, the content variations of extracellular proteins and polysaccharides of L. monocytogenes were measured, the formation of L. monocytogenes biofilms was observed by using crystal violet staining, and the effects of brominated 2(5 H)-furanones on the adhesion of L. monocytogenes to Caco-2 cells were studied at the concentrations of MIC, 5 MIC and10 MIC, respectively. Results: The results showed that the MIC and MBC of brominated 2(5 H)-furanones to L. monocytogenes were 25 μg/mL and 400 μg/mL, respectively. The inhibitory rate of brominated 2(5 H)-furanones on L. monocytogenes biofilm formation were 18.83%, 39.98% and 83.27%, extracellular proteins and polysaccharides of L. monocytogenes had a decreasing direction, and the inhibition of L. monocytogenes adhesion to Caco-2 cells were 44.41%, 67.68%and 85.28% at the brominated 2(5 H)-furanones working concentrations of MIC, 5 MIC and 10 MIC, respectively. Conclusion: This study confimed brominated 2(5 H)-furanones could play an important inhibitting role in L. monocytogenes biofilm formation and the adhesion of L. monocytogenes to Caco-2 cells, and the effects could be strengthened with the increase of its concentration.
引文
[1] MBANDI E, SHELEF L A. Enhanced antimicrobial effects of combination of lactate and diacetate on Listeria monocytogenes and Salmonella spp. in beef biology[J]. International Journal of Food Microbiology, 2002, 76(3):191-198.
[2] JADHAV S, BHAVE M, PALOMBO E A. Methods used for the detection and subtyping of Listeria monocytogenes[J]. Journal of Microbiological Methods, 2012, 88(3):327-341.
[3] JUSTIN D, JIM M L. Human Listeria monocytogenes infections in Europe——an opportunity for improved European surveillance[J]. Eurosurveillance,2008, 13(13):1854-1861.
[4] JADHAV S, BHAVE M, PALOMBO E A. Methods used for the detection and subtyping of Listeria monocytogenes[J]. Journal of Microbiological Methods, 2012, 88(3):327-341.
[5]叶正兴,王艳,李培京,等.单增李斯特菌1/2a血清型菌株生物膜形成能力及相关基因差异研究[J].中国人兽共患病学报, 2013, 29(4):354-360.
[6]单志英.铜绿假单胞菌生物被膜形成早期相关基因的研究[D].天津:南开大学, 2004.
[7] NITHYA C, BEGUM M F, PANDIAN S K. Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1[J]. Applied Microbiology and Biotechnology,2010, 88(1):341-358.
[8]李夏,邓璐霞,吴桂霞,等. HMGN2对大肠埃希氏菌黏附Hela细胞的影响[J].西部医学, 2010, 22(5):788-792.
[9] RENIER S, HEBRAUD M, DESVAUX M. Molecular biology of surface colonization by Listeria monocytogenes:an additional facet of an opportunistic Gram-positive foodborne pathogen[J]. Environmental Microbiology, 2011, 13(4):835-850.
[10] HENTZER M, WU H, ANDERSEN J B, et al. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors[J]. EMBO Journal, 2003,22(15):3803-3815.
[11] TEPLITSKI M, CHEN H, RAJAMANI S, et al.Chlamydomonas reinhardtii seeretes cornpounds that mimic baeterial signals and interfere with quorum sensing regulation in baeterial[J]. Plant Physiology,2004, 134:137-146.
[12]翟有朋.群体感应抑制剂对海洋生态功能菌生物膜形成的影响[D].扬州:扬州大学, 2013.
[13]郭嘉亮,李冰洲,刘伟,等.溴代2(5H)-呋喃酮对绿脓杆菌生物膜形成的抑制作用[J].生命科学研究,2009, 13(1):38-41.
[14]叶联华,许赓,黄云超,等.溴代呋喃酮对胸心外科聚氯乙烯材料表面表皮葡萄球菌生物膜形成的影响[J].中国胸心血管外科临床杂志, 2010, 17(4):312-316.
[15]王玉真.几种天然呋喃酮化合物的合成及其细菌生物被膜抑制活性研究[D].广州:暨南大学, 2011.
[16]张拥军主编.食品卫生与检验[M].北京:中国计量出版社, 2011:189-190.
[17] WANG M Z, WANG H R, CAO H H, et al. Effects of limiting amino acids on rumen fermentation and microbial community in vitro[J]. Agricultural Sciences in China, 2008, 7(12):1524-1531.
[18]富瑶,杨洪英,马玉蕊,等.砷离子胁迫下浸矿细菌胞外多糖含量变化的研究[C].北京:中国有色金属学会学术年会, 2008.
[19]孙焕,杨宏军,胡桂学,等.金黄色葡萄球菌荚膜多糖的提取及其多糖含量的测定[J].中国农学通报,2009, 25(6):1-4.
[20]黄迅辰.荧光假单胞菌分离鉴定、生物膜形成及免疫磁珠检测技术的研究[D].杭州:中国计量学院,2015.
[21] COSTERTON J W, STEWART P S, CREENBERG E P. Bacterial biofilm:a common cause of persis tent infections[J]. Science, 1999, 284(5418):1318-1322.
[22] POTERA C. Forginga link between biofilms and disease[J]. Science, 1999, 283(5409):1837-1839.
[23] COSTERTON J W, LEWANDOWSKI Z, CALDWELL D E, et al. Microbial biofilms[J]. Annual Review of Microbiology, 1995, 49(1):711-745.
[24]王丽英.生物膜的形成与控制[J].食品科学,1999,(1):10-12.
[25] MANEFIELD M, RASMUSSEN T B, HENZTER M,et al. Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover[J]. Microbiology,2002, 148(4):1119-1127.
[26]郭嘉亮,陈卫民.细菌群体感应信号分子与抑制剂研究进展[J].生命科学, 2007, 19(2):224-232.
[27] MARTINELLI D, GROSSMANN G, SEQUIN L, et al. Effects of natural and chemically synthesized furanones on quorum sensing in Chromobacterium violaceum[J]. BMC Microbiology, 2004, 4(1):1-10.
[28] HENTZER M, WU H, ANDERSEN J B, et al. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors[J]. EMBO Journal, 2003,22(15):3803-3815.
[29] MILLER S T, XAVIER K B, CAMPAGNA S R, et al. Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2[J]. Molecular Cell, 2004, 15(5):677-687.
[2] JADHAV S, BHAVE M, PALOMBO E A. Methods used for the detection and subtyping of Listeria monocytogenes[J]. Journal of Microbiological Methods, 2012, 88(3):327-341.
[3] JUSTIN D, JIM M L. Human Listeria monocytogenes infections in Europe——an opportunity for improved European surveillance[J]. Eurosurveillance,2008, 13(13):1854-1861.
[4] JADHAV S, BHAVE M, PALOMBO E A. Methods used for the detection and subtyping of Listeria monocytogenes[J]. Journal of Microbiological Methods, 2012, 88(3):327-341.
[5]叶正兴,王艳,李培京,等.单增李斯特菌1/2a血清型菌株生物膜形成能力及相关基因差异研究[J].中国人兽共患病学报, 2013, 29(4):354-360.
[6]单志英.铜绿假单胞菌生物被膜形成早期相关基因的研究[D].天津:南开大学, 2004.
[7] NITHYA C, BEGUM M F, PANDIAN S K. Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1[J]. Applied Microbiology and Biotechnology,2010, 88(1):341-358.
[8]李夏,邓璐霞,吴桂霞,等. HMGN2对大肠埃希氏菌黏附Hela细胞的影响[J].西部医学, 2010, 22(5):788-792.
[9] RENIER S, HEBRAUD M, DESVAUX M. Molecular biology of surface colonization by Listeria monocytogenes:an additional facet of an opportunistic Gram-positive foodborne pathogen[J]. Environmental Microbiology, 2011, 13(4):835-850.
[10] HENTZER M, WU H, ANDERSEN J B, et al. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors[J]. EMBO Journal, 2003,22(15):3803-3815.
[11] TEPLITSKI M, CHEN H, RAJAMANI S, et al.Chlamydomonas reinhardtii seeretes cornpounds that mimic baeterial signals and interfere with quorum sensing regulation in baeterial[J]. Plant Physiology,2004, 134:137-146.
[12]翟有朋.群体感应抑制剂对海洋生态功能菌生物膜形成的影响[D].扬州:扬州大学, 2013.
[13]郭嘉亮,李冰洲,刘伟,等.溴代2(5H)-呋喃酮对绿脓杆菌生物膜形成的抑制作用[J].生命科学研究,2009, 13(1):38-41.
[14]叶联华,许赓,黄云超,等.溴代呋喃酮对胸心外科聚氯乙烯材料表面表皮葡萄球菌生物膜形成的影响[J].中国胸心血管外科临床杂志, 2010, 17(4):312-316.
[15]王玉真.几种天然呋喃酮化合物的合成及其细菌生物被膜抑制活性研究[D].广州:暨南大学, 2011.
[16]张拥军主编.食品卫生与检验[M].北京:中国计量出版社, 2011:189-190.
[17] WANG M Z, WANG H R, CAO H H, et al. Effects of limiting amino acids on rumen fermentation and microbial community in vitro[J]. Agricultural Sciences in China, 2008, 7(12):1524-1531.
[18]富瑶,杨洪英,马玉蕊,等.砷离子胁迫下浸矿细菌胞外多糖含量变化的研究[C].北京:中国有色金属学会学术年会, 2008.
[19]孙焕,杨宏军,胡桂学,等.金黄色葡萄球菌荚膜多糖的提取及其多糖含量的测定[J].中国农学通报,2009, 25(6):1-4.
[20]黄迅辰.荧光假单胞菌分离鉴定、生物膜形成及免疫磁珠检测技术的研究[D].杭州:中国计量学院,2015.
[21] COSTERTON J W, STEWART P S, CREENBERG E P. Bacterial biofilm:a common cause of persis tent infections[J]. Science, 1999, 284(5418):1318-1322.
[22] POTERA C. Forginga link between biofilms and disease[J]. Science, 1999, 283(5409):1837-1839.
[23] COSTERTON J W, LEWANDOWSKI Z, CALDWELL D E, et al. Microbial biofilms[J]. Annual Review of Microbiology, 1995, 49(1):711-745.
[24]王丽英.生物膜的形成与控制[J].食品科学,1999,(1):10-12.
[25] MANEFIELD M, RASMUSSEN T B, HENZTER M,et al. Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover[J]. Microbiology,2002, 148(4):1119-1127.
[26]郭嘉亮,陈卫民.细菌群体感应信号分子与抑制剂研究进展[J].生命科学, 2007, 19(2):224-232.
[27] MARTINELLI D, GROSSMANN G, SEQUIN L, et al. Effects of natural and chemically synthesized furanones on quorum sensing in Chromobacterium violaceum[J]. BMC Microbiology, 2004, 4(1):1-10.
[28] HENTZER M, WU H, ANDERSEN J B, et al. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors[J]. EMBO Journal, 2003,22(15):3803-3815.
[29] MILLER S T, XAVIER K B, CAMPAGNA S R, et al. Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2[J]. Molecular Cell, 2004, 15(5):677-687.