该文研究了副溶血弧菌在32、10 ℃下生物被膜的形成,并采用结晶紫染色法、四唑鎓盐(XTT)法、叠氮溴化丙锭与荧光定量PCR结合技术、激光共聚焦显微镜观察的手段以评估不同浓度的薰衣草精油和4种常用抗菌剂对副溶血弧菌成熟生物被膜的清除效果,进一步分析了2种温度下形成的生物被膜对不同浓度的薰衣草精油的抗性。研究结果表明,副溶血弧菌在两种温度下均能形成稳定的生物被膜,32 ℃下形成的生物被膜量显著高于10 ℃;低温生物被膜对抗菌剂的抗性更强;在5种抗菌剂中,薰衣草精油对副溶血弧菌生物被膜的清除效果最佳。1/2最低抑菌浓度(minimum inhibitory concentration,MIC)~4 MIC的薰衣草精油对副溶血弧菌32 ℃下形成的常温生物被膜和10 ℃下形成的低温生物被膜的清除率分别为36.39%~72.38%和58.20%~67.62%,使代谢活性分别下降35.72%~71.38%和42.06%~50.48%。激光共聚焦显微镜图像显示,经薰衣草精油处理的生物被膜结构稀疏,胞外多糖减少,死菌数量增加。此外,薰衣草精油对成熟生物被膜细胞具有良好的杀伤效果,随着其处理浓度的增加,生物被膜内活细胞和可培养细胞数量逐渐减少。该研究对防控副溶血弧菌污染,拓宽植物精油在食品工业中的应用具有积极的作用。
In this study, the biofilm formations of Vibrio parahaemolyticus at 32 ℃ and 10 ℃ were investigated, and crystal violet staining, XTT assay, propidium monoazade(PMA)-qPCR and confocal laser scanning microscopy (CLSM) were used to evaluate the elimination effects of different concentrations of lavender essential oil (LEO) and four common antibacterial agents on mature biofilms of V. parahaemolyticus. And the resistance of biofilms formed at two temperatures to different concentrations of LEO was further analyzed. The results showed that V. parahaemolyticus could form mature biofilms at 32 ℃ and 10 ℃, and the amount of biofilm formed at 32 ℃ was significantly higher than that at 10 ℃. The low-temperature biofilms formed at 10 ℃ were more resistant to antibacterial agents compared with the room-temperature biofilms formed at 32 ℃. Moreover, LEO exhibited the most excellent elimination effect on the mature biofilms among the tested antimicrobial agents. At 1/2 minimum inhibitory concentration (MIC) to 4 MIC, the removal rates of LEO on the low-temperature biofilms and room-temperature biofilms were 36.39%-2.38% and 58.20%-67.62%, respectively, and the metabolic activity decreased by 35.72%-71.38% and 42.06%-50.48%, respectively. CLSM images revealed that the treated biofilm structure was sparse, the extracellular polysaccharide decreased and the number of dead cells increased. In addition, LEO presented a strong killing effect on mature biofilm cells. With the increase of the treatment concentration, the number of viable cells and culturable cells in biofilms gradually decreased. This study would be of positive significance to prevent and control V. parahaemolyticus pollution and broaden the application of plant essential oil in food industry.
[1] ASHRAFUDOULLA M, MIZAN M F R, PARK H, et al.Genetic relationship, virulence factors, drug resistance profile and biofilm formation ability of Vibrio parahaemolyticus isolated from mussel[J].Frontiers in Microbiology, 2019, 10:513-526.
[2] 刘欢, 谢婷, 何美珊, 等.广州市售海产品中副溶血弧菌分离菌株的鉴定及其耐药性分析[J].食品与发酵工业, 2018, 44(11):28-34.
LIU H,XIE T,HE M S, et al.Isolation, identification and antibiotic resistance analysis of Vibrio parahaemolyticus in seafood from Guangzhou[J].Food and Fermentation Industries, 2018, 44(11):28-34.
[3] 张德福, 安慧, 张健, 等.副溶血弧菌耐药及其机制的研究进展[J].食品工业科技, 2018, 39(9):311-317;324.
ZHANG D F, AN H, ZHANG J, et al.Research progress of the drug-resistance and its mechanism of Vibrio parahaemolyticus[J].Science and Technology of Food Industry, 2018, 39(9):311-317;324.
[4] MIZAN M F R, JAHID I K, HA S D.Microbial biofilms in seafood:A food-hygiene challenge[J].Food Microbiology, 2015, 49:41-55.
[5] HALL-STOODLEY L, STOODLEY P.Evolving concepts in biofilm infections[J].Cellular Microbiology, 2009, 11(7):1 034-1 043.
[6] BRINDHADEVI K, LEWISOSCAR F, MYLONAKIS E, et al.Biofilm and quorum sensing mediated pathogenicity in Pseudomonas aeruginosa[J].Process Biochemistry, 2020, 96:49-57.
[7] CROSSLEY K B.Staphylococci in Human Disease[M].2nd ed.Blackwell, 2010.
[8] 何美珊, 丁楠, 卢承蓉, 等.两种抗菌剂对不同温度下形成的单增李斯特菌生物膜的清除作用[J].食品与发酵工业, 2019, 45(14):84-90.
HE M S, DING N, LU C R, et al.Elimination effects of two antibacterial agents on Listeria monocytogenes biofilms formed at different temperatures[J].Food and Fermentation Industries, 2019, 45(14):84-90.
[9] 谢军, 孙晓红, 潘迎捷, 等.酸性电解水及其在食品工业中的应用[J].食品工业科技, 2010, 31(2):366-368;373.
XIE J, SUN X H, PAN Y J, et al.Acidic electrolyzed water and its application in the food industry[J].Science and Technology of Food Industry, 2010, 31(2):366-368;373.
[10] 雷欢, 谢婷, 魏宇清, 等.4种防腐剂对副溶血弧菌生物膜形成的抑制作用[J].食品科学, 2017, 38(7):6-10.
LEI H, XIE T, WEI Y Q, et al.Inhibitory effects of four kinds of food preservatives on biofilm formation by Vibrio parahaemolyticus[J].Food Science, 2017, 38(7):6-10.
[11] ADASZYŃSKA-SKWIRZYŃSKA M, SZCZERBIŃSKA D.The antimicrobial activity of lavender essential oil (Lavandula angustifolia) and its influence on the production performance of broiler chickens[J].Journal of Animal Physiology and Animal Nutrition, 2018, 102(4):1 020-1 025.
[12] SOHEILI M, SALAMI M.Lavandula angustifolia biological characteristics:An in vitro study[J].Journal of Cellular Physiology, 2019, 234(9):16 424-16 430.
[13] BETT-GARBER K L, GREENE J L, LAMIKANRA O, et al.Effect of storage temperature variations on sensory quality of fresh-cut cantaloupe melon[J].Journal of Food Quality, 2011, 34(1):19-29.
[14] CHUNG K H, PARK M S, KIM H Y, et al.Growth prediction and time-temperature criteria model of Vibrio parahaemolyticus on traditional Korean raw crab marinated in soy sauce (ganjang-gejang) at different storage temperatures[J].Food Control, 2019, 98:187-193.
[15] WONG H, CHEN L, YU C.Survival of psychrotrophic Vibrio mimicus, Vibrio fluvialis and Vibrio parahaemolyticus in culture broth at low temperatures[J].Journal of Food Protection, 1994, 57(7):607-610.
[16] BURNHAM V E, JANES M E, JAKUS L A, et al.Growth and survival differences of Vibrio vulnificus and Vibrio parahaemolyticus strains during cold storage[J].Journal of Food Science, 2009, 74(6):314-318.
[17] BRO ŻYNA M, PALECZNY J, KOZ ŹOWSKA W, et al.The antimicrobial and antibiofilm in vitro activity of liquid and vapour phases of selected essential oils against Staphylococcus aureus[J].Pathogens, 2021, 10(9):1 207-1 232.
[18] RAMIC' D, BUCAR F, KUNEJ U, et al.Antibiofilm potential of lavandula preparations against Campylobacter jejuni[J].Applied and Environmental Microbiology, 2021, 87(19):e0109921.
[19] DE RAPPER S, VILJOEN A, VAN VUUREN S.The in vitro antimicrobial effects of Lavandula angustifolia essential oil in combination with conventional antimicrobial agents[J].Evidence-Based Complementary and Alternative Medicene, 2016, 2016(7):1-9.
[20] CHERRAT L, DUMAS E, BAKKALI M, et al.Effect of essential oils on cell viability, membrane integrity and membrane fluidity of Listeria innocua and Escherichia coli[J].Journal of Essential Oil Bearing Plants, 2016, 19(1):155-166.
[21] FERRO S, AMORICO T, DEO P.Role of food sanitising treatments in inducing the ‘viable but nonculturable’ state of microorganisms[J].Food Control, 2018, 91:321-329.
[22] 谷立慧, 钟青萍, 方祥, 等.“活的非可培养态”食源致病细菌的研究进展[J].食品与发酵工业, 2016, 42(2):270-274.
GU L H, ZHONG Q P, FANG X, et al.Research progress on the viable but non-culturable state of food-borne pathogenic bacteria[J].Food and Fermentation Industries, 2016, 42(2):270-274.
[23] NOWAKOWSKA J, OLIVER J D.Resistance to environmental stresses by Vibrio vulnificus in the viable but nonculturable state[J].FEMS Microbiology Ecology, 2013, 84(1):213-222.
[24] 黄韵, 余以刚, 黄秀丽, 等.单增李斯特菌非可培养状态的诱导与PMA-qPCR检测[J].食品工业科技, 2014, 35(1):137-140;149.
HUANG Y, YU Y G, HUANG X L, et al.Induction and PMA-qPCR detection of Listeria monocytogenes in viable but non-culturable state[J].Science and Technology of Food Industry, 2014, 35(1):137-140;149.
