大黄鱼源腐败菌的黏附特性与生物膜特性分析
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摘要
以分离纯化自冰鲜大黄鱼的3株希瓦氏菌(MA1-5、MA1-7、MA1-13)和3株假单胞菌(R3-1、R3-2、R3-5)为供试菌株,研究鱼源腐败菌的表面疏水性、自凝聚能力、生物膜和腐败菌对鱼体(鱼肠、鱼鳃和体表)黏液的黏附能力,探明黏附特性与不同部位黏附能力的相关性,并进一步研究生物膜在不同因素下的形成特性。研究表明,希瓦氏菌具有更强的表面疏水性和自凝聚能力,对鱼体黏液的黏附性强,并具有更强的生物膜形成能力。主成分分析和聚类分析说明黏附能力在属间存在差异性,属内具有相似性。腐败菌对鱼肠和鱼鳃的黏附能力与自凝聚能力和生物膜形成能力具有较高的相关性,对体表黏附能力只与生物膜形成能力有关。腐败菌生物膜的形成受初始菌浓度、培养时间、温度、pH值、盐含量等多种环境因素影响。希瓦氏菌在初始菌浓度106 CFU/mL以上、37℃、pH 7～8、0.8%NaCl时形成的生物膜量最多;生物膜在12～24 h期间快速形成,并在36 h达到峰值后,随培养时间延长而逐渐下降。经鱼体黏液包被后,生物膜形成量受到显著影响,鱼鳃黏液促进生物膜形成,鱼肠黏液抑制生物膜形成。
Three Shewanella sp. strain and three Pseudomonas sp. strains isolated and purified from iced Pseudosciaena crocea were evaluated in this study for their surface hydrophobicity, auto-aggregation ability and biofilm formation ability together with their correlation with adhesion characteristics to fish mucus(gut, gill and surface). Meanwhile, the characteristics of biofilm formation were investigated under different conditions. The results showed that compared with Pseudomonas sp., Shewanella sp. had stronger surface hydrophobicity and auto-aggregation ability and enhanced ability to adhere to fish mucus and form biofilm. Principal component analysis(PCA) and clustering analysis(CA) showed an inter-genus difference but intra-genus similarity in bacterial adhesion ability. Both the auto-aggregation ability and biofilm formation ability were highly correlated with the adhesion ability to gut and gill mucus, while the surface mucus adhesion ability was correlated exclusively with the biofilm formation ability. In addition, biofilm formation was related to initial concentration, incubation time, temperature, pH, NaCl concentration and other environmental factors. The spoilage bacteria formed biofilm rapidly from 12 to 24 h of culture and biofilm biomass reached the highest peak at 36 h, and then gradually reduced with incubation time. The optimum conditions for biofilm formation by Shewanella were as follows:bacterial suspension greater than 106 CFU/mL, temperature 37 ℃, NaCl concentration 0.8% and pH 7–8. Biofilm formation was significantly influenced by fish mucus. Gill mucus promoted biofilm formation, whereas the opposite effect was true for gut mucus.
Three Shewanella sp. strain and three Pseudomonas sp. strains isolated and purified from iced Pseudosciaena crocea were evaluated in this study for their surface hydrophobicity, auto-aggregation ability and biofilm formation ability together with their correlation with adhesion characteristics to fish mucus(gut, gill and surface). Meanwhile, the characteristics of biofilm formation were investigated under different conditions. The results showed that compared with Pseudomonas sp., Shewanella sp. had stronger surface hydrophobicity and auto-aggregation ability and enhanced ability to adhere to fish mucus and form biofilm. Principal component analysis(PCA) and clustering analysis(CA) showed an inter-genus difference but intra-genus similarity in bacterial adhesion ability. Both the auto-aggregation ability and biofilm formation ability were highly correlated with the adhesion ability to gut and gill mucus, while the surface mucus adhesion ability was correlated exclusively with the biofilm formation ability. In addition, biofilm formation was related to initial concentration, incubation time, temperature, pH, NaCl concentration and other environmental factors. The spoilage bacteria formed biofilm rapidly from 12 to 24 h of culture and biofilm biomass reached the highest peak at 36 h, and then gradually reduced with incubation time. The optimum conditions for biofilm formation by Shewanella were as follows:bacterial suspension greater than 106 CFU/mL, temperature 37 ℃, NaCl concentration 0.8% and pH 7–8. Biofilm formation was significantly influenced by fish mucus. Gill mucus promoted biofilm formation, whereas the opposite effect was true for gut mucus.
引文
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[9]尹清干,程俊茗,刘腾飞,等.环境因子对鳗弧菌生物膜形成的影响[J].微生物学通报,2018,45(1):138-145.DOI:10.13344/j.microbiol.china.170214.
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[12]张雯,倪莉,黄志清,等.冰鲜大黄鱼肠腔细菌类群鉴定及菌群结构分析[J].中国食品学报,2013,13(12):188-196.
[13]GE Y,ZHU J,YE X,et al.Spoilage potential characterization of Shewanella and Pseudomonas isolated from spoiled large yellow croaker(Pseudosciaena crocea)[J].Letters in Applied Microbiology,2017,64(1):86-93.DOI:10.1111/lam.12687.
[14]杨振泉,靳彩娟,张咪,等.高黏附性戊糖片球菌的筛选、标记及其表面疏水与自凝聚性特征[J].食品与生物技术学报,2015,34(9):926-934.DOI:10.3969/j.issn.1673-1689.2015.09.005.
[15]RAHMAN M M,KIM W S,KUMURA H,et al.Autoaggregation and surface hydrophobicity of bifidobacteria[J].World Journal of Microbiology&Biotechnology,2008,24:1593-1598.DOI:10.1007/s11274-007-9650-x.
[16]VINDEROLA C G,MEDICI M,PERDIGON G.Relationship between interaction sites in the gut,hydrophobicity,mucosal immunomodulating capacities and cell wall protein profiles in indigenous and exogenous bacteria[J].Journal of Applied Microbiology,2004,96(2):230-243.
[17]LARSEN M H,LARSEN J L,OLSEN J E.Chemotaxis of Vibrio anguillarum to fish mucus:role of the origin of the fish mucus,the fish species and the serogroup of the pathogen[J].FEMS Microbiology Ecology,2001,38(1):77-80.DOI:10.1111/j.1574-6941.2001.tb00884.x.
[18]DJORDJEVIC D,WIEDMANN M,MCLANDSBOROUGH L A.Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation[J].Applied and Environmental Microbiology,2002,68(5):2950-2958.DOI:10.1128/AEM.68.6.2950-2958.2002.
[19]黄翔峰,方正,黄薇,等.MATH法表征环境微生物细胞表面疏水性的研究进展[J].微生物学通报,2015,15(1):200-206.DOI:10.13344/j.microbiol.china.140397.
[20]CHEN Q,YAN Q,WANG K,et al.Portal of entry for pathogenic Vibrio alginolyticus into large yellow croaker Pseudosciaena crocea,and characteristics of bacterial adhesion to mucus[J].Diseases of Aquatic Organisms,2008,80(3):181-188.DOI:10.3354/dao01933.
[21]LIN S Q,YANG L,CHEN G,et al.Pathogenic features and characteristics of food borne pathogens biofilm:biomass,viability and matrix[J].Microbial Pathogenesis,2017,111:285-291.
[22]任真真.以斑马鱼为模型筛选高黏附性乳酸菌及其免疫调节作用的研究[D].杭州:浙江工商大学,2015:19-29.
[23]STOODLEY P,SAUER K,DAVIES D G,et al.Biofilms as complex differentiated communities[J].Annual Reviews in Microbiology,2002,56(1):187-209.DOI:10.1146/annurev.micro.56.012302.160705.
[24]尹清干,程俊茗,刘腾飞,等.环境因子对鳗弧菌生物膜形成的影响[J].微生物学通报,2018,45(1):138-145.DOI:10.13344/j.microbiol.china.170214.
[25]O’TOOLE G A,KOLTER R.Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development[J].Molecular Microbiology,1998,30(2):295-304.DOI:10.1046/j.1365-2958.1998.01062.x.
[26]SAUER K,CAMPER A K,EHRLICH G D,et al.Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[J].Journal of Bacteriology,2002,184(4):1140-1154.DOI:10.1128/jb.184.4.1140-1154.2002.
[27]LEBLANC L,LEBOEUF C,LEROI F,et al.Comparison between NaCl tolerance response and acclimation to cold temperature in Shewanella putrefaciens[J].Current Microbiology,2003,46(3):157-162.DOI:10.1007/s00284-002-3837-z.
[28]陆文伟,林炳谕,尹一婷,等.双歧杆菌生物膜成膜规律及特性研究[J].中国食品学报,2017,17(12):42-49.DOI:10.16429/j.1009-7848.2017.12.006.
[29]MIZAN M F R,ASHRAFUDOULLA M,SADEKUZZAMAN M,et al.Effects of NaCl,glucose,and their combinations on biofilm formation on black tiger shrimp(Penaeus monodon)surfaces by Vibrio parahaemolyticus[J].Food Control,2018,89:203-209.
[2]ELLIS A E.Innate host defense mechanisms of fish against viruses and bacteria[J].Developmental&Comparative Immunology,2001,25(8):827-839.DOI:10.1016/S0145-305X(01)00038-6.
[3]VAN DEN ABBEELE P,MARZORATI M,DERDE M,et al.Arabinoxylans,inulin and Lactobacillus reuteri 1063 repress the adherent-invasive Escherichia coli from mucus in a mucosacomprising gut model[J].NPJ Biofilms and Microbiomes,2016,2:2055-5008.DOI:10.1038/npjbiofilms.2016.16.
[4]LEE J S,DAMTE D,LEE S J,et al.Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel(Anguilla japonica)for its use in aquaculture[J].Aquaculture Nutrition,2015,21(4):444-456.DOI:10.1111/anu.12176.
[5]DEL RE B,SGORBATI B,MIGLIOLI M,et al.Adhesion,autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum[J].Letters in Applied Microbiology,2000,31(6):438-442.DOI:10.1111/j.1472-765X.2000.00845.x.
[6]许女,史改玲,程宏乾,等.牛源乳酸菌的黏附特性及对奶牛乳房炎大肠杆菌的黏附抑制研究[J].中国食品学报,2017,17(1):20-30.DOI:10.16429/j.1009-7848.2017.01.003.
[7]郭庆昕,苏锦治,卢海景.铜绿假单胞菌黏附力与生物膜形成能力试验研究[J].中华医院感染学杂志,2015,25(22):5069-5071.DOI:10.11816/cn.ni.2015-140764.
[8]龚虹,王海霞,马征途.乳酸菌黏附力与生物膜,疏水性和自凝集特性的研究[J].中国微生态学杂志,2016,28(9):1026-1028.
[9]尹清干,程俊茗,刘腾飞,等.环境因子对鳗弧菌生物膜形成的影响[J].微生物学通报,2018,45(1):138-145.DOI:10.13344/j.microbiol.china.170214.
[10]DALGAARD P.Qualitative and quantitative characterization of spoilage bacteria from packed fish[J].International Journal of Food Microbiology,1995,26(3):319-333.DOI:10.1016/0168-1605(94)00137-U.
[11]GRAM L,DALGAARD P.Fish spoilage bacteria-problems and solution[J].Current Opinion in Biotechnology,2002,13(3):262-266.DOI:10.1016/S0958-1669(02)00309-9.
[12]张雯,倪莉,黄志清,等.冰鲜大黄鱼肠腔细菌类群鉴定及菌群结构分析[J].中国食品学报,2013,13(12):188-196.
[13]GE Y,ZHU J,YE X,et al.Spoilage potential characterization of Shewanella and Pseudomonas isolated from spoiled large yellow croaker(Pseudosciaena crocea)[J].Letters in Applied Microbiology,2017,64(1):86-93.DOI:10.1111/lam.12687.
[14]杨振泉,靳彩娟,张咪,等.高黏附性戊糖片球菌的筛选、标记及其表面疏水与自凝聚性特征[J].食品与生物技术学报,2015,34(9):926-934.DOI:10.3969/j.issn.1673-1689.2015.09.005.
[15]RAHMAN M M,KIM W S,KUMURA H,et al.Autoaggregation and surface hydrophobicity of bifidobacteria[J].World Journal of Microbiology&Biotechnology,2008,24:1593-1598.DOI:10.1007/s11274-007-9650-x.
[16]VINDEROLA C G,MEDICI M,PERDIGON G.Relationship between interaction sites in the gut,hydrophobicity,mucosal immunomodulating capacities and cell wall protein profiles in indigenous and exogenous bacteria[J].Journal of Applied Microbiology,2004,96(2):230-243.
[17]LARSEN M H,LARSEN J L,OLSEN J E.Chemotaxis of Vibrio anguillarum to fish mucus:role of the origin of the fish mucus,the fish species and the serogroup of the pathogen[J].FEMS Microbiology Ecology,2001,38(1):77-80.DOI:10.1111/j.1574-6941.2001.tb00884.x.
[18]DJORDJEVIC D,WIEDMANN M,MCLANDSBOROUGH L A.Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation[J].Applied and Environmental Microbiology,2002,68(5):2950-2958.DOI:10.1128/AEM.68.6.2950-2958.2002.
[19]黄翔峰,方正,黄薇,等.MATH法表征环境微生物细胞表面疏水性的研究进展[J].微生物学通报,2015,15(1):200-206.DOI:10.13344/j.microbiol.china.140397.
[20]CHEN Q,YAN Q,WANG K,et al.Portal of entry for pathogenic Vibrio alginolyticus into large yellow croaker Pseudosciaena crocea,and characteristics of bacterial adhesion to mucus[J].Diseases of Aquatic Organisms,2008,80(3):181-188.DOI:10.3354/dao01933.
[21]LIN S Q,YANG L,CHEN G,et al.Pathogenic features and characteristics of food borne pathogens biofilm:biomass,viability and matrix[J].Microbial Pathogenesis,2017,111:285-291.
[22]任真真.以斑马鱼为模型筛选高黏附性乳酸菌及其免疫调节作用的研究[D].杭州:浙江工商大学,2015:19-29.
[23]STOODLEY P,SAUER K,DAVIES D G,et al.Biofilms as complex differentiated communities[J].Annual Reviews in Microbiology,2002,56(1):187-209.DOI:10.1146/annurev.micro.56.012302.160705.
[24]尹清干,程俊茗,刘腾飞,等.环境因子对鳗弧菌生物膜形成的影响[J].微生物学通报,2018,45(1):138-145.DOI:10.13344/j.microbiol.china.170214.
[25]O’TOOLE G A,KOLTER R.Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development[J].Molecular Microbiology,1998,30(2):295-304.DOI:10.1046/j.1365-2958.1998.01062.x.
[26]SAUER K,CAMPER A K,EHRLICH G D,et al.Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[J].Journal of Bacteriology,2002,184(4):1140-1154.DOI:10.1128/jb.184.4.1140-1154.2002.
[27]LEBLANC L,LEBOEUF C,LEROI F,et al.Comparison between NaCl tolerance response and acclimation to cold temperature in Shewanella putrefaciens[J].Current Microbiology,2003,46(3):157-162.DOI:10.1007/s00284-002-3837-z.
[28]陆文伟,林炳谕,尹一婷,等.双歧杆菌生物膜成膜规律及特性研究[J].中国食品学报,2017,17(12):42-49.DOI:10.16429/j.1009-7848.2017.12.006.
[29]MIZAN M F R,ASHRAFUDOULLA M,SADEKUZZAMAN M,et al.Effects of NaCl,glucose,and their combinations on biofilm formation on black tiger shrimp(Penaeus monodon)surfaces by Vibrio parahaemolyticus[J].Food Control,2018,89:203-209.