创伤弧菌生物被膜形成能力及影响因素的研究

高梦迪; 宁喜斌

doi:10.13386/j.issn1002-0306.2022080256

创伤弧菌生物被膜形成能力及影响因素的研究

doi: 10.13386/j.issn1002-0306.2022080256

高梦迪^1,,
宁喜斌^{1, 2, 3,*, ,}

1.
上海海洋大学食品学院，上海 201306
2.
农业部水产品贮藏保鲜质量安全风险评估试验室（上海），上海 201306
3.
国家淡水水产品加工技术研发分中心（上海），上海 201306

详细信息

作者简介:
高梦迪（1996−），女，硕士研究生，研究方向：食品微生物，E-mail：1404843089@qq.com

通讯作者:
宁喜斌（1964−），男，博士，教授，研究方向：微生物学、食品安全，E-mail：xbning@shou.edu.cn

中图分类号: TS201.3
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- 文章访问数: 18
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- 被引次数: 0
出版历程
- 收稿日期: 2022-08-24
- 刊出日期: 2023-05-15

Biofilm Forming Ability and Influencing Factors of Vibrio vulnificus

GAO Mengdi^1
,,
NING Xibin^{1, 2, 3,*
, ,}

1.
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2.
Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, China
3.
National R&D Branch Center for Freshwater Aquatic Products Processing Technology (Shanghai), Shanghai 201306, China

摘要

摘要: 研究26株创伤弧菌（Vibrio vulnificus，Vv）产生物被膜情况及影响生物被膜形成的因素，为有效控制创伤弧菌形成生物被膜提供理论依据。本研究采用刚果红平板法、改良试管法及改良微孔板法分析25株创伤弧菌分离株及1株标准菌株形成生物被膜的能力，从中选出一株产膜能力最强的菌株，并研究不同初始菌浓度、温度及时间、pH、NaCl浓度、金属阳离子以及接触材料对其生物被膜形成的影响。结果显示，所选菌株中具有生物被膜形成能力的有25株（96.15%）。其中菌株VvK产膜能力最强，在25 ℃条件下，初始菌浓度为10⁸ CFU/mL，含3% NaCl、pH8~9培养24 h时，生物被膜形成量最大。而添加一定浓度的金属阳离子（Cu²⁺、Mn²⁺、Ca²⁺、Mg²⁺）后，生物被膜的形成受到不同程度的抑制，其抑制能力依次降低。菌株VvK在接触亲水性表面（不锈钢和玻璃）时生物被膜形成量显著高于疏水性表面（聚苯乙烯），且在不锈钢表面形成量最大。不同创伤弧菌生物被膜形成能力具有较大的差异，且在不同培养条件下具有特定的规律，需引起重点关注。
- 创伤弧菌 /
- 生物被膜 /
- 培养条件 /
- 影响因素 /
- 产膜能力
Abstract: The biofilm produced by 26 strains of Vibrio vulnificus (Vv) and the factors affecting biofilm formation were studied to provide theoretical basis for effective control of biofilm formation by Vv. In this study, Congo red plate method, improved test tube method and improved microplate method were used to analyze the ability of 25 isolated Vv strains and 1 standard strain to form biofilm, and one strain with the strongest ability to produce biofilm was selected from them. The effects of different initial bacterial concentration, temperature, culture time, pH, NaCl concentration, metal cations and contact materials on the formation of biofilm were investigated. The results showed that 25 strains (96.15%) had biofilm forming ability among the selected strains and VvK had the strongest ability to produce biofilm. At 25 ℃, the initial bacterial concentration was 10⁸ CFU/mL, 3% NaCl and pH8~9 for 24 h, the biofilm quantity was the largest. However, the formation of biofilm was inhibited by certain concentration of metal cations (Cu²⁺, Mn²⁺, Ca²⁺, Mg²⁺) definitely, and the inhibition ability of above cations decreases in turn. When VvK contacted hydrophilic surfaces (stainless steel and glass), the amount of biofilm formation was significantly higher than hydrophobic surfaces (polystyrene), and the amount of biofilm formation on stainless steel was the largest. The biofilm forming ability of different Vv was quite different, and had specific rules under different cultivation conditions, which should be paid more attention.
- Vibrio vulnificus /
- biofilm /
- culture conditions /
- influence factor /
- film production capacity

HTML全文

图 1 刚果红平板鉴定生物被膜

注：左：PIA阳性菌株VvK；右：PIA阴性菌株Vv Bq。

Figure 1. Congo red plate for biofilm identification

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图 2 改良试管法鉴定生物被膜

注：第3排第1管为对照管，其余为试验管。

Figure 2. Biofilm identification by modified tube method

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图 3 初始菌浓度对VvK生物被膜的影响

Figure 3. Effect of initial concentration on VvK biofilm

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图 4 NaCl浓度对VvK生物被膜的影响

Figure 4. Effect of NaCl concentration on VvK biofilm

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图 5 pH对VvK生物被膜的影响

注：不同小写字母表示差异显著（P<0.05），相同小写字母表示差异不显著（P>0.05）。

Figure 5. Effect of pH on VvK biofilm

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图 6 温度和时间对VvK生物被膜的影响

Figure 6. Effects of temperature and time on VvK biofilm

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图 7 金属阳离子对VvK生物被膜的影响

Figure 7. Effect of metal cations on VvK biofilm

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图 8 不同接触材料对VvK生物被膜的影响

Figure 8. Effects of different contact materials on VvK biofilm

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表 1 创伤弧菌生物被膜测定结果

Table 1. Results of Vibrio vulnificus biofilm determination

生物被膜粘附程度	测定值范围（OD_570nm）	菌株数目（占比%）
+++	0.477±0.008～1.877±0.035	9（34.6%）
++	0.322±0.032～0.466±0.018	13（50%）
+	0.214±0.007～0.230±0.020	3（11.6%）
−	0.088±0.009	1（3.8%）
	0.088±0.009	空白对照
注：+++：强粘附，++：中等粘附，+：弱粘附，−：无粘附。

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表 2 强生物被膜形成能力菌株

Table 2. Strong ability of biofilm formation strains

菌株名称	OD_570nm值（平均值±标准差）
VvK	1.877±0.035^a
Vv 18s	0.658±0.022^b
Vv 61	0.656±0.026^b
Vv Ds	0.572±0.005^c
Vv Cs	0.519±0.038^d
Vv Js	0.518±0.030^d
Vv 52	0.513±0.019^d
Vv Y5	0.503±0.023^d
Vv CICC21615	0.477±0.009^d
注：不同小写字母表示差异显著（P<0.05），相同小写字母表示差异不显著（P>0.05）。

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参考文献(33)

[1]	王明义, 胡成进. 创伤弧菌致病性及其毒力因子研究进展[J]. 中国微生态学杂志,2017,29(12):1470−1473. [WANG M Y, HU C J. Research progress on the pathogenicity and virulence factors of Vibrio vulnificus[J]. Chinese Journal of Micro Ecology,2017,29(12):1470−1473. doi: 10.13381/j.cnki.cjm.201712026
[2]	黄春兰, 余洪立, 马丽梅. 创伤弧菌感染1例[J]. 中国感染与化疗杂志,2022,22(1):95−97. [HUANG C L, YU H L, MA L M. One case of Vibrio vulnificus infection[J]. Chinese Journal of Infection and Chemotherapy,2022,22(1):95−97.
[3]	ELBASHIR S, PARVEEN S, SCHWARZ J, et al. Seafood pathogens and information on antimicrobial resistance: A review[J]. Food Microbiology, 2018, 70: 85-93
[4]	唐旭华, 郭鹏豪, 周晖, 等. 创伤弧菌感染引起的坏死性筋膜炎2例[J]. 皮肤性病诊疗学杂志,2020,27(4):274−277. [TANG X H, GUO P H, ZHOU H, et al. Necrotizing fasciitis caused by Vibrio vulnificus infection in 2 cases[J]. Journal of Dermatology and Venereology,2020,27(4):274−277. doi: 10.3969/j.issn.1674-8468.2020.04.015
[5]	JONES M K, OLIVER J D. Vibrio vulnificus: disease and pathogenesis[J]. Infection & Immunity,2009,77(5):1723−1733.
[6]	LI J H, ZHAO X H. Effects of quorum sensing on the biofilm formation and viable but non-culturable state[J]. Food Research International,2020,137(7):109742.
[7]	MUHSIN J, WISAL A, SAADIA A, et al. Bacterial biofilm and associated infections[J]. Journal of the Chinese Medical Association,2018,81(1):7−11. doi: 10.1016/j.jcma.2017.07.012
[8]	FLEMMING H C, WINGENDER J, SZEWZYK U, et al. Biofilms: an emergent form of bacterial life[J]. Nature Reviews Microbiology,2016,14(9):563−575. doi: 10.1038/nrmicro.2016.94
[9]	JOO H S, OTTO M. Molecular basis of in vivo biofilm formation by bacterial pathogens[J]. Chemistry & Biology,2012,19(12):1503−1513.
[10]	SAXENA P. Biofilms: Architecture, resistance, quorum sensing and control mechanisms[J]. Indian Journal of Microbiology,2019,59(1):3−12. doi: 10.1007/s12088-018-0757-6
[11]	COSTERTON J W, LEWANDOWSKI Z, CALDWELL D E, et al. Microbial biofilms.[J]. Annual Review of Microbiology,1995,49(1):711−745. doi: 10.1146/annurev.mi.49.100195.003431
[12]	蔡瑞昭, 祁少海. 创伤弧菌生物特性及临床研究进展[J]. 中华损伤与修复杂志(电子版),2020,15(6):490−494. [CAI R Z, QI S H. Biological characteristics and clinical research progress of Vibrio vulnificus[J]. Chinese Journal of Injury and Repair (electronic version),2020,15(6):490−494.
[13]	吴谦. 副溶血弧菌生物菌膜形成特性及亚甲基蓝光动力灭活作用研究[D]. 广州: 暨南大学 WU Q. Study on the characteristics of biofilm formation of Vibrio parahaemolyticus and photodynamic inactivation of methylene blue[D]. Guangzhou: Jinan University.
[14]	THAÍS DIAS LEMOS KAISER A, B E M P, KÁTIA REGINA NETTO DOS SANTOS C, et al. Modification of the Congo red agar method to detect biofilm production by Staphylococcus epidermidis[J]. Diagnostic Microbiology and Infectious Disease,2013,75(3):235−239. doi: 10.1016/j.diagmicrobio.2012.11.014
[15]	KUNWAR A, SHRESTHA P, SHRESTHA S, et al. Detection of biofilm formation among Pseudomonas aeruginosa isolated from burn patients[J]. Burns Open,2021,5(2):125−129.
[16]	STEPANOVIC S. A modified microtiter-plate test for quantification of staphylococcal biofilm formation[J]. J Microbiol Methods,2000:40.
[17]	CHRISTENSEN G D, SIMPSON W A, J J YOUNGER, et al. Adherence of coagulase-negative Staphylococci to plastic tissue culture plates: A quantitative model for the adherence of Staphylococci to medical devices[J]. Journal of Clinical Microbiology,1986,22(6):996−1006.
[18]	贾玲华, 宁喜斌, 张徐晶. 溶藻弧菌生物被膜形成能力及特性分析[J]. 食品工业科技,2015,36(20):201−205. [JIA L H, NING X B, ZHANG X J. Analysis of biofilm formation ability and characteristics of Vibrio alginolyticus[J]. Science and Technology of Food Industry,2015,36(20):201−205.
[19]	FEY P D, OLSON M E. Current concepts in biofilm formation of Staphylococcus epidermidis[J]. Future Microbiology,2010,5(6):917−933. doi: 10.2217/fmb.10.56
[20]	张晓婷, 邓一秒, 董冬丽, 等. 培养条件对奇异变形杆菌生物膜生长的影响[J]. 食品工业科技,2020,41(12):6. [ZHANG X T, DENG Y M, DONG D L, et al. Effect of culture conditions on the biofilm of Proteus mirabilis[J]. Science and Technology of Food Industry,2020,41(12):6.
[21]	沈津宇, 倪莹, 高璐. 盐度对副溶血性弧菌生物特性的影响[J]. 现代食品,2018(6):91−94. [SHEN J Y, NI Y, GAO L. Effect of salinity on biological characteristics of Vibrio parahaemolyticus[J]. Modern Food,2018(6):91−94.
[22]	刘文竹, 李红月, 范学亭, 等. 环境因素对溶藻弧菌HN08155生物膜形成的影响[J]. 海南大学学报(自然科学版),2015,33(4):365−371. [LIU W Z, LI H Y, FAN X T, et al. Effect of environmental factors on biofilm formation of Vibrio alginolyticus HN08155[J]. Journal of Hainan University (Natural Science Edition),2015,33(4):365−371.
[23]	YIN W, WANG Y, LIU L, et al. Biofilms: The microbial ''Protective Clothin'' in extreme environments[J]. International Journal of Molecular Sciences,2019,20(14):3423. doi: 10.3390/ijms20143423
[24]	BLANCO Y, ALFONSO RIVAS L, GONZALEZ-TORIL E, et al. Environmental parameters, and not phylogeny, determine the composition of extracellular polymeric substances in microbial mats from extreme environments[J]. Science of The Total Environment, 2018, 650 （ PT.1 （ 1-834 ）） : 384−393.
[25]	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):8. [YIN Q G, CHENG J M, LIU T F, et al. Effects of environmental factors on biofilm formation in Vibrio anguillus[J]. Bulletin of Microbiology,2018,45(1):8.
[26]	ZOU M, LIU D. Effects of carbon sources and temperature on the formation and structural characteristics of food-related Staphylococcus epidermidis biofilms[J]. Food Science and Human Wellness,2020,9(4):370−376. doi: 10.1016/j.fshw.2020.05.007
[27]	ÇAM S, BRINKMEYER R. The effects of temperature, pH, and iron on biofilm formation by clinical versus environmental strains of Vibrio vulnificus[J]. Folia Microbiol,2020,65(3):557−566. doi: 10.1007/s12223-019-00761-9
[28]	ZHANG B Z. Bacterial adhesion and biofilms on surfaces[J]. Progress in Natural Science,2008(9):1049−1056.
[29]	TANG D, GAO Q, ZHAO Y, et al. Mg²⁺ reduces biofilm quantity in Acidithiobacillus ferrooxidans through inhibiting type IV pili formation[J]. FEMS Microbiology Letters,2018,365(4):fnx266.
[30]	JESSE G, WILLIAM Y H, CYNTHIA G, et al. Critical shear stresses of Pseudomonas aeruginosa biofilms from dental unit waterlines studied using microfluidics and additional magnesium ions[J]. Physics of Fluids,2022,34(2):021902. doi: 10.1063/5.0076737
[31]	熊富忠, 赵小希, 廖胤皓, 等. 材料表面特征对生物膜形成的影响及其应用[J]. 微生物学通报,2018,45(1):155−165. [XIONG F Z, ZHAO X X, LIAO Y H, et al. Effect of material surface characteristics on biofilm formation and its application[J]. Bulletin of Microbiology,2018,45(1):155−165.
[32]	WANG H H, CAI L L, LI Y H, et al. Biofilm formation by meat-borne Pseudomonas fluorescens on stainless steel and its resistance to disinfectants[J]. Food Control,2018,91(9):397−403.
[33]	李燕杰, 苏新国, 姚玉静, 等. 不同类型表面材料对单增李斯特菌生物被膜形成的影响[J]. 郑州轻工业学院学报(自然科学版),2015,30(Z2):33−37. [LI Y J, SU X G, YAO Y J, et al. Effect of different surface materials on biofilm formation of Listeria monocytogenes[J]. Journal of Zhengzhou Institute of Light Industry (Natural Science Edition),2015,30(Z2):33−37.