阪崎肠杆菌生物膜形成条件及超声波对其去除作用
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摘要
通过96/6孔板培养法进行生物膜体外模型建立,比浊法测定生物膜含量,结晶紫染色法观察生物膜形态,平板菌落计数法评价超声波对玻璃表面生物膜的去除作用效果,激光共聚焦显微镜观察超声波处理后生物膜脱落效果,研究阪崎肠杆菌生物膜的形成条件和超声波处理对其去除作用效果。结果表明,阪崎肠杆菌生物膜形成的最适温度37℃,最适pH 7.5,最适盐度为2.5%。CaCl_2(0.50%)、MgCl_2(1.50%)对阪崎肠杆菌生物膜形成具有明显促进作用。Cu~(2+)(1.50%)、EDTA(2.5%)对阪崎肠杆菌生物膜形成具有抑制作用。超声波处理能够使玻璃表面上附着的阪崎肠杆菌生物膜脱落,超声波处理初始温度30℃,时间14 min条件下去除效果最好。激光共聚焦显微镜可直接观察到超声波处理后盖玻片上生物膜中阪崎肠杆菌活菌数量变化,可用于证实和评价超声处理对玻璃表面阪崎肠杆菌生物膜的去除效果。
The formation condition of Cronobacter sakazakii biofilm and its elimination by ultrasound treatment on glass surfaces were investigated through biofilm establishment in vitro model by 96/6 orifice culture method and turbidimetric determination of biofilm content. The morphology of biofilm was observed by crystalline violet staining. The ultrasonic shedding was evaluated by plate colony counting method and the elimination effect of ultrasonic treatment was observed by laser scanning confocal microscopy. The optimal temperature and pH for Cronobacter sakazakii formation was 37 ℃ and 7. 5. NaCl( 2. 5%),CaCl_2( 0. 50%) and MgCl_2( 1. 50%) added in medium promoted biofilm formation obviously,Cu~(2+)( 1. 50%) and EDTA( 2. 5%) added in medium inhibited bioflm formation. Cronobacter sakazakii biofilm attached to the glass surface was eliminated by ultrasonic treatment. The elimination effect was the best when initial temperature was 30 ℃ and time was 14 min. The change of living cells number on biofilm after ultrasound treatment was observed by laser scanning confocal microscopy to prove and evaluate the elimination effect of ultrasonic treatment.
The formation condition of Cronobacter sakazakii biofilm and its elimination by ultrasound treatment on glass surfaces were investigated through biofilm establishment in vitro model by 96/6 orifice culture method and turbidimetric determination of biofilm content. The morphology of biofilm was observed by crystalline violet staining. The ultrasonic shedding was evaluated by plate colony counting method and the elimination effect of ultrasonic treatment was observed by laser scanning confocal microscopy. The optimal temperature and pH for Cronobacter sakazakii formation was 37 ℃ and 7. 5. NaCl( 2. 5%),CaCl_2( 0. 50%) and MgCl_2( 1. 50%) added in medium promoted biofilm formation obviously,Cu~(2+)( 1. 50%) and EDTA( 2. 5%) added in medium inhibited bioflm formation. Cronobacter sakazakii biofilm attached to the glass surface was eliminated by ultrasonic treatment. The elimination effect was the best when initial temperature was 30 ℃ and time was 14 min. The change of living cells number on biofilm after ultrasound treatment was observed by laser scanning confocal microscopy to prove and evaluate the elimination effect of ultrasonic treatment.
引文
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[19]LEE Y D,PARK J H,CHANG H I.Detection,antibiotic susceptibility and biofilm formation of Cronobacter spp.From various foods in Korea[J].Food Control,2012,24(1/2):225-230.
[20]魏奇,龚小丽,林佳扬,等.阪崎肠杆菌的生物学特性及其在婴幼儿配方奶粉中的预测模型[J].中国食品学报,2017,17(4):58-64.
[21]CHEN X,STEWART P S.Role of electrostatic interactions in cohesion of bacterial biofilms[J].Applied Microbiology and Biotechnology,2002,59(6):718-720.
[22]袁海兰,苏建,胡鲲,等.环境因子对水霉菌生物被膜形成的影响[J].微生物学通报,2014,41(9):1 829-1 836.
[23]SONG B.LEFF L G.Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens[J].Microbiological Research,2006,161(4):355-361.
[24]李姝.Cu2+对生物膜及其胞外多聚物的作用规律研究[D].昆明:昆明理工大学,2014.
[25]ROBERTSON E J,WOLF J M,Casadevall A.EDTA inhibits biofilm formation,extracellular vesicular secretion,and shedding of the capsular polysaccharide glucuronoxylomannan by Cryptococcus neoformans[J].Applied&Environmental Microbiology,2012,78(22):7 977-7 984.
[2]LECLERCQ A,WANEGUE C,BAYLAC P.Comparison of fecal coliform agar and violet red bile lactose agar for fecal coli form enumeration in foods[J].Applied&Environmental Microbiology,2002,68(4):1 631-1 638.
[3]HIMELRIGHT I,HARRIS E,Lorch V,et al.Enterobacter sakazakii infections associated with the use of powdered infant formula-Tennessee,2001[J].Morbidity&Mortality Weekly Report,2002,51(14):297-300.
[4]TERAMOTO S,TANABE Y,OKANO E,et al.A first fatal neonatal case of Enterobacter sakazakii infection in Japan[J].Pediatrics International,2010,52(2):312-313.
[5]VALERIA C,ASATO,VIVIANA E,et al.First clinical isolates of Cronobacter spp.(Enterobacter sakazakii)in Argentina:Characterization and subtyping by pulsed-field gel electrophoresis[J].Revista Argentina De Microbiologia,2013,45(3):160-164.
[6]Jackson E E,Flores J P,Fernandez-Escartin E,Forsythe SJ.Reevaluation of a suspected Cronobacter sakazakii outbreak in Mexico[J].Journal of Food Protection,2015,78(6):1 191-1 196.
[7]NAZAROWEC-WHITE M,FARBER J M.Enterobacter sakazakii:a review[J].International Journal of Food Microbiology,1997,34(2):103-113.
[8]张欣强,庞杏林,刘俊华,等.广州市售国产婴幼儿配方粉中阪崎肠杆菌污染调查[J].中国卫生检验杂志,2010,1(1):183-185.
[9]权玉玲,胡晓宁,兰光,等.甘肃省地产婴幼儿配方奶粉阪崎肠杆菌污染调查分析[J].中国卫生检验杂志,2013,23(4):990-993.
[10]袁丹茅,金建潮,李筱玲.龙岩市市售婴幼儿配方奶粉中阪崎肠杆菌污染调查[J].基层医学论坛,2016,20(2):233.
[11]RENNER L D,WEIBEL D B.Physicochemical regulation of biofilm formation[J].MRS Bulletin,2011,36(5):347-355.
[12]CARPENTIER B,CERF O.Biofilms and their consequences with particular reference to hygiene in food industry[J].Journal of Applied Bacteriology,2003,75(6):499-511.
[13]ISLAM M A,WOON C W,ETHIRAJ B,et al.Ultrasound driven biofilm removal for stable power generation in microbial fuel cell[J].Energy&Fuels,2017,31(1):968-976.
[14]DROR N,MANDEL M,HAZAN Z,et al.Advances in microbial biofilm prevention on indewelling medical devices with emphasis on usage of acoustic energy[J].Sensors,2009,9(4):2 538-2 554.
[15]BAUMANN A R,MARTIN S E,HAO F.Removal of Listeria monocytogenes biofilms from stainless steel by use of ultrasound and ozone[J].Journal of Food Protection,2009,72(6):1 306-1 309.
[16]TORLAK E,SERT D.Combined effect of benzalkonium chloride and ultrasound against Listeria monocytogenes biofilm on plastic surface[J].Letters in Applied Microbiology,2013,57(3):220-226.
[17]周汉洪,田巍威,李红.2010~2013年达州市婴幼儿食品阪崎肠杆菌监测结果分析[J].现代预防医学,2015,42(19):3 501-3 502.
[18]WONG H C,CHUANG Y C,YU J A.Attachment and inactivation of vibrio parahaemolyticus on stainless steel and glass surface[J].Food Microbiology,2002,19(4):341-350.
[19]LEE Y D,PARK J H,CHANG H I.Detection,antibiotic susceptibility and biofilm formation of Cronobacter spp.From various foods in Korea[J].Food Control,2012,24(1/2):225-230.
[20]魏奇,龚小丽,林佳扬,等.阪崎肠杆菌的生物学特性及其在婴幼儿配方奶粉中的预测模型[J].中国食品学报,2017,17(4):58-64.
[21]CHEN X,STEWART P S.Role of electrostatic interactions in cohesion of bacterial biofilms[J].Applied Microbiology and Biotechnology,2002,59(6):718-720.
[22]袁海兰,苏建,胡鲲,等.环境因子对水霉菌生物被膜形成的影响[J].微生物学通报,2014,41(9):1 829-1 836.
[23]SONG B.LEFF L G.Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens[J].Microbiological Research,2006,161(4):355-361.
[24]李姝.Cu2+对生物膜及其胞外多聚物的作用规律研究[D].昆明:昆明理工大学,2014.
[25]ROBERTSON E J,WOLF J M,Casadevall A.EDTA inhibits biofilm formation,extracellular vesicular secretion,and shedding of the capsular polysaccharide glucuronoxylomannan by Cryptococcus neoformans[J].Applied&Environmental Microbiology,2012,78(22):7 977-7 984.