组合处理对双菌种生物被膜的清除和杀灭效果
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摘要
该研究建立了食源性病原菌(液化沙雷菌S1和腐生葡萄球菌S2)双菌种生物被膜研究模型,采用微孔板法和MTT法检测单一、组合处理方式对双菌种生物被膜的清除和杀灭效果。研究发现,采用24孔板37℃于TSB培养基中培养24 h可获得稳定的液化沙雷菌-腐生葡萄球菌双菌种生物被膜,组合处理方式优于单一处理效果,处理顺序的不同也会影响生物被膜的清除和杀灭效果,1%Na OH处理20 min再用0.3%Na Cl O处理20 min清除效果最好,双菌种生物被膜的量由1.11(OD595 nm)减少至0.08(OD595 nm),杀灭率达95.73%。
The study mainly focused on the establishment of a dual-species biofilm model formed by two foodborne pathogenic bacteria Serratia liquefaciens S1 and Staphylococcus saprophyticus S2. Microtitor-plate method and MTT assay were used to measure the removal effect and killing efficacy of single or combination treatments on dual-species biofilm formation. The results showed that the stable dual-species biofilms, consisting of S. liquefaciens S1 and S. saprophyticus S2, could be obtained in tryptone soy broth(TSB) at 37 ℃ for 24 h. The effects of combined treatments were better than single treatments. Moreover, in combined treatments, the removal effect and killing efficacy were also influenced by treatment orders.Results indicated that when the dual-species biofilms were treated with 1% Na OH for 20 min firstly and then disposed with 0.3% Na Cl O for another20 min, the removal effects of biofilms were better than that under other conditions, while the dual-species biofilms quantity decreased from 1.11(OD595 nm)to 0.08(OD595 nm) and the killing efficacy reached to 95.73%.
The study mainly focused on the establishment of a dual-species biofilm model formed by two foodborne pathogenic bacteria Serratia liquefaciens S1 and Staphylococcus saprophyticus S2. Microtitor-plate method and MTT assay were used to measure the removal effect and killing efficacy of single or combination treatments on dual-species biofilm formation. The results showed that the stable dual-species biofilms, consisting of S. liquefaciens S1 and S. saprophyticus S2, could be obtained in tryptone soy broth(TSB) at 37 ℃ for 24 h. The effects of combined treatments were better than single treatments. Moreover, in combined treatments, the removal effect and killing efficacy were also influenced by treatment orders.Results indicated that when the dual-species biofilms were treated with 1% Na OH for 20 min firstly and then disposed with 0.3% Na Cl O for another20 min, the removal effects of biofilms were better than that under other conditions, while the dual-species biofilms quantity decreased from 1.11(OD595 nm)to 0.08(OD595 nm) and the killing efficacy reached to 95.73%.
引文
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[2]FLEMMING H C,WINGENDER J.The biofilm matrix[J].Nat Rev Microbiol,2010,8(9):623-633.
[3]BROOKS J D,FLINT S H.Biofilms in the food industry:problems and potential solutions[J].Int J Food Sci Technol,2008,43(12):2163-2176.
[4]COSTERTON J W,LEWANDOWSKI Z,DEBEER D,et al.Biofilms,the customized microniche[J].J Bacteriol,1994,176(8):2137-2142.
[5]YANG L A,LIU Y,WU H,et al.Current understanding of multi-species biofilms[J].Int J Oral Sci,2011,3(2):74-81.
[6]HABIMANA O,MORETRO T,LANGSRUD S,et al.Micro ecosystems from feed industry surfaces:a survival and biofilm study of Salmonella versus host resident flora strains[J].Bmc Vet Res,2010,6:48-58.
[7]GIAOURIS E,CHORIANOPOULOS N,DOULGERAKI A,et al.Co-culture with Listeria monocytogenes within a dual-species biofilm community strongly increases resistance of Pseudomonas putida to benzalkonium chloride[J].Plos One,2013,8(10):e77276.
[8]LIU N T,NOU X W,LEFCOURT A M,et al.Dual-species biofilm formation by Escherichia coli O157:H7 and environmental bacteria isolated from fresh-cut processing facilities[J].Int J Food Microbiol,2014,171:15-20.
[9]BEHNKE S,PARKER A E,WOODALL D,et al.Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single-and dual-species cultures[J].Appl Environ Microbiol,2011,77(20):7176-7184.
[10]BRIDIER A,SANCHEZ-VIZUETE P,GUILBAUD M,et al.Biofilmassociated persistence of food-borne pathogens[J].Food Microbiol,2015,45(Pt B):167-178.
[11]GIAOURIS E,HEIR E,HEBRAUD M,et al.Attachment and biofilm formation by foodborne bacteria in meat processing environments:Causes,implications,role of bacterial interactions and control by alternative novel methods[J].Meat Sci,2014,97(3):298-309.
[12]KOSTAKI M,CHORIANOPOULOS N,BRAXOU E,et al.Differential biofilm formation and chemical disinfection resistance of sessile cells of Listeria monocytogenes strains under monospecies and dual-species(with Salmonella enterica)conditions[J].Appl Environ Microbiol,2012,78(8):2586-2595.
[13]SREY S,JAHID I K,HA S D.Biofilm formation in food industries:A food safety concern[J].Food Control,2013,31(2):572-585.
[14]孙纪录,陈小雪,韩北忠.金黄色葡萄球菌生物被膜的控制方法研究进展[J].中国酿造,2011,30(6):1-3.
[15]FERNANDES M D,KABUKI D Y,KUAYE A Y.Biofilms of Enterococcus faecalis and Enterococcus faecium isolated from the processing of ricotta and the control of these pathogens through cleaning and sanitization procedures[J].Int J Food Microbiol,2015,200:97-103.
[16]MATHIEU L,BERTRAND I,ABE Y,et al.Drinking water biofilm cohesiveness changes under chlorination or hydrodynamic stress[J].Water Res,2014,55:175-184.
[17]HA J H,HA S D.Synergistic effects of sodium hypochlorite and ultraviolet radiation in reducing the levels of selected foodborne pathogenic bacteria[J].Foodborne Pathogen Dis,2011,8(5):587-591.
[18]DI BONAVENTURA G,PICCOLOMINI R,PALUDI D,et al.Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces:relationship with motility and cell surface hydrophobicity[J].J Appl Microbiol,2008,104(6):1552-1561.
[19]方蓉,李芳秋,武建国.MTT比色法的条件探讨[J].临床检验杂志,2003(1):34-35.
[20]边兴艳.MTT比色法及其应用[J].国外医学:临床生物化学与检验学分册,1998(2):83-85.
[21]MORIMATSU K,HAMANAKA D,TANAKA F,et al.Effect of temperature fluctuation on biofilm formation with bacterial interaction between Salmonella enterica and Pseudomonas putida[J].J Fac Agr Kyushu U,2013,58(1):125-129.