乳酸钠对单增李斯特菌生物被膜形成的抑制作用
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摘要
为初步研究乳酸钠对单增李斯特菌生物被膜形成的抑制作用,作者首先采用结晶紫染色法检测不同质量浓度乳酸钠(0、2.5、5、10、20 g/dL)对单增李斯特菌生物被膜形成的抑制效果,其次探究乳酸钠对其生物被膜结构、胞外多糖和胞外蛋白、膜内细菌细胞活性及细胞膜完整性的影响,并采用荧光实时定量PCR(quantitative real-time PCR,qRT-PCR)检测其生物被膜相关基因motB、mogR、degU、flgE、dnaK、prfA及sigB的表达水平。结果表明,随着乳酸钠浓度的增高(2.5%～20%),其对单增李斯特菌生物被膜形成的抑制效果显著增强(p<0.05),抑制率分别为8.34%、32.2%、46.6%、55.2%。显微镜观察结果显示,经5 g/dL乳酸钠处理后,单增李斯特菌生物被膜结构明显变稀疏,厚度减少;同时胞外多糖和胞外蛋白质的形成量显著降低(p<0.05)。此外,该质量浓度下的乳酸钠可抑制膜内细菌细胞活性及破坏细胞膜的完整性,从而抑制新生物被膜的形成;实时定量PCR结果进一步显示,与单增李斯特菌生物被膜形成相关基因的表达显著下调(p<0.05)。本研究为乳酸钠可有效抑制单增李斯特菌生物被膜的形成提供科学依据。
The objective of this study was to investigate the inhibition effet of sodium lactate(NaL)on biofilm formation by Listeria monocytogenes. First of all,the crystal violet staining assay was used to evaluate the inhibition effect of Na L at different concentrations(0%,2.5%,5%,10% and 20%) on the biofilm formation of L. monocytogenes. Furthermore,the effets of NaL on its biofilm structure,polysaccharides,extracelluar proteins,cell activity and membrane integrity of L. monocytogenes biofilm cells were also investigated. Finally,the detection of the expression level of the biofilm-associated genes motB,mogR,degU,flg E,dnaK,prfA and sigB was performed by using quantitative real-time PCR(qRT-PCR). With the increase of the NaL concentration(2.5～20%),biofilm formation of L. monocytogenes was inhibited significantly(p<0.05) and the inhibition rate was 8.34 %,32.2 %,46.6 % and 55.2 %,respectively. Results of microscopy analysis showed that after 5 % Na L treatment,the structure of L. monocytogenes biofilm was forming instead of small clumps with reduction of thickness,and a significant decrease(p< 0.05) was determined in total production of polysaccharides and extracelluar proteins of L. monocytogenes biofilm cells. In addition,5 % Na L inhibited the cell activity and membrane integrity of L. monocytogenes biofilm cells to suppress the development of new biofilms. qRT-PCR results revealed that NaL could downregulate biofilm-associated genes in L. monocytogenes and therefore to inhibit its biofilm formation. In conclusion,this study could provide a scientific basis for the effective inhibition of NaL on L. monocytogenes biofilm formation.
The objective of this study was to investigate the inhibition effet of sodium lactate(NaL)on biofilm formation by Listeria monocytogenes. First of all,the crystal violet staining assay was used to evaluate the inhibition effect of Na L at different concentrations(0%,2.5%,5%,10% and 20%) on the biofilm formation of L. monocytogenes. Furthermore,the effets of NaL on its biofilm structure,polysaccharides,extracelluar proteins,cell activity and membrane integrity of L. monocytogenes biofilm cells were also investigated. Finally,the detection of the expression level of the biofilm-associated genes motB,mogR,degU,flg E,dnaK,prfA and sigB was performed by using quantitative real-time PCR(qRT-PCR). With the increase of the NaL concentration(2.5～20%),biofilm formation of L. monocytogenes was inhibited significantly(p<0.05) and the inhibition rate was 8.34 %,32.2 %,46.6 % and 55.2 %,respectively. Results of microscopy analysis showed that after 5 % Na L treatment,the structure of L. monocytogenes biofilm was forming instead of small clumps with reduction of thickness,and a significant decrease(p< 0.05) was determined in total production of polysaccharides and extracelluar proteins of L. monocytogenes biofilm cells. In addition,5 % Na L inhibited the cell activity and membrane integrity of L. monocytogenes biofilm cells to suppress the development of new biofilms. qRT-PCR results revealed that NaL could downregulate biofilm-associated genes in L. monocytogenes and therefore to inhibit its biofilm formation. In conclusion,this study could provide a scientific basis for the effective inhibition of NaL on L. monocytogenes biofilm formation.
引文
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[15]LUNDEN J,AUTIO T,KORKEALA H.Transfer of persistent Listeria monocytogenes contamination between food-processing plants associated with a dicing machine[J].Journal of Food Protection,2002,65(7):1129-1133.
[16]JONES W L,SUTTON M P,MCKITTRICK L,et al.Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms[J].Biofouling,2011,27(2):207-215.
[17]RICHARD J,DUBER M M,BARBARA N,et al.Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation[J].Scientific Reports,2015,5:9856.
[18]SUTHERLAND I W.The biofilm matrix-an immobilized but dynamic microbial environment[J].Trends in Microbiology,2001,9(5):222-227.
[19]BRANDA S S,CHU F,KEARNS D B,et al.A major protein component of the Bacillus subtilis biofilm matrix[J].Molecular Microbiology,2006,59(4):1229-1238.
[20]HAIBO M,AMIN Z,LIN Z,et al.Inhibitory effects of chitosan in combination with antibiotics on Listeria monocytogenes biofilm[J].Food Control,2014,38:215-220.
[21]LI L,SHI Y,CHESEREK M J,et al.Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes[J].Applied Microbiology and Biotechnology,2013,97(4):1711-1723.
[22]MONDS RD,O’TOOLE G A.The developmental model of microbial biofilms:ten years of a paradigm up for review[J].Trends in Microbiology,2009,17(2):73-87.
[23]LEMON K P,HIGGINS D E,KOLTER R.Flagellar motility is critical for Listeria monocytogenes biofilm formation[J].Journal of Bacteriology,2007,189(12):4418-4424.
[24]GUTTENPLAN S B,KEARNS D B.Regulation of flagellar motility during biofilm formation[J].FEMS Microbiology Reviews,2013,37(6):849-871.
[2]FLEMMING H C,WINGENDER J.The biofilm matrix[J].Nature Reviews Microbiology,2010,8(9):623-633.
[3]ZHOU Wenyuan,ZHANG Hongmei,JIANG Yan et al.Inhibition effect of three nature products on biofilm formation[J].Journal of Food Science and Biotechnology,2014,33(2):171-176.(in Chinese)
[4]DAVIES D.Understanding biofilm resistance to antibacterial agents[J].Nature Reviews Drug Discovery,2003,2(2):114-122.
[5]USDA-FSIS.FSIS to increase permissible levels of food ingredients used as antimicrobials and flavoring agents[J].Fed Regist,2000,65:3121-3123.
[6]VASAVADA M,CARPENTER C E,CORNFORTH D P.Sodium levulinate and sodium lactate effects on microbial growth and stability of fresh pork and turkey sausages[J].Journal of Muscle Foods,2003,14(2):119-129.
[7]LIPSY C,GURDEEP S,KAUTILYA K J,et al.Sonorensin:A new bacteriocin with potential of an anti-biofilm agent and a food biopreservative[J].Scientific Reports,2015,5:13412.
[8]KIM H S,PARK H D.Ginger extract inhibits biofilm formation by Pseudomonas aeruginosa PA14[J].Plos One,2013,9(8):e76106.
[9]SCHILLACI D,ARIZZA V,DAYTON T,et al.In vitro antibiofilm activity of Boswellia spp.oleogum resin essential oils[J].Letters in Applied Microbiology,2008,47(5):433-438.
[10]CHANG Y,GU W,FISCHER N,et al.Identification of genes involved in Listeria monocytogenes biofilm formation by mariner-based transposon mutagenesis[J].Applied Microbiology and Biotechnology,2012,93(5):2051-2062.
[11]STIJIN van der Veen,TJAKKO A.Importance of Sig B for Listeria monocytogenes static and continuous-flow biofilm formation and disinfectant resistance[J].Applied and Environmental Microbiology,2010,23(76):7854-7860.
[12]MIRJAMI M,MIIA L,PANU S,et al.Role of flhA and motA in growth of Listeria monocytogenes at low temperatures[J].International Journal of Food Microbiology,2011,148(3):177-183.
[13]VIJAY K J.Delayed Clostridium perfringens growth from a spore inocula by sodium lactate in sous-vide chicken products[J].Food Microbiology,2006,23(2):105-111.
[14]WANG Jinghui,HAN Jianzhong,QU Daofeng.Hazard and control of bacterial biofilm in the meat industry[J].Food Science,2012,21(33):351-354.(in chinese)
[15]LUNDEN J,AUTIO T,KORKEALA H.Transfer of persistent Listeria monocytogenes contamination between food-processing plants associated with a dicing machine[J].Journal of Food Protection,2002,65(7):1129-1133.
[16]JONES W L,SUTTON M P,MCKITTRICK L,et al.Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms[J].Biofouling,2011,27(2):207-215.
[17]RICHARD J,DUBER M M,BARBARA N,et al.Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation[J].Scientific Reports,2015,5:9856.
[18]SUTHERLAND I W.The biofilm matrix-an immobilized but dynamic microbial environment[J].Trends in Microbiology,2001,9(5):222-227.
[19]BRANDA S S,CHU F,KEARNS D B,et al.A major protein component of the Bacillus subtilis biofilm matrix[J].Molecular Microbiology,2006,59(4):1229-1238.
[20]HAIBO M,AMIN Z,LIN Z,et al.Inhibitory effects of chitosan in combination with antibiotics on Listeria monocytogenes biofilm[J].Food Control,2014,38:215-220.
[21]LI L,SHI Y,CHESEREK M J,et al.Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes[J].Applied Microbiology and Biotechnology,2013,97(4):1711-1723.
[22]MONDS RD,O’TOOLE G A.The developmental model of microbial biofilms:ten years of a paradigm up for review[J].Trends in Microbiology,2009,17(2):73-87.
[23]LEMON K P,HIGGINS D E,KOLTER R.Flagellar motility is critical for Listeria monocytogenes biofilm formation[J].Journal of Bacteriology,2007,189(12):4418-4424.
[24]GUTTENPLAN S B,KEARNS D B.Regulation of flagellar motility during biofilm formation[J].FEMS Microbiology Reviews,2013,37(6):849-871.