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Preliminary Study On The Stress Response Of Vibrio Parahaemolyticus And Listeria Monocytogenes Biofilms To Different Modified Atmospheres

Posted on:2021-04-16Degree:MasterType:Thesis
Country:ChinaCandidate:H QianFull Text:PDF
GTID:2370330611961524Subject:Biology
Abstract/Summary:PDF Full Text Request
As we all know,Vibrio parahaemolyticus?VP?and Listeria monocytogenes?LM?can easily attach to various food related surfaces,grow by various interactions and produce many extracellular polymers?EPS?.Among them,EPS surrounds bacteria to produce 3D membrane materials with high tissue structure,which is called biofilm?BF?.The biofilm of VP and LM has increasingly become an important threat to the safety of seafood.Bacteria strengthen their resistance by forming biofilm,which brings serious burden to food processing and production.In the field of food,in order to keep the loss of taste and nutritional value of food to a minimum,different proportions of atmospheres are usually filled to ensure the quality of food.It has been reported that different proportions of modified atmospheres can effectively affect the bacterial survival of foodborne pathogens such as VP and LM.Therefore,in this paper,under three different proportions of modified atmospheres?anaerobiosis 20%CO2:80%N2;micro-aerobiosis 20%O2:80%N2;aerobiosis 60%O2:40%N2;?,the influence and the preliminary mechanism of modified atmospheres on biofilm formation of VP and LM were studied.This study is of great significance to the food safety problems caused by foodborne pathogens,the development of food industry and the prevention and control measures for the future.In this paper,four cocktail VP and four cocktail LM were used as the main body.Firstly,the effects of different proportions of modified atmospheres on the biofilm formation of VP and LM were explored;secondly,the effects of different proportions of modified atmospheres on the structural characteristics of biofilm of VP and LM were explored;finally,RT-q PCR was used to investigate the effects of different proportions of modified atmospheres on the expression of related genes?biofilm,EPS and virulence?.The important contents and conclusions of this article are as follows:1.Effect of modified atmospheres on VP biofilm and LM biofilm.VP and LM are common food-borne pathogens,and the formation of their biofilm is of great significance for survival and transmission.Compared with the micro-aerobic conditions?control group?,the total amount of biofilm formed by VP and LM in anaerobic and aerobic conditions was measured by crystal violet staining;using chemical methods to analyze the change of extracellular DNA?e DNA?,extracellular protein and extracellular polysaccharide in the EPS formed under three different proportions of modified atmospheres.The results showed that for VP,OD600nm under anaerobic condition increased and OD600nm under aerobic condition decreased compared with the micro-aerobic condition?control group?;however,for LM,OD600nm under anaerobic condition increased and OD600nm under aerobic condition gradually increased.Compared with the micro-aerobic condition?control group?,the treatment of modified atmospheres significantly affected the EPS of VP and LM.The effect of modified atmospheres on VP'EPS was e DNA>extracellular protein>extracellular polysaccharide.However,the effect of the modified atmospheres on LM'EPS was extracellular protein>e DNA>extracellular polysaccharide.The results of correlation analysis showed that the modified atmospheres mainly affected the formation of biofilm by affecting e DNA and extracellular protein in EPS of VP and LM.2.Effect of modified atmospheres on structural characteristics of VP biofilm and LM biofilm.In this study,the morphological structure of biofilm was visually observed by SEM.Using CLSM imaging method and ISA-2,a structural analysis method was established to verify the effects of modified atmospheres on biofilm and its structural parameters.The results of SEM imaging's showed that:compared with the micro-aerobic condition?control group?,VP showed a well-formed biofilm network structure under anaerobic condition;when in aerobic condition,sporadic or even fewer biofilm cells could be observed scattered on the contact surface.For LM,compared with micro-aerobic condition?control group?,the biofilm formed under anaerobic and aerobic conditions showed the opposite performance with VP.For VP,compared with the micro-aerobic condition?control group?,the imaging results of CLSM showed that,the anaerobic condition resulted in a dense and well-organized biofilm structure,the biomass and average thickness significantly increase,while structure entropy and roughness decrease;the aerobic condition resulted in a sparse biofilm structure,the structure entropy and roughness increase,while the biomass and average thickness decrease significantly.For LM,compared with micro-aerobic condition?control group?,the anaerobic condition resulted in a sparse and loose biofilm structure,the structure entropy and roughness increase,while biomass and average thickness decrease significantly;and the aerobic condition resulted in a denser and better tissue structure biofilm structure,the biomass and average thickness significantly increase,while structure entropy and roughness decrease.Combined with the previous analysis of the influence of the modified atmospheres on the components of EPS of VP and LM,the modified atmospheres mainly affect the formation of biofilm structure by affecting e DNA of VP and extracellular protein of LM.3.Effect of modified atmospheres on the expression level of genes related to VP biofilm and LM biofilm.In the third part of this study,RT-q PCR was used to analyze the expression level of biofilm,EPS,virulence and other related genes to further reveal the primary mechanism of modified atmospheres affecting biofilm.Compared with the micro-aerobic condition?control group?,the results showed that flagella,fimbria and QS-related genes were down-regulated in the biofilm of VP under aerobic conditions;Compared with the micro-aerobic condition?control group?,the flagella-related genes were down-regulated in the biofilm of LM under anaerobic conditions.The above results show that different proportion of modified atmospheres affect the formation of biofilm by affecting the expression of biofilm related genes.When the biofilm of VP and LM were treated with modified atmospheres,EPS related genes also changed.The results showed that modified atmospheres could affect EPS production by affecting the expression of EPS related genes of VP and LM.Compared with the micro-aerobic condition?control group?,the T3SS-related genes of VP and virulence genes of LM were regulated by different proportions of modified atmospheres.The results show that different proportion of modified gas will affect the pathogenicity of the strain by affecting the expression of virulence gene.
Keywords/Search Tags:Vibrio parahaemolyticus, Listeria monocytogenes, Modified atmospheres, Stress response, Biofilm, Extracellular polymeric substances (EPS), Structure, RT-qPCR
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