Related Factors And Regulatory Mechanisms Of Listeria Monocytogenes Biofilm Formation

Listeria monocytogenes is a common foodborne pathogen,which can secret Listeria hemolysin(LLO)and cause a zoonosis,listeriosis.Listeriosis can not only make animals sick and cause huge economic losses;but also cause food poisoning and infection in human,inducing severe diseases such as sepsis and meningitis.Listeria monocytogenes is widespread in nature,especially in food.Dairy products,meat,and aquatic products are often contaminated by Listeria monocytogenes due to improper operations during the production process.The outbreak of Listeria monocytogenes contamination is a worldwide food safety issue which have occurred in many parts of the world such as the Germany,Brazil,United States,and South Africa.In food processing environment,Listeria monocytogenes often attaches to the surface of food processing equipment and forms biofilm,which increases its resistance to sterilization and disinfectants.Due to the difficulty of removal,biofilm cells often cause repeated occurrence of food contamination.Controlling biofilm is the first step to prevent continuous contamination of Listeria monocytogenes,and the research on biofilm formation is the basis of controlling contamination.Therefore,the biofilm formation and regulation mechanism has become a hot topic in the field of pathogenic bacteria in recent years.The aim of this research is to study the influence factors and regulation mechanism of Listeria monocytogenes biofilm formation.We first studied the effects of food environmental factors and cell characteristics on biofilm formation.Secondly,through gene knockout technology,a preliminary study on the relationship between biofilm formation and chemotaxis in Listeria monocytogenes was conducted.Finally,the related genes of biofilm formation were analyzed through the study of genomics and transcriptomics,which provided a direction for the food industry to control Listeria monocytogenes contamination.The main results obtained are as follows:By measuring the biofilm formation capicity of 26 Listeria monocytogenes strains,six strains representing strong,moderate and weak biofilm forming strains were selected to study the factors affecting biofilm formation.It was found that the response of biofilm formation to environmental factors is strain-specific.But in most strains,the addition of glucose and salt promoted the formation of biofilms,and low nutrient concentrations limited the formation of biofilms in all six strains.37? is the optimum temperature for biofilm formation and 7 is the optimum pH for biofilm formation.The environmental factor that has the greatest effect on biofilm formation was temperature,followed by pH.Through the study of cell characteristics,it has been found that bacterial growth characteristics had nothing to do with the capacity of biofilm formation;hydrophobicity has a positive correlation with the amount of biofilm produced by cells.At 37?,hydrophobicity had a greater impact on biofilm formation;and at 30?,a positive correlation between motility and biofilm production was exhibited.The relative gene expression of flagellin gene flaA,rotor gene motB,and the chemotactic two-component system gene cheA/Y in biofilm and planktonic cells were measured by fluorescence relative quantitative PCR.The results showed that,compared with the planktonic cells,the expression of flaA and motB in the biofilm cells were significantly up-regulated;the expression of the cheA/Y genes in the biofilm cells were also up-regulated,indicating that the biofilm formation is affected by motility and chemotaxis systems.Then a ?cheY mutant strain of LMB 33426 was constructed by two-step homologous recombination exchange technology.We found that,under the same culture conditions,the biofilm production of mutant straindecreased significantly;the motility and hydrophobicity of the mutant strain also decreased,but the growth cruve,acid-base tolerance,and drug resistance haven't changed due to the lack of chemotaxis genes.Transcriptomics analysis showed that at 37?,546 genes expressed differentially in biofilm cells compared to planktonic cells,including 356 up-regulated genes and 190 down-regulated genes.Among them,genes involved in glycolysis/gluconeogenesis pathway,tricarboxylic acid cycle,pentose phosphate pathway,and fructose and mannose metabolism were significantly down-regulated in biofilm bacteria after 36h culture,while genes encoding five Clp-HSP100 ATPase and four penicillin-binding protein were significantly up-regulated.Penicillin binding protein gene lmo 1438,heat strike protein 90 gene lmo 0942,superoxide dismutase gene sod are the three genes with the most extensive regulatory network among the differentially expressed genes.