| In china, there is an old saying "Hunger breeds discontentment”, which shows that food is the basic material prerequisites for the people’s right of existence, and first of all, food safety issues should be taken into account. After 30 years development of reform and opening up, food-related industries have been gradually become an independent system and also served as one of the pillar domain in national economy. However, since the 21 st century, China’s food safety incidents were reported more frequently, which not only display a negative effect on the development of industries, but also become a threat for people’s health and safety. Food safty has become a major livelihood issues of social concern. The foodborne disease caused by microorganisms is one of the most important factors in food safety. During the food chain, food processing surfaces are generally humid and covered with nutrients, which provides bacterial with an appropriate condition for their biofilm formation. The conventional sterilization methods can not remove biofilm cells efficiently and thoroughly due to its high antibiotic resistance, which has become a great risk for food security. Therefore, it should be an emergency necessary to preven and control the biofilm formation of pathogens.Listeria monocytogenes is one of the most common foodborne pathogens, which can easily form biofilm in various food contact surfaces such as stainless steel, glass and polystyrene. Recently, there major research directions on biofilm formation of L. monocytogenes are described in brief as follows:(1) the effects of environmental conditions on biofilm formation, such as temperature, nutrition and food contact materials.(2) Comparison analysis of biofilm-forming ability of L. monocytogenes isolated from different sources or serotypes.(3) Comparison analysis of biofilm-forming ability of wild-type and its mutant strains, aimed to understand the associated genes and molecular mechanism of biofilm formation. Many effective methods are commonly applied to study on biofilm formation, in recent years, it has been attracted more attention to use bioinformatics technology. L. monocytogenes can be widely found in soil, water, food and animal forage and other conditions, which is closely related to the most important environmental stress response factor σ. Although, studies have been shown that sigB plays an important role in L. monocytogenes biofilm formation, but its molecular mechanism remains to be unclear. The role of a single gene in bacterial biofilm formation can provide a great significance understanding to reveal its pathogenic mechanism.As mentioned above, the wild-type L. monocytogenes WaX12 and its mutant WaX12-△sigB were used in this study. The objective was to investigate(1) the effects of different conditions(Temperature, p H and Na Cl concentration) on biofilm-forming ability of L. monocytogenes WaX12 and WaX12-△sigB;(2) Using transcriptome technology to analyze the role of major environmental stress response factor sigB in L. monocytogenes biofilm formation, which was aimed to reveal its pathogenic mechanism. Thus, this study will provide theoretical research foundation on efficiently preventing and controlling L. monocytogenes biofilm formation. The main content of this study were summarized as follows: 1. Effects of Sig B on Biofilm Formation by Listeria monocytogenes under Various Culture ConditionsThe role of sigB, a major transcriptional regulator of stress response genes of L. monocytogenes, was assessed in biofilm formation of wild-type strain(WaX12) and sigB deletion mutant(WaX12-△sigB) as affected by temperatures(4 oC, 15 oC, 25 oC and 37 oC), p H values(4, 5, 6 and 7) and Na Cl concentrations(0.5 %, 2.5 %, 4.5 % and 6.5 %). Results showed that the biofilm biomass was significantly reduced in L. monocytogenes mutant lacking sigB(p < 0.05). According to the coefficient of variation analysis, all three of the culture conditions tested(i.e., temperature, p H and Na Cl concentration) appeared to affect the strain variability of the biofilm formation of WaX12 and WaX12-△sigB, the increase in the strain variability of biofilm formation caused by temperature is much greater than that caused by Na Cl or p H. The ability of forming biofilm by WaX12-△sigB was more vulnerable to the effects of external culture conditions. There was a significant difference of biofilm biomass between WaX12 and WaX12-△sigB at 37 oC, p H 6 and 2.5 % Na Cl, respectively, so the further research was observed under these culture conditions. Notably, results showed that the relative amount of total polysaccharides and extracellular proteins were much lower in WaX12-△sigB biofilm(p < 0.05). However, there was little influence on live cells. In conclusion, this study could provide a scientific basis for further investigation of the important role of sigB in L. monocytogenes biofilm formation. 2. Effects of sodium lactate on biofilm formation by Listeria monocytogenes WaX12 and WaX12-△sigBThe objective of this study was to investigate the effets of sodium lactate(Na L) at different concentrations(0 %, 2.5 %, 5 %, 10 % and 20 %) on biofilm formation by L. monocytogenes WaX12 and WaX12-△sigB. First of all, the significant difference of biofilm biomass produced by WaX12 and WaX12-△sigB strains was observed at culture condition with 5 % Na L. This condition was chosen to be used in subsequent studies, results showed that the structure of WaX12-△sigB biofilm was forming instead of network structure with reduction of thickness, and a significant decrease(p < 0.05) was determined in total production of polysaccharides, extracelluar proteins, as well as cell activity and membrane integrity in WaX12-△sigB biofilm cells. Finally, the detection of the expression level of the biofilm-associated genes mot B, mog R, deg U, flg E, dna K, and prf A was performed by using quantitative real-time PCR(q RT-PCR), result suggested that the relative quantitative expression of genes associated with flagellar motility and synthesis displayed an significant decrease in WaX12-△sigB biofilm cells. 3. Preliminary study on molecular mechanism of environmental stress response factor sigB in biofilm formation by Listeria monocytogenesThe bioinformatics technology was used to analyze the role of major environmental stress response factor sigB in biofilm formation by L. monocytogenes in this study, and the biological functions of differentially expressed genes of WaX12 and WaX12-△sigB biofilm cells were classified according to GO, KEGG pathway and COG database. Total numbers of 2847 differentially expressed genes were observed. 701 genes showed significant difference expression of two samples, consisted of up-regulated genes(55.92 %), down-regulated genes(44.08 %) and function unknown genes(59.5 %). Analysis of GO database showed that all differentially expressed genes were classified to biological process(Ribosome biogenesis) and molecular function(structural constituent of ribosome). The enrichment of differentially expressed genes according to COG database was determined into two function parts such as Amino acid transport and metabolism(8.9 %), Carbohydrate transport and metabolism(12.2 %). Finally, 74 different KEGG pathways related to the role of sigB in biofilm formation of L. monocytogenes were investigated, and showed a significant correlation with Ribosome metabolic and Pentose and glucuronate interconversions metabolic. |
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