Formation Mechanism And Control Of Salmonella Typhimurium Biofilm

Salmonella is one of the most common foodborne pathogens and has the highest infection rate among all foodborne pathogens.Food poisoning incidents caused by Salmonella contamination were frequently exposed around the world every year.Therefore,the prevention and control of Salmonella contamination has been paid much attention.Among all Salmonella genera,Salmonella typhimurium is the most common of all serotypes isolated.It is a zoonotic disease with strong aggression to poultry,mammals and humans.The moisture in the food processing industry,such as sediment environment is a good medium biofilm formation,usually Salmonella typhimurium in gas–liquid surface to form a layer of dense membrane or adhesion in food processing equipment and utensils,such as the surface of stainless steel,plastic,etc.If food processing personnel during operation or improper cleaning disinfection is not complete,bacteria can then enter the processing of products,leading to the occurrence of foodborne illness.After the formation of the biofilm,the bacteria were wrapped in a large number of extracellular polymers,making the bacteria show stronger tolerance to various disinfectants,detergents,antibiotics and some adverse environments such as high pressure,high temperature,acid and alkali environment,which is also the main reason for incomplete disinfection and sterilization.Therefore,it is of great guiding significance to study the formation and regulation mechanism of Salmonella typhimurium biofilm,evaluate and search for methods that can inhibit the formation of biofilm and have effective removal effect on mature biofilm for the prevention and control of food contamination caused by biofilm.In this study,Salmonella typhimurium was used as the research object to explore the regulation and mechanism of biofilm formation and explore effective measures to control biofilm formation and clearance from different perspectives.11 strains of Salmonella typhimurium strains existing in the laboratory were selected.Firstly,the changes of biofilm formation rules under different environmental conditions were explored,and the characteristics of bacteria were measured to analyze and compare the relationship between the biofilm formation ability and the biofilm formation ability.Then,the biofilm and planktonic states of CDC 3 and CVCC 3384,two strains with different biofilm formation ability,were sequenced with high throughput,and the differences in the regulation of biofilm formation between them were compared from the transcriptional level.Finally,17 kinds of scavengers were selected to study their effects on the formation of biofilm,the removal effect on mature biofilm and the number of viable bacteria in biofilm..The main results were as follows:The law of biofilm formation under the environmental conditions of temperature,p H,salt concentration and sugar concentration was determined by crystal violet staining method.It was found that the optimal temperature for biofilm formation was30℃ instead of 37℃ for bacterial growth.The amount of biofilm formation did not change much in the range of p H 5.0–8.0.Biofilm was still generated in alkaline environment,which indicated that Salmonella had a good adaptability to acid and alkali environment.Bacteria were more conducive to the formation of biofilm in the environment of low sugar and low salt,indicating that biofilm is more likely to form in the environment of malnutrition.ST 19 was different from other strains,and the increase of glucose concentration is favorable for this strain,which can form more biofilm.On this basis,two strains of strong,medium and weak biofilm forming ability were selected respectively for the subsequent study of cell characteristics.In the study of the characteristics of the cells,it was found that the motor ability was determined by the genetic factors of the cells themselves and had little relationship with the state they were in,and the strains with strong biofilm forming ability had relatively strong motor ability.There was no obvious correlation between cell surface hydrophobicity and self–polymerization and biofilm formation ability.The number of living cells in the biofilm is more than that in the biofilm.There was a positive correlation between the amount of extracellular polysaccharide,extracellular protein and biofilm.The main components of the biofilm,curli and cellulose,can be observed by naked eye through the Congo Red – Coomass Bright Blue plate,and can reflect the ability of biofilm formation to a certain extent.RNA–Seq sequencing results showed that in the strain biofilm CDC 3 cells,highly expressed genes mainly have a few kdp ABC family genes,as well as mgt C and drug–resistant genes encoding multiple bhs A stress protein gene,while lower expression is primarily a flagellum synthesis and bacterial chemotaxis related genes,and the preceding chapter of biofilm formation ability and is not consistent with motility is related to the conclusion that may exist in the CDC 3 strains of other signaling pathways to control biofilm formation and movement ability;The comparative analysis of CDC 3 biofilm cells and planktonic cells showed that in addition to the above mentioned up–regulated genes,genes related to bacterial pathogenicity and infectivity were also up–regulated.It can be seen that once the bacteria formed a biofilm,the pathogenicity and infection rate would be higher.Strain CVCC 3384 in biofilm cells,a series of genes encoding fimbriae protein related csg ABCDEG,Ⅲ secretion system and Salmonella infection related gene expression were raised,this and the previous research conclusion was consistent,namely fimbriae protein coding genes in the process of biofilm formation plays an important role;Finally,the comparison between CDC 3 and CVCC 3384 showed that the differentially expressed genes were mainly unknown genes.According to the influence of environmental factors on the biofilm formation ability of Salmonella typhimurium,it was found that strain ST19 could still produce a large amount of biofilm under high sugar content(1.5%),while strain CDC3 did not form biofilm.On the basis,we sequenced the transcriptome of ST19 and CDC3 biofilm cells and planktonic cells under high glucose concentration.The results showed that gat C,fru A,gat B and other genes involved in the regulation of PTS system were down regulated in st19 biofilm cells under high glucose concentration,In the quorum sensing system,the related operon lsr ABCDRK,which regulates AI-2signal molecules,is also downregulated.The existence of biofilm in food industry has brought great burden to cleaning staff in terms of cleaning and disinfection,and also caused great economic loss.CDC3 and ST 34 strains with strong biofilm–forming ability were used as the research objects to explore the control effect of different scavengers on biofilm.It is concluded that interfering with the biofilm formation process is more effective and thorough to avoid biofilm fouling than cleaning the mature biofilm.Among the 17 scavengers selected,hydrogen peroxide has significant advantages in inhibiting the formation of biofilms or in clearing mature biofilms.Moreover,hydrogen oxide treatment can effectively kill the activity of cells in the biofilm and reduce the number of living cells in the biofilm.In addition,the addition of ethanol,benzalkonium chloride,benzalkonium bromide and essential oil of carvol,cinnamaldehyde and thyme during the culture process can also effectively interfere with the formation of biofilm,but their removal effect on the mature biofilm is slightly inferior.