Factors Affecting The Formation Of Biofilm By Listeria Monocytogenes And Basic Research On The Formation Mechanism Of Suspended Aggregates

Listeria monocytogenes(LM)is a kind of pathogenic microorganism which can grow in low temperature environment and has low nutritional requirements.It is widely distributed in food processing,storage,transportation and other places contacting with food.Compared with planktonic bacteria,LM secretes extracellular polymer to form microbial aggregation,which has significantly increased resistance to many adverse conditions such as drying,extreme temperature,antibacterial drugs and disinfectants.It is of great significance to study the influencing factors of biofiltn formation and the formation mechanism of suspended aggregates for further exploring the mechanism of LM resistance to adverse environment and the prevention or control of pathogenic microorganisms.In this study,a LM strain with strong biofilm formation ability was selected as the research object to explore the influence of various environmental factors on biofilm formation and the conditions of inducing LM to form suspended aggregates by Ralstonia insidiosa(RI).The possible mechanism of LM to form suspended aggregates was preliminarily revealed by RNA-seq technology.The main research results are as follows:(1)Nine LM strains used in this study can still maintain the ability of growth and biofilm formation under the conditions of low temperature and poor nutrition.The biofilm formation amount of all the tested strains is the highest under the condition of normal nutrition,cultured at 37℃ for 48 hours.Compared with the normal state of nutrition,the amount of biofilm formation in the poor state of nutrition decreased.Among them,the strain no.5 isolated form milk had the strongest ability to form biofilm,and the amount of biofilm in the nutrient-poor state was 33.46%less than that in the nutritionally normal state.At low temperature(4℃),the amount of biofilm formation decreased by 30.61%compared with that at 37℃.(2)Weak acid,alkaline condition,low concentration of sodium chloride and RI can all significantly promote LM to form biofilm,but the amount of biofilm formation in nutritionally barren state is reduced compared with that in nutritionally normal state.Several common carbohydrates promoted the formation of biofilm to some extent,especially xylitol and konjac powder,which caused the growth rate of biofilm as high as 75.00%and 96.67%,respectively.Rhamnosus and soluble starch had no obvious effect on the formation of biofilm by LM-Confocal laser scanning microscope microscope(CLSM)was used to observe that the space structure of biofilm formed by LM and RI is different.(3)At 28℃ and 37℃:when LM is cultured alone,neither static culture nor low-speed culture can form suspended aggregates;when RI is cultured in static culture,it can form small and many suspended aggregates;when the two kinds of bacteria are cultured in different proportion,no formation of suspended aggregates is observed in static culture,and at low-speed co-culture,the formation of suspended aggregates was observed,and the smaller the proportion of LM inoculation was,the greater the aggregation degree of the formed suspended aggregates was.Compared with the suspended aggregates formed at 37℃,the suspension aggregate formed at 28℃ is more obvious and lasts longer.Further study showed that the supernatant of RI culture could induce LM to form suspended aggregates,while the inactivated RI culture could not induce LM to form suspended aggregates,which indicated that the formation of LM suspended aggregates was induced by some substances secreted during RI growth.(4)RNA-seq was used to analyze the gene expression differences between LM and RI co cultured and LM planktonic bacteria induced by RI culture supernatant.The results showed that there were 511 gene expression differences between LM and LM planktonic bacteria,273 of which were up-regulated Among them,210 genes were up-regulated and 240 genes were down regulated.The results of GO enrichment showed that the two groups of differentially expressed genes were mainly enriched in translation,ribosome,rRNA binding and glycolysis.The results of KEGG enrichment showed that the differentially expressed genes were mainly involved in pyrimidine metabolism,glycolysis/gluconeogenesis.Combined with relevant references,It was analyzed that the formation of LM suspended aggregates may be regulated by two-component system.chemotaxis of bacteria,swarm induction,phosphotransferase system,flagellum assembly,ABC transporter,glycolysis/gluconeogenesis.In addition,some genes(sigB,rsbW,adhE,groL,ampC,hfq and ccpA)that may be related to the formation of suspended aggregates were screened out,and these genes were involved in coding RNA polymerase,acetaldehyde/ethanol dehydrogenase,molecular chaperone,penicillin binding protein,RNA chaperone and catabolite control protein A,respectively.