| Listeria monocytogenes(LM)can invade the three major barriers of human and animal organism,mainly causing symptoms such as gastroenteritis,meningitis,miscarriage,and sepsis.The distribution of the disease is global and the incidences is increasing year by year.It is an important pathogen of zoonotic and food-borne diseases.Biofilm(BF)is an extracellular polymer of bacteria.Due to its special three-dimensional multilayer structure,it enhances the resistance of bacteria and leads to the symbiosis of multiple bacteria,which further pollutes the food processing environment and food.It seriously harms humans health,public health safety and the development of livestock.The study of transcriptomics will help to discover the influence of BF formation on the expression of LM gene and reveal the regulation mechanism of BF formation.Based on this,this study has completed the following research work:(1)The formation of Listeria monocytogenes BF was determined under different culture conditions by test tube culture method,slide culture method and microplate quantitative method,and its formation conditions were optimized.The results showed that during the process of BF formation in vitro,a small amount of bacteria initially attached and microcolonies formed from 6 h to 24 h.Mature and dense BF appeared and the microcolonies were connected into a network structure after 24 h.From 48 h to 72 h,the BF aggregated and superimposed,forming a complex clusters.The most suitable BHI medium concentration,temperature,acid base vale,Na Cl concentration and glucose concentration for the BF formation were 100%,37?,p H 7.5,5.0 g/L and 22.0 g/L.(2)By study of transcriptomics for BF and planktonic of LM,the results showed that BF bacteria had 1 588 differentially expressed genes(DEGs)at 12 h,including 797 gene expression up-regulation and 791 gene expression down-regulation.BF bacteria had1 517 DEGs at 24 h,including 758 gene expression up-regulation and 759 genes were down-regulated.BF bacteria had 1462 DEGs at 48 h,including 736 genes up-regulated and 726 genes down-regulated.Among them,1 123 genes were differentially expressed in these 3 time periods.(3)GO function enrichment and KEGG pathway enrichment analysis were performed on the 1 123 DEGs.The result found that there were 3 biological processes(metabolism,biosynthesis and translation process),3 cellular components(organelles,protein complexes and cytoplasm)and 4 molecular functions(structural molecules,ligases,catalytic and transport activities)in GO function annotation.It mainly involves the phosphotransferase system(PTS),flagella assembly,microbial metabolism in different environments,bacterial chemotaxis,bacterial secretion system and quorum sensing(QS)pathways in the KEGG pathway analysis.(4)Some DEGs were selected to verification using real-time quantitative PCR and Northern blot.The results show that the fold change obtained by real-time quantitative PCR and Northern blot hybridization results were consistent with the trend of RNA-seq mostly,indicating that the transcriptomics analysis results were stable and reliable.In a word,the dynamic changes in the growth of BF bacteria were firstly confirmed in this study.Secondly,the suitable conditions were optimized for the formation of Listeria monocytogenes BF.Thirdly,the differentially expressed genes were obtained and enriched analysis by study of transcriptomics at BF and planktonic of LM.Finally,some genes were selected for verification.This study preliminary understood the molecular mechanism of the BF bacteria in metabolism,drug resistance and secretion,deeply understood the regulation mechanism of BF formation,and provided theoretical basis for effective control of BF formation and find BF inhibitors in the future. |
PDF Full Text Request