Inhibitory Effect Of Organic Acid Treatment On Biofilm Formation Of Enterobacter Cloacae And Escherichia Coli

Enterobacter cloacae and Escherichia coli are gram-negative bacteria,which are the most common pathogenic bacteria in the digestive tract of livestock and poultry.Extracellular substances produced by bacteria during growth can form biofilms.Biofilms are easy to be found on the slaughtering and processing of livestock and poultry,as well as the surfaces of such pathogenic bacteria remaining in eggs and milk,and the microorganisms wrapped in biofilms have resistance to antibiotics and bacteriostats and it is not easy to completely inactivated.As a viscous membrane layer containing microbial colonies covering the surface of the object,biofilm is of great significance to the growth,reproduction and survival of spoilage bacteria,and is not conducive to the preservation of food.Phenyllactic acid(PLA),lactic acid(HL),and citric acid(CA)are three natural organic acids,among which PLA has broad-spectrum bacteriostasis,HL and CA also have certain inhibitory effects on pathogenic bacteria and are often used as food additives.At present,there are few reports on the effect of organic acids on biofilms.In this paper,Enterobacter cloacae CY4(E.cloacae CY4)and Escherichia coli K12(E.coli K12)were isolated from fresh sheep carcass surface as target strains to study the inhibitory effect of three organic acids on the strain biofilm,and make a preliminary exploration of the molecular-level inhibition mechanism.In this study,the traditional bacterial isolation methods are used to determine the composition of the major bacterial groups during lamb processing,and the dominant strains with strong filmforming ability are screened for biofilm inhibition experiments.Observe the treatment of organic acids on biofilm formation inhibition by Minimum Inhibitory Concentration(MIC),migrate ability,crystal violet staining,scanning laser confocal microscope(CLSM)and scanning electron microscope(SEM),and the significantly expressed protein is showed by proteome results that is used in related gene knockout strains for transcriptome analysis,and finally,the possible inhibition mechanism of organic acid treatment on biofilm formation is analyzed.This experiment showed that the MIC of PLA,HL and CA to E.cloacae CY4 and E.coli K12 are both 5mg/m L.However,for the same organic acid at the same concentration,the tolerance of E.coli K12 is weaker than E.cloacae CY4,and the effect of PLA in the organic acid with the same concentration is more obvious than the other two acids.1/2 MIC and 1/4 MIC organic acids have a significant inhibitory effect on the migrate ability of the strain;1/2 MIC and 1/4 MIC organic acids generally inhibit biofilm formation,but this inhibition will decrease at 48 h.The organic acid treatment reduced the polysaccharide content,which greatly reduced the thickness of biofilm,the surface pores increased,the bacterial structure was loose,and the attachment on the surface of the strain was greatly reduced;the fluorescence quantitative PCR results further confirmed that organic acids reduced the transcription level of genes related to adhesion and polysaccharide production in bacteria.It was found that the protein expression of the E.cloacae CY4 biofilm in the presence of phenyllactic acid changed by proteome analysis.Among them,the expression of 21 proteins was significantly up-regulated,and the expression of 58 proteins was significantly down-regulated.The differentially expressed proteins were mainly related to metabolic pathways such as amino acids,carbohydrates,lipids,energy,enzymes and signal transduction,and also contains transport and regulation proteins that affect bacterial acid resistance,growth and protein secretion,transport metabolism and membrane growth.Through proteome data,it was found that the bacterial cells in the E.cloacae CY4 biofilm in the presence of phenyllactic acid would be stressed to produce a Mar R regulatory protein related to bacterial acid tolerance.In order to further study how the transcriptional regulatory protein Mar R affects the growth and biofilm formation of E.cloacae CY4 cells under organic acid,the changes of E.coli K12?mar R mutant and wild bacteria during biofilm formation were first analyzed.It was found that K12?mar R significantly decreased in migrate ability and film-forming ability,and K12?mar R mutant bacteria were less likely to form biofilms in the presence of phenyllactic acid.Transcriptome analysis of upstream and downstream proteins related to the transcribed protein Mar R,it is found that after mar R gene knockout,a total of 40 genes were up-regulated and 133 genes were down-regulated in K12?mar R.Among which the upregulated genes are related to pantothenic acid,Co A biosynthesis,fructose metabolism,glycine,serine and threonine metabolism,etc.The down-regulated genes are related to the expression of Mar A protein,quorum sensing system,?-lactam resistance,two-component system,various biosynthesis and metabolic processes.In addition,the acid tolerance of the cells decreased significantly after mar R knockout.Studies have shown above that PLA,HL,and CA have significant inhibitory effects on biofilm formation of E.cloacae CY4 and E.coli K12.Fluorescence quantification,proteome,and transcriptome also indicate that organic acid treatment will inhibit the expression of related genes related to motility,synthesis and secretion of extracellular substances,carbohydrate and amino acid metabolism,energy metabolism.At the same time,it also induced the expression of acid stress regulatory gene mar R,to form a protective mechanism for the strain.