The Effect And Mechanism Of Ferulic Acid On The Planktonic Growth And Biofilm Formation Of Shigella Flexneri

Shigella flexneri(Sh.flexneri)is an important human pathogen causing shigellosis and primarily endemic in developing countries.In recent years,the abuse of antibiotics for disease treatment has led to the emergence of superbug.Development and spread of multidrug-resistant Sh.flexneri have become a global public health concern.Planktonic Sh.flexneri has the ability to attach,grow,and form a biofilm on food and the surfaces of food processing equipment.Biofilms greatly enhance the resistance of Sh.flexneri to biocides and antibiotics,and consequently lead to persistent bacterial contamination.It is extremely urgent to develop an effective method that controls Sh.flexneri and its biofilm.Ferulic acid(FA)has been considered a natural,nutritional,and safe alternative to synthetic antiseptics and preservatives for their wide range of sources,multiple functions and high consumer acceptance.In this study,Sh.flexneri cells were treated with FA extracted from a by-product of rice processing.Thus,the objectives of this study were to understand the mechanism of the antibacterial and anti-biofilm effects of FA against planktonic Sh.flexneri and biofilm formation.We hope that the results presented here provide a theoretical basis for the control of planktonic and biofilm-associated Sh.flexneri infections in the food industry,and the development of FA-derived food antiseptics and preservatives.These results also lend new insight into the development and utilization of grain processing by-product resources.The main findings are as follows1.FA exhibited moderate antibacterial effects against planktonic Sh.flexneri,as characterized by the decrease in maximum population density and maximum growth rate.Furthermore,the cell membranes of Sh.flexneri exhibited irreversible damage after the FA treatment,and this disruptive effect was evidenced by decreased cell viability,increased cell membrane permeability,increased leakage of intracellular biomacromolecules,prevented protein synthesis,accelerated ATP consumption,cell membrane depolarization,and cellular morphological changes.All these effects of FA on planktonic Sh.flexneri may eventually contribute to bacterial death2.FA exerted significant inhibitory effects on Sh.flexneri initial adhesion and biofilm formation,and has a dual effect of targeting inhibition of bacterial community and extracellular polymeric substances(EPS)of biofilm.This inhibitory effects as confirmed by the results that FA not only reduced the number of viable cell,the activity of metabolic,and ATP content in the biofilm,but also inhibited the content of three main components(extracellular polysaccharides,extracellular proteins,and extracellular DNA)that constitute the EPS of biofilm.In addition,FA significantly inhibited and inactivated the mature biofilm formed by Sh.flexneri on three surfaces(polystyrene,stainless steel,and glass).3.A total of 702 differentially expressed genes(DEGs)were identified by transcriptome sequencing and bioinformatics analysis of biofilm-type Sh.flexneri treated with FA at sub-inhibitory concentration,in which 169 DEGs were up-regulated and 533 DEGs were down-regulated.KEGG pathway enrichment analysis showed that DEGs were mainly enriched in Ribosome,Citrate cycle(TCA cycle),and ABC transporters.Moreover,FA inhibited the expression of genes involved in the bacterial adhesion and the synthesis and transport of main components of Sh.flexneri biofilm.RT-qPCR analysis showed that the expression trends of biofilm-forming genes of Sh flexneri such as csgD,mdoC,yojN,rcsC,rcsB,and waaG were consistent with those of transcriptome sequencing,further confirmed that the transcriptome sequencing results can be trusted.