Screening And Control Effects Of Quorum Sensing Inhibitors Of Listeria Monocytogenes

Listeria monocytogenes(L.m)has a wide range of life and can grow under alkaline,acidic and low temperature conditions,and has strong resistance to stress.In addition,various virulence factors such as internalizing hormone(InlA and InlB)and hemolysin(LLO)are highly likely to cause high mortality,and biofilms(BF)which are easy to form are also suitable for processing tools and storage environments.The cleaning is inconvenient and enhances the tolerance of L.m to the physical and chemical factors of the external environment.The strain has been defined by the World Health Organization as one of the four foodborne pathogens.The high morbidity and mortality caused by L.m pollution are mainly caused by biofilm and virulence factors regulated by bacterial quorum sensing system(QS).Therefore,exogenous compounds can be used as quorum sensing inhibitors(QSIs)to block the binding of signaling molecules to receptors,targeting the normal function of interfering with bacterial QS function,and then controlling the hazards of L.m.This experiment detects the strength of bioluminescence of Vibrio harveyi BB170,4-hydroxy-2,5-dimethyl-3(2H)-furanone(DMHF)and 16 marine lactic acid bacteria metabolites.For the screening of QSIs-like,a reporter strain expressing green fluorescent protein was constructed by plasmid cloning technique.Agr QSIs were screened for a synthetic peptide and Bacillus metabolite by detecting the intensity of green fluorescence.The effects of QSIs on the QS interference of L.m were evaluated by the growth of L.m,the formation of biofilm and kinetics,the determination of virulence and virulence gene expression.The main experimental contents and results are as follows:1 The AI-2 QSIs were screened by the biochemical luminescence experiment of the indicator strain Vibrio harveyi BB170 on the crude extracts of 16 marine lactic acid bacteria and the food flavor DMHF preserved in our laboratory.The QSIs were screened for the AgrQS system of L.m.The pII-EGFP fragment was designed and synthesized by gene synthesis,then cloned into the vector plasmid p ERL3 by SalI/XbaI and constructed by T4 DNA ligase.The recombinant plasmid p ERL3-PII-EGFP was recombined and transformed into the L.m wild strain EGDe,and the constructed plasmid and the reporter strain were identified by PCR using the designed specific primer pair.The screening materials were screened for Agr QSIs by observing the luminescence intensity of the constructed reporter strains using a fluorescence inverted microscope.The screening materials were 17 synthetic Bacillus supernatants preserved in the laboratory and synthetic peptides of AIP structural analogs designed according to the literature.The results showed that the inhibition rate of lactic acid bacteria from L.m was higher than 75%,accounting for 35% of the screening materials,especially the inhibition rate of AI-2 activity was higher than 98.5.%.DMHF with food-grade flavor of 100 ?g/mL has an inhibition rate of L.m AI-2 of up to 80%;Successfully constructed reporter strain carrying recombinant plasmid L.m p ERL3-PII-EGFP,and two strains of Bacillus supernatant were screened.It has Agr QS inhibitory activity,accounting for11.8% of the screening material,and the synthetic peptide can completely inhibit the luminescence of the reporter strain.2 The growth curve of L.m under QSIs was quantitatively determined by optical density value,and the antibacterial effect of the selected QSIs was evaluated.The semi-solid puncture culture method was used to detect the dynamics of L.m under the action of low concentration of QSIs.The staining method and optical microscopy were used to determine and observe the biofilm content and morphology of L.m under different QSIs.The results showed that using QSIs to target the QS system,200 ?g/mL of DMHF,500 nM of synthetic peptide,500 ?g/mL of Pediococcus pentosaceus ethyl acetate extract B-1,and starch were obtained.The B.amyloliquefaciens supernatant extracts HY-4 and HY-5 can effectively control the growth of pathogenic bacteria,the formation of biofilm and the expression of virulence factors at a lower dose.Delaying the L.m into the growth period and effectively reducing its final growth concentration,and inhibiting the movement of flagella,the formation of the biofilm structure loosely controls the infection and pathogenicity of L.m.The 50 nM synthetic peptide can inhibit the luminescence of the reporter strain,which proves that it has certain Agr sensation inhibition effect,but it can not observe its inhibitory effect on the growth,dynamics andbiofilm of L.m.3 In order to explore the effect of QSIs on the virulence of L.m,the hemolytic plate method was used to visually detect the change of L.m in the hemolytic ring,and the QSIs were tested by RT-qPCR.Expression of L.m key virulence genes,including hemolysin hly,internalization factor inl A,actin Glucose ActA and AgrD genes.At the same time,the expression of the AgrD gene was analyzed to further determine the accuracy of the screening results of the constructed Agr reporter strains.The results showed that the hemolysis plate experiment visually showed the inhibitory effect of QSIs on the expression of L.m hemolysin LLO.RT-qPCR was used to quantitatively detect the expression of virulence genes.Compared with the negative control,the inhibition rate of hly and inlA virulence genes by 100?g/mL DMHF was 50%,500 ?g/mL Pseudomonas pentrea B-1 significant inhibition of hly,inlA,actA virulence genes up to 80%;500 nM synthetic peptide had a good inhibitory effect on hly and inlA virulence gene expression,but not on act A gene;The inhibition effect of 500?g/mL B.amyloliquefaciens HY-5 was better than HY-4;and the significant inhibition of agrD gene verified that synthetic peptides and Bacillus amyloliquefaciens HY-5,HY-4 was Agr QSIs.