| Proteus mirabilis(PM)is an important zoonotic pathogen.It is widely distributed in nature.It often infects humans or animals through food,water,feces,etc.It can cause food poisoning,diarrhea,meningitis,otitis media and other diseases.In recent years,with the increase in the incidence of livestock and poultry caused by the bacteria and the decline in the effectiveness of antibiotics,it is urgent to carry out research on the prevention and control of the bacteria.Biofilm(BF)is that bacteria adhere to the surface of a substance and are wrapped by extracellular matrix to form a tightly structured multicellular population.BF can increase the applicability of bacteria to the environment.Its formation is affected by nutrition,temperature,Time,osmotic pressure and other factors are regulated.In addition,the density sensing system(quorum sensing,QS)is also an important factor affecting the formation of bacterial biofilms.QS means that after bacteria reach a certain concentration in the environment,they can regulate the growth status,motility,drug resistance,BF formation,and virulence gene expression of the microbial population through the production of autoinducers(AI)and many other physiological characteristics.The LuxS/AI-2 quorum sensing system of bacteria involves the methionine metabolism pathway and can regulate multiple functions of bacteria by producing AI-2 signal molecules.At present,more than 70 kinds of bacteria have been found to have quorum sensing systems,and the LuxS/AI-2 quorum sensing system of poultry source PM has not yet seen relevant research,exploring the existence of avian PM quorum sensing system and what methionine metabolic pathway it uses can provide new ideas for the prevention and control of this bacteria.In order to study the prevalence of PM in chicken farms,the pattern of PM biofilm formation,and the types of quorum sensing systems,the following studies were carried out:1.Molecular Epidemiological Investigation of Proteus mirabilis from Chicken.In order to study the molecular epidemiology of chicken-derived PM,this study isolated and identified 52 PMs from diseased chickens from 2019 to 2020.Study the biological characteristics of the isolated strains such as pathogenicity,drug resistance,virulence factors and biofilm formation.The results showed that the PM separation rate was 15.29%;The test results of 16 antibiotics showed that 52 isolated strains were resistant to clindamycin,azithromycin,erythromycin,tetracycline and rifampicin.Multi-drug resistance analysis showed that 10 strains were resistant to all 16 drugs tested,and 19 strains could develop 15 levels of drug resistance;There are 23 strains of bacteria that can produce 14-fold drug resistance,indicating that the isolated strains have a wide range of drug resistance;The detection of 13 virulence genes of PM showed that all strains could detect hpmA,hpmB,rpoA,mrpA,fliL,zapA,ureC,atfC,atfA,pmfA,and the detection rates of ucaA and rsbA were 19.2%(10/5 2)and 48.1%(25/5 2),hlyA was not detected.The results of this study show that PM from chickens carries a variety of virulence genes,and the drug resistance patterns are diverse and increasingly serious,suggesting that the monitoring of PM in animal pathogenicity and drug resistance should be strengthened to reduce the risk of infection.2.The formation of Proteus mirabilis biofilm and its influencing factorsThe growth of bacteria is affected by the surrounding environment such as temperature,nutritional conditions,osmotic pressure and other factors.In order to explore the impact of these different conditions,this section has designed different temperatures,different cultivation times,different nutritional conditions and different osmotic pressures to explore its Influence on the formation of PM biofilm.The results of testing the biofilm formation ability of 52 strains of PM showed that all strains can form biofilms with medium strength(1.14?OD<2.28)and 15.38%(8/52)of the isolated strains have strong biofilm formation ability(OD?2.28),and the film-forming ability is stronger under the temperature of 25?.The strain Prom 34 with strong BF formation ability was selected to carry out the research on the influence of different culture conditions on the formation of PM biofilm.The results showed that glucose,galactose,lactose,sucrose,and sucrose were added to the M 9 medium with a final concentration of 0.2%.After fructose,mannitol,and maltose,BF can only be produced in the case of glucose and galactose;while adding the above-mentioned concentration and type of sugar to LB,all groups can produce BF,and there is no significant difference in the BF-forming ability of each group;Different concentrations(0.5%,1.0%,2.0%,3.0%and 4%)of sodium chloride or potassium chloride affect the formation of BF in PM.The test shows that the concentration is 0.5%and 1.0%sodium chloride or chlorinated Potassium significantly promotes the formation of BF(p<0.05),but as the salt ion concentration increases,the BF forming ability of PM decreases(p<0.05).Bacteria make a lot of changes to the water and solutes in the cell(including sodium ions and potassium ions K in the cytoplasm),so that they can adapt to osmotic pressures up to 100 times or more,but they can adapt to different conditions by absorbing and releasing sodium/potassium from the surrounding medium.The osmotic conditions will cause excessive ion concentration in the cell,which will have a negative impact on protein function,protein-DNA binding and protein synthesis.These effects will cause changes in flagella expression,cell membrane,quorum sensing and other related gene transcription.In order to further study the effect of different concentrations of sodium ions on the formation of PM biofilm,fluorescence quantitative PCR was used to detect the genes involved in the regulation of PM biofilm formation(sodB,filD,rsbA,umoC,ompF hfq,luxS.mtnN),and the results showed that the filD genes related to the activation of flagellar transcription were up-regulated by 1.22,5.95,9.5,respectively.However,the transcription levels of other genes(sodB,rsbA,umoC,ompF,hfq,luxS,mtnN)are not significantly changed.It is speculated that in a high osmotic environment,PM may inhibit the transcription of PM flagella to reduce its BF formation.The results of this study show that the isolated chicken PM has a higher BF formation ability.Only glucose and galactose can make PM form a more obvious biofilm,and the formation of BF decreases with the increase of osmotic pressure.3.Proteus mirabilis luxS/AI-2 type methionine metabolic pathway explorationLuxS/AI-2 quorum sensing system participates in the regulation of multiple functions including the formation of bacterial BF,In order to verify the presence of LuxS/AI-2 quorum sensing system in PM,this study used Vibrio harveyi BB 170 bioluminescence method to detect PM signal molecule AI-2,the results show that PM can produce self-inducing signal molecule AI-2 outside the cell.To explore whether PM's luxS and mtnN genes(also called pfs in E.coli)are involved in the synthesis of signal molecule AI-2,the luxS gene and mtnN of PM were supplemented to the luxS-deficient strain(DE17?luxS)and mtnN gene-deficient strain(DE17?pfs)of E.coli DE17,respectively,to construct E.coli heterologous complement strains DE17?luxS-PM-luxS and DE17?pfs-PM-mtnN,The AI-2 signal molecule detection of the heterologous complementary strains showed that all the above-mentioned complementary strains can produce the signal molecule AI-2,indicating that the luxS and mtnN genes of PM are involved in the synthesis of the signal molecule AI-2.In order to further verify the in vitro catalytic activity of PM luxS and mtnN,this study successfully constructed prokaryotic expression plasmids pCold I-PM-luxS and pCold I-PM-mtnN expressing PM's luxS and mtnN genes,transform into E.coli,and successfully express recombinant PM-luxS and PM-mtnN after induction.In order to verify its in vitro catalytic activity,PM-luxS and PM-mtnN were co-catalyzed by S-adenosyl homocysteine(SAH),the results show that catalyzing 1 mmol of SAH substrate can produce DPD of 228.29 ?mol,which further proves that the luxS and mtnN genes of PM are involved in the regulation of LuxS/AI-2 quorum sensing system.The results of this study show that PM strains from chickens have strong biofilm formation ability,carry a variety of virulence genes,and drug resistance patterns are diverse and increasingly serious.The monitoring of PM in animal pathogenicity and drug resistance should be strengthened to reduce the risk of infection.In addition,PM can generate signal molecule AI-2 through LuxS/AI-2 quorum sensing system.This research provides a new idea for PM prevention and control from the perspective of quorum sensing. |
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