| The luminescent bacteria are able to generate a unique optical signal by synthesizing the luciferase and its corresponding luminescent substrate encoded by the lux CDABE gene cluster carried by itself.Due to its low autofluorescence background,non-destructive,and easily detectable,lux CDABE gene has been widely used as a reporter gene for model studies of various microorganisms in food and the environment.P.aeruginosa is a conditional pathogen as its strong environmental adaptability,rapid rate of division,and low requirements for growth environment(nutrition,humidity,etc.).P.aeruginosa is widely distributed in the environment and causes safety problems such as food spoilage or human poisoning.In this study,recombinant luminescent P.aeruginosa were constructed based on the lux CDABE gene cluster derived from Photobacterium luminescens.The author then carried out studies on its luminescent properties,antibacterial evaluation,and application in the investigation of biofilm formation mechanisms.The specific research results are as follows:(1)Based on the lux CDABE from Photobacterium luminescens,two recombinant P.aeruginosa strains PAO1-CE and PA27853-CE that successfully expressed bioluminescence were developed.The optimum culture enviroment for the illumination of the two biosensors is 37?,p H at 7.0 and a Na Cl concentration of 1.0 mg/m L.The results show that PAO1-CE and PA27853-CE have good luminescence stability and room temperature stability.Since the luminescence expression of PAO1-CE was about 5 times that of PA27853-CE,PAO1-CE was selected for subsequent studies.(2)Using PAO1-CE as a tool,the model for quantitative detection of sensitive antibacterial substances of P.aeruginosa against Str and Car was established are exponential model:y=96.1138(1-e(-0.1393x)),y=95.2735(1-e(-0.1362x))(Sensitive range:1.00?50.00?g/m L,1.00?47.50?g/m L),while the quantitative model against Amp,Ter,Tmp and Cip were:y=42.8581+0.0283x,y=41.7624+0.5885x,y=14.311+0.436x,y=2.0228+137.28x(Linear range:56.25?1800.00?g/m L,6.75?100.00?g/m L,500.00?2000.00?g/m L,0.05?0.7?g/m L).Except for Amp,the determination coefficients R~2 of each fitted model is greater than 0.98.Antibacterial effects of Str,Car,Amp,Ter,Tmp and Cip on P.aeruginosa were determined by MIC,growth model parameters,ATP levels,AKP enzyme activity,intracellular/external membrane permeability,cell membrane integrity and FESEM.It was concluded that six antibiotics have an effect on the normal metabolism and destroyed the of membrane and cell wall integrity P.aeruginosacell.PAO1-CE can be used as a rapid and sensitive test tool for evaluating the sensitivity of P.aeruginosa to new antibacterial compounds.(3)PAO1-CE was used to quantitatively evaluate the antibacterial effect of SAN and EGN on P.aeruginosa.The results showed that the mathematical models of PAO1-CE quantitative detection of SAN and EGN were y=11.3398+0.5822x(R~2=0.9321)and y=17.856+0.5862x(R~2=0.9665),indicating that the toxic effects of SAN and EGN on P.aeruginosa were closely related to the concentration of action,confirming the feasibility of the PAO1-CE for quantitative assessment of the cytotoxic effect of a substance on P.aeruginosa.By further exploring the bacteriostatic mechanism of SAN and EGN against P.aeruginosa,it was concluded that SAN inhibits P.aeruginosa by destroying the integrity of its cell membrane.EGN can change the permeability of the inner and outer cells of P.aeruginosa.However,it does less damage to the integrity of cell membranes than SAN.Both SAN and EGN have a good inhibitory effect on P.aeruginosa.(4)The biological conditions of P.aeruginosa biofilm formation model were determined by measuring the biofilm formation of P.aeruginosa under different media types(LB/TSB/M9),different temperatures(25?/37?)and different inoculum concentrations(10~6/10~7/10~8 CFU/m L).The biofilm mass was determined by the crystal violet method after inoculation at a concentration of 10~6 CFU/m L in LB medium at 37?for 3 days or 7 days.Under the optimized modeling conditions,the six antibiotics and the two nanoparticles showed effective inhibition of P.aeruginosa biofilm formation at the concentrations of 1/2 SICs and SICs.The results of silver staining and FESEM observations confirmed the ability of various substances to inhibit the biofilm formation of P.aeruginosa.Based on the result of crystal violet method,the best inhibition of biofilm formation among the eight substances were Str and Car.Therefore,Str and Car wrer selected to explore the quantitative relationship between the IR%and the amount of biofilm formation,and the mechanism of inhibition of P.aeruginosa.(5)When the linear range of 0?14?g/m L,the mathematical model of predicting biofilm mass(3 days or 7 days)of Str using IR%(5 h)by Sigma Plot 10.0 were fitted as y=-0.0412x+3.0817(R~2=0.8874)and y=-0.0265x+1.4294(R~2=0.9072),while the Car were:y=-0.0538x+3.3640(R~2=0.8972)and y=-0.0202x+1.1796(R~2=0.8667).The results of specific promoter-reporter assays and RTq-PCR validation shows that Str and Car could reduce the formation of P.aeruginosa biofilm by inhibiting the expression of the extracellular polysaccharide synthesis gene psl M/pel A/alg A/bdl A/ppy R.Therefore,the mechanism by which Str and Car inhibited the biofilm formation of P.aeruginosa was to inhibit the formation of its extracellular polysaccharides Psl and Pel,change bacterial chemotaxis,elastase activity and bacterial motility and swimming. |
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