| Aeromonas is ubiquitous in aquatic ecosystems and poses a significant threat to both aquaculture and human health.As an opportunistic pathogen of human-fish-zoonosis,Aeromonas can cause a variety of fish diseases,such as septicemia,ulcers,hemorrhagic fecal disease,etc.It is the main pathogenic bacteria group that infects freshwater fish,and these diseases have caused great financial losses to the aquaculture sector.Meanwhile,it may also cause acute gastroenteritis,colitis and other diseases in humans.In this study,water samples from Wanzhou Section of the Three Gorges Reservoir and commercially available healthy fish and dead fish were taken as research objects.Biological analysis of the bacteria was carried out by plate culture,physiological and biochemical identification,drug sensitivity test and 16 S r RNA molecular identification,and the pathogenicity and toxicity of the strains were analyzed by C.elegans and cell tests;Subsequently,the extraction process of lemon essential oil was optimized by single factor and response surface tests,the composition of essential oil was analyzed by GC-MS,the biological activity of the essential oil was also studied by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)radical scavenging method,inhibition circle size determination,methyl thiazolyl blue tetrazolium bromide(MTT)colorimetric method and protein immunoblotting(western blotting,WB);Finally,the antibacterial activity of lemon essential oil against Aeromonas was investigated,and the preliminary study of the antibacterial mechanism of lemon essential oil was carried out by measuring the minimum inhibitory concentration(MIC),biofilm,FDA fluorescence intensity and intracellular substance leakage.A total of 84 strains of bacteria were isolated from water and fish,including 14 genera such as Acinetobacter,Aeromonas,Pseudomonas and Bacillus.Further study on Aeromonas by morphological difference,physiological and biochemical characteristics and phylogenetic evolutionary tree,the 23 strains of Aeromonas were determined to contain A.vickeri,A.hydrophila,A.caviae,A.salmonicide and other species.It was found that Aeromonas was sensitive to penicillins and tetracycline drugs and resistant to aminoglycosides.Using the lifespan and locomotion behavior of C.elegans as evaluation indexes,23 strains of Aeromonas were established as infection models.The test results showed that A.hydrophila S2 and A.caviae A7 were two strains of highly pathogenic Aeromonas.Compared with other strains,the decline of locomotion behavior ability and the shortening of lifespan of C.elegans were more intense.Compared with standard strain1.2017,S2 infected normal human colorectal cancer cells,the cell shrinkage and deformation were more significant,and the apoptosis rate was higher after S2 treatment by flow cytometry.In this study,the optimal extraction process of lemon essential oil was optimized by single factor combined with response surface as follows: liquid-solid ratio 12:1,ultrasonic temperature 35℃,distillation time 138 min,sodium chloride addition 6.200 g/L.Under this process,the extraction yield could reach 2.810%.There were 102 compounds in essential oil,among which the content of alkenes was the highest,reaching 62.890%.Lemon essential oil has a good scavenging ability for DPPH free radicals.When the concentration of essential oil was 343 mg/m L,the scavenging rate reached 92.680%,which is close to VC.The antibacterial activity test showed that lemon essential oil had inhibitory effect on all the four tested bacteria,and the inhibitory effect on A.Vickerii was the best.The MTT assay combined with the half-inhibitory concentration(IC50)showed that lemon essential oil exhibited significant anti-cancer activity,especially on the proliferation viability of colorectal cancer cells,and WB assay on colorectal cancer cell cycle and autophagy-related proteins revealed that lemon essential oil inhibited the proliferation of colorectal cancer cells by cycle blocking and promoting autophagy.In this study,the mechanism of inhibiting Aeromonas by lemon essential oil was preliminarily investigated.The minimum inhibitory concentrations of lemon essential oil against Aeromonas S2 and A7 were 1.75mg/m L and 2mg/m L respectively.SEM showed that lemon essential oil destroyed the morphology of cells,and the cell surface was damaged and morphologic distortion occurred.The crystalline violet staining test revealed that the absorbance at 590 nm showed a decreasing trend with the increase of lemon essential oil concentration.Lemon essential oil inhibited the formation of Aeromonas biofilm,increased the permeability of cell membrane,led to the loss of luciferin,and reduced the fluorescence intensity of FDA.Finally,due to the action of lemon essential oil,the integrity of Aeromonas cell membrane was damaged,a large number of nucleic acid and proteins are released,and the growth and reproduction of the bacteria are blocked,and even lead to the death of the bacteria.In summary,pathogenic Aeromonas was present in water bodies in the Wanzhou section of the Three Gorges Reservoir and in fish bodies sold in Wanzhou,and lemon essential oil could inhibit Aeromonas.This study provides a scientific basis for further identification of the pathogenicity of Aeromonas,as well as for the prevention and control of the bacterium. |
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