| Campylobacter jejuni is a gram-negative,elongated,spirally coiled,and microaerobic bacteria that exists in the gastrointestinal tract of many poultry and mammals.According to reports,Campylobacter caused 96 million cases of disease in 2010,making it the second largest food-borne pathogen in the world.At present,the main method to prevent and control Campylobacter jejuni in poultry breeding is to add antibiotics to the feed.However,with the excessive use of antibiotics,the multi-drug resistance of Campylobacter jejuni continues to increase.Therefore,there is an urgent need for new prevention and control methods that can replace antibiotics.Nowadays,there have been many scientific and clinical studies on the immune activity,antioxidant activity and antibacterial activity of garlic essential oil compounds,as well as the protective activity against infection and inflammation.Garlic bulbs contain a large amount of y-glutamyl cysteine,which can be oxidized into an inactive derivative of allicin,which is released by allicinase in the process of shredding or cutting,and further metabolized into an active derivative of garlic Element(diallyl thiosulfate).Allicin can quickly decompose into sulfur-rich compounds,including diallyl sulfide(DAS),diallyl disulfide(DADS)and diallyl trisulfide(DATS).These organic sulfur compounds are considered to be the active ingredients in plants that inhibit the growth of bacteria and are the main source of the antibacterial activity of garlic essential oil.The number of sulfur atoms determines the antibacterial ability of garlic essential oil.DATS is a trisulfide compound with a molecular weight of 162.Compared with DAS and DADS,DATS has more sulfur atoms,indicating that it has stronger antibacterial activity.In this study,the natural compound diallyl trisulfide(DATS)in garlic was used as the object of the research.The antibacterial ability of DATS on Campylobacter jejuni and the effect on the biological characteristics of Campylobacter jejuni were measured in vitro to explore the effect of DATS on the jejunum.DATS was microencapsulated in order to be able to apply DATS to the chicken breeding process as a method to prevent and control Campylobacter jejuni infection.1.The effect of DATS on the biological characteristics of Campylobacter jejuniFirstly,the in vitro bacteriostatic ability of DATS against Campylobacter jejuni was studied.Four reference strains of Campylobacter jejuni(81-176,11168,12662,33560)were selected to determine the minimum inhibitory concentration(MIC).The results showed that DATS had an effect on Campylobacter jejuni.The MIC is 32-64 μg/mL.Subsequently,the chicken isolates with a resistance background of 5-6 resistance were selected for MIC determination.The results showed that DATS also had a good antibacterial effect on the chicken isolates of Campylobacter jejuni,and the MIC range was 1-64 μg/mL.The growth curve measurement of Campylobacter jejuni 81-176 showed that the number of viable bacteria can be reduced by 1.5 log at 16 μg/mL DATS,and the number of viable bacteria can be reduced by 2 log at 32 μg/mL DATS.Secondly,the effect of DATS on the biological characteristics of Campylobacter jejuni was studied.The results of motile experiments showed that when 16 μg/mL DATS was added,it had no significant effect on the motility of Campylobacter jejuni,but when 32 μg/mL DATS was added,the motility of the treatment group was significantly lower than that of the control group.In chemotaxis experiments,it was shown that DATS is not a harmful substance to be avoided by Campylobacter jejuni.The results of biofilm experiments showed that DATS of 32 μg/mL DATS had no effect on the biofilm formation of Campylobacter jejuni,while 64μg/mL DATS could significantly reduce the biofilm formation of Campylobacter jejuni.Subsequently,the effect of DATS on the adhesion and invasion of Campylobacter jejuni was studied at the cellular level.Cytotoxicity experiments show that DATS has no inhibitory effect on the growth of Caco-2 cells.The results of the adhesion experiments showed that after treatment with 32 μg/mL DATS,the amount of bacteria attached was reduced by 25%compared to the control group.The results of the invasion experiment showed that after treatment with 32 μg/mL DATS,the amount of invaded bacteria was reduced by 80%compared to the control group.Finally,the effect of DATS on the morphology of Campylobacter jejuni cells was explored using scanning electron microscopy.The results showed that after DATS treatment,the morphology of Campylobacter jejuni cells deformed,shrank,and the cell membrane was damaged,and the degree of damage showed a dose-dependent relationship,it can cause cell disintegration at high concentrations.2.The preliminary exploration of DATS on the antibacterial mechanism of Campylobacter jejuniThe antibacterial mechanism of DATS on Campylobacter jejuni was preliminarily defined.In Raman spectroscopy experiments it were shown that compared with the control group,DATS destroys the spatial structure of the protein in Campylobacter jejuni,causing nucleic acid and protein damage,and this damage presents a dose-dependent relationship.The RNA-seq results showed that after 16μg/mL DATS treatment,94 genes were up-regulated and 116 genes were down-regulated.Among them,14 genes were related to plasma membrane,6 genes were related to periplasmic space,and 4 genes were related to periplasmic space.Genes are involved in transport across membranes.The results of KEGG pathway analysis showed that the main functions of DATS were nitrogen metabolism,ABC transport system,flagella system and cell membrane formation.The qRT-PCR results were consistent with those of RNA-seq,The deletion of the five genes related to the ABC transport system was constructed and verified that after these genes were deleted,the DATS changed the MIC value of the deleted strain.The results showed that the deletion of these genes can reduce the MIC value by 2 to 4 times.Based on the above results,we preliminarily summarized the effect of DATS on Campylobacter jejuni 81-176.First of all,the antibacterial groups of DATS include trisulfide bonds and reactive oxygen species produced by olefinic bonds.When DATS comes into contact with bacteria,the expression of genes related to the bacteria including FlgB,FlgK,FlgL and the ABC transport system will be up-regulated.Then when DATS enters the bacterial cell,it will destroy the DNA and RNA in the bacterial body,and at the same time change the phospholipid structure in the cell membrane,thereby changing the cell morphology.After 8-24 hours of exposure,it will inactivate the bacteria.3.DATS microencapsulation and its preliminary application in chicken breedingIn order to better apply DATS to the chicken breeding process,sodium alginate and chitosan were used to encapsulate DATS,and DATS-containing microcapsules were successfully prepared using freeze-d rying technology.The embedding rate of DATS in microcapsules was 70%～75%in element analyzer.It was observed that the prepared microcapsules are spherical under scanning electron microscopy and belong to micron-sized microcapsules.The release curve results show that the microcapsules can release the embedded DATS at 1.5 h.Furthermore,the antibacterial ability of the microcapsules was evaluated,and the results showed that sodium alginate has certain antibacterial ability,but the antibacterial ability is not obvious at this concentration(p>0.5),and DATS has the main antibacterial ability in the microcapsules.In animal experiments,after the chickens were challenged by Campylobacter jejuni,they would first excrete a large amount of bacteria,and then the number of colonization gradually increased.At 25 days of age,the positive rate reached 100%,and the number of colonization became stable.The results of quantitative detection showed that the amount of campylobacter excretion in chickens and the load of campylobacter in the intestine and intestinal contents of chickens were all after feeding DATS and microcapsules at 28 days,35 days,and 42 days.Significantly reduced. |
PDF Full Text Request