Study On The Adhesion Of Enterococci, Citrobacter And Bacillus To The Carp Foregut Mucus In Vitro

To investigate the adhesion characteristics of bacteria to carp(Cyprinus carpio) intestine and established a efficacious screening method of carp intestinal probiotics. Used immobilized mucosal model in vitro,combined with the isotope tracing method,the adhesion characteristics and mechanisms of nine bacteria and the inhibition to the pathogens were investigated.Twenty bacteria were isolated from the cultivation pool,aquarium of Animal Micro-ecology Lab and the gut of carp in teaching farm of Sichuan Agricultural University(CM2,WP1,WP2,WP3,GL1,GL2,CI1,WL1,C3,Z2, D1,D2,D3,D4,E1,E2,E3,L1,L2,L3),eight bacteria were stored byAnimal Micro-ecology Lab(JS01,D9401,ND1,ND2,ND3,ND4,ND5,ND6) were totally used in this test.By inhibition to morbigenous Escherichia coli and Aeromonas hydrophila in vitro and heat treatment,five bacteria were selected which could resist heat somewhat(Z3,C3,L2,E2,D2).By 16S rRNA sequencing,they were identified as Bacillus,Enterococcus and Citrobacter.In this study,the isotopeγ-³²p-ATP was used as the tracing material,so the influence on the growth of bacteria was invested.The result showed,after addedγ-³²p -ATP,the number of the bacteria were declined,but the difference is unnotable,even the number of some bacteria rised.Generally,all the bacteria were influenced byγ-³²P -ATP,but the number of them were between 10⁸ to 10⁹ cfu/ml.The adhesion characteristics of Enterococci,Citrobacter isolated from carp intestinal and Bacillus isolated from water to the carp foregut mucus including the characteristics of mucus receptor,lectin and the interaction between pathogens and Bacillus,Enterococci and Citrobacter were investigated. Results showed that the relative adhesion population of homologous Enterococci were higher than heterogenous bacteria'(p＜0.01).The relative adhesion population of homologous Citrobacter and the Bacillus differed unnotable(p＞0.05).The relative adhesion population of pathogens which has the stronger pathogenicity is higher than others(p＜0.01).After the modification with proteolytic enzymes or sodium metaperiodate,The relative adhesion percentages of five bacteria were declined extreme significantly after they were treated with sodium metaperiodate(p＜0.01),but it was affected less by the proteolytic enzymes treatment(p＞0.05).After the modification with proteolytic enzymes or sodium metaperiodate of mucus.The relative adhesion percentages of part of five bacteria were declined significantly after the mucus treated with proteolytic enzymes(p＜0.05),but it was affected significantly by the sodium metaperiodate treatment(p＜0.05).The relative adhesion population were different led by the different of lectins on the surface of bacteria.So to be the homologous bacteria of carp intestine,the relative adhesion population of Enterococci L2 was higher than the others extremely(p＜0.01).According to the interactions such as exclusion,competition,displacement,part of the five bacteria can reduce the relative adhesion percentages of pathogens significantly(p＜0.05),and the relative adhesion percentages of pathogens were improved sometimes(p＜0.05),this was because the bacteria changed the surface construction of the mucus.In conclusion,all of the bacteria had the ability to adhere to carp foregut mucus immobilized on the cell culture plates.The main lectins existing on the cell surface of the five bacteria are glycoprotein compounds.The special receptors of the microorganisms are protein compounds.The inhibition of displacement to the pathogens is best,but the disparity was led by the different bacteria.