| Alcaligenes faecalis(A.faecalis)is a subgroup of Alcaligenes.At present,there have studies shown that the extracellular serine protease(Esp)contained in the bacterium has the function of dissolving the body wall of insects,especially the nematode.Therefore,Alcaligenes faecalis is considered to have the potential of microbial soil bacterial agent.In this study,the suspected bacteria were isolated in tick collected from natural grazing sheep in Hulunbuir,Inner Mongolia,Normal method and molecular biology were used for species identification and analyze the virulence factors.The details are as follows:1.Alcaligenes faecalis IMH isolation and identificationAfter the tick was completely crushed,the broken tissue fluid was inoculated with the LB high salt agar medium for bacterial isolation and culture.Alcaligenes faecalis IMH was identified by the test of Gram staining and physiological and biochemical tests.This was in accordance with the molecular identification results.Based on the results of the 16S rRNA sequence,this strain was the closest to Alcaligenes sp.CSB1(GenBank accession number KX289442.1;homology 99%,based on 16S rRNA),which is one of a group of bacteria isolated from tomb soil.A new Alcaligenes faecalis was successfully isolated and renamed Alcaligenes faecalis IMH(A.faecalis IMH).The 16S rRNA gene was reregistered in GenBank,and the registration number was LC213619.1.2.Alcaligenes faecalis IMH biological characteristics analysisIn this study,the Alcaligenes faecalis IMH characteristics included hemolytic,antibiotic sensitivity.The results was that Alcaligenes faecalis IMH can erythrolysis to 5%defibrinated sheep red blood cell.The drug sensitivity analysis showed it were sensitive to gentamycin,erythromycin,cefotaxime,amoxicillin,ciprofloxacin and resistance to sulfamethoxazole,kanamycin,streptomycin,penicillin and amphotericin B.They were motility and tolerance to 10%NaCl that characteristics of high salt tolerance.Saccharose,mannitol,maltose,lactose,glucose,indolis,nitrate(reduction),gelatin test showed negative.Urea,oxidation type and fermentation test,simon citrate test,oxidase test results positive.3.Study on pathogenicity of Alcaligenes faecalis IMH against miceThe results of A.faecalis IMH test in mice showed that mice with high dose that is1.0×10~111 CFU/mL intraperitoneal injection had transient blindness after infection of about15 h.The mice showed a different degree of depression,loss of appetite,temperature and weight loss significant.After 31 h injection of bacteria,the mice were dead,dissecting dead mice.The main clinical pathological changes of the tissues and organs are the yellow gelatinous substance of the small intestine;the splenomegaly and the atrophy of the brain;there is no obvious pathological changes in the heart,liver and lungs.The tissue section showed that the necrosis of the brain cells,the hemorrhage of the meninges,the moderate swelling of the liver cells,the small nuclear disintegration of the lymphocytes in the white marrow,but no obvious change in the cardiac myocytes.4.Cloning and correlation analysis of main pathogenic factors of Alcaligenes facealis IMHSequence analysis of Esp that a major pathogenic factor of Alcaligenes faecalis IMH.The esp gene is 2.8kbp.The physicochemical properties of the protein indicate that Esp is a stable hydrophilic protein.The secondary structure of the protein is predicted to be random coil,accounting for 48.97%of the total structure,alpha helix is 22.26%,extended strand is22.48%,beta turn is 6.30%.Compare virulence factor Esp tertiary structure of A.faecalis IMH with A.faecalis ZD02,at the 96-98 amino acid and 106-111 amino acid are alpha helix,302-309 amino acid is beta fold,and 213-219 amino acid is random curls.The prediction of the tertiary structure of A.faecalis IMH virulence factor Esp,at the 63-351amino acid location,there is S8 family autotransporter active region and at 392-917 amino acid location,there is autotransporter superfamily active region. |
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