Study On The Bionomics Of Enterococci Originated From Infected Swine

Enterococci are gram-positive bacteria, globular or orbicular-ovate, catenation.Since the recognition of Enterococcus as a separate genus in 1984, it increased from the original two species to forty-one species. Enterococci not only may cause the peritonitis, endocarditis, urethritis and meningitis of hospital patients, but also may cause animal's infections,such as swine, sheep, chicken,duck, and so on . And infected animal's enterococci may also infect human directly, enterococci in the pollution food may also cause human's food poisoning, therefore enterococci has grave significance in public health. Forty-two isolates collected from invasion swine of septicaemia, meningitis and arthritis in Xu Chang, Nanyang, Zhengzhou, Luoyang in Henan province, from the year 2003 to 2007, carried on the conventional morphology observation, tolerant experiment, biochemistry identification and whole-cell protein profiles (WCPP) analysis, and has carried on 16S rRNA and RAPD molecular level approach, as providing the reference for further studies on the multiplicity and clinical treatment and the public health significance of swine-derived enterococci.All of forty-two isolates are G+, orbicular-ovate or globular,and catenation length or short. Enterococci can grow under the condition of 6.5%NaCl bouillon, pH 9.6 glucose bouillon and 10℃, 45℃, 60℃30 min. Thirty-four strains of forty-two were identified successfully by Vitek-32 complete automatic bacterium appraisal system, the appraisal rate was 81.0%, inclueding E. faecalis 10 strains, E. faecium 2 strains, E. casseliflavus 22 strains,and eight isolates wrer not appraised at the specific level; Whole-cell protein profiles analysis result indicated that, two complete different kinds of cluster type of whole-cell protein strap of forty-two isolates. On these grounds,forty-two isolates may divide into two kinds of types, their molecular weight difference of electrophoresis bandings mainly in 97KDa, 90KDa, 67KDa and 46KDa, one kind of type has 90 KDa bandings of the protein molecular weight, another kind of type has 46KDa, 67 KDa and 97 KDa three bandings.Therefore, the 90KDa banding and 46KDa, 67 KDa and 97 KDa three bandings may regard as the mark differentiated banding of the two kinds of enterococci. 16S rRNA gene sequence analysis has became the standard method of bacterium genera and genus identification and classification.We are using the bacteria universal 16S rRNA primer to amplify complete16S rRNA gene of enterococci of infected swine , then cloning to the pTG19-T vector, sequenced,the 16S rRNA gene sequences were compared with correlated specific sequences deposited in GenBank by the software of BLAST. The result indicate that, the 16S rRNA gene sequences of forty-two enterococci have highly homology with various kinds of enterococci in GenBank . Carring on the sequence homology analysis of forty-two enterococci of infected swine and thirty-two kinds of 16S rRNA sequences of enterococci from GenBank using the DNAstar software, all of forty-two isolates were identified at the specific level, the homology of 12 strains of enterococci of infected swine with 4 E. faecalis mode strains(AF515223,AJ301831,EU728747,NC₀04668)was between 99.3%～99.9%; the homology of 30 strains of enterococci of infected swine with 3 E.faecium mode strains(EU547780,DQ411813,Y18294)was between 99.3%～100%. All of 16S the rRNA gene sequence of forty-two enterococci were registed to GenBank, the accession number of HN-N1～HN-N42 was FJ378656～FJ378697. Comparing with the publicated species specificity probe sequences of enterococci, all of 16SrRNA gene sequence of forty-two enterococci included species specificity probe sequences of E.faecium and E.faecalis.forty-two isolates were identified by the technique of random amplified polymorphic DNA(RAPD)with 19 random primers . The result indicated that, better polymorphism straps wree obtained by amplifying all the isolates with 4 random primers, the number of each strain DNA amplification straps were between 1to10, the fragment size of different strains were between 200 bp to 4000 bp, all of forty-two strains observed 673 limpid stripes, each primer generate 168.3 amplification fragments average,each enterococcus generate 4 amplification fragments average. The similarity coefficient and genetic distance index was calculated by SPSS13.0 software, drawing phylogenetic tree with the method of nearest neihbor. The result indicated that, the genetic distance index of HN-N3 and HN-N6 was 0.000, the two strains supposed maybe the homologization strain.At level 23 of rescaled distance cluster combine of cluster analysis, 42 tested strains were clustered into 2 clustering groups, the identification result has completely coincidence with 16SrRNA, indicated that at level 23 of rescaled distance cluster combine of cluster analysis can identify enterococcal strains. At level 17 of rescaled distance cluster combine of cluster analysis, 30 E.faecium were clustered into 7 clustering groups; At level 19 of rescaled distance cluster combine of cluster analysis, 12 E.faecalis and 3 positive referrence strain(sATCC29212,ATCC33186,CMCC(B)32223)of E.faecalis were clustered into 4 groups.