| ABSTRACT Enterococci are globular or orbicular-ovate, catenulate gram-positive bacteria. Since Enterococcus was separated from Streptoococcus as a new genus in 1984, its members have increased from the original two species to forty-one. Enterococci may not only cause peritonitis, endocarditis, urethritis and meningitis in hospital patients, but may lead to infection in animals such as swine, sheep, chicken, duck, and so on. Enterococcus that infects animals may directly infect humans, and Enterococcus-contaminated food may also cause food poisoning, therefore Enterococcus is of great significance in public health. During the years 2003 to 2008,43 Enterococcus-like isolates were obtained from dead pigs, respectively from farms in Xuchang, Nanyang, Zhengzhou and Luoyang of Henan province, which showed symptoms of septicaemia, meningitis and arthritis. Conventional morphological observation, tolerance experiments, Vitek-32 automatic biochemical bacteria identification and whole-cell protein profiles (WCPP) analysis were carried out, followed by the 16S rRNA and RAPD molecular approaches. Upon these bases, virulence factor genes carried by those strains were investigated and mice peritonitis models were set up with selected isolates, and further analysis was done on the relevance between pathogenicity of the isolates and virulence genes, virulence phenotypes, and antibiotic tolerance, so that information could be provided on epidemiology, pathogenicity, and eventually prevention and control of swine enterococcal infections.The conventional morphological observation and tolerance experiments showed the characteristics of the isolates were:gram-positive, globular or orbicular-ovate, catenulately arrayed, tolerant to 6.5% NaCl broth,pH 9.6 glucose broth, and temperatures of 10℃,45℃and 60℃in 30 minutes.35 among the 43 isolates were identified to species level by the Vitek-32 method, with a result of 11 strains of E. faecalis,2 of E. faecium and 22 of E. casseliflavus, whereas 8 others remained unidentified. The whole-cell protein profiles (WCPP) analysis presented a different conclusion that the predominant difference among the 43 isolates dwelled at 97KDa, 90KDa, 67KDa and 46KDa bands of protein molecular weight in electrophoresis. Accordant to the reference strains ATCC29212 and ATCC33186, thirteen isolates presented the 97KDa band, whilst the others had 46KDa,67 KDa and 97 KDa ones, implying another group of Enterococcci.The results of 16S rRNA gene sequence analysis indicated a high homology between the trial strains and those registered in the GenBank database. By comparison through the software DNAStar, all the 43 clinical isolates were successfully identified at the species level, among which 13 trial strains showed homology of 99.3%～99.9% to 4 E. faecalis type strains (AF515223, AJ301831, EU728747, NC004668), and the other 30 isolates except HE25 presented homology of 99.3%～100% to 3 E. faecium strains (EU547780, DQ411813, Y18294), the HE25 strain showing 99%～99.4% homology the other 29 identified E.faecium strains. The 16S rRNA gene sequences of forty-two enterococci(HN-Nl to HN-N42) were registered to GenBank, with accession numbers FJ378656 to FJ378697 respectively.Nineteen random primers were synthesized and the genomic polymorphism of the isolates was analysed so as to clarify in genomic level the identification results, and then to collect reference information for epidemiological purpose. It showed that all the isolates performed good polymorphism straps when amplified with 4 of the random primers. The similarity coefficients and genetic distance index were calculated by SPSS11.5 software, and the phylogenetic tree was drawn with the method of nearest neighbor. The result indicated that the genetic distance index of HN-N3 and HN-N6 was 0.000, suggesting an identical origin of them. When the rescaled distance cluster combine was 21, the test strains could be identified to species level, and they dropped into four clustering groups, namely E.faecalis, E.faecium and E. avium groups, and the forth which had a single member of HN-25. Under a rescaled distance cluster combine of 19, the cluster of E. faecalis could be further divided into four subgroups, whereas the E. faecium members except HN-25 belonged to one large cluster.Genes of twelve virulence factors, which were predominant in E. faecalis and E. faecium, were detected on test strains of different origins -clinically infected pigs, fresh pork and 15 healthy-looking feces. Generally, E. faecalis strains carried more virulence factor genes than the E. faecium ones(7.83:3.73); clinically originated strains carried the highest numbers of virulence genes, pork strains being the second and fecal ones the fewest. Among the clinical E.faecalis isolates, the virulence genes gelE, efaA and sprE were 100% detectable, and the percentage of ace,cylA and esp genes were 84.6%,84.6% and 69.2%, which were much higher than those of other origins; likewise, the percentages of virulence genes cylA and gelE (83.3% and 66.7%) in clinical E.faecium isolates were also higher than the other counterparts.In the mice peritonitis models, pathogenicity of E. faecalis strains were more potent than the E.faecium bacteria, and different E. faecalis isolates also varied in their capacity of infection, among which HN-41was the most potent strain (LD50 of 106.01/ml), while HN-1 the weakest (LD50 of 107.01/ml). Similarly, E. faecium HN-32presented a higher LD50 than HN-15 did. Analysis implied a positive correlation between the pathogenecity of an isolate and its virulence gene numbers; different kinds of genes contributed unequally to the bacterial pathogenicity; the influence of virulence phenotypes on pathogenicity depended on the species of Enterococcus, and within the species theβ-hemolysis phenotype was more predominant than glutin liquefaction; the antibiotic resistance of an isolate, by contrast, did not show clear correlation with its potential, which remains to further study.
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