| Enterococcus faecalis is one of the main pathogen causing Nosocomial infections and resistant to multiple antibiotics. Although it has caused worldwide concern in human infection, there is a little literature about Enterococcal infection in swine, Exceptionally,We made a study on characteristics and diagnosis method of Enterococcus faecalis isolates.Enterococcus faecalis strains (n=42) was isolated from clinical diseased pig in Hunan province from 2003 to 2008, The colonial morphology and the shape of Cram strain bear a close analogy to ATCC 29212 strain, Lancefield test showed that 97.6% (41/42) strains belongs to group D. Biochemistric characteristics of those strains are concordant with Enterococcus faecalis characteristics recorded in Bergey's manual of systematic bacteriology. The sequence analysis of 16S rDNA revealed that all Hunan strains share a high identity with reference strain ATCC 29212 (≥99.2%) and the homology ranged from 98.7% to 100% when comparing with selected reference sequences. The 16S rDNA sequence of Hunan isolates had highest identity with E. faecalis published in genebank by BLAST in NCBI. Finally,42 isolates were identified as Enterococcus faecalis and the sequencing result was submited to genebank. The phyletic evolution of all compared E. faecalis strains have no connection with the geographic distribution and host, more variation was observed in the 16S rDNA between E. faecalis and others four Enterococcus, including E.faecium,E.canis,E.avium,E.hirae, but variation region are conservative in those four species.PCR assay based on 16S rDNA was developed because of 16S rDNA sequencing is time-consuming and expensive for daily detection. Specific primer was designed by comparing the difference between E. faecalis and others'enterococcus. The PCR assay presented good specificity and sensitivity after compared detection result with 16S rDNA sequencing, used others genus as negative controls, amplification fragment sequencing and sensitivity testing. In addition, the method needs no special treatment of PCR templates when bacterial culture was detected and can detect sample directly。For 42 strains, the existence of esp,cylA,asa1,ace,efaA,EF0591,EF3314,gelE genes were detected by PCR scanning. EF3314 (100%) and efaA (92.86%) are commonly found in those E. faecalis, The incidence is similar to Italianate strains reported by Creti's, but obvious difference was discovered in ace gene,100%(Creti's report) and 59.52%(Hunan) respectively. The esp gene positive rate of Hunan strain (n=42) is 38.10%, the proportion is more like human strains reported by Creti and Shankar but differ greatly to swine strains (8%, Shankar). To the same strains, a-hemolysis more likely occurred in sheep blood agar plates, but in rabbits blood mainly presentedβ-hemolysis. Genotype is nonconsistent with phenotype in hemolysis test, which cylA+strains failed to decompose erythrocyte, cylA—strains decomposed erythrocyte. For most strains (34/42), genotype is consistent with phenotype in gelatin degradation test, which gelE+strains decompose gelatin but gelE-not. However, there are still 4 gelE+strains failed to decompose gelatin,4 gelE—strains decomposed gelatin. In addition, the multiplex PCR for virulence-related genes detection was developed to simplify detection procedure in this study.The growth curve determination of strain HN45 show that the bacterial comes to log phase growth after 3.5 hour cultured and continuing to 5.5 hour, and then reached plateau phase. HN45 had lower toxicity (LD50=5.76×109) to mice, but It is amazing that the bacterial was detected in heart, liver, spleen, lungs, kidney by Gram staining and PCR assay after intraperitoneal challenge 1 hour,2 hour later detected in brain and 3h in tail vein blood. Obvious pathological change was observed in heart, liver, lungs of dead mice, as such hemorrhage, hyperemia, the degeneration and necrosis of kidney epithelial cells and hepatic cells, hyperplasia of interalveolar septum and serous effusion.
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