Detection And The Correlation Study On Virulence Genes Of Enterococcus Faecalis Isolated From Different Animals

Enterococcus is a Gram-positive,commensal bacterium including more than 40 species.Among them,Enterococcus faecalis as one of the most important opportunistic pathogens,which can cause life-threatening infections in humans,such as Endocarditis,sepsis,urinary tract infections,meningitis and other infections.The key factor for opportunistic infections is a variety of virulence factors involved in the infection and unique mechanism of drug resistance.This study used PCR method to detect the carrying rate of 9 virulence factors in 287 strains of Enterococcus faecalis.Ascertain the assortment of virulence factors and the relationship between the pathogenicity of E.faecalis isolated from different animals and drug resistance by infected mice to study the pathogenicity of isalates with different virulence gens.Drug resistance group and drug sensitive group were selected to test the growth curve,LD₅₀,determination of relationship between CFU and OD₆₀₀,pathogenicity changes of infected mice.ERIC-PCR method combined with statistical analysis were used in this experience to discover the genotype of Enterococcus faecalis isolated from different animals.Atypical resistant strain?Erythromycin sensitive strains?were selected to induce resistance test in vitro to observe the effect of drug resistance change on virulence genes in different animals.The result is as follows: 1.Distribution and sequence analysis on virulence factors in different isolatesResult were: ace?6.27%?,asa1?6.97%?,asa373?5.92%?,efaA?4.18%?,cylA?5.92%?,gelE?42.86%?,sprE?20.91%?,esp?11.85%?and hyl?6.62%?.The result of virulence genotypes distribution shows that there were 104 strains did not carry any virulence factor involved in this study,23 strains carried 3 kinds of them,9 carried 4 kinds of them,1 carried 5 kinds of them and 2 carried 6 kinds.The homology rate of the same virulence gene carried by different animals were above 95%.2.Pathogenicity of isolates with different virulence genotypesThe representative strains with different virulence factors were selected,pig origin JZ15?gelE,hyl?,chicken origin J13?asa373,gelE?,bovine origin D1?ace,gelE,sprE?,sheep origin 62?ace,asa1,cylA,gelE,sprE?,horse origin M14?ace,gelE,sprE,esp?and dung beetle origin S3?gelE,sprE,esp?were used to infect the mice and the hemolysis test.Observed the death time,death numbers and the pathological changes after death,result shows that pig origin were all dead by 16 hour,3 died in the dung beetle and 1 died in sheep origin,bovine origin and chicken origin respectively.No death in horse origin.After 48 h,no death occurred in each group.All the tissues of death mice were sectioned and observed,result shows that each tissue showed varying degrees of pathological changes,mainly characterized by hepatic steatosis,hepatocellular necrosis,myocardial waxy necrosis,splenic infarction,renal interstitial hemorrhage,pulmonary lymphocytic infiltration,encephaledema,neuronophagia and necrotic foci.There were no obvious hemolysis loops in each group in hemolysis test.The results of pathogenicity test show that,pathogenicity from strong to weak were: pig,hung beetle,sheep,bovine,chicken and horse.And all the group contain gelE gene,but pathogenicity of pig was most strong,which suggest that hyl played the most important role in Enterococcus faecalis infection.3.Relationship between drug resistance and virulence of isolates from different animalsDrug resistance group pig origin JZ15 and hung beetle origin S1,and drug sensitive group JZ3 and S3 were selected to test the growth curve,LD₅₀,determination of relationship between CFU and OD₆₀₀,pathogenicity changes of infected mice.Results shows that 4 strains get a logarithmic growth phase on 3-4h,and a flat period on the 7th hour.The LD₅₀ of 4 group were: JZ15 LD₅₀=7.079×109;S1 LD₅₀=4.467×10¹⁰;JZ3 LD₅₀=1.778×10¹⁰;S3 LD₅₀=7.079×10¹⁰.There was no obvious difference between each group from the result of relationship determination.Combining the pathological section,result can be obtained that the toxicity of resistant group was stronger than the non-resistant group in same animal origin,and in same level of drug resistant,swine-derived stronger than dung beetle-derived.4.Preliminary study on the relationship between drug resistance and virulence ofEnterococcus faecalis from different animalsERIC-PCR method combined with statistical analysis were used in this experience to discover the genotype of Enterococcus faecalis isolated from different animals.The result shows that isolated from the same animal were clustered into one class,strains with similar virulence genes or drug resistance genes were clustered into one class,and isolated from the same animal of the same area were clustered into one class.which suggest that there was risk of opportunistic infections.The results of inducing resistance test in vitro showed that the increase of drug reistance could change the combination of virulence gene in Enterococcus faecalis,indicated that there was a correlation between drug resistance and virulence.