Multilocus Sequence Typing And Virulence Analysis Of Enterococcus Faecalis In Chicken

Enterococcus faecalis is not only a probiotic that enhances human disease resistance and animal performance,but also a conditional pathogen that causes zoonotic diseases.It is not clear whether this is caused by the virulent difference or colonization sites difference of Enterococcus faecalis.In this experiment,multilocus sequence typing(MLST),virulence genes and virulence analysis were used to investigate the relationship between genotype and virulence of Enterococcus faecalis isolated from chickens.This study will lay the foundation for the safety assessment of Enterococcus faecalis as probiotic and the prevention and treatment of Enterococcus faecalis as pathogen.Enterococcus faecalis isolates were be classified by multilocus sequence typing(MLST)referring to the http://pubmlst.org/efaecalis/.The results were analyzed and a Splits Tree was drawn using Phyloviz software.A total of 17 STs were obtained from 71 isolates by MLST,among which 2 STs were new type.Two clonal complexes and 12 independent STs were formed by the 17 STs.Among the 71 isolates,ST273 accounts for 26.8%(19 strains),ST202 15.5%(11 strains),ST16 11.3%(8strains),STnew1 9.9%(7 strains),ST245 and ST466 5.6%(4 strains of each ST),ST180 and STnew2 4.2%(3 strains of each ST),ST631,ST63 and ST81 2.8%(2strains of each ST),ST409,ST474,ST228,ST82,ST746 and ST21 1.4%(1 strain of each ST).The ST273,ST202,and ST16 were more advanced STs.The 35 isolates from egg shell showed 11 STs.The 36 isolates from liver showed 12 STs.ST273 and ST202 are the common dominant STs of egg shell source and liver source.Five virulence genes were amplified by designing specific primers and PCR to investigate the number of virulence genes carried by 71 Enterococcus faecalis isolates.Detection rate of virulence genes was 94.37%(67/71).The detection rates of five virulence genes were different,among which the two high were 85.92% for ace and84.51% for asa1,11.27% for cylA,the two low were 2.82% for hyl and agg.Three virulence genes were simultaneously detected in 6 strains(8.45%),two virulence genes were simultaneously detected in 54 strains(76.06%),of which the main virulence gene combination was asa1+ace;only a virulence gene was detected in 7strains(9.86%),and no virulence genes were detected in 4 strains.The results suggested that the number and species of virulence genes carried by different strains are different.Virulence of 8 Enterococcus faecalis isolates with different sources,genotypes and pathogenic genes were analyzed by the embryo lethality assays to explore the relationship between the virulence and the source,genotype and pathogenic genes.The results showed that Enterococcus faecalis(bacterial liquid concentration 730CFU/mL,dose 0.2 mL/egg)inoculated in the allantoic cavity blocked and weaken the chicken embryo,caused obvious congestion and hemorrhage and death of chicken embryo.The mortality was 0%-75%.Moderately virulent strains are STnew2 and ST273 mainly derived from the liver of sick chickens,and the embryonic mortality rate(43%-75%)of them are greater than 40%;Avirulent strains are ST 245,ST202 and ST16 mainly derived from the eggshell,and the embryonic mortality rate(0%-38%)of them are less than 40%.The results suggest that the embryo lethality assays can be used for the virulence analysis of Enterococcus faecalis,and the virulence of different strains is different;the virulence of Enterococcus faecalis is related to the source and genotype.