Detection Of The Vancomycin-resistant Phenotype And Genotype In Enterococci And Study Of Its Homology In Animal And Water Source

Objective: The isolation rate of Vancomycin resistant enterococcus which from animals (pet hospitals,farms,pig farms),water (Minjiang River, community ponds,fish ponds in school,cesspool,sewer) was invertigated, and the resistance phenotype,genotype testing and homology was studied in this paper,which lay solid foundation to the reasonably use of drug and curb VRE spreading to the human.Methods: Agar screening method (ADSP method) was used to separate vancomycin-resistant enterococci;According to the methods which recommended by Clinical and Laboratory criteria (CLSI) of American, broth microdilution was used detect to make the sensitivity to six kinds of commonly used antimicrobia.The detection was made for the vancomycin-resistant enterococci which was isolated from animal and water;Multiple PCR method was used to identify the drug resistance gene and the enterococci levels;used the method of Repetitive extragenic palindromic elements-PCR to analyse molecular typing and homology of the Vancomycin resistant enterococcus which isolated from animal and water.Results:(1) 30 strains of Vancomycin resistant enterococcus were sereened out from 420 samples which were isolated from two pets hospitals, three large-scale pig farms and one large-scale chicken farm,screened out 17 strains of vancomycin-resistant enterococci from 200 samples which were isolated from the Minjiang River,a pond in living area, fish ponds at school,cesspool,sewer;between different swine in different pig farms , the separation rate was different: the higest was the breast-feeding pig,followed by conservation pig. Different sources of water had different Separation rate too: sewer was the higest,followed by Cesspool. Sensitivity test results showed that oxytetracycline had the highest resistance rate in vancomycin- resistant enterococci which isolated from animal and water,the resistance rate were 93.3% and 90%,and ampicillin had the higest sensitive rate which were 80% and 88.2%.(2) PCR method detected the vancomycin-resistant genes,in 30 vancomycin-resistant enterococci strains from animals , two VanA -positive , 12 VanB-positive ,7 VanC1-positive,5 VanC2/3-positive,4 vancomycin-resistant enterococci strains no Van gene amplification;in 17 vancomycin-resistant enterococci strains from water,3 VanB-positive,9 VanC1-positive,5 VanC2/3-positive,non-amplified VanA genes.(3) REP-PCR results showed that 30 vancomycin-resistant enterococci strains from animal were divided into 13 types,signed by A-M.Type A was divided into A1-A6 subtype , F-type was divided into F1-F3-subtype , J type was divided into J1-J3subtype,B,C,D,E,G,H,I,K,L,M hadn't subtypes.Phylogenetic analysis showed that 100% homology in No.27,No.28,No.29 strain,No.21 and 22, No.1 and 7,No.4 and 9,No.17 and 18,No.11 and 14,No.6 and 8 strains,had 100% homology with each other.17 water origin VIE were amplified 3-9 bands,sized between 200bp-2000bp,were divided into five types,expressed with A-E.Type A was divided into A1-A3 subtype,B,C,D,E had no subtypes.Subtype A1 was the main type in 5 genotypes.Subtype A1 had 6 strains,subtype A2 had 1 strain,subtype A3 had 2 strains,subtype B3 had 3 strains,type D had 3 strains,type C,E all had 1 strain.No.9,10,11,14,16 and No.17, No.5,12 and No.13, No.3,4 and 7, No.1 and 15, had 100% homology with each other.Conclusion:(1) vancomycin-resistant enterococci could be isolated from animal and water,these vancomycin-resistant enterococci resistant to multiple antibiotics,and drug resistance phenotype consistent with genotype.(2) The homology analysis showed that the vancomycin-resistant enterococci which separated from the same pig farm had high homology;the vancomycin-resistant enterococci which separated from cesspool and sewer had high homology;the vancomycin-resistant enterococci which separated from animal and water had varying degrees of hybrid Cloning,horizontal transmission might exist.(3) Investigating the vancomycin resistance and transmission of enterococci which separated from animal and water can lay solid foundation to the reasonably use of drug and curb VRE spreading to the human.