Study On Molecular Epidemiological Of Drug-resistant Enterococci From Captive Wild Animals

Objective: To investigate the drug resistance of the high-level aminoglycoside -resistant enterococcus and vancomycin-resistant enterococcus which isolated from the captive wild animals (Zoo,Panda House), environment (water samples,soil samples) and humans (breeders,veterinarians and the general population), and drug-resistant phenotype, genotype testing and homology were studied for the isolated drug-resistant strains in this paper, so as to provide theoretical basis for reasonably use of drug and control the transmitte of drug resistance.Methods: Agar screening method(ADSP method)was used to isolate high-level aminoglycoside-resistant enterococcus(HLARE), High-level streptomycin -resistant enterococcus, vancomycin-resistant enterococcus (VRE); According to the method which recommended by Clinical and Laboratory Standards Institute (CLSI) of American, broth microdilution was used to detect the sensitivity to six kinds of antimicrobia. The detection was made for the resistant strains which were isolated from different sources; PCR method was used to identify the enterococci levels of HLARE and the drug resistance gene of HLGRE; Multiple PCR method was used to identify the drug resistance gene and the enterococci levels of VRE which were isolated from different sources; The method of REP-PCR was used to analyse molecular typing and homology of HLGRE, VIE which isolated from different sources.Results:(1) 165 strains of HLGRE, 39 strains of VIE were screened out from 283 samples which were isolated from zoo(captive wild animals,water samples and soil samples,breeders,veterinarians), Panda House (pandas)and the general population. 38 strains of HLSRE were screened out from 74 samples which were isolated from zoo(part of captive wild animals,breeders,veterinarians), Panda House (pandas)and the general population. The separation rate was different: the separation rate of HLGRE and HLSRE in different animal sources is the hihgest, accounting for 65.92%, 64.86%, but the rate of HLGRE, HLSRE in general population is only 23.81%, 19.05% respectively. The rates of VIE were very different, between 0%~37.5% from different sources.Sensitivity test results showed that the erythromycin and oxytetracycline had the highest drug-resistance rate, but the ampicillin had the lowest drug-resistance rate in HLARE and VIE,which isolated from different sources. The antimicrobial resistance of E. faecium was higher than that of E. faecalis for the HLGRE.(2) PCR method detected the resistant genes of HLGRE: the result showed that among the different sources of 165 strains HLGRE, the positive rate of aac (6 ')-Ie-aph (2 ")-Ia gene was 88.48%(146/165). For the HLGRE strains which isolated from the captive wild animals, environment and human, the positive rates of aac (6 ')-Ie-aph (2 ")-Ia gene were 88.98% (105/118), 84.37% (27/32) and 93.33% (14/15) respectively. Non-amplified aph (2 ")-Ib; aph (2")-Ic and aph (2 ")-Id genes。Multiple PCR method detecte the resistant genes of VRE: the result showed that in 26 strains of animal-derived VIE, 2 VanB-positive, 9 VanC1-positive, 12 VanC1/C2-positive, non-amplified VanA genes, 3 vancomycin-resistant enterococci strains no above Van gene amplified; In 7 strains of environment-derived VIE, 2 VanB-positive, 1 VanC1-positive, 3 VanC1/C2-positive, non-amplified VanA genes, 1 vancomycin-resistant enterococci strain no above Van gene amplified. In 6 strains of human-derived VIE, 4 VanB-positive, 2 VanC1-positive, non-amplified VanA and VanC1/C2 genes.(3)REP-PCR results showed that the HLGRE, VIE which were isolated from different sources could be divided into 2~ 3 types, and different types can be divided into 2 ~ 4 subtypes. Homology analysis showed that 6 groups have 100% homology respectively in different sources of HLGRE, including No.5 and No. 8 which isolated from the Panda House ; No.1 and No.4 strains which isolated from carnivores; No.2,No.4 and No.5 which isolated from reptiles and amphibians; No.7 and No.11 which isolated from the general human; No.5 and No. 8, No.6 and No.7 which isolated from zoo keepers. In different sources of VIE, No.2 and No.3, No.1, No.8 and No.9 which isolated from the zoo environment, No.4 and No.7, No.14 and No.15, No.1, No.3, and No.10 which isolated from different zoo animals have 100% homology.Conclusion:(1) It's very common for HLARE which isolated from different sources, most of HLGRE strains were mediated by aac (6 ')-Ie-aph (2 ")-Ia gene, and the resistance of E.faecium was significantly higher than E.faecialis. The separation rate of VIR was relatively high, the captive wild animals(animals, environment) were mainly mediated by the VanC-type, the human sources were mainly mediated by VanB-type, and drug resistance phenotype consistent with genotype. Erythromycin and oxytetracycline had the highest drug-resistance rate and ampicillin had the lowest drug-resistance rate in resistant strains.(2) The homology analysis showed that the HLGRE, VIE strains which isolated from different captive wild animals and enviroment had 100% homology in the same species animals, different species of animals and different environments. Besides, the same phenomenon also existed in breeders or general humans, Taking into the specificity of the captive wild animals and the contact of different humans might exist, the results indicated that different levels of horizontal transmission or clon dissemination might have been happened.(3) Studied on the drug-resistant present situations and the drug-resistance mechanisms of HLGRE, VIE which isolated from the captive wild animals (animals, environment) and humans (breeders, veterinarians, general humans), it could provide rational guide of prevention and clinical treatment of antibiotics to animals, and reveal the possible routes of transmission of resistance genes and establish basis for public health.