Effects Of Three Antibiotics On The Escherichia Coli Drug Resistance And Caecum Flora Structure In Rex Rabbits

Antibiotics have been widely used for the prevention and the treatment of human and animal diseases,and they are utilized as fed additives for agricultural animals to promote growth.In the present study,we analyzed the drug resistance characteristics of E.coli and explored the effects of chlortetracycline,colistin sulfate and zinc bacitracin on cecal microflora and immune function in rex rabbits.The experiments selection 80 rex rabbits were randomly divided into four groups:control group(B,basal diet),chlortetracycline group(C,50 mg/kg),colistin sulfate group(S,20 mg/kg)and zinc bacitracin group(Z,40 mg/kg).For a period of 28 days experiment.The major results were demonstrated as follows:1.Colistin sulfate and zinc bacitracin increased the average daily gain(ADG)of rex rabbits,but not significantly(P>0.05).Furthermore,the abilities of colistin sulfate and zinc bacitracin to improve weaning rex rabbits weight were better than chlortetracycline.2.A total of 21 strains E.coli were isolated from faeces samples of the C group rex rabbits.In addition,10 strains E.coli were isolated from faeces samples of the S group rex rabbits.Evaluation of the E.coli isolates resistance phenotype to.10 antibiotics commonly used in clinical was undertaken by K-B disk diffusion tests.The results showed that as high as 90%resistance rates to ampicillin,bactrim,rifampicin and amoxicillin/clavulanic acid of the isolated E.coli in both C and S groups.In addition,disk diffusion test results showed that the S group of E.coli isolated existed more extensive resistance phenotype than those isolates in C group.All isolates had multiple drug resistance that they were all resistant to two or more tested antibiotics.PCR was used to detect the drug resistance genes,which showed that all isolates were carrying tetA,and most of them were carrying pmrA and pmrB genes.In this experiment,we successfully obtained 13 transconjugants of which 11 strains of the donors were from C group and 2 were from S group.Compared with the donor strains,the resistance of transconjugants were generally decreased in this experiment antibiotics.Furthermore,its chlortetracycline and colistin sulfate MIC levels were significantly decreased.The transconjugants had higher resistance to rifampicin(100%).Meanwhile,all transconjugants were able to amplify tetA,pmrA and pmrB genes.3.PCR-DGGE results showed that there were no significant changes in the shannon,richness and evenness indices of cecal microflora among groups(P>0.05).The dendrograms from the total samples illustrated that the samples in B group and C group were clustered into a large branch.In addition,PCA2 was distinguished the B and C group from S and Z group.4.Caecum microbial communities of rex rabbits from the four groups were analyzed by Illumina Miseq platform.Alpha diversity showed no significantly difference between control and experiment groups(P>0.05).Bacterial communities were clustered by using PCoA that showed C group and B group had higher similarities bacterial members than S and B,and Z and B through clustering closely on the two-dimensional PCoA plot.At the phylum level,a total of 26 bacteria forming by representatives of 5 phyla were identified,including Firmicutes,Bacteroidetes,Verrucomicrobia,Proteobacteria and Tenericutes.The majority of obtained sequences belonged to Firmicutes,which followed by Bacteroidetes,and these two phyla constituted roughly 83%of the caecum microbiota.The ratio of Bacteroidetes/Firmicutes in B,C,S and Z groups was 31.78%,42.31%,26.45%and 21.84%,respectively.In comparison to the B group,dietary supplementation with chlortetracycline increased Bacteroidetes/Firmicutes ratio,while were a decreased in S and Z groups.Proteobacteria and Lactobacillus spp.abundance were significantly decreased in Z group,however,Lactobacillus spp.abundance was significantly increased in S group(P<0.05).We had analyzed the lower taxonomical genus levels showing that the predominant of Ruminococcus spp.Ruminococcus albus in S group and Z group were significantly higher than that in B group(P<0.05).The proportion of Coprococcus in the Z group was significantly increased(P<0.05).5.Q-PCR analysis of the specific bacteria in cecum of the weaning rex rabbits,and the results indicated that chlortetracycline significantly increased the abundance of Prevotella-porphyromonas and Bacteroides(P<0.05),and significantly reduced the number of lactobacillus(P<0.05).Colistin sulfate significantly increased the abundance of Clostridium cluster ??a,Enterococcus spp.and Streptococcus spp.(P<0.01 or P<0.05),and significantly reduced the abundance of Clostridium cluster IV(P<0.05).Zinc bacitracin significantly increased the number of Firmicutes(P<0.05),and significantly reduced the number of Clostidium luster ??a and Bacteroides(P<0.05).6.The results of serum cytokine test showed that chlortetracycline and zinc bacitracin increased the levels of IL-1,IL-2,IL-4,IL-6,IL-10,IL-12,IFN-? and TNF-?,while the levels of IL-2,IL-4 and TNF-? were significantly increased(P<0.05 or P<0.01)in C group and Z group.Furthermore,zinc bacitracin significantly upregulated the IL-1 level(P<0.05).Colistin sulfate increased the levels of IL-1,IL-2 and IL-4,and the mRNA expression level of IL-4 was significantly increased(P<0.05),but the levels of IL-6,IL-10,IL-12,IFN-? and TNF-? were decreased.The results of TLR2 and TLR4 mRNA expression levels showed colistin sulfate upregulated liver TLR2 mRNA and caecal TLR4 mRNA expressions(P<0.05).Zinc bacitracin significantly promoted the TLR2 mRNA expression in the liver(P<0.05),but the TLR2 mRNA and TLR4 mRNA expressions were downregulated in the spleen(P<0.05).