| The meat duck bio-bed is a kind of new raising system to degrade the organic excreta by microbes,and to control the pollution effectively.Antibiotics have been widely used in animal production more than half a century to prevent and treat diseases,to promote animal growth and increase feed efficiency.Zinc as one of the essential metal elements is often used as mineral additives in animal production.It involved in many physiological functions,and reduce disease,improve performance.In feed antibiotic and metal will accumulate in the bio-bed in line with animal raising on it.So far,how this increased level of antibiotic and metal affects the structure of microflora in bio-bed,and antibiotic or zinc resistance of Escherichia coli is not clear.Therefore,this study focused on above issues and the thesis is constituted in the following four sections.The first section aimed to character the succession of bacterial community in the litter during the fermentation of duck bio-bed.The experiment was conducted in one meat duck bio-bed farm of Jiangsu province.The new litter（DO）,used litter from 4th（D4）;8th（D8）meat duck flock and feces from duck at 34 d age were sampled.PCR/denaturing gradient gel electrophoresis（PCR-DGGE）,16S rRNA gene sequencing and real-time PCR were used to monitor the composition changes of microbiota in the bio-bed.DGGE profiles showed that,the similarity between D4 and D8 was 81.93%,which was significantly higher than the similarity between D4,D8 and DO（68.81%and 70.82%,respectively）（P<0.05）.Bands 6 and 8（the closest relatives:Leqionella tunisiensis and Pedobacter bauzanensis,respectively）were dominant in all litter samples.Band 10（the closest relative:Rummeliibacillus suwonesis）was only dominant in used litters.Bands 12 and 13（the closest relatives:Psychrobacter sp.PRwf-1,lamia majanohamensis,respectively）commonly presented in all litters and feces.Real-time PCR results showed that the number of Escherichia coli in duck feces was significantly higher than that in D4 and D8 litters（P<0.05）,while no significant difference was observed between duck feces and DO litter（P>0.05）.In conclusion,both the application time of bio-bed and duck feces origin microorganisms can affect the bacterial community and the number of E.coli in the bio-bed,and the bacterial community structure tends to be stable in line with the application time of the bio-bed.The second section was conducted to study the antibiotic and zinc resistance of E.coli isolated from meat duck’s bio-bed,and to provide some suggestions on the rational use of drugs and the management of bio-bed by discussing the relationship between those two different resistances.152 of E.coli were isolated from new litter（DO）,used litter at 4th（D4）and 8th（D8）meat duck flock,which antibiotic and zinc resistance were detected by broth micro-dilution method or agar dilution method according to the recommendation of the CLSI.The remarkable antibiotic resistance of isolated E.coli to selected antibiotics was observed,the percentage of resistance isolates to individual antibiotic was ranged from 21.05%to 95%.The percentage of antibiotic resistance isolates from D8 litter to ceftiofur and quinolones were significantly higher than that form D4 litter（P<0.05）.98.03%of all isolates possessed multi-drug resistance（MDR）,among which majority were MDR to five classes of antibiotics.The percentage of resistance isolates to zinc was extremely high（100%）and the MIC value increased over time.Nevertheless,this study hasn’t found the correlation between zinc and antibiotic resistance of E.coli isolated from duck bio-bed.The third section was to investigate the prevalence of plasmid-mediated enrofloxacin and zinc resistance genes.Plasmid-mediated enrofloxacin resistance genes（qnrS,qepA,oqxAB,aac（6’）-lb-cr）and zinc resistance gene（ZntA）in 66 high resistant（MIC ≥ 32 μg/mL）and 11 sensitive（MIC<0.25 μg/mL）isolates were detected to explore the relationship between enrofloxacin resistance and zinc resistance in gene level.The detected plasmid-mediated enrofloxacin resistance genes were oqxAB（57.14%）,aac（6’）-lb-cr（38.96%）,qnrS（33.77%）,whileqepA gene was not detected.77.92%of isolates were at least carrying one of the plasmid-mediated enrofloxacin resistance genes.By combining with the resistance phenotype,we found that nine isolates with sensitive phenotype were detected carrying at least one resistance gene;however,15 strains with high resistant phenotype were not detected the plasmid-mediated resistance genes.On the other hand,98.70%（76/77 strains）of strains with zinc-resistant phenotype carryed ZntA genes.In this study,resistant phenotype and resistance genes in strains were not exactly the same,it may suggest that there are other mechanisms involving in the enrofloxacin resistance,except the plasmid-mediated mechanism.The fourth section aimed to explore the effects of low concentration of enrofloxacin and zinc on sensitivity of E.coli strains in vitro model.We selected four enrofloxacin sensitive strains（MIC = 0.5 μg/mL）,and cultured them in 1/2 of MIC（0.25 μg/mL）enrofloxacin MH broth（E group）,1/3 of MIC（1.33 μg/mL）zinc chloride MH broth（Zn group）and both 1/2 of MIC enrofloxacin and 1/3 of MIC zinc chloride in MH broth（E+Zn group）in vitro.We determinated the enrofloxacin and zinc MIC values of 0,3,6,9,12,15,18 generations and detected the resistance genes of 0 and 18 generation strains.The results showed that:the enrofloxacin MIC value increased significantly in E group（MIC value up to four times than parents strains）;the MIC vaule of enrofloxacin also increased in Zn group and E+Zn group,but there was no significant difference.We also found that:only qnrS could be detected in the parent strains,while oqxAB,aac（6’）-lb-cr,qnrS could be detected in the induced strains.The MIC values of zinc increased in all group,ZntA gene was detected in all strains.In conclusion,the appearance of oqxAB,aac（6’）-lb-cr genes might be related to the increase of MIC value to enrofloxacin.ZntA gene was existed in all strains,but the mechanism of zinc resistance and the relationship between antibiotic and zinc need to study in the further. |
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