| In animal husbandry,antibiotics can be used to prevent and cure disease,or added in feed as a growth-promoting agent,make a great contribution to the development of the modem animal husbandry,but they also lead to a serious antibiotic resistance problem of animal gut microorganisms.Copper is an essential micro-mineral both to the body and bacterial cell,it can also inhibit the bacterial growth.Therefore,copper is often added in piglets feed to inhibit diarrhea and promote growth;however,it can induce bacterial copper resistance.Some studies indicated that there is a complicated relation of metal and antibiotics resistance,but whether copper in feed have co-selection to the formation of antibiotic resistance in E.coli.If it exists,what is the possible mechanism?These problems are not clear at present,so this thesis intends to focus on these two key scientific problems in-depth study and is constituted in the following four sections:In the first section,the experiment was conducted to compare the difference of antibiotic and copper resistance among E.coli isolated from sows and piglets before and after weaning.In the experiment,224 of E.coli were isolated from lactation sows,suckling and weaned piglets,which antibiotic resistance to 7 antimicrobial agents and copper resistance was detected using agar dilution method according to the recommendation of the Clinical Laboratory Standards Institute(CLSI).The remarkable antibiotic resistance of isolated E.coli to 7 selected antibiotics was observed,the percentage of resistance isolates to individual antibiotic was ranged from 28.13%to 93.30%.70.09%of all isolates possessed multi-drug resistance(MDR).The antibiotic resistance of E.coli isolates from sows and piglets was highly similar,except resistance to ampicillin and gentamicin.However,the antibiotic resistance of isolates from weaned pigs changed significantly.The antibiotic resistance to ceftiofur,florfenicol and chloramphenicol increased very significantly(P<0.01),resistance to ampicillin increased significantly(P<0.05).Comparing to sow and suckling piglets,the copper resistance of isolates from weaned pigs increased very significantly(P<0.01).In addition,when the copper resistance of E.coli isolates changed,the antibiotic resistance also changed,there might be a correlation between copper and antibiotic resistance.Drug-resistance bacteria in the gut of sows and in feed antibiotics contribute significantly to the development of antibiotics of E.coli in the gut of piglets.Moreover,it initially reveals the presence of a certain relationship between in feed copper and antibiotic resistance change of E.coli isolates from piglets.In the second section,the experiment was conducted to study the effect of in feed copper and Enrofloxacin on antibiotic resistance and copper resistance of E.coli in the porcine gut.In the experiment ten weaned Xiang-pigs,a kind of local miniature pig with low percentage of antibiotic-resistant isolates in their feces,were divided into two treatment groups,low copper group(4 mg/kg)and high copper group(125 mg/kg),five piglets in per group.The sub-therapeutic Enrofloxacin was added into the feed of two treatment groups at the fifteenth day during the experiment for ten days,and stop this in feed addition at the twenty-fifth day.Fecal samples were collected from piglets at 0,14,19,24,34,48 days during the experimental period to isolate and identify E.coli,which antibiotic resistance to Enrofloxacin and copper resistance were detected using agar dilution method according to the recommendation of the CLSI.The results showed that the numbers of E.coli isolates declined very significantly in two groups because of the use of in feed copper and Enrofloxacin(P<0.01).During the additional period of Enrofloxacin in feed,the Enrofloxacin resistance firstly increased very significantly(P<0.01)and then decreased very significantly(P<0.01)in two treatment groups.Both the percentage of resistant E.coli isolates to Enrofloxacin in high copper group before adding(49.44%&4.05%,P<0.01)and after withdrawing(14.86%&2.70%,P<0.05)Enrofloxacin in feed were significantly higher than that in low copper group.The percentage of copper resistant E.coli isolates in high copper group was significantly higher than that in low copper group(P<0.05),and this high percentage in high copper group maintained overall expeimental period,however the percentage of copper resistant E.coli isolates in low copper group constantly decreased.There was a correlation between copper and Enrofloxacin resistance.In the high copper group,the Enrofloxacin resistance of E.coli isolates was positively correlative to the copper resistance(P<0.01).In low copper group,this positive correlation appeared after in feed Enrofloxacin addition(P<0.01).In conclusion,there is significant effect of in feed copper and Enrofloxacin on the appearance of antibiotic resistance and copper resistance of E.coli,high copper in feed may facilitate the appearance and maintenance of antibiotic resistance of E.coli.In the third section,the experiment was conducted to investigate the correlation between quinolone and copper resistance of E.coli on the plasmid-mediated gene level,and also to study the interrelation between the phenotypes and the genotypes of resistance.Based on MIC value of E.coli isolates detected in the previous chapter,125 E.coli isolates sorted into four different phenotypes were selected,which were high antibiotic and high copper resistant isolates(Enrofloxacin MIC>32μg/mL,copper MIC≥24 mM),high antibiotic and low copper resistant isolates(Enrofloxacin MIC>32 pg/mL,copper MIC<24 mM),low antibiotic and high copper resistant isolates(Enrofloxacin MIC<0.125μg/mL,copper MIC>24 mM),low antibiotic and low copper resistant isolates(Enrofloxacin MIC<0.125 pg/mL,copper MIC<24 mM).Four plasmid-mediated quinolone resistant genes(qnrS、qepA、oqxAB、aac(6’)-lb-cr)and two copper resistant genes(pcoA、pcoD)in all selected isolates were detected by PCR.The results showed that except for qepA,the high prevalence of other resistant genes were observed.The prevalence rate of quinolone resistant genes,oqxAB,qnrS,and aac(6’)-lb-cr was 95.2%,92.0%and 81.6%respectively,and that of copper resistant genes,pcoA and pcoD was 48.8%and 66.4%respectively.111 of 125 detected isolates carried 3 or more plasmid-mediated resistant genes.There was a correlation between phenotype and genotype of detected isolates,the isolates with higher resistant phenotype got higher prevalence rate of resistant gene.The prevalence rate of aac(6’)-lb-cr gene increased very significantly after adding Enrofloxacin in feed(P<0.01).Positive correlation between the prevalence rate of quinolone resistant genes and that of copper resistant genes were observed(P<0.05).In conclusion,E.coli isolates can carry a variety of plasmid mediated resistance gene.The phenotypes and the genotypes of resistance are not entirely consistent.The prevalence of plasmid-mediated quinolone and copper resistant genes has the positive correlation.In the fourth section,the experiment was conducted to study the effect of copper on Enrofloxacin resistance of E.coli in vitro.2 sensitive isolates(M1,M2)of E.coli(Enrofloxacin MIC =0.5μg/mL,copper MIC=20mM)from weaned Xiang pigs were cultured in MH broth with three different additive,Enrofloxacin(E),copper sulfate(C),and Enrofloxacin plus copper sulfate(E+C).The minimum inhibitory concentration(MIC)of the E.coli in the 0th,3rd,6th,9th,12th,15th,18th passage number was measured by the agar dilution method,and the plasmid-mediated quinolone resistant and copper resistant genes of E.coli in the 0th and 18th passage number were also detected.The results showed in E group,MIC value of two isolates to Enrofloxacin in the 18th passage number was increased 8 times than that in the 0th passage number,while in E+C group,it was increased 4 times.For the MIC value to copper,M2 reached to 28 mM in E group,and both two strains reached to 24mM in E+C group.The qnrS,one of plasmid-mediated quinolone resistant genes,was not observed in the 0th but appeared in the 18th passage E.coli.In conclusion,low level of Enrofloxacin has a remarkable effect on MIC value of E.coli to Enrofloxacin.Copper has not showed remarkable effect on facilitating the appearance and maintenance of antibiotic resistance of E.coli in vivo,and needs to be further explored. |
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