Effect Of In Feed Copper And Stress Hormone On Resistant Plasmids Conjugational Transfer Of Animal Gut Escherichia Coli

Antimicrobial resistance has caused a wide concern,which lead to urgently standardize the use of antibiotic.Stress is common in domestic livestock and poultry farming,and gastrointestinal norepinephrine and other catecholamines are main hormones that respond to acute stress in the host.Bacteria face the dual pressures of heavy metals and antibiotics,it is easy to become resistant to them.Besides,some studies indicated that elevated dietary copper may facilitate and maintain antibiotic resistance of gut microorganisms,but whether in feed copper have co-selection to the formation of antibiotic resistance in Escherichia coli.Whether the addition of high copper increasing the level of resistance gene horizontal transfer?Whether the stress hormone participate in the pathogen adaptation and evolution in the host intestinal environment?So this thesis intends to focus on these three key scientific problems in-depth study and is continued in the following three sections:In the first section,the aim at this paper was to evaluate the potential effects of acute stress factors on the level of the horizontal transfer of bacterial resistant genes in the animal production.Two isolates from weaned Xiang pigs were selected as test strains.The optimal growth promotion concentration of NE for E.coli was determined by measuring the growth curves of the OD600.Four isolates(A,B,C,D)from weaned Xiang pigs were used to carry plasmid-mediated enrofloxacin resistant genes(aac(6’)-lb-cr,oqxAB,qnrS),the transfer rate of the conjugation between the donors(induced by NE or not)and the recipient(E.coli J53)were calculated in filter mating.The MICs of the transconjugants,donor strains and the recipient strain to 6 antibiotics were determined by the Kirby.Bauer test and the agar dilution method.The plasmid-mediated enrofloxacin resistant genes of the transconjugants,donor strains and the recipient strain were detected by PCR.[Results]The E.coli growth was promoted by NE dose-dependently,the optimal growth promotion concentration was determined by the administration of 100 μmol/L NE.The transfer rates of the conjugation between three donors(A,B,C)induced by NE(100 μmol/L)and the recipient were significantly higher than those of control groups(the donors without exposure to NE)(P<0.01).Three plasmid-mediated enrofloxacin resistant genes(aac(6’)-lb-cr,oqxAB,qnrS)were detected in all transconjugants.The MICs of the transconjugants to 6 antibiotics were the same as or lower than those of the donor strains.NE increased the transfer frequency of resistant plasmids of isolates in vitro,in addition,the E.coli growth was promoted by NE dose-dependently.Suggesting that the management of stress should be strengthened in the animal production and breeding in order to reduce the horizontal transfer of bacterial resistant genes.In the second section,rats fed with different levels of copper were orally inoculated with an rifampicin-resistant E.coli EC600 strain harboring a conjugative plasmid carry a gene conferring resistance blaCTX-M-1.This experiment aimed to evaluate the impact of high level of long-term in feed copper and ceftiofur on horizontal transfer of blaCTX-M-1 among the E.coli and the significance for different copper levels on the number of sensitive Enterobacterium and the Enterobacterium isolated from CTX-Mac plates before and after using ceftiofur,the difference of the transfer frequency at different copper levels were compared in vitro.According to the different copper content in the basal diet and ceftiofur inoculation or not,32 male rats at 21 days of age were randomly assigned to two control groups(C-:Cu:6 mg/kg,without ceftiofur treatment or C+:Cu:6 mg/kg,with ceftiofur treatment),and two treatment groups(H-:Cu:240 mg/kg,without ceftiofur treatment or H:Cu:240 mg/kg,with ceftiofur treatment).All rats were orally inoculated with an rifampicin-resistant E.coli EC600 strain harboring a conjugative plasmid carry a gene conferring resistance blaCTX-M-1 at 20,22,24 days.The ceftiofur(CTO)groups(C+ and H+)were given 10 mg/kg of body weight ceftiofur hydrochloride at 26,27,28 days,while the control groups(C-and H-)were given salad oil at the same dose.Fecal samples obtained from rats were used for enumeration of total Enterobacterium on Mac plates and resistant Enterobacterium on cefotaxime(CTX)-Mac plates.The detection on blaCTX-M-1 gene in Enterobacterium isolated from CTX-Mac plates were also conducted.After administration of ceftiofur,the counts of CTX-resistant Enterobacterium in CTO group were significantly higher than that in corresponding control group(C＋vs C-;H+ vs H-)(P<0.01).Compared with the corresponding control group,the detection rate of blaCTX-M-1 gene was maintained at high level.The number of the blaCTX-M-1 gene in H-group was higher compared with the C’ group,and the rate of disappearance of the blaCTX-M-1 gene was faster in the C" group compared with the H-group after the treatment of ceftiofur.In vitro test took the inoculcation strain as donor and four strains of susceptible Enterobacterium isolated from rats as recipient.The results showed the transfer rates of the conjugation induced by high copper(12 mmol/L)were significantly higher than that of low copper(2 mmol/L)group(P<0.01).In conclusion,high copper facilitates the horizontal transfer and maintenance of the resistant genes.However,in the presence of high copper and antibiotics,antibiotics for the maintenance effect is more obvious.In the third section,this experiment aimed to analyze the homology among the Enterobacterium isolated from rat’s fecal samples and trace the recipient strains of the blaCTX-M positive Enterobacterium.Forthermore,test analyzed the transmission mechanism of conjugative plasmid in E.coli by S1-PFGE and Southern-blot hybridization.Based on the animal design in last chapter,the cefotaxime sensitive or resistant isolates from rat’s fecal samples of C" and H-groups were analyzed the homology.PFGE results showed that 9 different PFGE types were detected among the 34 sensitive Enterobacterium isolated from rat’s fecal samples in C" group(similarity is less than 85%),while 9 different PFGE types were detected among the 31 sensitive Enterobacterium isolated from rat’s fecal samples in H-group.In the two groups,we found the recipient strains(the sensitive nterobacterium)corresponding to the transconjugants(the blaCTX-M positive Enterobacterium).The transconjugants and the corresponding recipient strains,as well as the inoculcation strain TC4-2,the donor strain No.4(the donor strain of the inoculcation strain)were subjected to S1-PFGE and Southern-blot hybridization.S1-PFGE results showed that the blaCTX-M-1 gene was present on the plasmid of about 67kb.Southern-blot hybridization showed that the hybridization strip appeared in the No.4 strain,TC4-2,and the blaCTX-M positive Enterobacterium,but not in the correspongding recipient strains.In conclusion,bacteria such as E.coli can transfer conjugative plasmids carrying a gene conferring resistance blaCTX-M-1 to the recipient through conjugation,so as to spread the resistant genes among different bacteria.