| The applications of antibiotics for food animals could lead to the existence of drug resistant bacteria, and the bacteria might spread via contact or food chain or environment, to humans and cause human infection. It could bring an awareness of potential threats to human health. This thesis is to study the horizontal transfer inhibition of resistane gene in foodbore bacteria by determining the efficiency of IntI1-mediated food-borne resistance gene horizontal transmission in vitro and in mouse intestines, and investigating the effect of siRNA interference, then to set up a method for the inhibition of resistane gene from horizontal transfer in foodbore bacteria and the estimation of inhibited efficiency. The content and results are as follows:(1) The efficiency of IntI1-mediated resistance gene horizontal transfer in vitroR388 is the recipient plasmid that carried attI1 within class 1 integron, In3. R388 was transformed into BL21(DE3) cells to create the donor strain; pGC-1~4 were donor plasmids which carried dfrA1-aadA1, aadA1, aadB and cmlA resistance gene cassettes. The pGC-1~4 were transformed into DH5αcells to create the recipient strain. Recipient and donor strains were co-cultured without antibiotic selective presure, screened by antibiotic plate and PCR tested using intI1-F and several cassette-specific primers, recombination site was determined sequentially. The IntI1-mediated recombination efficiency of R388 and pGC-1~4 were 4.6×10-3, 6.8×10-4, 8.7×10-5 and 9.9×10-5 cfu/donor strain respectively. The two serial cassettes dfrA1-aadA1 in pGC-1 were integrated by IntI1 in the form of dfrA1 and aadA1. The resistance gene cassettes were integrated specifically into GTT consensus site of attI1.(2) The horizontal transmission of IntI1 mediated drug resistance gene in vivoMice without carrying bacteria resistant to any one of these antibiotic were selected for the experiments. All of the selected mice were orogastrically inoculated with 0.5 mL of a fresh overnight culture of donor strain with a concentration of 109 cfu/mL. After the colonization of donor strain reached a level of 108 cfu/g stool sample in the intestines of mice, 0.5 mL of recipient strain was administered at a concentration of 109 cfu/mL. Fresh stools of mice were collected on days 1, 3, 5, 7, 14, 21, 28, 35 and 42 after orogastric inoculation. Each stool sample was diluted, homogenized and resuspended with physiologic saline, selected with antibiotic plate, then PCR verification and sequeced. Results showed that, 1 day after orogastrically inoculated of recipient strain, the recombination efficiency of recipient and donor strain carried pGC-1~4 respectively were 4.6×10-2,8.1×10-3,2.2×10-4 and 7.2×10-3 cfu/donor strain, the recombination efficiency in vivo was highter than that in vitro. It was showed by sequencing that all recombination sites took place between attI1×attC. On the 42nd day all of cointegrates colonizing on the intesines of mice were negative.(3) Design, screening of siRNA and the inhibition of recombination efficiency in vitro5 different 21bp specific sequences of siRNA were designed for use to target upon the intI1 sequence, and they were co-cultured with recipient strain contained pGC-2 and donor strain contained R388 to determine the recombination efficiency. Results showed that inhibition rates of siRNA-1 and siRNA-2 were 52.1% and 50.7% respectively. The optical interference condition was: the siRNA was added for 3 times, the final concentration reached 0.2μM, one addition every 2 hours, 8 hours for all, and the inhibition efficiency treated by siRNA-1 and siRNA-2 was reached to 60.8% and 58.1%.(4) Determination of effects of siRNA on the integration efficiency and the intI1 mRNA level by PCR testThe method of one step SYBR Green I real-time PCR was used to relatively quantify and absolutely quantify the changes of integration efficiency and the level of intI1 mRNA before and after co-cultured with siRNA. It was found from the relative quantification that compared with cramble siRNA, after treated by siRNA-1 and siRNA-2 the integration efficiency reduced 52% and 55% respectively. It was indicated from the absolute quantification that cramble siRNA and siRNA-1 were 8.1×10-4 cfu/donor strain and 3.84×10-4 cfu/donor strain respectively. The intI1 mRNA level was reduced 58% after treatment by siRNA-1.
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