食源微生物耐药基因水平传播抑制研究
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摘要
在食用动物中使用抗生素会导致耐药菌的出现,动物感染的耐药菌可以通过直接接触、食物链和环境污染将其耐药性及耐药基因传播给人类,这直接威胁人类生命健康。本论文通过建立体外和体内小鼠肠道中耐药基因水平传播模型,在基因水平上探讨耐药菌的来源、传播途径和第一类整合子整合酶(IntI1)介导的食源性耐药基因在体外和体内小鼠肠道中的水平传播效率及小干扰RNA(small interference RNA,siRNA)对其抑制作用,提出一种将siRNA用于控制IntI1介导的耐药基因水平传播及评价其抑制效果的方法。论文主要内容有以下四个部分:
(1)IntI1介导的耐药基因体外水平传播
采用含有完整第一类整合子的受体质粒R388和分别含有dfrA1-aadA1、aadA1、aadB和cmlA耐药基因的供体质粒pGC-1~4,分别转化到BL21(DE3)和DH5α中形成供体菌和受体菌。无抗生素选择压力下,受体菌和供体菌进行接合实验,抗生素平板筛选,以intI1-F和系列基因盒引物进行PCR验证,最后测序验证重组位点。IntI1介导下,R388与pGC-1~4的重组效率分别为:4.6×10-3、6.8×10-4、8.7×10-5和9.9×10-5 cfu/(供体菌)。pGC-1中dfrA1-aadA1两个串联基因盒,以dfrA1和aadA1形式分别被IntI1整合。测序后比对发现,体外重组反应都发生在attI1和attC之间且IntI1介导耐药基因盒同源重组位点是attI1位点simple site中的G和TT之间。
(2)IntI1介导的耐药基因在小鼠肠道的传播
筛选肠道中不含干扰本实验的耐药菌的小鼠为模型。先灌胃0.5mL供体菌(109 cfu/mL),待供体菌在小鼠肠道中定殖水平达到108 cfu/(g粪便)时,再灌胃0.5mL受体菌(109 cfu/mL)。在第一周内隔天、之后每周取小鼠新鲜粪便样本,生理盐水悬浮,涂抗生素平板,菌落PCR验证,测序比对。结果显示:灌胃受体菌1天后,含有R388的供体菌与含有pGC-1~4的受体菌的重组效率分别为:4.6×10-2、8.1×10-3、2.2×10-4和7.2×10-3 cfu/(供体菌), pGC-1的两个基因盒dfrA1-aadA1被分别整合。与体外实验相比,体内实验显示了更高的耐药基因水平传播效率。测序发现,重组位点全部发生在attI1×attC之间。到42天时,全部整合菌株在小鼠肠道中为阴性。
(3)siRNA的设计、筛选及体外抑制耐药基因水平传播
以intI1为靶基因,设计5对特异的21bp siRNA。通过siRNA与含有R388的供体菌和含有pGC-2的受体菌共培养,考察整合效率的变化。结果显示: siRNA-1和siRNA-2对IntI1介导的整合率的抑制效率最高分别为52.1%和50.7%。siRNA干扰的最优条件为:分三次将siRNA加入共培养体系,使终浓度为0.2μmol/L,每隔2小时添加一次,共培养8h时,siRNA-1和siRNA-2处理后,对IntI1介导整合效率的抑制率分别为:60.8%和58.1%。
(4)Real-time PCR检测siRNA对整合效率和intI1 mRNA水平的影响
以一步法real-time PCR相对定量和绝对定量检测siRNA共培养前后整合效率和intI1 mRNA水平的变化。相对定量结果显示:与无siRNA相比,siRNA-1和siRNA-2处理后,整合效率分别下降了52%和55%;绝对定量结果显示:对照样品(阴性siRNA)和siRNA-1处理后样品的整合效率分别为8.1×10-4cfu/(供体菌)和3.84×10-4cfu/(供体菌),经siRNA-1处理后,intI1 mRNA水平降低了58%。表明siRNA能有效抑制IntI1介导的耐药基因水平传播。
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 vitro
R388 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 vivo
Mice 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 vitro
5 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 test
The 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.
(1)IntI1介导的耐药基因体外水平传播
采用含有完整第一类整合子的受体质粒R388和分别含有dfrA1-aadA1、aadA1、aadB和cmlA耐药基因的供体质粒pGC-1~4,分别转化到BL21(DE3)和DH5α中形成供体菌和受体菌。无抗生素选择压力下,受体菌和供体菌进行接合实验,抗生素平板筛选,以intI1-F和系列基因盒引物进行PCR验证,最后测序验证重组位点。IntI1介导下,R388与pGC-1~4的重组效率分别为:4.6×10-3、6.8×10-4、8.7×10-5和9.9×10-5 cfu/(供体菌)。pGC-1中dfrA1-aadA1两个串联基因盒,以dfrA1和aadA1形式分别被IntI1整合。测序后比对发现,体外重组反应都发生在attI1和attC之间且IntI1介导耐药基因盒同源重组位点是attI1位点simple site中的G和TT之间。
(2)IntI1介导的耐药基因在小鼠肠道的传播
筛选肠道中不含干扰本实验的耐药菌的小鼠为模型。先灌胃0.5mL供体菌(109 cfu/mL),待供体菌在小鼠肠道中定殖水平达到108 cfu/(g粪便)时,再灌胃0.5mL受体菌(109 cfu/mL)。在第一周内隔天、之后每周取小鼠新鲜粪便样本,生理盐水悬浮,涂抗生素平板,菌落PCR验证,测序比对。结果显示:灌胃受体菌1天后,含有R388的供体菌与含有pGC-1~4的受体菌的重组效率分别为:4.6×10-2、8.1×10-3、2.2×10-4和7.2×10-3 cfu/(供体菌), pGC-1的两个基因盒dfrA1-aadA1被分别整合。与体外实验相比,体内实验显示了更高的耐药基因水平传播效率。测序发现,重组位点全部发生在attI1×attC之间。到42天时,全部整合菌株在小鼠肠道中为阴性。
(3)siRNA的设计、筛选及体外抑制耐药基因水平传播
以intI1为靶基因,设计5对特异的21bp siRNA。通过siRNA与含有R388的供体菌和含有pGC-2的受体菌共培养,考察整合效率的变化。结果显示: siRNA-1和siRNA-2对IntI1介导的整合率的抑制效率最高分别为52.1%和50.7%。siRNA干扰的最优条件为:分三次将siRNA加入共培养体系,使终浓度为0.2μmol/L,每隔2小时添加一次,共培养8h时,siRNA-1和siRNA-2处理后,对IntI1介导整合效率的抑制率分别为:60.8%和58.1%。
(4)Real-time PCR检测siRNA对整合效率和intI1 mRNA水平的影响
以一步法real-time PCR相对定量和绝对定量检测siRNA共培养前后整合效率和intI1 mRNA水平的变化。相对定量结果显示:与无siRNA相比,siRNA-1和siRNA-2处理后,整合效率分别下降了52%和55%;绝对定量结果显示:对照样品(阴性siRNA)和siRNA-1处理后样品的整合效率分别为8.1×10-4cfu/(供体菌)和3.84×10-4cfu/(供体菌),经siRNA-1处理后,intI1 mRNA水平降低了58%。表明siRNA能有效抑制IntI1介导的耐药基因水平传播。
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 vitro
R388 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 vivo
Mice 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 vitro
5 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 test
The 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.
引文
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