猪场土壤中5种四环素抗性基因的检测和定量

Quantification of Five Tetracycline Resistance Genes in Soil from a Swine Feedlot

近年来由于四环素在畜禽养殖业中的大量使用,诱导了环境中四环素抗性微生物的产生,然而有关四环素抗性基因在土壤中存在、迁移和扩散的研究目前还很少.论文提取了北京一规模化养猪场周边土壤的微生物DNA,利用普通PCR检测到5种四环素抗性基因(tet(B/P)、tet(M)、tet(O)、tet(T)、tet(W)),这5种基因都属于编码核糖体保护蛋白的一类.进一步建立了定量PCR程序对这5种基因进行了定量.结果显示,除tet(T)和tet(M)含量接近外(p=0.367),其余几种基因含量之间均差异显著(p<0.05),其中tet(W)的含量最高((2.16±0.20)×108copies·g-1(干土)),比含量最低的tet(B/P)((2.89±0.54)×106copies·g-(1干土))高出约两个数量级.抗性基因tet(W)、tet(T)、tet(M)、tet(O)含量均较高,为猪场土壤中优势抗性基因.这些基因曾被报道广泛存在于猪和牛的肠道中,显示抗性基因很可能是通过基因横向转移(HGT)等机制从养殖动物体内传播到周围土壤中土著微生物体内的.论文所建立的实验方法为进一步系统研究抗生素抗性基因在土壤中的环境行为及其生态风险提供了基础.

In recent years, the prevalence of tetracycline-resistant bacterial in environment are influenced by the wide use of tetracyclines in livestock production facilities. However, the survival, fate and dissemination of tetracycline resistance （Tcr）genes in soil are still poorly understood. Polymerase -chain-reaction （PCR）assays were conducted for five Tcr genes encoding ribosomal protection proteins（tet（B/P）, tet（M）, tet（O）, tet（T）, tet（W））, which were found in total DNA extracted from soil around a swine feedlot in Beijing. Furthermore, a real -time quantitative PCR method was developed for quantifying these five specific Tcr genes. Except for the similar copy numbers between tet（T）and tet（M）（p=0.367）, the copy numbers of other Tcr genes in the soil were significantly different （p0.05）. The highest copy numbers of tet（W） （（2.16±0.20）×108 copies·g-1（dry soil））were about two orders of magnitude higher than the lowest copy numbers of tet （B/P）（（2.89±0.54）×106 copies·g-1（dry soil））. tet（M）, tet（O）, tet（T）, tet（W）were predominant in soil and these genes had been reported to be dominant in the gastrointestinal tracts of pigs and cattles, indicating that the swine feedlot had an effect on the surrounding soil and these Tcr genes might be transferred from animals to the indigenous bacterial in soil through horizontal gene transfer （HGT）. The established method in this study provides the basis for further studying the environmental behavior and the associated risk of antibiotic resistance genes in soil.