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由于抗生素在农业[1]和医疗[2]环境的频繁使用,使得抗生素抗性成为21世纪6大难题之一[3]。抗生素的持续性选择压力,使得抗生素抗性基因(Antibiotic resistance genes,ARGs)不仅出现在人类活动频繁的环境[1-2, 4-5],在远离人类的地区也有报道[6-7],因此人类对ARGs的不断扩散非常担忧。冰川环境因抗生素选择压力比较小,能反应微生物对抗生素的固有抗性,因此是抗生素抗性风险评估的重要环境。该环境ARGs的研究有2种不同的结果:VAN GOETHEM et al[8]使用宏基因组测序技术,在南极Mackay冰川表层土壤中检测到的ARGs序列与其他环境具有不同的聚类分枝,这些ARGs是其进化的结果。然而,WANG et al[9]对南极冈瓦纳研究站的研究结果表明,人类的长期和短期活动都会影响ARGs在南极的传播,但长期影响和短期影响的结果差不多。OKUBO et al[10]在1200~1400年的南极冰芯检测到sul2,虽然赋予人工合成的磺胺甲恶唑抗性的基因在抗生素时代之前就已经存在,但sul2-strA-strB基因簇与当今细菌具有非常高的序列相似性,该结果与VAN GOETHEM et al[8]的报道相反。
ARGs不仅会受到抗生素的选择压力,重金属同样也会对其产生影响[11–12],如Cu会选择临床上重要的抗生素—万古霉素的耐药性[13]。重金属污染具有持久性,能在环境中存在数百年,其对ARGs的选择压力甚至比抗生素自身还要严重。重金属同样也会选择重金属抗性基因(Heavy metal resistance genes,HMRGs),因此ARGs和HMRGs可同时出现,使得微生物产生多重抗性。这类研究多集中在人类活动频繁的环境,如污水处理厂[14]、活性污泥[15]、农业土壤[1]、医院废水[2]、肥料[16]、城市污水和饮用水[17]、富营养化的地表水中[18],但尚未见到冰川环境HMRGs的研究。而且上述人类活动频繁的环境HMRGs的研究多集中在Cu、Zn、Hg、Ag、As、Ni和Cd等的抗性基因,关于Te和Mn等的抗性基因尚未见报道。
冰川雪冰中重金属的浓度非常低,有时候甚至低于pg/g(10−12 g/g)水平[19],给其研究带来诸多困难。由于HMRGs的丰度与重金属浓度的密切相关性[20],以及荧光定量PCR(Real-time Quantitative PCR,qPCR)的敏感性,HMRGs可代替重金属,从侧面反应冰川中重金属的存在状况。作者之前通过宏基因组测序技术,在青藏高原西昆仑山的崇测冰帽冰川沉积物检测出Zn(9.13%)、Cu(22.64%)、Fe(14.76%)、As(7.35%)、Ni(0.61%)、Mo(9.86%)、Mn(9.16%)、Co(5.38%)、Hg(2.40%)、Cr(6.36%)、Cd(5.50%)、W(0.75%)和Te(6.09%)这13种HMRGs,其中Te和Mn所占百分比并不低,位于第4和第8位。本研究以距离崇测冰帽比较近的青藏高原喀喇昆仑山脉德普昌达克冰川,以及南极中山站为研究位点,通过qPCR,检测代表Hg、Cu、Zn、Cr、Te、Mn和Cd等金属的抗性基因,人工合成的氟喹诺酮类、磺胺类的抗性基因,天然的四环素和红霉素类抗性基因,以及在耐药性传递方面起重要作用的I型整合子(class I integron,intI),一方面初步判断ARGs与HMRGs在不同研究位点存在与否及相对丰度,另一方面了解ARGs、HMRGs与intI之间的相关性。该研究有助于了解冰川环境ARGs的存在状况,以及重金属和intI对其影响。
德普昌达克冰川和中山站抗生素抗性基因及影响因素研究
Antibiotic resistance genes and their influencing factors in Depuchangdake Glacier and Zhongshan Station
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摘要: 为了解冰川环境抗生素抗性基因(Antibiotic Resistance Genes,ARGs)的存在状况及其与重金属和I型整合子(class I integron,intI)的关系,通过荧光定量PCR(Real-time Quantitative PCR,qPCR),研究青藏高原德普昌达克冰川和南极中山站雪样ARGs、重金属抗性基因(heavy metaI resistance genes,HMRGs)和intI的相对丰度及其相关性。结果表明,大部分基因的相对丰度在德普昌达克冰川小于中山站。德普昌达克冰川tetA、czcD、pcoA、znuA、intI1和intI2的相对丰度随海拔高度的增加而降低;南极的merA、zntA、znuA、sul2、tetA和intI2距离中山站建筑区域越近,相对丰度越高。2个研究位点ARGs均受Zn抗性基因znuA的影响,且znuA与intI之间显著相关。冰川环境虽然远离人类活动,但其ARGs同时受intI和与人类活动有关的重金属Zn的影响。Abstract: To investigate the occurrence of the antibiotic resistance genes (ARGs) and the relationship between the ARGs and the heavy metals or type I integron (intI) in the glacial environment, the Real-time Quantitative PCR (qPCR) was used to analyze the relative abundance of the ARGs, heavy metal resistance genes (HMRGs) and intI, as well as their correlation in the glacial snow samples from Depuchangdake Glacier on the Qinghai-Tibet Plateau and Zhongshan Station in Antarctica. The results showed that the relative abundance of most genes detected in Depuchangdake Glacier was lower than that in Zhongshan Station. The relative abundance of tetA, czcD, pcoA, znuA, intI1 and intI2 in Depuchangdake Glacier decreased with the increasing of the altitude. The relative abundance of merA, zntA, znuA, sul2, tetA and intI2 were higher with a closer distance to the building area in Zhongshan Station. ARGs in the 2 sites were affected by Zn resistance gene znuA, which was significantly and positively correlated with intI. Thus, ARGs in glacial environments were affected by both intI and Zn, which was closely related to human activities.
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表 1 qPCR引物名称、序列、片段扩增长度和退火温度
目标基因 目的基因 引物序列(5′→3′) 片段长度/bp 退火温度/℃ 文献 HMRGs merA F: GTGCCGTCCAAGATCATG
R: GGTGGAAGTCCAGTAGGGTGA321 60 [21] zntA F: GGTCGGGTCTGGCATTGAAG
R: TTGCAGCATCGGCGCGCAGGGTA263 60 [22] czcD F: TCATCG CCG GTGCGATCATCAT
R:TGT CAT TCA CGA CAT GAA CC272 55 [19] pcoA F: GCTGCAGATGGCCAGTATGTAAA
R: CCCTCGAGCGTAACCGGTCC147 60 [23] znuA F: TCATCAGTAGCGGTTTCACA
R: CACAGGTTGCTGAACTCGCC213 58 [24] terD F:AGTAAAGCAGCTCCGTCAAT
R: CCGAACAGCATGGCAGTCT434 55 [25] chrA F: TCACGCCGGAATATAACTAC
R: CGTACCCTGATCAATCACTT229 55 [26] czcA F:TCGACGGBGCCGTGGTSMTBGTCGAGAA
R: GTVAWSGCCAKCGGVBGGAACA232 63 [22] mntH F: TCACCGTGGCATACAGTGACACAC
R: TGAAATTGTTTTAGCGCACGACCT187 63 [27] ARGs aac(6')-Ib-cr F: TTGCGATGCTCTATGAGTGGCTA
R: CTCGAATGCCTGGCGTGTTT482 55 [28] qnrA F: AGAGGATTTCTCACGCCAGG
R: GCAGCACTATKACTCCCAAGG619 58 [29] tetY F:GCTGATATTTGCGGGTTTCTA
R:CGTCAAGCCTGTTAAAGTTCC177 58 [30] tetM FF:ACAGAAAGCTTATTATATAAC
R:TGGCGTGTCTATGATGTTCAC171 55 [31] tetA F:GCTACATCCTGCTTGCCTTC
R: CATAGATCGCCGTGAAGAGG210 64 [32] sul1 F:CGCACCGGAAACATCGCTGCAC
R: TGAAGTTCCGCCGCAAGGCTCG163 62 [33] sul2 F:TCCGGTGGAGGCCGGTATCTGG
R:CGGGAATGCCATCTGCCTTGAG191 62 [33] ermB F: TAAAGGGCATTTAACGACGAAACT
R:TTTATACCTCTGTTTGTTAGGGAATTGAA172 58 [34] intI intI1 F: GGCTTCGTGATGCCTGCTT
R: CATTCCTGGCCGTGGTTCT148 59 [35] intI2 F: GTTATTTTATTGCTGGGATTAGGC
R: TTTTACGCTGCTGTATGGTGC166 55 [36] intI3 F: GGATGTCTGTGCCTGCTTG
R: GCCACCACTTGTTTGAGGA100 60 [37] 16S rRNA 338F: ACTCCTACGGGAGGCAGCAG
518R: ATTACCGCGGCTGCTGG200 55 [38] 表 2 ARGs、HMRGs和intI之间斯皮尔曼相关性分析(n=3)
基因名称 德普昌达克冰川 中山站 sul2 tetA intI1 intI2 intI3 sul2 tetA intI1 intI2 intI3 merA −0.50 −1.00** −0.50 −1.00** −0.50 1.00** 1.00** −1.00** 1.00** 0.50 zntA −0.50 0.50 −0.50 0.50 −0.50 1.00** 1.00** −1.00** 1.00** 0.50 czcD 0.50 1.00** 0.50 1.00** 0.50 −0.50 −0.50 0.50 −0.50 −1.00** pcoA 0.50 1.00** 0.50 1.00** 0.50 0.50 0.50 −0.50 0.50 1.00** znuA 0.50 1.00** 0.50 1.00** 0.50 1.00** 1.00** −1.00** 1.00** 0.50 terD 1.00** 0.50 1.00** 0.50 1.00** 0.50 0.50 −0.50 0.50 1.00** chrA −0.50 −1.00** −0.50 −1.00** −0.50 0.50 0.50 −0.50 0.50 1.00** czcA 1.00** 0.50 1.00** 0.50 1.00** 0.50 0.50 −0.50 0.50 1.00** mntH −0.50 −1.00** −0.50 −1.00** −0.50 0.50 0.50 −0.50 0.50 1.00** sul2 1.00 0.50 1.00** 0.50 1.00** 1.00 1.00** −1.00** 1.00** 0.50 tetA 0.50 1.00 0.50 1.00** 0.50 1.00** 1.00 −1.00** 1.00** 0.50 注:**表示极显著相关(P<0.01,双边检验)。 -
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