Meta-transcriptomic analysis of the response of river biofilms to pharmaceutical products using anonymous DNA microarray

Low concentrations of pharmaceutical compounds were shown to induce transcriptional responses in isolated microorganisms, which could have consequences on ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two distinct rivers and supplemented with environmentally relevant doses of four pharmaceutical products (erythromycin-ER, gemfibrozil-GM, sulfamethazine-SN and sulfamethoxazole-SL). To follow the expression of functional genes, we constructed a 9,600 features anonymous DNA microarray platform onto which cDNA from the various biofilms was hybridized. The reactor design for biofilm development has been previously described (Lawrence et al., 2004; Lawrence et al., 2000). Two duplicate experiments were carried out, with reactors being inoculated with either water from the WC (nutrient rich) or the SSR (nutrient poor). Treatments consisted in the addition of various pharmaceutical compounds: 1 µg l-1 erythromycin (ER), 1 µg l-1 gemfibrozil (GM), 0.5 µg l-1 sulfamethazine (SN), 0.5 µg l-1 sulfamethoxazole (SL). Nothing was added to control reactors (CO). All treatments were replicated independently three times. A reference sample (composite sample from Wascana Creek reactors used to construct the microarray) was hybridized (Cy5) on each slide.

研究表明，低浓度药物化合物可诱导分离培养微生物产生转录应答反应，这可能对生态系统动态产生影响。为验证这类转录应答是否同样存在于复杂的河流微生物群落中，本研究采用取自两条不同河流的水体接种生物膜反应器，并向反应器中投加环境相关剂量的四种药物化合物：红霉素（erythromycin-ER）、吉非贝齐（gemfibrozil-GM）、磺胺二甲嘧啶（sulfamethazine-SN）以及磺胺甲恶唑（sulfamethoxazole-SL）。为追踪功能基因的表达水平，本研究构建了包含9600个特征位点的匿名化DNA微阵列（DNA microarray）平台，将不同生物膜样品的互补DNA（complementary DNA，cDNA）与该平台进行杂交。生物膜培养所用反应器的设计方案已在既往研究中发表（Lawrence等，2004；Lawrence等，2000）。本研究开展两组重复实验，反应器分别接种取自WC（富营养）或SSR（贫营养）的水体。实验组投加不同组合的药物化合物：1 μg·L⁻¹红霉素（erythromycin-ER）、1 μg·L⁻¹吉非贝齐（gemfibrozil-GM）、0.5 μg·L⁻¹磺胺二甲嘧啶（sulfamethazine-SN）以及0.5 μg·L⁻¹磺胺甲恶唑（sulfamethoxazole-SL）；对照组反应器（CO）不添加任何药物化合物。所有处理组均独立重复三次。每张微阵列玻片均将参考样品（用于构建该微阵列的Wascana Creek反应器混合样品）以Cy5荧光染料标记后进行杂交。