不同营养水平下混合抗生素调控铜绿微囊藻水华的基因组学机制

Ecological hazard of antibiotic pollutants at environmentally relevant concentrations remains unclear, except for inducing tolerance/resistance of bacteria. Cyanobacteria are an important class of prokaryotes with bacteria-like structures. A previous study of our laboratory found that antibiotic pollutants could stimulate the growth of a typical cyanobacterium (Microcystis aeruginosa) and the production of cyanotoxins during static culture. In this study, M. aeruginosa was chronically exposed to binary, ternary and quarternary mixtures of spiramycin, amoxicillin, ciprofloxacin and sulfamethoxazole. The long-term effects and cellular mechanism of mixed antibiotics on the growth of M. aeruginosa and the production of microcystins were studied through semi-continuous culture at currently reported contamination levels. The action mechanism of mixed antibiotics in M. aeruginosa was interpreted at the genomic level through microarray based-comparative genomic hybridization. Differentially expressed genes in antibiotic-treated groups were further verified by real-time polymerase chain reaction (PCR) and differentially expressed proteins, according to which antibiotic-responding genes were recognized. Mutation of antibiotic-responding genes was then screened. Resistance of M. aeruginosa to mixed antibiotics was checked on the basis of genomic responses. At last, combined regulation effects and mechanism of mixed antibiotics and nutrient factors on the bloom of M. aeruginosa were studied. The experimental design of this study was close to the actual conditions in aquatic environments. The obtained results could contribute to a better knowledge of the ecological hazard of antibiotics and provide reference for dictating discharge standards for antibiotics and controlling cyanobacterial bloom with co-existing antibiotics.

除诱导产生抗药/耐药细菌外，抗生素的其他生态危害尚不完全清楚。蓝藻具有和细菌相似的原核细胞结构。前期研究发现抗生素在现有污染浓度下对蓝藻的生长和产毒具有短期促进作用。本项目选定典型抗生素螺旋霉素、阿莫西林、环丙沙星和磺胺甲恶唑为目标化合物，依据水环境污染现状构建多元混合物。通过半连续培养继续研究低浓度抗生素混合物对典型蓝藻（铜绿微囊藻）的长期调控效应，并阐述其细胞学机制。同时以基因芯片为载体利用比较基因组杂交技术，从全基因组水平分析混合抗生素对微囊藻的作用机制，并通过实时定量PCR和差异表达蛋白进行多角度验证，确认特异性响应基因。再从特异性响应基因入手筛查抗生素引发的基因突变，探索微囊藻对抗生素的抗性。最后模拟实际水环境引入营养因子，阐明混合抗生素和营养因子对微囊藻的联合调控效应与机制。研究结果可以深化对抗生素生态危害的认知，为抗生素排放标准制定和复合污染条件下蓝藻水华治理提供依据。

痕量抗生素污染物在短期静态培养条件下可以促进蓝藻模式种铜绿微囊藻的生长和产毒，存在促进铜绿微囊藻爆发进而危害水生态安全的潜在可能。但究竟该可能性在天然水环境中是否可以成为现实，仍需验证抗生素对铜绿微囊藻的促进作用是否具有持续性，并揭示抗生素对铜绿微囊藻的作用机制，分析抗生素与常规水环境因子的联合调控效应。鉴于水环境中多种痕量抗生素同时共存的污染现状，本项目以螺旋霉素、阿莫西林、环丙沙星、磺胺甲恶唑和四环素等5类经常检出的典型抗生素混合物为目标物质，通过30天的半连续培养试验，分析混合抗生素对铜绿微囊藻的长期效应。研究结果表明，150ng/L和200ng/L的二元混合抗生素和60-200ng/L的三元、四元、五元混合抗生素持续促进藻细胞生长和提升藻细胞叶绿素合成量（p <0.05）。60-200ng/L的二元、三元、四元、五元混合抗生素暴露下，藻细胞的光合活性、POD酶活性、ATP合成酶活性、藻毒素合成和释放能力得到显著提升（p <0.05），且上述提升作用具有持续性。通过对藻细胞进行转录组和蛋白质组测序，发现藻细胞中参与活性氧清除、光合作用、碳固定、电子传递、氧化磷酸化和生物合成（包括叶绿素合成和微囊藻毒素合成）等过程的23个差异基因/蛋白对抗生素混合物具有特异性响应，是混合抗生素在藻细胞中的可信作用靶位。靶基因/蛋白的生物信息学分析表明藻细胞的光合作用、碳代谢和能量代谢之间通过电子传递链耦联，共同防御混合抗生素产生的氧化压力，进而产生一系列适应性响应。高通量测序结果表明抗生素并未诱导藻细胞产生基因突变和抗性基因，抗生素对藻细胞的刺激作用主要归结于毒物兴奋效应。由于营养因子是水华形成最重要的水环境因子，本项目继续研究营养因子（氮和磷）和抗生素对微囊藻的联合调控效应和机制。结果表明，随着营养水平的下降，抗生素对藻细胞的刺激作用也下降。混合抗生素暴露浓度与营养水平之间存在交互作用（p < 0.05）。在不同的营养水平下，psaA、psbB、cbbL基因表达量均受混合抗生素诱导上调，光合作用在藻细胞对抗生素的应激性响应中发挥最核心的作用。当每种抗生素的浓度降低至5ng/L的安全浓度阈值以下时，抗生素混合物对铜绿微囊藻无显著刺激作用（p > 0.05）。蓝藻水华治理过程中有必要同步治理共存的抗生素污染物。