• 关键词：抗性基因  大肠杆菌  绿色荧光蛋白  重金属  基因转移

• 基金项目：国家自然科学基金（No.41671481）；广东省应用型科技研发专项资金项目（No.2016B020240003）

• 李亚飞
• 广东工业大学环境科学与工程学院, 广州 510006

• 许燕滨
• 广东工业大学环境科学与工程学院, 广州 510006

• 凌嘉茵
• 广东工业大学环境科学与工程学院, 广州 510006

• 周家乐
• 广东工业大学环境科学与工程学院, 广州 510006

• 何晓林
• 广东工业大学环境科学与工程学院, 广州 510006

• 黄禄
• 广东工业大学环境科学与工程学院, 广州 510006

• 郑莉
• 广东工业大学环境科学与工程学院, 广州 510006

• 乔庆霞
• 广东工业大学环境科学与工程学院, 广州 510006

• 摘要：为探究抗生素和重金属对抗性转移及接合子抗性基因丰度的影响，借助绿色荧光蛋白标记质粒，采用滤膜接合配对方法研究了胁迫条件对细菌接合频率的影响，定量检测了转移后的AmpC β-内酰胺酶类抗性基因丰度的变化.结果表明，头孢噻肟钠及重金属共胁迫促进了AmpC β-内酰胺酶类抗性基因从复杂群落到Escherichia coli BL21的转移.3 μg·L^-1头孢噻肟钠胁迫下，细菌接合频率为空白组的2.06倍，其中，接合子耐药基因CIT、FOX和EBC的相对丰度分别升高了7.47、4.30和1.60倍.同时，以3 μg·L^-1头孢噻肟钠胁迫组为对照，研究了重金属汞、铜和锌共胁迫对抗性转移的影响，考虑到头孢噻肟钠的水解特性和接合子克隆性扩增，接合时间定为3 h.结果发现，汞、铜、锌与头孢噻肟钠共胁迫组的细菌接合频率分别比对照组增长了2.57、1.60和1.74倍.汞共胁迫对接合子中7种AmpC β-内酰胺酶类抗性基因丰度无显著影响；铜共胁迫效应因抗生素抗性基因（ARGs）种类不同而不同，其中，对抗性基因EBC相对丰度的影响最大；锌共胁迫效应受锌离子浓度和ARGs种类影响，100 mg·L^-1锌共胁迫使得EBC和CIT丰度比对照组分别增加3.56和2.76倍.可见，抗生素与重金属共胁迫条件可促进环境中AmpC β-内酰胺酶类抗性基因的转移，值得关注.

• Abstract：In order to investigate the effects of antibiotics and heavy metals on antibiotics resistance gene(ARG) transfer and the abundance of ARGs, the transfer frequency of the AmpC β-lactamase gene under stress condition was studied by filter mating, and the quantitative detection of the transferred AmpC β-lactamase resistance gene was performed, with the aid of enhanced green fluorescent protein (EGFP)-tagged plasmid. Results show that co-stress of cefotaxime sodium and heavy metal contributed to a higher horizontal transfer frequency of AmpC β-lactamase genes from mixed microbial community to Escherichia coli BL21. Bacterial conjugation frequency of the stress group exposure to cefotaxime sodium of 3 μg·L^-1 is 2.06 times higher than that of control group, the resistant gene of CIT, FOX, EBC increased 7.47, 4.30, 1.60 times, respectively. Considering the time for cefotaxime sodium hydrolysis properties and transconjugants' clonal growth, conjugation time was designed to 3 h to investigate the effects of mercury(Ⅱ), copper(Ⅱ), zinc(Ⅱ) on resistance transfer with cefotaxime sodium concentration of 3 μg·L^-1. The transfer frequency of bacterial was 2.57, 1.60, and 1.74 times higher than that of 3 μg·L^-1 cefotaxime sodium under heavy metal mercury, copper, zinc and antibiotic stress conditions, respectively. However, mercury was found to have no significant effect on abundance of seven AmpC β-lactamase-resistant genes in the transconjugants. With the co-stress of copper, the relative abundance was different from ARGs to ARGs and the relative abundance of EBC changed most obviously. The co-stress of zinc could vary the relative abundance with both zinc concentration and ARGs diversity, and the abundance of EBC and CIT increased by 3.56 and 2.76 times compared with the control group with zinc at 100 μg·L^-1. Consequently, co-stress of antibiotics and heavy metal could promote the transfer of the AmpC β-lactamases resistance gene-in-environment, which was worth serious concerns.

• Key words：antibiotic resistance  Escherichia coli  EGFP  heavy metal  gene transfer

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