摘要

      环境中的抗生素耐药基因（ARGs）由于其在细胞分裂过程中的复制和被病原体获得的潜力，已成为人们日益关注的问题。据报道，水平基因转移（HGT）是ARGs在微生物中传播的主要原因，但由于检测手段的不足，HGT在环境生物膜中的特性尚不清楚。为了应对这一挑战，我们采用了一种新型的微流控系统，通过不断提供营养和细胞间的紧密接触来培养薄的生物膜。类似于开放环境中生物膜的生存状态，该芯片显示了ARG编码质粒RP4和pKJK5向受体（如活性污泥细菌）的转移。经流式细胞仪测定，RP4寄主的假单胞菌KT2440到活性污泥菌的平均质粒转移频率（T/R）为2.5 ××10-3，pKJK5寄主的大肠杆菌MG1655的平均质粒转移频率（T/R）为8.9 ××10-3，两种寄主菌株的平均供体频率（T/D）不同，分别为4.3×10-3和1.4×10-1。质粒转移动力学解释了T/R和T/D之间的差异，暗示了这两种计算的特殊目的。最后，我们通过荧光活化细胞分类收集了这些转接蛋白，并进一步测序它们的16srdna。蛋白质细菌门和硬壁菌的细菌比拟杆菌科的细菌更易被转运。实验结果表明，微流控系统在生物膜HGT研究中具有优势，可以为环境ARG的控制提供更多的信息。

       Antibiotic resistance genes (ARGs) in environment have become a growing public concern, due to their potential to be obtained by pathogens and their duplication along cell division. Horizontal gene transfer (HGT) was reported to be responsible for ARGs dissemination in microbes, but the HGT feature in environmental biofilm was still unclear due to insufficient assay tools. To address this challenge, we applied a novel microfluidic system to cultivate thin biofilm by continuous supply of nutrients and close contact between cells. Resembling the living state of biofilm in open environment, this chip visualized the transfer of ARG-encoded plasmids RP4 and pKJK5 to the receptors, e.g., activated sludge bacteria. The average plasmid transfer frequency per receptor (T/R) from RP4-hosted Pseudomonas putida KT2440 to activated sludge bacteria was quantified to be 2.5 × 10^-3 via flow cytometry, and T/R for pKJK5-hosted Escherichia coli MG1655 was 8.9 × 10^-3, while the corresponding average frequencies per donor (T/D) were diverse for the two host strains as 4.3 × 10^-3 and 1.4 × 10^-1 respectively. The difference between T/R and T/D was explained by the plasmid transfer kinetics, implying specific purposes of the two calculations. Finally, we collected the transconjugants by fluorescent activated cell sorting and further sequenced their 16S rDNA. Bacteria from phyla Proteobacteria and Firmicutes were found more susceptible to be transconjugants than those from Bacteroidetes. Our work demonstrated that microfluidic system was advantageous in biofilm HGT study, which can provide more insights into environmental ARG control.

        https://www.sciencedirect.com/science/article/pii/S0048969718320746?via%3Dihub