Uncovering bacterial hosts of class 1 integrons in an urban coastal aquatic environment with a single-cell fusion-polymerase chain reaction technology

Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbour class 1 integrons. We developed a modified version of epicPCR (emulsion, paired isolation and concatenation PCR) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first application of epicPCR fo..., Isolation of bacteria from coastal seawater 
Coastal water samples were collected in three 50 mL biological replicates after rain in a rocky intertidal zone downstream from a stormwater outlet at Shark Point (location coordinates: -33.91, 151.27) near Sydney, NSW, Australia (Supplementary Figure 1). The samples were filtered using EASYstrainer cell strainers with a mesh size of 40 µm (Greiner AG, Germany) and centrifuged at 3220g for 15 minutes. Cell pellets were resuspended in phosphate buffered saline (PBS) solution at pH 7.4. Resuspended cells were stored as 25% glycerol stocks.
Bacterial cell counts by fluorescence microscopy 
Approximately 105–106 cells were fixed and stained in PBS containing 4% formaldehyde and 2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 30 min in the dark with rotation at room temperature. Re-suspended cells were diluted in PBS, transferred to a haemocytometer and imaged under the x40 lens of an Olympus BX63 fluorescence microscope. Differential interfere..., The sequencing data generated in this study are available in this repository. The nine FASTQ files that are available for download are as follows:
S1_1_Replicate_1.fastq;  S1_1_Replicate_2.fastq; S1_1_Replicate_3.fastq;  S1_2_Replicate_1.fastq; S1_2_Replicate_2.fastq; S1_2_Replicate_3.fastq; S1_3_Replicate_1.fastq; S1_3_Replicate_2.fastq; S1_3_Replicate_3.fastq
The downloaded .ZIP file can be decompressed with the unzip command in the UNIX/Linux environment or using the macOS Terminal. All Nanopore sequence filtering and processing steps for epicPCR amplicon reads were carried out using an in-house pipeline (https://github.com/timghaly/Int1-epicPCR). First, the pipeline quality-filters Nanopore reads using NanoFilt v2.8.0 (1), removing those with an average read quality of less than 7 and read length less than 670 bp, which represents the minimum length of an epicPCR product with a cassette-less integron. Quality-filtered reads are then oriented and trimmed with the final nested epicPCR...

水平基因转移（Horizontal gene transfer, HGT）是通过跨类群遗传物质传递推动细菌进化的关键驱动力。1类整合子是一类与人为污染强相关的遗传元件，可通过水平基因转移促进抗菌耐药性（antimicrobial resistance, AMR）基因的传播。尽管其对人类健康具有重要意义，但目前仍缺乏可靠的无培养监测技术，用以鉴定携带1类整合子的未培养环境类群。本研究改良了乳液配对分离串联聚合酶链式反应（emulsion, paired isolation and concatenation PCR, epicPCR）技术，该技术可在乳化水相液滴中，将从单个细菌细胞扩增得到的1类整合子与同一细胞的分类学标记物进行关联。利用该单细胞基因组学方法结合纳米孔测序（Nanopore sequencing），我们成功将主要携带抗菌耐药性基因的1类整合子基因盒阵列，匹配至受污染的近岸海水样本中的宿主菌。本研究首次将epicPCR应用于……，从近岸海水中分离细菌。
近岸海水样本采集自澳大利亚新南威尔士州悉尼附近鲨鱼点（坐标：-33.91, 151.27）雨水过后的暴雨排污口下游岩质潮间带，设置3组50 mL生物学重复样本（补充图1）。使用孔径40 μm的EASYstrainer细胞过滤器（Greiner AG，德国）过滤样本，随后以3220g离心15分钟。将细胞沉淀重悬于pH 7.4的磷酸盐缓冲液（phosphate buffered saline, PBS）中，重悬后的细胞以25%甘油储存液形式保存。
细菌细胞荧光显微镜计数：取约10^5–10^6个细胞，在含4%甲醛和2 μg/mL 4′,6-二脒基-2-苯基吲哚（4′,6-diamidino-2-phenylindole, DAPI）的PBS中固定染色30分钟，室温避光旋转孵育。将重悬细胞用PBS稀释后转移至血细胞计数板，使用奥林巴斯BX63荧光显微镜的x40物镜成像。差分干涉……
本研究产生的测序数据可在本仓库获取。可供下载的9个FASTQ文件如下：
S1_1_Replicate_1.fastq; S1_1_Replicate_2.fastq; S1_1_Replicate_3.fastq; S1_2_Replicate_1.fastq; S1_2_Replicate_2.fastq; S1_2_Replicate_3.fastq; S1_3_Replicate_1.fastq; S1_3_Replicate_2.fastq; S1_3_Replicate_3.fastq
下载的.ZIP压缩包可在UNIX/Linux环境下使用unzip命令或通过macOS终端解压。针对epicPCR扩增子读段的所有纳米孔序列过滤与处理步骤，均通过自研流程完成（https://github.com/timghaly/Int1-epicPCR）。该流程首先使用NanoFilt v2.8.0对纳米孔读段进行质量过滤，移除平均读段质量低于7、读长小于670 bp的序列——该读长阈值对应不含基因盒的整合子epicPCR产物的最小长度。经过质量过滤的读段随后将进行方向校正与截切，最终得到嵌套式epicPCR……