PDF(Pubmed)
Abstract：
Avian pathogenic Escherichia coli (APEC) cause avian colibacillosis and accurately distinguishing infectious isolates is critical for controlling its transmission. Multilocus sequence typing (MLST) is an accurate and efficient strain identification method for epidemiological surveillance. This research aimed to develop a fast and high-throughput workflow that simultaneously sequences the Achtman typing scheme\'s 7 housekeeping genes of multiple E. coli isolates using the Oxford Nanopore Technologies (ONT) platform for large-scale APEC study. E. coli strains were isolated from poultry farms, the housekeeping genes were amplified, and amplicons were sequenced on an R9.4 MinION flow cell using the Nanopore GridION sequencer (ONT, Oxford, UK) following the initial workflow (ONT-MLST). Moreover, the workflow was revised by introducing large-scale DNA extraction and multiplex PCR into the ONT-MLST workflow and applied to 242 new isolates, 18 isolates from the previous workflow, and 5 ATCC reference strains using Flongle flow cell on the Nanopore MinION Mk1C sequencer (ONT, Oxford, UK). Finally, the sequence type (ST) results of the 308 isolates collected from infected chickens and poultry farm environments were reported and analyzed. Data indicated that E. coli belonging to ST159, ST8578, and ST355 have the potential to infect multiple organs in broiler. In addition, zoonotic STs, ST69, ST10, ST38, and ST131, were detected from poultry farms. With the advantages of the high throughput of ONT, this study provides a rapid workflow for large-scale E. coli typing and identified frequently isolated sequence types related to APEC infection in poultry.
摘要：
禽致病性大肠杆菌（APEC）引起禽大肠杆菌病，准确区分感染性分离株对于控制其传播至关重要。多位点序列分型(MLST)是一种准确、高效的流行病学监测毒株鉴定方法。这项研究旨在开发一种快速和高通量的工作流程，使用牛津纳米孔技术（ONT）平台对多个大肠杆菌分离株的Achtman分型方案的7个管家基因进行测序，以进行大规模的APEC研究。大肠杆菌菌株从家禽养殖场中分离出来，管家基因被扩增，和扩增子在R9.4MinION流动池上使用NanoporeGridION测序仪（ONT，牛津,英国)遵循初始工作流程(ONT-MLST)。此外，通过在ONT-MLST工作流程中引入大规模DNA提取和多重PCR来修改工作流程，并将其应用于242个新分离株，从以前的工作流程中分离出18个，和5个ATCC参考菌株在NanoporeMinIONMk1C测序仪上使用Flongle流动池(ONT，牛津,英国）。最后,报告并分析了从受感染的鸡和家禽养殖场环境中收集的308个分离株的序列类型(ST)结果.数据表明,属于ST159、ST8578和ST355的大肠杆菌具有感染肉鸡的多个器官的潜力。此外,人畜共患STs，从家禽养殖场检测到ST69、ST10、ST38和ST131。凭借ONT高吞吐量的优势，这项研究为大规模大肠杆菌分型提供了一个快速的工作流程，并确定了与家禽中APEC感染相关的频繁分离的序列类型。
