Long-read sequencing data from pure cultures of Escherichia coli O157:H7 and ground beef inoculated with E. coli O157:H7

Foodborne pathogens are a significant cause of illness and infection with Shiga toxin-producing Escherichia coli (STEC) has the potential to produce life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate using long-read sequencing to detect STEC in ground beef. The objectives of the project included: establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on Oxford Nanopore Technologies’ MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, rrsC) were detected in DNA extracted from STEC pure cultures within 1 hour of sequencing, and 30X coverage was obtained within 2 hours. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. Growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef. The sequencing data from this project has been uploaded.

食源性病原体是导致疾病和感染的重要因素，其中产志贺毒素的大肠杆菌（STEC）具有引发致命并发症的潜力。目前识别肉类中STEC的方法包括基于培养、分子和蛋白质组学的检测，且整个过程至少需耗时四天。通过采用长读长全基因组测序来识别食源性病原体，可缩短这一时间。因此，本项目旨在评估利用长读长测序技术检测碎牛肉中的STEC。项目目标包括：确立最佳的测序参数，确定所有目标STEC致病基因在纯培养和掺假碎牛肉中的检测限，以及评估选择性测序以增强碎牛肉中STEC的检测。测序文库在牛津纳米孔技术公司的MinION测序仪上运行。在MinKNOW中采用默认参数获得最佳测序输出，仅将最小读长设置为1 kb。所有目标基因（eae、stx1、stx2、fliC、wzx、wzy、rrsC）在测序后1小时内从STEC纯培养中提取的DNA中均被检测到，且在2小时内获得了30倍覆盖度。所有致病基因在STEC DNA含量低至12.5 ng时均被可靠地检测到。在接种STEC的碎牛肉中，软件控制的选择性测序提高了致病基因的检测；然而，由于样本中牛DNA浓度较高，导致某些致病基因未能检测到。在mTSB中接种的肉样进行生长富集后，与未富集样本相比，致病基因的检测量提高了100倍。本项目的研究结果表明，长读长测序协议的进一步开发可能导致检测碎牛肉中STEC的更快、更节省劳动力的方法。本项目的测序数据已上传。