鸟枪宏基因组学通过提供对微生物污染物的快速和全面的见解,在食源性暴发的调查中被证明是有效的。然而,样品的培养富集仍然是一个先决条件,尽管生长竞争对病原体检测有潜在影响。为了规避文化丰富的需要,我们探索了使用自适应采样使用各种数据库进行靶向纳米孔测序,与单独的猎枪宏基因组学相比。
■自适应采样方法首先在与先前与食源性爆发有关的金黄色葡萄球菌菌株的DNA混合的土豆泥的DNA上进行了测试。选择性测序用于耗尽马铃薯测序读数或富集病原体测序读数,并与猎枪测序进行了比较。然后,活的金黄色葡萄球菌以105CFU掺入25g土豆泥中。测试了三个DNA提取试剂盒,结合使用自适应采样的富集,全基因组扩增后。经过数据分析,用不同的测序和提取方法表征污染物的可能性,没有文化丰富,被评估。
■总的来说,自适应采样优于鸟枪测序.虽然使用宿主去除DNA提取试剂盒和使用食源性病原体数据库的靶向测序允许快速检测病原体,当仅使用金黄色葡萄球菌数据库与常规DNA提取试剂盒结合使用时,可以实现最完整的表征,能够在爆发病例的同时将菌株准确地放置在系统发育树中。
■该方法显示出在无需培养物富集的情况下对食源性暴发进行菌株水平分析的巨大潜力,从而使更快的调查和促进精确的病原体表征。适应性采样与宏基因组学的整合为更有效和有针对性地分析食源性暴发中的微生物群落提供了有价值的策略,有助于改善食品安全和公共卫生。
UNASSIGNED: Shotgun metagenomics has previously proven effective in the investigation of foodborne outbreaks by providing rapid and comprehensive insights into the microbial contaminant. However, culture enrichment of the sample has remained a prerequisite, despite the potential impact on pathogen detection resulting from the growth competition. To circumvent the need for culture enrichment, we explored the use of adaptive sampling using various databases for a targeted nanopore sequencing, compared to shotgun metagenomics alone.
UNASSIGNED: The adaptive sampling method was first tested on DNA of mashed potatoes mixed with DNA of a Staphylococcus aureus strain previously associated with a foodborne outbreak. The selective sequencing was used to either deplete the potato sequencing reads or enrich for the pathogen sequencing reads, and compared to a shotgun sequencing. Then, living S. aureus were spiked at 105 CFU into 25 g of mashed potatoes. Three DNA extraction kits were tested, in combination with enrichment using adaptive sampling, following whole genome amplification. After data analysis, the possibility to characterize the contaminant with the different sequencing and extraction methods, without culture enrichment, was assessed.
UNASSIGNED: Overall, the adaptive sampling outperformed the shotgun sequencing. While the use of a host removal DNA extraction kit and targeted sequencing using a database of foodborne pathogens allowed rapid detection of the pathogen, the most complete characterization was achieved when using solely a database of S. aureus combined with a conventional DNA extraction kit, enabling accurate placement of the strain on a phylogenetic tree alongside outbreak cases.
UNASSIGNED: This method shows great potential for strain-level analysis of foodborne outbreaks without the need for culture enrichment, thereby enabling faster investigations and facilitating precise pathogen characterization. The integration of adaptive sampling with metagenomics presents a valuable strategy for more efficient and targeted analysis of microbial communities in foodborne outbreaks, contributing to improved food safety and public health.