Metagenomic driven isolation of poorly culturable species in food

Although isolating microorganisms from food microbiota may appear less challenging than from the gut or environmental sources, recovering all representative species from food remains a difficult task. Here, we showed by metagenomic analysis that several abundant species had escaped isolation in a previous study of ten cheeses, including several previously uncharacterized species. To address this gap, we designed a novel strategy integrating metagenomics-based probes targeting the species of interest, coupled with an incremental culturing approach using pooled samples. As proof of concept, we applied this strategy to two cheeses containing species that were not isolated in our previous study, with the objective of isolating all species present at levels above 2% and, in particular, potential novel food species. Through this approach, we successfully performed the targeted isolation of two Psychrobacter and two Vibrio species from the first cheese, and four Halomonas and two Pseudoalteromonas species from the second one. Notably, P. undina and V. litoralis represented, as far as we know, the first cheese isolates characterized for these species. However, we were unable to isolate a novel species of Pseudoalteromonas, with no characterized representative to date, and Marinomonas foliarum, previously isolated from marine environment. Using metagenome-assembled genomes (MAGs) and metagenomic analysis, we discussed the possible reasons for their non-recovery.尽管从食品微生物群中分离微生物看似比从肠道或环境来源中分离难度更低，但从食品中获取所有代表性物种仍是一项艰巨的任务。 本研究通过宏基因组分析（metagenomic analysis）发现，在一项针对10种奶酪的既往研究中，多个丰度较高的物种未被成功分离，其中还包含若干此前从未被表征的物种。 为填补这一研究空白，我们设计了一种全新策略：整合针对目标物种的宏基因组探针技术，并结合使用混合样本的增量培养方法。 为验证该策略的可行性，我们将其应用于两种此前研究中未分离到目标物种的奶酪，目标为分离出所有丰度占比超过2%的物种，尤其是潜在的新型食品源物种。 通过该方法，我们成功从第一种奶酪中靶向分离出2种不动杆菌属（Psychrobacter）和2种弧菌属（Vibrio）物种，从第二种奶酪中分离出4种盐单胞菌属（Halomonas）和2种假交替单胞菌属（Pseudoalteromonas）物种。 值得注意的是，据我们所知，undina假交替单胞菌（P. undina）与海岸弧菌（V. litoralis）是这两个物种首次在奶酪中获得分离并完成表征的菌株。 但我们未能分离出一种迄今尚无已表征代表菌株的新型假交替单胞菌属物种，以及此前仅从海洋环境中分离得到的叶海单胞菌（Marinomonas foliarum）。 我们借助宏基因组组装基因组（metagenome-assembled genomes，MAGs）与宏基因组分析手段，探讨了这两类物种未能被成功分离的潜在原因。