三种核果包装设施的环境微生物群落组成随季节变化而变化，其中包含指示李斯特菌污染的分类群。
The composition of environmental microbiota in three tree fruit packing facilities changed over seasons and contained taxa indicative of L. monocytogenes contamination.
机构信息
Department of Food Science, The Pennsylvania State University, University Park, PA, 16802, USA.
Microbiome Center, The Pennsylvania State University, University Park, PA, 16802, USA.
出版信息
Microbiome. 2023 Jun 5;11(1):128. doi: 10.1186/s40168-023-01544-8.
BACKGROUND
Listeria monocytogenes can survive in cold and wet environments, such as tree fruit packing facilities and it has been implicated in outbreaks and recalls of tree fruit products. However, little is known about microbiota that co-occurs with L. monocytogenes and its stability over seasons in tree fruit packing environments. In this 2-year longitudinal study, we aimed to characterize spatial and seasonal changes in microbiota composition and identify taxa indicative of L. monocytogenes contamination in wet processing areas of three tree fruit packing facilities (F1, F2, F3).
METHODS
A total of 189 samples were collected during two apple packing seasons from floors under the washing, drying, and waxing areas. The presence of L. monocytogenes was determined using a standard culturing method, and environmental microbiota was characterized using amplicon sequencing. PERMANOVA was used to compare microbiota composition among facilities over two seasons, and abundance-occupancy analysis was used to identify shared and temporal core microbiota. Differential abundance analysis and random forest were applied to detect taxa indicative of L. monocytogenes contamination. Lastly, three L. monocytogenes-positive samples were sequenced using shotgun metagenomics with Nanopore MinION, as a proof-of-concept for direct detection of L. monocytogenes' DNA in environmental samples.
RESULTS
The occurrence of L. monocytogenes significantly increased from 28% in year 1 to 46% in year 2 in F1, and from 41% in year 1 to 92% in year 2 in F3, while all samples collected from F2 were L. monocytogenes-positive in both years. Samples collected from three facilities had a significantly different microbiota composition in both years, but the composition of each facility changed over years. A subset of bacterial taxa including Pseudomonas, Stenotrophomonas, and Microbacterium, and fungal taxa, including Yarrowia, Kurtzmaniella, Cystobasidium, Paraphoma, and Cutaneotrichosporon, were identified as potential indicators of L. monocytogenes within the monitored environments. Lastly, the DNA of L. monocytogenes was detected through direct Nanopore sequencing of metagenomic DNA extracted from environmental samples.
CONCLUSIONS
This study demonstrated that a cross-sectional sampling strategy may not accurately reflect the representative microbiota of food processing facilities. Our findings also suggest that specific microorganisms are indicative of L. monocytogenes, warranting further investigation of their role in the survival and persistence of L. monocytogenes. Video Abstract.
背景
李斯特菌可以在寒冷潮湿的环境中生存，例如水果包装厂，它与水果产品的爆发和召回有关。然而，对于与李斯特菌共同存在的微生物群及其在水果包装环境中季节性的稳定性，我们知之甚少。在这项为期 2 年的纵向研究中，我们旨在描述三个水果包装厂（F1、F2、F3）湿处理区微生物群落组成的空间和季节变化，并确定指示李斯特菌污染的分类群。
方法
在两个苹果包装季节中，共从清洗、干燥和打蜡区下方的地板上采集了 189 个样本。使用标准培养方法确定李斯特菌的存在，使用扩增子测序描述环境微生物群。使用 PERMANOVA 比较两个季节设施之间的微生物群落组成，使用丰度-占据分析确定共享和时间核心微生物群。应用差异丰度分析和随机森林检测指示李斯特菌污染的分类群。最后，对三个李斯特菌阳性样本进行了基于 Nanopore MinION 的 shotgun 宏基因组测序，作为直接检测环境样本中李斯特菌 DNA 的概念验证。
结果
李斯特菌的检出率从第 1 年的 28%显著增加到第 2 年的 46%（F1），从第 1 年的 41%显著增加到第 2 年的 92%（F3），而第 2 年在 F3 中所有样本均为李斯特菌阳性。在两个年份中，来自三个设施的样本的微生物群落组成均有显著差异，但每个设施的组成都随时间而变化。一组细菌分类群包括假单胞菌、寡养单胞菌和微杆菌，以及真菌分类群包括隐球酵母属、库尔茨曼氏酵母属、节丛孢属、拟青霉属和毛孢子菌属，被鉴定为监测环境中李斯特菌的潜在指示生物。最后，通过直接对从环境样本中提取的宏基因组 DNA 进行 Nanopore 测序，检测到了李斯特菌的 DNA。
结论
本研究表明，横断面采样策略可能无法准确反映食品加工设施的代表性微生物群。我们的研究结果还表明，特定的微生物是李斯特菌的指示生物，值得进一步研究它们在李斯特菌生存和持续存在中的作用。
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