Microbiome-based environmental monitoring of a dairy processing facility highlights the challenges associated with low microbial-load samples

Abstract

Food processing environments can harbor microorganisms responsible for food spoilage or foodborne disease. Efficient and accurate identification of microorganisms throughout the food chain can allow the identification of sources of contamination and the timely implementation of control measures. Currently, microbial monitoring of the food chain relies heavily on culture-based techniques. These assays are determined on the microbes expected to be present in the environment, and thus do not cater for unexpected contaminants. Many culture-based assays are also unable to distinguish between undesirable taxa and closely related harmless species. Furthermore, even when multiple culture-based approaches are used in parallel, it is still not possible to comprehensively characterize the entire microbiology of a food-chain sample.High throughput DNA sequencing represents a potential means through which microbial monitoring of the food chain can be enhanced. While sequencing platforms, such as the Illumina MiSeq, NextSeq and NovaSeq, are most typically found in research or commercial sequencing laboratories, newer portable platforms, such as the Oxford Nanopore Technologies (ONT) MinION, offer the potential for rapid analysis of food chain microbiomes. In this study, having initially assessed the ability of rapid MinION-based sequencing to discriminate between different microbes within a simple mock metagenomic mixture of related food spoilage, spore-forming microorganisms. Subsequently, we proceeded to compare the performance of both ONT and Illumina sequencing for environmental monitoring of an active food processing facility.Overall, ONT MinION sequencing provided accurate classification to species level, which was comparable to Illumina-derived outputs. However, while the MinION-based approach provided a means of easy library preparations and portability, the high concentrations of DNA needed to run the rapid sequencing protocols was a limiting factor, requiring the random amplification of template DNA in order to generate sufficient material for analysis.

Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study

“…Additionally, for shotgun sequencing with ONT, MDA amplification was needed to generate sufficient quantities of DNA required for sequencing, which has been previously found to introduce some biases (McHugh et al, 2021). Despite all these points, as improvements are made to the sequencing technology and analytical tools, coupled with its advantages in portability, time effectiveness and the possibility of real-time analysis, there is great potential for the use of ONT sequencing in characterising microbial communities in food and foodrelated environments.…”

A Round Trip to the Desert: In situ Nanopore Sequencing Informs Targeted Bioprospecting

“…Nanopore sequencing has previously been used in range of real-time applications, such as pathogen detection and surveillance (Quick et al, 2016;Charalampous et al, 2019;Chan et al, 2020); forensic identification (Tytgat et al, 2020;Vasiljevic et al, 2021); or industrial process monitoring (Hardegen et al, 2018;McHugh et al, 2021). Among all the potential uses of MinION, in situ sequencing is especially interesting for those situations where no alternative analyses are feasible due to a lack of equipment (i.e., second-generation sequencing platforms, qPCR instruments.).…”