Biology subjects

Crispie, F.

Publications and source records attributed to Crispie, F..

3 recordsLinked to original sources

bioRxiv2022

Development of an amplicon-based high throughput sequencing method for genotypic characterisation of norovirus in oysters

Norovirus is a highly diverse RNA virus often implicated in food-borne outbreaks, particularly shellfish. Shellfish are filter feeders, and when harvested in bays exposed to wastewater overflow or storm overflows, they can harbour various pathogens, including human pathogenic viruses. The application of Sanger or amplicon-based High Throughput Sequencing (HTS) technologies to identify human pathogens in shellfish faces two main challenges i) distinguishing multiple genotypes/variants in a single sample and ii) low concentrations of norovirus RNA. Here we have assessed the performance of a novel norovirus capsid amplicon HTS method. We generated a panel of spiked oysters containing various norovirus concentrations with different genotypic compositions. Several DNA polymerase and Reverse Transcriptase (RT) enzymes were compared, and performance was evaluated based on i) the number of reads passing quality filters per sample, ii) the number of correct genotypes identified, and iii) the sequence identity of outputs compared to Sanger-derived sequences. A combination of the reverse transcriptase LunaScript and the DNA polymerase AmpliTaq Gold provided the best results. The method was then employed, and compared with Sanger sequencing, to characterise norovirus populations in naturally contaminated oysters. ImportanceWhile foodborne outbreaks account for approximately 14% of norovirus cases (Verhoef L, Hewitt J, Barclay L, Ahmed S, Lake R, Hall AJ, Lopman B, Kroneman A, Vennema H, Vinje J, Koopmans M. 2015. 1999-2012. Emerg Infect Dis 21:592-599), we do not have standardised high-throughput sequencing methods for genotypic characterisation in foodstuffs. Here we present an optimised amplicon high- throughput sequencing method for the genotypic characterisation of norovirus in oysters. This method can accurately detect and characterise norovirus at concentrations typically detected in oysters. It will permit the investigation of norovirus genetic diversity in complex matrices and contribute to ongoing surveillance of norovirus in the environment.

microbiology↗

bioRxiv2020

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

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.

microbiology↗

bioRxiv2020

Fermented food metagenomics reveals substrate-associated differences in taxonomy, health-associated- and antibiotic resistance-determinants

Fermented foods have been the focus of ever greater interest as a consequence of purported health benefits. Indeed, it has been suggested that the consumption of these foods that help to address the negative consequences of industrialization of the human gut microbiota in Western society. However, as the mechanisms via which the microbes in fermented foods improve health are not understood, it is necessary to develop an understanding of the composition and functionality of the fermented food microbiota to better harness desirable traits. Here we considerably expand the understanding of fermented food microbiomes by employing shotgun metagenomic sequencing to provide a comprehensive insight into the microbial composition, diversity and functional potential (including antimicrobial resistance, carbohydrate-degrading and health-associated gene content) of a diverse range of 58 fermented foods from artisanal producers from around the Globe. Food type, i.e., dairy-, sugar- or brine-type fermented foods, was to be the primary driver of microbial composition, with dairy foods found to have the lowest microbial diversity. From the combined dataset, 127 high quality metagenome-assembled genomes (MAGs), including 10 MAGs representing putatively novel species of Acetobacter, Acidisphaera, Gluconobacter, Lactobacillus, Leuconostoc and Rouxiella, were generated. Potential health promoting attributes were more common in fermented foods than non-fermented equivalents, with waterkefirs, sauerkrauts and kvasses containing the greatest numbers of potentially health-associated gene clusters (PHAGCs). Ultimately, this study provides the most comprehensive insight into the microbiomes of fermented foods to date, and yields novel information regarding their relative health-promoting potential. ImportanceFermented foods are regaining popularity in Western society due in part to an appreciation of the potential for fermented food microbiota to positively impact on health. Many previous studies have studied fermented microbiota using classical culture-based microbiological methods, older molecular techniques or, where deeper analyses have been performed, have involved a relatively small number of one specific food type. Here, we have used a state-of-the-art shotgun metagenomic approach to investigate 58 different fermented foods of different type and origin. Through this analysis, we were able to identify the differences in the microbiota across these foods, the factors that drove their microbial composition, and the relative potential functional benefits of these microbes. The information provided here will provide significant opportunities for the further optimisation of fermented food production and the harnessing of their health promoting potential.

microbiology↗