High-Throughput 16S rRNA Sequencing to Assess Potentially Active Bacteria and Foodborne Pathogens: A Case Example in Ready-to-Eat Food

Abstract

Technologies to detect the entire bacterial diversity spectra and foodborne pathogens in food represent a fundamental advantage in the control of foodborne illness. Here, we applied high-throughput 16S rRNA sequencing of amplicons obtained by PCR and RT-PCR from extracted DNA and RNA targeting the entire bacterial community and the active bacterial fraction present in some of the most consumed and distributed ready-to-eat (RTE) salad brands in Europe. Customer demands for RTE food are increasing worldwide along with the number of associated foodborne illness and outbreaks. The total aerobic bacterial count in the analyzed samples was in the range of 2–4 × 106 CFU/g (SD ± 1.54 × 106). Culture validated methods did not detect Salmonella spp., Escherichia coli, and other fecal coliforms. 16S rRNA gene Illumina next-generation sequencing (NGS) data were congruent with these culture-based results and confirmed that these and other well-known foodborne bacterial pathogens, such as Listeria, were not detected. However, the fine-resolution of the NGS method unveiled the presence of the opportunistic pathogens Aeromonas hydrophyla and Rahnella aquatilis (relative frequency of 1.33–7.33%) that were metabolically active in addition to non-pathogenic, active members of Yersinia spp. (relative frequency of 0.0015–0.003%). The common ail and foxA marker genes of Yersinia enterocolitica were not detected by qPCR. Finally, our NGS data identified to non-pathogenic Pseudomonas spp. as the most abundant and metabolically active bacteria in the analyzed RTE salads (53–75% of bacterial abundance). Our data demonstrate the power of sequencing, in parallel, both 16S rRNA and rDNA to identify and discriminate those potentially and metabolically active bacteria and pathogens to provide a more complete view that facilitates the control of foodborne diseases, although further work should be conducted to determine the sensitivity of this method for targeting bacteria

A Systematic Review and Meta-Analysis of the Effects of Food Safety and Hygiene Training on Food Handlers

“…Food safety and hygiene are critical in all steps in the farm-to-fork chain. In an ideal scenario of the farm-to-fork continuum, a total absence of foodborne pathogens and opportunistic bacteria is obviously desired [120]. Nevertheless, despite good knowledge, attitude, and self-reported practices, there may be poor performance in hygiene [121] and food safety practices.…”

“…Nevertheless, despite good knowledge, attitude, and self-reported practices, there may be poor performance in hygiene [121] and food safety practices. Bacteria might exist in nature in a range of different metabolic stages, such as dormant, active, and growing; thus, it is important to detect bacteria and ascertain whether they are potentially active [120]. Despite the central role that food workers' hands play in bacterial transfer among food and various surfaces [81], only one study assessed the number of bacteria growing on cultures obtained from the hands [86], while another demonstrated cross-contamination with hand hygiene sessions using GloGerm ® powder and UV light [91].…”

16S rRNA gene-based assessment of common broiler chicken sampling methods: Evaluating intra-flock sample size, cecal pair similarity, and cloacal swab similarity to other alimentary tract locations

“…Using a culture-free approach to survey a microbial community allows for the identification of both culturable and non-culturable microorganisms. Inevitably, there are also limitations to this approach, such as the inability to distinguish between live and dead cells, the use of relative abundance data (as opposed to absolute numbers such as enumeration of bacterial colonies on a plate), and difficultly in identifying less abundant bacterial taxa ( Mira Miralles et al, 2019 ; Weinroth et al, 2022 ).…”

Análise molecular da comunidade bacteriana ativa nas infecções endodônticas

“…Como uma alternativa, a análise NGS baseada em RNA ribossômico (rRNA) tem sido utilizada para apresentar uma visão mais compreensiva das bactérias que estão ativas e que podem contribuir para a patogênese de uma doença (41). Além disso, como as bactérias ativas apresentam uma maior abundância de rRNA do que DNA (42), a razão rRNA/DNA dos dados de NGS tem sido utilizada pra revelar a atividade bacteriana em diversos ecossistemas (24,(43)(44)(45)(46). No entanto, a comunidade bacteriana ativa das infecções endodônticas não foi desvendada até o momento.…”

“…Da mesma forma, a presença de DNA contaminante em pontas de papel estéril pode fornecer resultados falso-positivos na análise do microbioma de amostras clínicas (117). Portanto, uma estratégia integrada de rRNA e DNA foi proposta como um método mais preciso para avaliar a atividade bacteriana e, assim, destacar a presença de células ativas em uma comunidade bacteriana (24,(44)(45)(46).…”