Retail-Level Microbiomes of Organic and Conventional Fresh Produce: A Multi-Kingdom Analysis of Amoeba-Associated Bacterial Viability

The increasing consumption of fresh organic produce has given rise to concerns regarding the microbiological safety of minimally processed foods. Organic cultivation may be associated with increased exposure to environmental microorganisms due to soil-based inputs and reduced chemical interventions, including both beneficial taxa and potential foodborne pathogens. Fresh produce is known to harbour complex microbial ecosystems, which are shaped by farming practices, plant physiology, handling, packaging and storage, particularly in raw-consumed products such as leafy greens and strawberries. In this study, bacterial (16S rRNA) and eukaryotic (18S rRNA) communities were characterized by amplicon sequencing. In parallel, an amoeba-associated bacterial microbiome was analyzed and DVC-FISH was used to assess the viability and metabolic activity of pathogenic bacteria internalized within free-living amoebae (FLA). No significant differences in alpha or beta diversity were observed between organic and conventional products, suggesting microbiome convergence at the retail stage driven by post-harvest handling and processing. Potentially pathogenic genera, including Pseudomonas, Stenotrophomonas, and Acinetobacter (bacterial), as well as Tilletiopsis, Candida, and Naegleria (eukaryotic), were identified in both organic and non-organic microbiomes. The viability of FLA-internalized Pseudomonas spp. was confirmed by DVC-FISH, demonstrating that FLA act as reservoirs, enhancing pathogen persistence in fresh produce. This integrated assessment of organic and conventional fruits and vegetables at the retail stage highlights the importance of post-harvest handling and retail conditions in shaping microbiological safety. The integration of microbiome profiling with targeted viability analyses demonstrates that downstream stages are critical control points for food safety and consumer exposure, beyond the influence of the production system alone.