Characterization of the Microbiota Dynamics in Cold-Smoked Salmon Under Cold Chain Disruption Using 16S rRNA Amplicon Sequencing

Background/Objectives: Cold-smoked salmon (CSS) is a ready-to-eat product with minimal preservation hurdles and a microbiota shaped by raw-material contamination and processing environments. Short breaks in refrigeration commonly occur during shopping and transport, yet their microbiological impact remains unclear. Here, we used ASV-resolved 16S rRNA gene metataxonomics to characterize storage-driven microbiota dynamics in CSS—quantifying ASV-level genetic diversity and phylogeny-aware (UniFrac) community structure—and to evaluate the effect of a brief, consumer-mimicking 2 h room-temperature cold-chain disruption. Methods: Three CSS types (organic, conventional Norwegian, and conventional Scottish) were stored at 5 °C for 35 days. On day 16, half of each batch was exposed to 2 h at room temperature (RT) before analysis; paired controls remained refrigerated. Culture-based counts (total mesophiles, lactic acid bacteria, Photobacterium spp.; indicator/pathogen screens) were performed per ISO methods. Community profiling used 16S rRNA (V3–V4) amplicon sequencing with QIIME 2/DADA2 and SILVA taxonomy. Linear mixed effects modelled alpha diversity; beta diversity by PERMANOVA on UniFrac distances; differential abundance by ANCOM-BC. Results: ASV-resolved 16S rRNA gene profiles of CSS were dominated by Pseudomonadota and Bacillota, with storage-driven shifts and taxon-specific trajectories (e.g., increasing Latilactobacillus). Both time and product type significantly explained phylogeny-aware community structure (unweighted and weighted UniFrac), consistent with storage-driven phylogenetic convergence across products. At day 16, ASV-level genetic diversity (Shannon/Observed features) and genus-level composition did not differ between RT-disrupted and continuously refrigerated samples. Culture-dependent counts increased from baseline to day 16 and largely plateaued by day 35, with lactic acid bacteria in Norwegian CSS continuing to rise; no systematic effect of the 2 h RT exposure was observed in culture-based comparisons. Indicator/pathogen screens detected no unexpected pathogenic species throughout the study period. Conclusions: Refrigerated storage drives pronounced, phylogeny-aware microbiota shifts and cross-product convergence in cold-smoked salmon, whereas a single 2 h RT interruption at mid-storage did not measurably alter ASV-level genetic diversity or community structure under the tested conditions. Integrating culture-based enumeration with ASV-resolved 16S rRNA gene metataxonomics provides complementary insights for shelf-life evaluation and risk assessment in ready-to-eat seafood.