Integrating genomics, phenotypes, and volatile profiling with cheese slurry trials reveals strain-dependent clostridial spoilage originating from silage.

Silage is a primary source of Clostridium spores entering the dairy chain; however, the contributions of spoilage beyond Clostridium tyrobutyricum remain undercharacterized. Here, we integrated whole-genome sequencing with fermentation phenotypes and controlled cheese slurry trials, including volatile profiling measured directly in inoculated slurries, to define strain- and species-specific spoilage modes originating from silage. 15 isolates recovered from silage were confirmed by 16S rRNA sequencing and whole-genome sequencing and analyzed alongside 10 ATCC reference strains for phylogenomic coverage. Across datasets, genomic pathway architecture aligned with measurable outcomes: organic-acid profiles, gas formation, physicochemical shifts in cheese slurries (pH, protein, moisture, salt-to-moisture ratio, fat), visible structural defects, and strain-specific volatile fingerprints. Genes supporting amino-acid catabolism and ammonia production were broadly distributed and corresponded to pH elevation and protein loss in the cheese matrix. Central carbon metabolism supported butyrate formation across species, while terminal butyrate pathway architecture explained acetate utilization patterns and differences in gas and butyrate phenotypes. Butyrate-rich and/or matrix-disruptive spoilage modes were prominent not only among grass-silage C. tyrobutyricum isolates but also in silage-derived C. sporogenes, C. beijerinckii, and C. sulfidigenes, demonstrating that severe spoilage is not restricted to C. tyrobutyricum. Volatile profiling further distinguished strain behaviors, with consistent ethylfuran increases and strain-dependent changes in butyric acid, higher alcohols, ketones, and esters. Together, these results support a multi-species, pathway-defined model of clostridial spoilage originating in silage and manifesting in dairy-relevant matrices.