Integrated profiling of β-lactam susceptibility and spoilage-associated enzyme activities in psychrotrophic bacteria from the raw milk production chain.

Psychrotrophic bacteria are important targets for microbiological control of cold-chain raw milk because of their ability to produce spoilage-associated enzymes and harbor antimicrobial resistance determinants. However, these two functional characteristics are often investigated separately, leaving their co-occurrence within the same isolate collection insufficiently characterized. To address this gap, 543 dominant culturable bacterial isolates were recovered from raw milk and dairy-farm-associated samples collected from six regions of China. Pseudomonas was the most prevalent genus, followed by Acinetobacter, Chryseobacterium, Serratia, and Psychrobacter. β-Lactam minimum inhibitory concentration (MIC) distributions varied substantially among genera, with Chryseobacterium exhibiting the highest proportion of β-lactamase-associated phenotype-positive isolates. Among 226 raw-milk isolates screened using ceftazidime, all 44 isolates showing inhibitor-responsive or carbapenemase-associated phenotypes were subjected to whole-genome sequencing. Genome-wide resistance-gene screening identified annotated β-lactamase genes predominantly in Chryseobacterium and Acinetobacter. In contrast, the resistance-related annotations retained in phenotype-positive Pseudomonas isolates were mainly associated with efflux systems and adaptive response mechanisms. In the integrated analysis, a relative MIC-enzyme risk-prioritization score ranked Serratia (0.88), Chryseobacterium (0.71), and Pseudomonas (0.67) as the three highest-ranking genera. The β-lactam MIC score showed weak positive correlations with the protease-associated score (Spearman's ρ = 0.297, P < 0.001) and with the lipase-associated score (ρ = 0.194, P < 0.001). These findings indicate that genus abundance alone is insufficient to characterize the functional heterogeneity of psychrotrophic bacteria in raw milk. Integrating β-lactam MIC patterns with spoilage-enzyme activity provides complementary information for genus-level prioritization within this isolate collection.