Phenotypic and Metabolic Diversity of Salmonella Isolates from Agricultural Creek Sediment

• Salmonella isolates from creek sediments show high variability in stress tolerance. • Hydrophobicity results suggest strains’ diversity in environmental surface affinity. • Isothermal calorimetry indicates adaptive metabolic shifts in several isolates. • Intermediate antimicrobial resistance links survival traits associated with AMR. • Results reveal need for improved risk assessment for preharvest safety. Salmonella is an important foodborne pathogen and its outbreaks are increasingly linked to environmental contamination in preharvest systems. Sediment in agricultural watersheds can provide a critical long-term microbial reservoir. However, the phenotypic and metabolic diversity in Salmonella populations hampers effective control. We assessed growth characteristics of 16 Salmonella isolates recovered from sediment of the Conococheague Creek, in Mid-Atlantic USA. We tested hydrophobicity, tolerance to acidic (pH 3.0), alkaline (pH 10.0), and oxidative (H 2 O 2 ) stress, metabolic activity using isothermal calorimetry, and antimicrobial susceptibility. Isolates showed variable hydrophobicity levels of 15% to 81%. Notably, serovar Newport isolates were grouped into two distinct clusters, indicating clonal variation. Isolate 5 (serovar Typhimurium) exhibited a high degree of acid resistance, whereas isolate 4 (serovar Holcomb) was highly susceptible to alkaline and oxidative stresses, indicating isolate-specific stress tolerance. Calorimetry analysis revealed variation in peak heat flow from 49.6 to 63.0 µW across isolates. In a subset of isolates, a secondary metabolic shift was observed at 24–26 h, pointing to the fact that these strains may adapt to fluctuating environmental conditions by utilizing alternative energy sources. Most isolates were susceptible to antimicrobials tested, while isolate 4 (serovar Holcomb) showed intermediate resistance to cefoxitin (MIC = 16 µg/mL), suggesting a potential mobile β-lactamase in this isolate, linking environmental survival to antimicrobial resistance (AMR) threats. We demonstrated distinctive characteristics of Salmonella isolates from the environment and this research provides a basis for improving risk assessment and preharvest food safety strategies.