Purpose: To examine the impact of filament-inducing conditions (reduced water activity) on physiological aspects of Salmonella relevant to existing detection techniques.
Methods: E. coli ATCC 25922, Salmonella enterica ser Typhimurium ATCC 13311 and two peanut butter outbreak-associated Salmonella spp. (Salmonella Typhimurium, Salmonella Tennessee) were grown under high osmotic conditions (Tryptic Soy Agar containing 8% NaCl) for up to two weeks at 37°C. Strains were examined at various intervals for growth on selective and non-selective media (XLT-4 agar and Tryptic Soy Agar, respectively) using light and fluorescence microscopy combined with Live/Dead staining (Invitrogen, Inc.) and using quantitative PCR or flow cytometry. Physiological responses to osmotic stress were compared across type and outbreak strains.
Results: Within four days of inoculation in TSB + 8% NaCl, all bacteria demonstrated a filamentous phenotype. Recovery of filamentous bacteria on selective or non-selective media was ~10-fold lower than expected from OD-adjusted cultures. Light microscopy and fluorescence microscopy (Live/Dead staining) indicated a greater persistence of a filamentous morphology and Live-staining (intact cell membrane) in outbreak-associated strains vs. E. coli and the Salmonella type strain. Both filamented and non-filamented cells were detectable via quantitative PCR.
Significance: Our results suggest a hardier phenotype for peanut butter outbreak-associated strains of Salmonella and demonstrate the utility of various physiological and molecular tools for characterization of differences between type and outbreak strains that may be important to understanding survival and persistence of Salmonella in foods having low water activity.