Sublethal concentrations of undissociated acetic acid may not always stimulate acid resistance in Salmonella enterica sub. enterica serovar Enteritidis Phage Type 4: Implications of challenge substrate associated factors

Acid adaptation enhances survival of foodborne pathogens under lethal acid conditions that prevail in several food-related ecosystems. In the present study, the role of undissociated acetic acid in inducing acid resistance of Salmonella Enteritidis Phage Type 4 both in laboratory media and in an acid food matrix was investigated. Several combinations of acetic acid (0, 15, 25, 35 and 45 mM) and pH values (4.0, 4.5, 5.0, 5.5, 6.0) were screened for their ability to activate acid resistance mechanisms of pathogen exposed to pH 2.5 (screening assay). Increased survival was observed when increasing undissociated acetic acid within a range of sublethal concentrations (1.9–5.4 mM), but only at pH 5.5 and 6.0. No effect was observed at lower pH values, regardless of the undissociated acetic acid levels. Three combinations (15mM/pH5.0, 35mM/pH5.5, 45mM/pH6.0) were selected and further used for adaptation prior to inoculation in commercial tarama (fish roe) salad, i.e., an acid spread (pH 4.35 ± 0.02), stored at 5°C. Surprisingly and contrary to the results of the screening assay, none of the acid adaptation treatments enhanced survival of Salmonella Enteritidis in the food matrix, as compared to non-adapted cells (control). Further examination of the food pH value, acidulant and storage (challenge) temperature on the responses of the pathogen adapted to 15mM/pH5.0, 35mM/pH5.5 and 45mM/pH6.0 was performed in culture media. Cells adapted to 35mM/pH5.5 were unable to induce acid resistance when exposed to pH 4.35 (tarama salad pH value) at 37°C and 5°C, whereas incubation under refrigeration (5°C) at pH 4.35 sensitized 45mM/pH6.0 adapted cells against the subsequent acid and cold stress. In conclusion, pre-exposure to undissociated acetic acid affected the adaptive responses of Salmonella Enteritidis Phage Type 4 in a concentration- and pH-dependent manner, with regard to conditions prevailing during acid challenge.