Effect of Citric Acid on Viability, Membrane Damage, Efflux Pump Activity, and Growth Recovery of Vibrio alginolyticus and Vibrio cholerae Strains

Abstract

Citric acid, a common food ingredient, can induce cellular stress in foodborne pathogens, potentially affecting their viability and recovery. This study evaluated the physiological responses of Vibrio alginolyticus and Vibrio cholerae cells in logarithmic and stationary growth phases after exposure to citric acid at pH 4.5, 5.0, and 5.5 for 60, 120, and 180 min. After exposure, viability, membrane damage, efflux pump activity, and recovery were evaluated. Culture media type and growth phase had no significant effect on cell viability (p > 0.05). V. cholerae strains were more sensitive to citric acid than V. alginolyticus strains, with a slight reduction (0.5 log CFU/mL) after 180 min of citric acid exposure (p < 0.05). Membrane damage was greater in stationary-phase cells than in logarithmic-phase cells after 60 min, whereas efflux pump activity increased over time and was independent of the growth phase (p < 0.05). Regardless of pH, cells exposed to citric acid for 60 min had longer detection times (7.92 ± 0.7 h) compared to those exposed for 180 min (6.68 ± 1.45 h), and cells grew faster at high pH values and at lower exposure time (p < 0.05). Overall, citric acid induced mild sublethal damage in both V. alginolyticus and V. cholerae, with cell recovery primarily attributed to efflux pump activity. These results provide valuable insights into the risk associated with Vibrio cells under mild citric acid stress, a condition similar to that occurring during the preparation of raw fish dishes marinated in lemon juice.