Citric acid can force Staphylococcus aureus into viable but nonculturable state and its characteristics

Pathogens in viable but nonculturable (VBNC) state can escape traditional detection methods based on culturable ability, thus bringing risks to food safety and human health. Considering Staphylococcus aureus as a kind of primary foodborne pathogen, this study attempted to investigate whether citric acid, a food additive commonly used, can force S. aureus into VBNC state along with low temperature. Treated with citric acid solution (pH 4.0) at 4 °C, S. aureus was confirmed to enter into VBNC state after induction for 18 days. Meanwhile, resuscitation was achieved in culture medium rather than in nutrition-free saline solution. In VBNC cells, ATP concentration still maintained at a high level, as about two-thirds of exponential-phase cells. For survival, intracellular structure of VBNC cells changed remarkably, including irregular cell shape, denser cytoplasm, space between cell wall and cell membrane, and decreased density of nuclear region. Notably, resistance of VBNC cells to simulated gastric fluid improved when compared with exponential-phase cells. What are noted above suggests that VBNC state adopted by S. aureus might be a survival strategy to the adverse environment (acidity stress and low temperature). In conclusion, our study sounds an alarm for the safety of citric acid-containing foods.