Formation of viable but nonculturable state of Staphylococcus aureus under frozen condition and its characteristics.

Viable but nonculturable (VBNC) state of microorganisms has attracted much attention due to its characteristics, including the difficulty in detection by culture-based methods, virulence retention, high resistance, and so on. As a foodborne pathogen, Staphylococcus aureus is widely distributed, and has been found to enter the VBNC state under some environmental stresses, posing a potential threat to human health. Freezing is a common condition for food storage. This study investigated whether citric acid, a common food additive, could induce S. aureus into the VBNC state at -20 °C. By measuring the number of culturable and viable cells, it was found that S. aureus entered the VBNC state after 72 days of induction in citric acid buffer at -20 °C. The VBNC cells were then successfully resuscitated at 37 °C in trypsin soybean medium (TSB) with or without heat shock treatment, and TSB supplemented with sodium pyruvate and Tween 80 after 48 h. Heat shock resulted in an excellent resuscitation effect. Observed by transmission electron microscopy, the internal structure of VBNC cells was found markedly changed, compared with that of exponential phase cells. API ZYM kit was used to compare the intracellular enzyme activity of S. aureus in the exponential phase with that in the VBNC state. The results showed that the enzyme activity decreased significantly in VBNC cells, and that the VBNC cells were more resistant to simulated gastrointestinal fluid through flow cytometry analysis. Quantitative reverse-transcription polymerase chain reaction results suggested that the ability of adhesion and biofilm formation of VBNC cells might be decreased due to the down-regulation of related genes. However, it should not be ignored the recovery potential of biofilm-forming ability of VBNC cells caused by the high expression of sarA. In conclusion, S. aureus could be induced into the VBNC state in citric acid buffer at -20 °C, which showed changes in some biological characteristics and could resuscitate successfully by many conditions. Food industry needs to pay attention to the potential hazard by VBNC S. aureus under frozen conditions.