Salmonella infection is one of the main causes of foodborne diseases and a major public health problem worldwide. Annually, Salmonella causes 200 million to over 1 billion infections worldwide, of which 85 % are food-linked, leading to 93 million cases of gastroenteritis and 155,000 deaths (Castro-Vargas et al., 2020; Chlebicz and Śliżewska, 2018; Hung et al., 2017). Li et al. (2020) analyzed the bacterial foodborne diseases in China from 2003 to 2017, and found that there were 899 outbreaks caused by Salmonella, resulting in 11,351 hospitalizations and 4 deaths. A considerable number of foodborne Salmonella outbreaks were caused by Salmonella contamination in low water activity (aw) foods. In 2022, 369 cases of confirmed and probable Salmonella Typhimurium linked to consumption of chocolate products produced in Belgium have been reported globally (Samarasekera, 2022). Outbreaks linked to contamination of powdered infant formula (PIF) by a variety of Salmonella serotypes have been reported in several countries (Jourdan et al., 2008; Park et al., 2004; Rodriguez-Urrego et al., 2010). In 2018, PIF produced by a French food enterprise was found continuously contaminated by Salmonella, causing the fourth outbreak of Salmonella linked to PIF in France (Jones et al., 2019). This highlights the risk of persistent Salmonella contamination in PIF manufacturing facilities, as PIF is not a sterile product and Salmonella contamination can occur at any time during production and storage (Angulo et al., 2008).

The viable but non-culturable (VBNC) state refers to a special state of bacteria that cannot be cultured on conventional media, but can be detected through non-culture detection methods, still has low metabolic activity, and can be restored to a cultivable state under certain conditions (Roszak et al., 1984; Xu et al., 1982). Most foodborne bacteria could be induced into VBNC state by a variety of adverse conditions (Morishige et al., 2017). However, the VBNC cells could still maintain a certain level of metabolic activity to maintain basic energy supply in harsh environment (Du et al., 2007). Factors that induce bacteria to enter VBNC state include low temperature (Chaisowwong et al., 2012), oxidative stress (Morishige et al., 2013; Oliver, 2010), nutritional starvation (Gupte et al., 2003), excessive nutrition (Morishige et al., 2014), low aw (Se et al., 2021), osmotic stress (Kusumoto et al., 2012), pH changes (Tholozan et al., 1999), food preservatives, antibiotics, and disinfectants (Oliver, 2010; Tian et al., 2017). Low temperature, low aw stress, oxidative stress and nutrition components are the most crucial factors for the induction of VBNC state in food processing (Aviles et al., 2013a; Du et al., 2007; Liu et al., 2010; Lothigius et al., 2010; Morishige et al., 2017). In addition, VBNC cells had the potential for recovery. Pathogenic bacteria successfully recovered might restore their original infectivity and invasiveness, or even became stronger, so we must pay enough attention to research on the resuscitation of VBNC bacteria. Induction conditions, resuscitation methods and the duration of entering VBNC state may influence the resuscitation of bacteria. It is necessary to investigate the survival and resuscitation ability of VBNC Salmonella under low aw in PIF, which can provide theoretical guidance for preventing and controlling Salmonella contamination in low aw foods.

There is still a lack of comprehensive and systematic research on the formation mechanism of VBNC cells. Most of the existing studies have been carried out based on analyses of specific genes or special functional proteins, showing their important role in the process of inducing VBNC cells (Dong et al., 2020), but so far there is no unified conclusion. RpoS regulates the physiological response of bacteria for their survival under various stress conditions (Bhagwat Arvind et al., 2006), which may be closely related to VBNC state during bacteria's adaptation to adverse environment (Ravel et al., 1994). Some studies have shown that the overexpression of RpoS is not conducive to the formation of bacterial VBNC state. For example, the Escherichia coli wild-type (WT) strain entered the VBNC state later than the rpoS-deletion (ΔrpoS) strain in the culture medium with 4 °C and poor nutrition (Boaretti et al., 2003). Kusumoto et al. (2012) also found that the formation of VBNC state Salmonella under high osmotic stress (7 % NaCl) would lead to the reduction of intracellular RpoS level. On the contrary, other studies suggest that the accumulation of RpoS may be associated to the transformation of bacteria into VBNC state. It was found that (p)ppGpp may be an inducer of VBNC state, and cells rich in (p)ppGpp are more likely to enter VBNC state (Chen et al., 2018b; Rowan-Nash Aislinn et al., 2019). (p)ppGpp is a signal molecule that regulates the expression of rpoS, and the increase of its content will lead to the increase of intracellular RpoS level (Kibbee and Örmeci, 2017). Chen et al. (2018b) also found that cells lacking (p)ppGpp had difficulty entering the VBNC state, indicating that the accumulation of RpoS may indirectly induce the formation of VBNC state. However, there are few reports on whether the formation of Salmonella in VBNC state in low aw food medium is related to the regulation of RpoS. In addition, there is still controversy about whether VBNC cells can still express virulence-related genes and produce toxins (Aviles et al., 2013a; Liu et al., 2010; Lothigius et al., 2010).

This study was conducted to explore the effect of rpoS on (1) bacterial survival; (2) morphological and physiological characteristics; and (3) VBNC state formation and resuscitation mechanism of Salmonella Enteritidis in PIF under different storage temperatures, so as to lay a research foundation for controlling Salmonella contamination in PIF in the future.