Formation and Control of the Viable but Non-culturable State of Foodborne Pathogen Escherichia coli O157:H7

As a common foodborne pathogen, Escherichia coli O157:H7 produces toxins causing serious diseases. However, traditional methods failed in detecting E. coli O157:H7 cells in the viable but non-culturable (VBNC) state, which poses a threat to food safety. This study aimed at investigating the formation, control, and detection of the VBNC state of E. coli O157:H7. Three factors including medium, salt, and acid concentrations were selected as a single variation. Orthogonal experiments were designed with three factors and four levels, and 16 experimental schemes were used. The formation of the VBNC state was examined by agar plate counting and LIVE/DEAD® BacLightTM bacterial viability kit with fluorescence microscopy. According to the effects of environmental conditions on the formation of the VBNC state of E. coli O157:H7, the inhibition on VBNC state formation was investigated. In addition, E. coli in the VBNC state in food samples (crystal cake) was detected by propidium monoazide–polymerase chain reaction (PMA-PCR) assays. Acetic acid concentration showed the most impact on VBNC formation of E. coli O157:H7, followed by medium and salt concentration. The addition of 1.0% acetic acid could directly kill E. coli O157:H7 and eliminate its VBNC formation. In crystal cake, 25, 50, or 100% medium with 1.0% acetic acid could inhibit VBNC state formation and kill E. coli O157:H7 within 3 days. The VBNC cell number was reduced by adding 1.0% acetic acid. PMA-PCR assay could be used to detect E. coli VBNC cells in crystal cake with detection limit at 104 CFU/ml. The understanding on the inducing and inhibitory conditions for the VBNC state of E. coli O157:H7 in a typical food system, as well as the development of an efficient VBNC cell detection method might aid in the control of VBNC E. coli O157:H7 cells in the food industry.

Bacteria in the VBNC state fail to form colonies in routine detection agar plates under certain environmental stresses. However, these cells have food spoilage and pathogenic capacity (Liu et al., 2017b). Factors including adverse nutrition levels, temperatures, and osmotic pressures, which are frequently encountered during food processing and storage environments, have been reported to induce bacteria entering the VBNC state. With the failure in detection by routine method and the capability in causing food safety problems, the VBNC state of E. coli O157:H7 is a major concern in the food industry (Liu et al., 2017b).
Crystal cake is a traditional Chinese snack that has the characteristics of rich nutrition. Its rich nutrients and sufficient water make it a natural medium for various pathogenic bacteria and spoilage bacteria, including E. coli. In this study, crystal cake was applied as a representative food system to study the formation, control, and detection of the VBNC state of E. coli O157:H7 under food processing and storage conditions.

MATERIALS AND METHODS
Cultivation of E. coli O157:H7 Escherichia coli O157:H7 ATCC25922 was stored at -80 • C in Luria-Bertani (LB) broth containing 20% (v/v) glycerol. It was streaked on LB agar plate and grown at 37 • C for 24 h. Then, a single colony was inoculated into 2 ml of LB broth and incubated at 37 • C for 12 h using a shaker incubator set at 150 r/min. The bacteria suspension was diluted 1:100 in fresh medium and cultured for 4 h to the log phase upon further experiments.

Induction of the VBNC State
According to the optimal culture conditions of E. coli O157:H7, and the principle of reverse adjustment, conditions that are unfavorable to bacterial growth were selected as candidate factors to induce the VBNC state. Three factors including nutritional state, salt concentration, and acidity were taken as a single variable. To investigate possible VBNC induction conditions, three factors and four levels of orthogonal experiments were designed, and 16 protocols ( Table 1) were applied. Log phase cells were centrifuged for 10 min at 5000 r/min and the pellet was washed once with 1 × phosphate buffer solution and then resuspended in the induced solution. The initial concentration of the bacterial solution was diluted to approximately 10 7 CFU/ml. To avoid the effects of repeated freezing and thawing, the bacterial suspension under each condition was mixed and divided into multiple 1.5-ml centrifuge tubes, followed by induction in refrigerators at 4 and −20 • C, respectively.

Determination of Culturable Cell Number During Storage
The culturable cell number in each of the VBNC inducing bacterial culture was determined every 3 days by a plate counting method. Briefly, the culture of E. coli O157:H7 was serial diluted in 0.9% NaCl, spread on LB agar plates, and incubated at 37 • C for 24 h. When the culturable number is 1 CFU/ml, the cells were considered non-culturable and possibly enter the VBNC state (Piao et al., 2019).

Determination of Viable Cell Number
To determine if the non-culturable cells are in the VBNC state after exposure to the respective treatment conditions, the  LIVE/DEAD R BacLight TM bacterial viability kit (Thermo Fisher Scientific, China) was used. Five hundred microliters of the nonculturable bacterial cell sample was centrifuged at 5000 r/min for 15 min, washed with saline twice, and resuspended in saline. Subsequently, 1.5 µl of SYTO 9 dye and 1.5 µl of propidium iodide were added to the sample, followed by mixing and 30 min incubation in the dark. After incubation, 5-µl cells were captured between a slide and a coverslip and used for fluorescence microscopy. The appearance of green cells indicates the existence of VBNC cells.

Inhibition of VBNC State by Acidity and Nutritional Conditions
According to the VBNC state induction process, appropriate conditions were selected to inhibit the formation of the VBNC state. The configured medium was resuspended, and the initial concentration of E. coli O157:H7 was adjusted to 10 7 CFU/ml. The bacterial solution with a volume of 30 ml was stored at 4 and −20 • C, respectively. The number of cultivable bacteria was measured by plate counting every 3 days (Tables 2, 3).

Elimination of the VBNC State in Crystal Cake
According to the National Food Safety standards (GB4789.3-2016) in China, 25 g of crystal cake was added to 225 ml of saline, and the concentration of this medium was determined to be 100, 25, and 50% of the food sample culture medium and is configured by dilution and used after autoclaving. Subsequently, E. coli O157:H7 was cultured to the logarithmic phase and centrifuged at 4 • C, the supernatant was discarded, and the suspension was resuspended three times with physiological saline. The bacterial cells were resuspended using the sterilized food sample medium, and the concentrations of E. coli O157:H7 were adjusted to 10 7 CFU/ml, respectively, and a sterile acetic acid solution was added to fix the volume fraction of acetic acid to 1.0%. The induction solution was stored at 4 • C, and the culturable number and activity were detected by a plate method combined with a LIVE/DEAD R BacLight TM bacterial viability kit.

PMA-PCR Assays
Twenty-five grams of crystal cake was mixed with 225 ml of physiological saline and sterilized, and E. coli in the VBNC state was added. Bacterial suspensions with initial concentrations of 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10 CFU/ml were obtained. Five hundred microliters of the bacteria suspension was added to a 1.5-ml centrifuge tube, and the PMA working solution was added to a final concentration of 5 µg/ml (to distinguish between the VBNC state and dead cells). After mixing, the samples were kept at room temperature for 10 min in the dark. Subsequently, the centrifuge tube was placed on ice, and 15 cm away from a 650 W halogen lamp for 5 min to complete the binding of PMA and DNA. The PMA-DNA was centrifuged at 10,000 r/min for 5 min, and the supernatant was discarded. DNA was then extracted using a bacterial group DNA extraction kit (Dongsheng Biotech, Guangzhou). The extracted DNA was detected by PCR. PCR assay was performed in a 25-µl volume and with 0.6 µM primers (rfbE-F: TTGGCATCGTGTGGACAGGGTAGGACCGCAGAGGAAAG A; rfbE-R: TGGGACAGGTGTGCTACGGTTTCCACGCC AACCAAGATC). The thermal profile for PCR mixtures was 94 • C for 5 min, followed by 30 cycles of 94 • C for 30 s, 52 • C for 60 s, and 72 • C for 90 s and a final extension cycle at 72 • C for 7 min. The amplified products (5 µl/well) were analyzed by gel electrophoresis in 2% agarose gels and stained with ethidium bromide for 10 min. A negative control was included using sterile water instead of culture or DNA template.

Formation of the VBNC State
To induce the formation of the VBNC state, 16 induction solutions were applied ( Table 1). In induction solution 1, when stored at 4 • C, the culturable cell number of E. coli O157:H7 was reduced to 5 × 10 4 CFU/ml in 66 days. When stored at −20 • C, the culturable cell number of E. coli O157:H7 dropped to 0 in 3 days ( Figure 1A). In induction solution 2, the number of culturable cells reduced to 0 after 18 days of storage at 4 • C, and the number of culturable cells reduced to 0 within 3 days of storage at −20 • C ( Figure 1B). In induction solution 10, the number of culturable cells reduced to 0 after storing at 4 and −20 • C for 39 and 33 days, respectively ( Figure 1F). In induction solution 13, the culturable cell number reduced to 0 in 48 and 39 days of storage at 4 and −20 • C, respectively ( Figure 1G).
In induction solution 14, the culturable cell number reduced to 0 in 57 and 66 days when stored at 4 and −20 • C, respectively ( Figure 1H). In induction solution 5, 7, and 9, although with a downtrend within 66 days, the number of culturable cells did not decrease to 0 (Figures 1C-E). In addition, in induction solutions 3,4,6,8,11,12,15, and 16, the number of culturable cells of E. coli O157:H7 reduced to 0 in 3 days.
For the induction solutions that were capable of inducing the culturable cell number of E. coli O157:H7 to 0 (Table 4), the existence of viable cells was determined. When the E. coli O157:H7 culture in induction solution 2 was stored at 4 • C for 18 days, the cells were all dead, indicating that E. coli O157:H7 did not enter the VBNC state (Figure 2). The fluorescence results of E. coli O157:H7 in induction solutions 1, 10, 13, and 14 showed that both dead and viable cells were detected, indicating that E. coli O157:H7 entered the VBNC state under these conditions.

Factors Affecting the Survival of E. coli O157:H7 Cells
In the 16 induction solutions, factors including salt, acetic acid, and medium concentrations were applied and analyzed on the effect on the survival of E. coli O157:H7 cells. First, the effect of salt and acid concentrations on the survival of E. coli O157:H7 cells was investigated. In induction solutions 3, 8, 9, and 14, the medium concentration is 50%. In induction solutions 3 (acid: 0.7%, salt: 0.9%) and 8 (acid: 1%, salt: 5%), the culturable cell number of E. coli O157:H7 reduced to 0 in 3 days. However, in induction solutions 9 (acid: 0, salt: 10%) and 14 (acid: 0.3%, salt: 15%), E. coli O157:H7 cells survived for more than 57 days. Especially in induction solution 9, the E. coli O157:H7 cells  survived for more than 66 days. In induction solutions 4, 10, and 13, the medium concentration is 100%. In induction solution 4 (acid: 1%, salt: 0.9%), E. coli O157:H7 all died in 3 days and did not enter the VBNC state. In induction solutions 10 (acid: 0.3%, salt: 10%) and 13 (acid: 0, salt: 15%), E. coli O157:H7 cells were able to survive for 33-48 days. These results suggest that the effect of acetic acid concentration on the survival of E. coli O157:H7 is stronger than that of salt concentration. Second, the effect of acid and medium conditions on the survival of E. coli O157:H7 cells under a certain salt concentration were investigated. In induction solutions 1, 2, 3, and 4, the salt concentration is 0.9%. In induction solutions 1 (medium: 0, acid: 0) and 2 (medium: 25%, acid: 0.3%), E. coli O157:H7 cells survived for more than 66 and 18 days at 4 • C, respectively. However, in induction solutions 3 (medium: 50%, acid: 0.7%) and 4 (medium: 100%, acid: 1%), the E. coli O157:H7 cells all died in 3 days. It showed the effect of acid on the survival of E. coli O157:H7 is stronger than that of nutrients. In addition, when the acid concentration is 1% in induction solutions 4, 8, 12, and 16, E. coli O157:H7 cells died in 3 days regardless of the medium and salt concentrations. Especially in induction solution 4 (medium: 100%, acid: 0.9%) with the premium centration of medium and acid, E. coli O157:H7 cells still died in 3 days, indicating that acidity has the most significant effect on the survival of E. coli O157:H7 cells.
Third, under the same acetic acid concentration, the effects of salt and medium concentrations on the survival of E. coli O157:H7 were investigated. In induction solutions 6, 10, and 14, the acetic acid concentration is 0.3%. In induction solution 6 (medium: 0, salt: 5%), E. coli O157:H7 cells died within 3 days. However, in induction solutions 10 (medium: 100%, salt: 10%) and 14 (medium: 50%, salt: 15%), although the salt concentration increased, E. coli O157:H7 cells were able to survive from 33 to 66 days due to the high medium concentration. It indicated that the increase in the medium concentration eliminated the adverse effect of the increase in salt concentration. Among the factors on the survival of E. coli O157:H7, the effect of the medium concentration might be greater than that of the salt concentration.
When the environment changes, porin is crucial for the survival of E. coli. The major external protein omp (ompF, ompC) of E. coli is regulated by envZ (osmotic pressure sensor protein) and ompR. Studies have showed that wild-type, ompF, and ompC mutant strain of E. coli can enter the VBNC state under environmental pressure (pH, osmotic pressure, and starvation stress conditions), but in the envZ mutant strain, they cannot enter the VBNC state. This shows that the envZ mutant strains cannot sense changes in the external environment. When the strains are exposed to adverse conditions, they cannot enter the VBNC state (Pienaar et al., 2016). The expression of (p) ppGpp (guanosine pentaphosphate or guanosine tetraphosphate) synthetic genes relA and spoT are identified to upregulate in the VBNC state of E. coli O157:H7 (Magnusson et al., 2005;Mishra et al., 2012). Compared with normal strains, mutants that failed to synthesize (p) ppGpp lost culturability more quickly, and their ability to enter the VBNC state was significantly reduced. When (p) ppGpp was overexpressed, the number of VBNC cells was significantly increased (Boaretti et al., 2003). When bacteria suffered from amino acid starvation, the expression of RelA or SpoT increased. (p) ppGpp synthesis increased and degradation decreased. In addition, exopolyphosphatase (PPX) was degraded by (p) ppGpp of polyphosphates (Ayrapetyan et al., 2018).

Effects of Acidity and Nutrition on Formation of the VBNC State
Among the factors affecting the survival of E. coli O157:H7, acetic acid concentration plays a major role, followed by medium and salt concentrations. Considering the actual situation in the food system and in the process of food processing, exploring the concentration of acetic acid and culture medium to control the normal state of E. coli O157:H7 and VBNC is meaningful.
In induction solutions 2, 3, 4, and 6, the culturable cell number of E. coli O157:H7 did not decrease to 0 in 3 days. However, the culturable cell numbers of E. coli O157:H7 reduced to 0 after storing at 4 and −20 • C for 3 days in induction solutions 1 and 5, and the cells were not viable. Thus, changing the acid concentration with cold storage might be efficient to control the formation of the VBNC state of E. coli O157:H7.

Elimination of the VBNC State in Crystal Cake
In the crystal cake food system with 100, 50, and 25% nutrient concentrations and 1.0% acetic acid, the culturable cell numbers of E. coli O157:H7 were 0 after storing at 4 and −20 • C for 3 days. The results of fluorescence microscopy showed that all the E. coli O157:H7 cells died, suggesting E. coli O157:H7 was not capable of entering into the VBNC state under these conditions (Figure 3). In the food system, without affecting the flavor and quality of the food, the normal and the VBNC state of E. coli O157:H7 was controlled and eliminated by adding 1% acetic acid.
The foodborne microorganisms in the VBNC state have food spoilage ability. Compared with normal state, VBNC state cells have reduced metabolic activity and increased resistance of the cell wall to external environmental stress, including high concentrations of antibiotics, heavy metal ions, high temperature, high salt, and higher acidity (Ayrapetyan and Oliver, 2016;Shekar et al., 2017;Schottroff et al., 2018). Due to the presence of resistance, E. coli O157:H7 in the VBNC state is more difficult to be completely eliminated. Under favorable conditions, E. coli O157:H7 in the VBNC state might be able to overcome this inactive state and become active again, thus being able to reproduce in the food system. During the processing of crystal cake, the surface of the processing equipment and the inside of the pipeline should be cleaned in time to ensure no residual nutrients, thereby inhibiting the survival of E. coli O157:H7. In addition, combined with a certain concentration of acetic acid treatment equipment surface (1.0% acetic acid), it ensures the elimination of E. coli O157:H7 and its VBNC state and avoid potential food safety risks.

Detection of the VBNC State of E. coli in Crystal Cake
The risk of food safety issues can be partially reduced by the efficient detection of the VBNC state of E. coli in food system. Propidium monoazide (PMA) is a photoreactive DNA-binding dye. Dead microorganisms lose their capability to keep their membranes intact, which leaves the "naked" DNA in the cytosol ready to react with PMA. DNA of living organisms are not exposed to the PMA, as they have a complete cell membrane. DNA extraction and PCR amplification of PMA-treated samples can effectively detect bacteria in the VBNC state. The detection limit was determined by detecting different concentrations of E. coli O157:H7 in the VBNC state. In this study, the detection limit of the VBNC state E. coli O157 in the crystal cake food system using PMA-polymerase chain reaction (PMA-PCR) was 10 4 CFU/ml, which can effectively avoid the potential threat brought by VBNC state bacteria.

CONCLUSION
The effect of external environmental conditions on the formation of the VBNC state of E. coli O157:H7 was as follows: acidity > nutritional state > salt concentration. When the nutrient concentration is higher than 50%, E. coli O157:H7 was killed by adding 1.0% acetic acid. In the crystal cake system, when the nutrient concentration is 25, 50, and 100%, E. coli O157:H7 cells died in 3 days by adding 1.0% acetic acid with no VBNC cells identified. In the food system, the VBNC state formation of E. coli O157:H7 was inhibited by adding 1.0% acetic acid. In addition, PMA-PCR assays can be utilized in the detection of the VBNC state of E. coli in the food system.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/supplementary material.

AUTHOR CONTRIBUTIONS
JL and KW conceived the study and participated in its design and coordination. YML and T-YH performed the experimental work. CY and LC analyzed the data. YL and JL prepared and revised this manuscript. All authors reviewed and approved the final manuscript.