Production of viable but nonculturable state in Salmonella isolates by combination of acidity, osmotic pressure and freezing

Introduction

Foodborne pathogenic bacteria are considered as one of the most critical sources of zoonotic diseases (Zhao et al., 2014). This group of pathogens can cause the spread of disease in human and livestock communities and in this way will cause a lot of damage (Pajohi Alamoti et al., 2022). Salmonella spp. is one of the most important foodborne pathogens, which are classified as zoonotic bacteria. Salmonella spp. cause infections and deaths worldwide (even in developed countries) every year. The main source of human contamination by this bacterium is its transmission through food of animal origin, such as meat, poultry and eggs (Manafi et al., 2020). Salmonella spp. can develop VBNC state. The capabilities of a bacterium to establish VBNC state can affect its abilities, such as biofilm formation ability (Li et al., 2020).

The VBNC state in bacteria was discovered and reported for the first time about four decades ago (Xu et al., 1982). Bacteria in the VBNC state do not grow under usual conditions and do not form any colonies in normal culture methods. If the conditions are appropriate, these bacteria are revived and continue to grow by forming colonies (Yan et al., 2021). When the bacterium is in the VBNC state, it becomes difficult to detect and isolate it. Also, the bacterium undergoes morphological and genetic changes in this state, which can further increase the ability to infect (Xu et al., 1982). Resuscitation of bacteria in VBNC state is one of the significant risks of these pathogens. In these cases, due to the changes they have had, bacteria can show more pathogenicity than before (Wei and Zhao, 2018). The number of microorganisms that have the ability to form the VBNC state has increased. One of the most important conditions that have increased this ability in microorganisms is the conditions related to food storage and processing (Dong et al., 2019).

Food-borne bacteria are exposed to the conditions of the food environment, such as osmotic pressure and temperature conditions; and these conditions affect the growth and behavioral characteristics of bacteria (Highmore et al., 2018). According to the studies conducted on the importance of pathogenicity of food-borne Salmonella spp. and also the increasing importance of the VBNC state in bacterial pathogenicity (Li et al., 2017), in this study, investigation of the effect of the conditions related to food storage and processing environments on the induction of VBNC state in Salmonella isolates of food origin have been done.

Materials and methods

Bacterial strains

Seven previously isolated Salmonella serotypes were used in the current study (Table 1). The bacterial strains were stored at -20 °C with 30 % glycerol. Before the experiments, the bacteria were re-cultured twice in TSB media (Trypticase Soy Broth, QUELAB, Canada) and incubated at 37 °C for 24 h.

Preparation of Bacterial cocktails

A previously described method was used to prepare the bacterial cocktail of each strain with some modifications (Lastra-Vargas et al., 2020). In the first step, the growth curves of all seven serotypes were plotted. After that, based on the growth curve, each strain was cultured in 10 ml of TSB, and then the cocktails were made by mixing 1 ml of the same serotype with a dilution of 1 × 10⁸ CFU/ml.

Osmotic pressure conditions

TSB media were firstly prepared with NaCl (4.5 %), and then 1 ml of fresh bacterial suspension was inoculated into 9 ml of these media and incubated at 37 °C for 24 h. After that, 100 µl of the above-mentioned culture was transferred to TSB and incubated at 37 °C for 20 h (Shah and Bergholz, 2020).

Acidity conditions

At first, TSB was prepared and acidified using pure lactic acid to pH of 4.5. Then, 1 ml of fresh bacterial suspension was inoculated into 9 ml of acidified media and incubated at 37 °C for 24 h. Finally, 100 µl of the culture mentioned above was transferred to normal TSB and incubated at 37 °C for 20 h (Kang et al., 2018).

Freezing conditions

The prepared bacterial suspensions were placed in closed tubes and stored at -20 °C for 96 h. After that, the samples were transferred to TSB and incubated at 37 °C for 20 h (Thongbai et al., 2006).

Combinatory conditions

Bacterial cocktails were exposed to environmental conditions in three treatments (Table 2). One group was also considered as a control group. Bacterial cocktails were inoculated into TSB media with 4.5% NaCl and the pH 4.5 (lactic acid) and then stored in a freezer at -20 °C for 96 hr.

Plate Counting Method

The plate counting method with slight modification was used to evaluate the VBNC state induction (Li et al., 2020; Zeng et al., 2013). At first, the bacteria were subjected to acid, osmotic pressure, and freezing treatments. After being exposed to the treatments, the samples were incubated in TSB (Qeulab, Canada) media for 24 h at 37°C, and then the samples were cultured in TSA (Quelab, Canada) media and the colonies were counted. Also, in parallel, the treated samples were incubated in TSB media with 5% of Tween 20^® (Polysorbate 20) at 37 °C for 24 h, and Tween 20 was used as a resuscitation agent. Then, the samples were cultured and counted again in TSA media. Differences between the numbers of recovered colonies were considered as bacteria suspected of VBNC status. Schematic of conditions for production and evaluation of VBNC state are shown in Figure 1.

Statistical analyses

SPSS version 20 software was used for statistical analysis of the obtained data. For this purpose, the Univariate Analysis of Variance and a significance level of P < 0.001 were used. All experiments were performed in three replicates.

Results and discussion

According to the results which are shown in Figure 2-4, Osmotic pressure (4.5% NaCl), along with freezing (-20 C for 96 h), could not cause a significant change in the growth rate of S. Typhi. However, in the case of S. Typhimurium and S. Enteritidis, it could increase the resuscitated bacteria. In the second treatment, which was the combination of freezing and acidic media (pH 4.5), none of the three strains grew, indicating bacterial death.

In the third treatment, combination of osmotic pressure, low pH, and freezing, no growth was observed without resuscitation. After using Tween 20^® and resuscitation, the growth in all three strains was observed, and this growth was higher in the case of S. Typhimurium, which shows the greater ability of this isolate in survival and formation of VBNC state.

The presence of tween 20 significantly increased the resuscitation of bacteria which was in the VBNC state. Also, in all three strains, the presence of osmotic pressure caused a decrease in bacterial growth even in the presence of resuscitation agent, which was significant in all three bacterial strains (P < 0.001).

Studies related to VBNC state in pathogenic bacteria are relatively novel and new cases that are developing day by day (Truchado et al., 2020). The inability to induce the VBNC state in high acidity has been seen in the case of Salmonella spp. (Li et al., 2020). Also, the mentioned study showed that low temperatures and osmotic pressure can induce VBNC state in Salmonella spp., which is consistent with the results of our study. The presence of temperature factors, osmotic pressure and acidity together cause changes in fatty acids in the bacterial cell membrane. This action, together with the changes in the membrane channels, reduces the volume of the cell, in which case the bacterial cell can adapt itself to harsh environmental and nutritional conditions. Therefore, the bacteria is alive in this state, but do not able to grow in an unfavorable environment (Yoon and Lee, 2019). The presence of the resuscitation agent causes the growth ability to return to the bacteria, which called as the recovery of VBNC, was observed in the present study. The resuscitation of bacteria entering the VBNC state has been reported (Zeng et al., 2013). This study showed that cold and freezing can be effective in induction of the VBNC state, which was similar to our finding.

It was reported that the presence of cold temperature along with salt causes the VBNC state in Vibrio parahemliticus, and the more the NaCl concentration, the greater number of bacteria entering in VBNC state (Yoon Jae-Hyun, Lee, 2019). In the case of Staphylococcus aureus, the existence of the VBNC state due to freezing in the bacteria reduces the enzyme activity of the bacteria. It was also shown that after the recovery of the bacteria, their resistance to gastrointestinal fluid increased (Yan et al., 2021).

Conclusion

Environmental conditions such as temperature, osmotic pressure, acidity, and nutritional deficiencies cause changes in the growth and behavior of bacteria, which can play an important role in induction of VBNC state. The results obtained from this study showed that freezing can be one of the most important factors in production of the VBNC state in food-borne Salmonella spp. Also, the presence of freezing along with other environmental factors of the food model (pH and osmotic pressure) play a role in creating of VBNC state. The induction of VBNC state in pathogenic bacteria in food at different stages of storage and processing can increases the prevalence of these bacteria; therefore, increasing studies in this field is one of the solutions for managing foodborne pathogens. Because the isolated serotypes had the ability to survive and create a VBNC state, they should be studied in order to develop methods of controlling them in food and reducing their pathogenic capabilities.

Acknowledgments

The authors would like to thank the Research Council and the Department of Food Hygiene and Quality Control of Urmia University for funding this research.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Ethical approval

Not applicable.