Effects of Cross-Resistance of Salmonella Enterica Serovar Enteritidis Induced by Sodium Hypochlorite to Environmental Stress

Abstract

To investigate the effects of repeated sodium hypochlorite stress on the resistance of Salmonella enterica Serovar Enteritidis (S. Enteritidis) LWCC1051. LWCC1051 was exposed to Trypticase Soy Broth (TSB) containing sodium hypochlorite concentrations of 9 mmol/L, 10 mmol/L, and 11 mmol/L. After 13 repeated transfers and incubations, three sodium hypochlorite resisted LWCC1051 strains were obtained. The D-values and colony morphologies of these strains were assessed. Their survival rates at 60 °C, 65 °C, 70 °C, 75 °C, and − 20 °C were determined and lethality curves at these temperatures were fitted using the Weibull model. Additionally, the Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for these strains in various chemicals, including malic acid, citric acid, ascorbic acid, acetic acid, lactic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, calcium chloride, sodium chloride, and potassium chloride were ascertained. Sodium hypochlorite concentrations of 9 mmol/L, 10 mmol/L, and 11 mmol/L in TSB induced sodium hypochlorite resistance in S. Enteritidis. D-value increased with the frequency of stress exposure. Higher concentrations of sodium hypochlorite resulted in greater D-values and noticeable differences in colony morphologies. The Weibull model accurately represented the temperature resistance curves of LWCC1051 at the specified temperatures. With increasing sodium hypochlorite stress, both high and low-temperature resistances of LWCC1051 improved. Furthermore, under acetic acid stress, the MIC and MBC values of LWCC1051 strains, post exposure to 9 mmol/L, 10 mmol/L, and 11 mmol/L sodium hypochlorite, doubled. Sodium hypochlorite stress enhances the cross-resistance of LWCC1051 to high temperature, low temperature, and acetic acid treatments.

Introduction

Salmonella enterica Serovar Enteritidis (S. Enteritidis) is a common foodborne pathogen with a wide range of serotypes that can cause human infections, such as septicaemia and gastroenteritis, by contaminating plants and animals [1, 2]. It can also adhere to food processing environments through its bacterial membrane, posing significant risks to food safety [3]. During food processing, storage, and marketing, Salmonella is exposed to repeated stresses from extreme temperatures, acids, alkalis, osmotic pressure, antibiotics, disinfectants, and more. These stresses can induce a series of adaptations, allowing the bacteria to cope with environmental changes and maintain survival [4]. Such adaptations can interfere with food processing disinfection measures, thereby increasing the likelihood of human infections [5].

Sodium hypochlorite is extensively utilized in food processing because of its efficiency and broad-spectrum disinfection capacities. It is used for disinfecting poultry meat, freshly cut fruits and vegetables, instruments, equipment, and other processing environments [6, 7]. In the USA, chlorine levels used in poultry processing facilities do not exceed 50 µg/mL to prevent the production of harmful by-products [8]. With the regular use of surface disinfectants and preservatives, many microorganisms gradually adapt to these conditions and increase their resistance to subsequent stresses [9, 10]. This resistance can amplify the severity and frequency of bacterial infections. If not addressed, this can lead to incomplete food sterilization and the persistence of resistant bacteria [11].

Consequently, there has been a surge in studies focusing on the interactions between sterilization methods and microbial resistance [12–14]. After repeated treatments with sublethal concentrations of diamyl dimethyl ammonium chloride, the majority of the 136 strains tested developed resistance to ampicillin, cefotaxime, ceftazidime, chloramphenicol, and ciprofloxacin [15]. Listeria monocytogenes demonstrated enhanced adaptability to sodium hypochlorite when adhered to a plate containing 3.5% sodium chloride for 7 days, followed by a 0.25% sodium hypochlorite exposure [16]. When Pseudomonas aeruginosa was treated with 1 mg/mL green tea polyphenols, the bacterium enhanced its tolerance to stress conditions including heat, citric acid, acetic acid, propionic acid, and lactic acid, potentially by amplifying catalase synthesis [17]. In adverse conditions, microorganisms can survive by modulating the expression of specific genes and proteins [18–20]. It remains unclear if S. Enteritidis can be conditioned to develop resistance to sodium hypochlorite after exposure to varied sodium hypochlorite concentrations, or if there are cross-protective effects under other stress conditions. Therefore, in this study, S. Enteritidis LWCC1051 was used to determine whether resistance to sodium hypochlorite and cross-protection to environments like high temperature, low temperature, acid, alkali, and salt could be induced by sodium hypochlorite stress. This investigation offers a valuable reference for the widespread use of sodium hypochlorite sterilization technology and is crucial for ensuring food processing and storage safety, effectively controlling foodborne pathogens, and establishing a robust food quality and safety control system.

Materials and Methods

Materials

Salmonella enterica Serovar Enteritidis LWCC1051 was purchased from the China General Microbial Strain Collection Management Centre. Plate Count Agar (PCA), Mueller-Hinton Broth (MHB), and Trypticase Soy Broth (TSB) were sourced from Qingdao Hope Bio-Technology Co., Ltd. (Shandong, China). Sodium hypochlorite (10%) was obtained from Guangdong Guanghua Technology Co., Ltd. (Guangdong, China). Hydrochloric acid (36–38%) and malic acid (98%) were sourced from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Citric acid (99.5%) was obtained from Xilong Chemical Co., Ltd. (Guangdong, China). Acetic acid (99.5%), sodium hydroxide (96%), potassium hydroxide (85%), and sodium chloride (99.5%) were sourced from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Lactic acid (90%) was procured from Tianjin Cometology Reagent Co., Ltd. (Tianjin, China). Calcium chloride (96%) and potassium chloride (99.5%) were obtained from Xilong Science Co., Ltd. (Guangdong, China).

Test Methods

Strain Activation and Pre-treatment

S. Enteritidis LWCC1051 was cultured in TSB. After activation, the test organism was inoculated into TSB and incubated for 24 h at 37 °C, with shaking at 120 r/min. This culture was then reserved as a control group for subsequent use.

Sodium Hypochlorite Stress Treatment of LWCC1051

• (i)Sodium Hypochlorite Stress Treatment: The activated LWCC1051 from Sect. 2.2.1 was inoculated into TSB medium at 1% concentration and then incubated for 24 h at 37 °C with shaking at 120 r/min until the stationary phase was reached. From this, 10 mL of culture solution was taken, centrifuged at 5000 r/min at 4 °C for 20 min, and the supernatant was discarded. The bacterial pellet was then resuspended in 10 mL of TSB containing sodium hypochlorite concentrations of 9 mmol/L, 10 mmol/L, and 11 mmol/L for stress treatment. Each treatment lasted 75 min at 25 °C. Following the sodium hypochlorite stress treatment, the LWCC1051 strains were inoculated into sterile TSB and incubated for 24 h at 37 °C, 120 r/min in a shaker. Another 10 mL of the culture solution was then centrifuged at 5000 r/min, 4 °C for 20 min. The supernatant was discarded, and the bacterial pellet was resuspended in 10 mL of TSB with sodium hypochlorite concentrations of 9 mmol/L, 10 mmol/L, and 11 mmol/L for the second round of stress treatment, with each treatment lasting 75 min. This culture and sodium hypochlorite stress treatment process was repeated 13 times in total.
• (ii)Obtaining Sodium Hypochlorite Resisted Strains of LWCC1051: 200 mL of the previously described LWCC1051 culture, treated 13 times with sodium hypochlorite at concentrations of 9 mmol/L, 10 mmol/L, and 11 mmol/L, was centrifuged at 5000 r/min, 4 °C for 15 min. The supernatant was discarded, and the pellet was washed twice with 0.85% sterile normal saline. The pellet was then resuspended in an equal volume of normal saline, adjusted to achieve a bacterial suspension of 10⁷ CFU/mL, and aliquoted into 10 mL centrifuge tubes for further processing.

Determination of the Resistance Parameter D for LWCC1051 to Sodium Hypochlorite

The microbial resistance parameter D for sodium hypochlorite signifies the time necessary to eliminate 90% of a specific bacterial population under a given lethal sodium hypochlorite condition. A greater D-value indicates stronger resistance to sodium hypochlorite; the D-value is directly proportional to the resistance strength under this lethal condition [21, 22].

The survival count of LWCC1051 at 15, 30, 45, 60, and 75 min under each sodium hypochlorite concentration, as outlined in Sect. 2.2.3, was determined, and colony counting was conducted on the PCA plate at each of these five time points. The sodium hypochlorite treatment time was plotted on the x-axis (abscissa), and the survival count of the bacteria was plotted on the y-axis (ordinate). Linear regression analysis was performed using these five time points, and the negative reciprocal of the slope of the regression line was taken as the D-value. D-values at sodium hypochlorite concentrations of 9 mmol/L (D₉), 10 mmol/L (D₁₀), and 11 mmol/L (D₁₁) were measured in TSB. The coefficient of determination, R², of the linear regression equation was used to assess the goodness of fit for the D-values.

Under the influence of different sodium hypochlorite concentrations, the D-value can be determined using Eq. (1):

$$D=\frac{t}{{log}_{10}\left(\frac{N_0}{N_t}\right)}$$ 1

The D-value represents the time (min) needed to decrease the count of LWCC1051 by 1 log unit under a specific sodium hypochlorite concentration. Here, t denotes the sodium hypochlorite treatment duration (min), while N₀ and N_t represent the initial microbial count at time 0 (before sodium hypochlorite treatment) and the microbial count after t minutes of sodium hypochlorite exposure, respectively.

Observation of the Morphology of LWCC1051 before and after Sodium Hypochlorite Stress

The original control strain from Sect. 2.2.1 and the three-sodium hypochlorite resisted strains obtained in Sect. 2.2.2 were cultured on PCA plates for 48 h. The morphology of LWCC1051 colonies was directly observed with the naked eye. A single colony was then picked using an inoculation loop. After staining with crystal violet and iodine solution, the individual morphology of LWCC1051 was observed using an optical microscope, following counterstaining with safranine.

High Temperature Treatment of the LWCC1051 Resisted Strains

The Weibull model was employed to fit the lethality curve of strain LWCC1051 at various high temperatures. The Weibull model is represented by Eq. (2).

$${log}_{10}\left(\frac{N_0}{N_t}\right)=-{\left(\frac{t}{\delta }\right)}^{\rho }$$ 2

where N₀ is the initial count of LWCC1051 (log CFU/mL); N_t is the count of surviving LWCC1051 after t minutes of high temperature treatment (log CFU/mL); t represents the high temperature treatment time (min); δ and ρ are the scale and shape parameters of the Weibull model, respectively. δ denotes the time (min) taken for the first Ten-fold reduction in the number of bacteriophages; ρ characterizes the profile of the high temperature lethality curve, when ρ = 1, the curve is linear; when ρ > 1, the curve shows a convex shape with a downward bending trend; when ρ < 1, the curve exhibits a concave shape with an upward bending trend [23, 24].

The original control strain and the three sodium hypochlorite-resisted strains were inoculated in TSB and incubated for 24 h with shaking at 37 °C and 120 r/min until the stationary phase was reached. Subsequently, 10 mL of the culture solution was transferred to sterile test tubes and subjected to high temperature in a thermostatic water bath at 60 °C, 65 °C, 70 °C, and 75 °C for 10 min each. Samples were taken at varying time intervals, determined by the survival rate of each strain at the respective temperatures. These samples, after being treated at different temperatures, were plated on PCA for colony counting. The survival rate of each strain at the varied high temperatures was computed using the formula: log₁₀ (N_t/N₀).

Low Temperature Treatment of LWCC1051 Resisted Strains

The original control strain and the three sodium hypochlorite-resisted strains were activated according to the method described in Sect. 2.2.5. Subsequently, 10 mL of the culture solution was dispensed into sterile test tubes and stored in a refrigerator at − 20 °C for a low temperature treatment over 15 days. Samples were collected every 3 days and streaked onto PCA plates for colony counting. The survival rate of each strain at − 20 °C was calculated using the formula log₁₀ (N_t/N₀). The lethality curve of the sodium hypochlorite-resisted strain LWCC1051 at − 20 °C was non-linearly fitted using the Weibull model.

Acid Treatment of LWCC1051 Resisted Strains

Following the methods of Karlowsky et al. [25] with relevant modifications, a broth microdilution approach was employed. Acids used for MIC and MBC experiments included malic, citric, acetic, lactic, and hydrochloric acid. Two-fold dilutions of the acid solutions were prepared using MHB to yield concentrations ranging from 200 mg/mL to 0.38 mg/mL and from 200 to 0.38 µL/mL. The original control strain and the three sodium hypochlorite resisted strains were activated according to the method described in Sect. 2.2.5. The bacterial suspension’s concentration was adjusted to 5 × 10⁵ CFU/mL using 0.85% sterile normal saline. Subsequently, 200 µL of the acid solutions were pipetted into the wells of 96-well plates. Then, 10 µL of the LWCC1051 strains was added to each well. The plates were incubated statically at 37 °C for 24 h. The positive control wells contained MHB medium with the tested bacterial concentrations to verify bacterial viability, while the negative control wells contained only sterile MHB to ensure the medium’s sterility. Results were interpreted when no growth was observed in the negative control wells. The MIC was defined as the lowest concentration of each acid solution that inhibited bacterial growth in comparison to the positive control wells. From wells showing no bacterial growth post-incubation, 100 µL of the culture solution was streaked onto PCA plates. These plates were then inverted and incubated at 37 °C for 24 h. The lowest concentration of each acid solution yielding no colonies on the PCA plates was recorded as the MBC. Acid tolerance was deemed to be significantly induced when the MIC or MBC of the sodium hypochlorite resisted strains was at least two-fold greater than that of the non-adapted strain [26].

Alkali Treatment of LWCC1051 Resisted Strains

Sodium hydroxide and potassium hydroxide were utilized in MIC and MBC experiments. A two-fold dilution of alkali solutions was performed using MHB to prepare concentrations ranging from 60 to 0.10 mg/mL. MIC and MBC values were determined according to the method outlined in Sect. 2.2.7. Alkali tolerance was deemed significantly induced if the MIC or MBC values of the sodium hypochlorite-resistant strains were at least two-fold higher than those of the non-adapted strain.

Salt Treatment of LWCC1051 Resisted Strains

Sodium chloride, potassium chloride, and calcium chloride were used in MIC and MBC experiments. Two-fold dilutions of the salt solutions were prepared using MHB, resulting in concentrations ranging from 300 to 0.59 mg/mL. MIC and MBC values were determined according to the method described in Sect. 2.2.7. Salt tolerance was deemed significantly induced if the MIC or MBC values of the sodium hypochlorite-resisted strains were at least two-fold higher than those of the non-adapted strain.

Data Processing and Statistical Analysis

In this study, the model was fitted using JMP 10.0 software, and data analysis was conducted with SPSS 22.0 software. The test data results were presented as mean ± standard deviation. Multiple comparisons between groups were conducted using Duncan’s new multiple range test (SSR), with P < 0.05 indicating significant differences. All experiments were repeated a minimum of three times on different working days.

Results and Analysis

Effect of Sodium Hypochlorite Stress Treatment on the D-value of S. Enteritidis LWCC1051

The changes in D-values of S. Enteritidis LWCC1051 after treatment with sodium hypochlorite at different concentrations are presented in Table 1. As observed from Table 1, the fit of the D-values was robust (R² > 0.93) for all treatment groups within the scope of this study. Under a 9 mmol/L sodium hypochlorite stress treatment, the D-value of LWCC1051 was approximately 20.5 min after the initial stress treatment. This value surged to 138.9 min after 13 treatments, representing an increase of about 6.8-fold from the first sodium hypochlorite treatment. For LWCC1051 under a 10 mmol/L sodium hypochlorite stress, the D-value started at 16.7 min and rose to around 142.9 min after 13 treatments, which is 8.5 times the initial value. At an 11 mmol/L sodium hypochlorite stress level, the D-value after the initial treatment was roughly 13.8 min, but it escalated to 185.2 min after 13 treatments, a 13.5-fold increase. The data indicates that after 13 repeated sodium hypochlorite stress treatments of LWCC1051 at concentrations of 9, 10, and 11 mmol/L, followed by transfer culture, the D-value consistently rose with increasing treatment iterations. Notably, a higher sodium hypochlorite concentration resulted in a larger D-value, signifying enhanced resistance to the disinfectant. Under the 11 mmol/L sodium hypochlorite stress condition, LWCC1051 exhibited the most rapid augmentation in resistance after consecutive stresses (P < 0.05).

Table 1.

Changes in the D-value of LWCC1051 under different concentrations of sodium hypochlorite stress

Times of sodium hypochlorite stress	9 mmol/L NaClO		10 mmol/L NaClO		11 mmol/L NaClO
	D9 value (min)	R 2	D10 value (min)	R 2	D11 value (min)	R 2
1	20.49 ± 0.18j	0.94–0.97	16.72 ± 0.22i	0.95–0.97	13.76 ± 0.08k	0.94–0.99
2	31.15 ± 0.35i	0.93–0.98	33.11 ± 0.65h	0.95–0.98	49.02 ± 0.10j	0.97–0.99
3	63.29 ± 0.29h	0.96–0.98	60.61 ± 0.48g	0.95–0.99	72.46 ± 0.22i	0.95–0.98
4	64.10 ± 0.28g	0.95–0.99	71.94 ± 0.21f	0.95–0.97	108.70 ± 0.23h	0.95–0.99
5	82.64 ± 0.17f	0.97–0.98	96.15 ± 0.26e	0.93–0.99	112.36 ± 0.23g	0.97–0.98
6	95.24 ± 0.25e	0.98–0.99	108.69 ± 0.57d	0.96–0.99	120.41 ± 0.23f	0.98–0.99
7	111.11 ± 0.21c	0.94–0.99	138.89 ± 0.11c	0.94–0.97	125.04 ± 0.18e	0.94–0.99
8	114.94 ± 0.30b	0.93–0.97	138.89 ± 0.39c	0.96–0.97	138.89 ± 0.52d	0.93–0.97
9	138.63 ± 0.24a	0.96–0.99	142.02 ± 0.17a	0.97–0.98	144.93 ± 0.09c	0.96–0.99
10	138.74 ± 0.11a	0.95–0.99	142.85 ± 0.40a	0.98–0.99	147.06 ± 0.07b	0.96–0.99
11	138.89 ± 0.43a	0.94–0.97	144.93 ± 0.13b	0.93–0.96	185.17 ± 0.07a	0.94–0.99
12	139.02 ± 0.18a	0.97–0.98	142.55 ± 0.32a	0.97–0.98	185.10 ± 0.08a	0.98–0.98
13	138.92 ± 0.26a	0.95–0.99	142.86 ± 0.11a	0.97–0.99	185.19 ± 0.23a	0.96–0.99

In the same column, different letters indicate significant differences (p < 0.05)

Colony Morphology of LWCC1051 before and after Sodium Hypochlorite Stress

The morphological changes of LWCC1051 colonies after 13 stress treatments in TSB medium with sodium hypochlorite concentrations of 9, 10, and 11 mmol/L are depicted in Fig. 1. As observed from Fig. 1: The colonies of the original control strain, after 48 h of incubation on PCA plates, were small, with a smooth surface, neat edges, and a consistent creamy white colour. At 9 mmol/L sodium hypochlorite, the colonies displayed wrinkles from the periphery to the centre, were mostly round with a dry surface. At 10 mmol/L sodium hypochlorite, in comparison to the non-adapted strain, the diameter of most colonies increased. The centres of these colonies appeared slightly sunken, the edges became irregular and serrated, and the surfaces were hazy. At 11 mmol/L sodium hypochlorite, the diameter of most colonies was notably larger than that of the non-adapted strain. These colonies exhibited significant crumpling from the periphery to the centre, had irregular serrated edges, and a dry surface.

Fig. 1.

Colonial morphology of S. Enteritidis LWCC1051 ( A Non- resisted of strain; B 9 mmol/L sodium hypochlorite resisted strain; C 10 mmol/L sodium hypochlorite resisted strain; D 11 mmol/L sodium hypochlorite resisted strain )

The morphological changes in LWCC1051 after 13 stress treatments in TSB medium with various sodium hypochlorite concentrations are illustrated in Fig. 2 under an optical microscope. From Fig. 2: The original control strains were short, rod-shaped with smooth ends, and the individual cells were relatively dispersed. Cells exposed to 9 mmol/L sodium hypochlorite were slightly shorter than the control strains, with a few becoming spherical in shape. For the 10 mmol/L and 11 mmol/L sodium hypochlorite treatments, there was a noticeable change in individual cells; most became spherical, and the interconnections between them appeared tighter. The magnitude of these changes became more pronounced with increasing sodium hypochlorite concentration.

Fig. 2.

Cellular morphology of S. Enteritidis LWCC1051stained with gram staining under optical microscope.  (10 × 100, A Control group without sodium hypochlorite stress treatment; B 9 mmol/L s sodium hypochlorite resisted strain; C 10 mmol/L sodium hypochlorite resisted strain; D 11 mmol/L sodium hypochlorite resisted strain)

Effect of Sodium Hypochlorite at Varying Concentrations on the Lethality of LWCC1051 Under High Temperature

The LWCC1051-resisted strains, obtained from 13 stress incubations in TSB containing various concentrations of sodium hypochlorite, and controls without sodium hypochlorite stress treatment, were assessed for lethality at 60 °C, 65 °C, 70 °C, and 75 °C, as depicted in Fig. 3.

Fig. 3.

Lethal curves of S. Enteritidis LWCC1051 grown in TSB containing different concentrations of sodium hypochlorite at − 20 °C—indicates the lethal curves of S . Enteritidis LWCC1051 at high temperatures, ○ indicates the observed survival rates of non- resisted of strain at high temperatures, ● indicates the observed survival rates of 9 mmol/L sodium hypochlorite resisted strain at high temperatures, □ indicates the observed survival rates of 10 mmol/L sodium hypochlorite resisted strain at high temperatures, ■ indicates the observed survival rates of 10 mmol/L sodium hypochlorite resisted strain at high temperatures

Figure 3 illustrates that the Weibull model was employed to fit the lethal curves of the three resisted strains and the original control strain of LWCC1051 under stress from TSB containing sodium hypochlorite at concentrations of 9 mmol/L, 10 mmol/L, and 11 mmol/L, respectively, at 60 °C, 65 °C, 70 °C, and 75 °C. The model fit was robust (R² > 0.9850). At varying high temperatures, the survival rates of the four strains decreased as treatment time increased. However, their survival rate was significantly higher than that of the non-adapted strain (P < 0.05). The survival rates of the strains differed significantly across temperatures. The high-temperature tolerance of LWCC1051-resisted strains, cultured to a stable phase under sodium hypochlorite stress, was distinct (P < 0.05), suggesting that the high-temperature resistance of LWCC1051 was influenced by the concentration of sodium hypochlorite in the stress culture.

The Weibull model was employed to conduct regression analysis on the lethal test data of three LWCC1051-resisted strains and the original control strain derived from sodium hypochlorite stress culture under high-temperature conditions. The resistance parameter δ was used to assess the heat resistance of the LWCC1051 strains. The high-temperature resistance parameters δ and ρ, obtained through JMP software fitting, are presented in Table 2.

Table 2.

Goodness of fit and parameters of high-temperature lethality model of LWCC1051 under different concentrations of sodium hypochlorite stress

Sodium hypochlorite stress culture concentration (mmol/L)	heat treatment temperature (°C)	Determination coefficient of Weibull model R2	δ(min)	ρ
No sodium hypochlorite stress	60	0.9973	5.06 ± 0.128d	1.31 ± 0.059a
9		0.9860	6.72 ± 0.239c	1.35 ± 0.142a
10		0.9903	9.24 ± 0.167b	2.08 ± 0.199b
11		0.9992	14.69 ± 0.218a	1.39 ± 0.033a
No Sodium hypochlorite stress	65	0.9972	0.27 ± 0.021c	0.49 ± 0.013c
9		0.9850	0.29 ± 0.029c	0.48 ± 0.016c
10		0.9904	0.55 ± 0.054b	0.55 ± 0.022b
11		0.9903	1.06 ± 0.062a	0.64 ± 0.020a
No sodium hypochlorite stress	70	0.9980	0.13 ± 0.010d	0.43 ± 0.009c
9		0.9966	0.17 ± 0.016c	0.44 ± 0.012c
10		0.9988	0.20 ± 0.011b	0.46 ± 0.008b
11		0.9958	0.28 ± 0.027a	0.49 ± 0.016a
No sodium hypochlorite stress	75	0.9961	0.02 ± 0.003d	0.31 ± 0.010c
9		0.9948	0.04 ± 0.007b	0.35 ± 0.013b
10		0.9976	0.06 ± 0.006b	0.38 ± 0.009a
11		0.9993	0.10 ± 0.005a	0.38 ± 0.005a

When LWCC1051 was treated at 60 °C for 10 min, there was a 2.43 log-reduction in the counts of the original control strain. In contrast, the counts of the strain resistant to 11 mmol/L sodium hypochlorite decreased by only 0.58 logs (Fig. 3). This indicates that as the concentration of sodium hypochlorite stress increased, the high-temperature resistance of LWCC1051, induced by sodium hypochlorite stress culture, also significantly increased (P < 0.05). Under high-temperature stress at 65 °C, the δ values of the three resisted strains cultured with 9 mmol/L, 10 mmol/L, and 11 mmol/L sodium hypochlorite were 1.07 times, 2.04 times, and 3.93 times that of the original control strain (Table 2), respectively. This suggests that the high-temperature resistance rose with the elevation in sodium hypochlorite stress concentration. When LWCC1051 was exposed to 70 °C for 10 min, the counts of strains cultured under the stress of 9 mmol/L, 10 mmol/L, and 11 mmol/L sodium hypochlorite decreased by 6.40, 6.11, 5.95, and 5.79 logs, respectively (Fig. 3). These results reveal that the strain induced by 11 mmol/L sodium hypochlorite stress culture exhibited the strongest high-temperature resistance. At 75 °C for 10 min, the high-temperature resistance parameters δ for the strains were 0.02, 0.04, 0.06, and 0.10 min, respectively. This signifies that the high-temperature resistance, induced by sodium hypochlorite stress culture, considerably increased with the rising concentration of sodium hypochlorite stress (P < 0.05). Except at 60 °C, the shape parameters ρ for both the original control strain and sodium hypochlorite-resisted strains were less than 1 under high-temperature treatment conditions. This led the Weibull curve to bend upwards, indicating that as time extended, the rate of bacterial kill-rate decline slowed for the LWCC1051 strains. In summary, augmenting the concentration of sodium hypochlorite in the stress culture of LWCC1051 could foster an increase in its high-temperature resistance.

Effect of Sodium Hypochlorite Concentrations on the Lethality of LWCC1051 at − 20 °C

The low-temperature lethality of the LWCC1051 original control strain and three sodium hypochlorite-resisted strains at − 20 °C is illustrated in Fig. 4.

Fig. 4.

Lethal curves at different high temperatures of S . Enteritidis LWCC1051 grown in TSB containing different concentrations of sodium hypochlorite (A The lethal curves at 60 °C; B The lethal curves at 65 °C; C The lethal curves at 70 °C; D The lethal curves at 75 °C )—indicates the lethal curves of S. Enteritidis LWCC1051 at high temperatures, ○ indicates the observed survival rates of non- resisted of strain at high temperatures, ● indicates the observed survival rates of 9 mmol/L sodium hypochlorite resisted strain at high temperatures, □ indicates the observed survival rates of 10 mmol/L sodium hypochlorite resisted strain at high temperatures, ■ indicates the observed survival rates of 10 mmol/L sodium hypochlorite resisted strain at high temperatures

As depicted in Fig. 4, the Weibull model was suitable for fitting the lethal curves of both the LWCC1051 original control strain and the three sodium hypochlorite-resisted strains at − 20 °C (R² > 0.9950). The survival rates of the LWCC1051 strain, when cultured under sodium hypochlorite stress and subsequently exposed to − 20 °C for 15 days, decreased. However, in comparison to the original control strain, the survival counts of the three resisted strains cultured under 9 mmol/L, 10 mmol/L, and 11 mmol/L sodium hypochlorite stress increased by 0.24, 0.37, and 0.52 logs, respectively.

The Weibull model was employed to fit the regression data on the lethality test of the three sodium hypochlorite-resisted strains of LWCC1051 and the original control strain at low temperatures. This was done to assess the LWCC1051 strain’s resistance to low temperatures at − 20 °C, using the resistance parameter δ. The low-temperature resistance parameters δ and ρ, determined by JMP fitting, are presented in Table 3. As Table 3 indicates, the shape parameter ρ for all four curves exceeds 1, resulting in downward-curving, convex curves. This suggests that the resistance model curve declined sharply as the low-temperature treatment duration extended, and the kill rate of LWCC1051 increased over time. In conclusion, within the experimental range of this study, elevating the concentration of sodium hypochlorite stress led to an enhancement in the low-temperature resistance of LWCC1051.

Table 3.

Fit and parameters of low-temperature lethality model of LWCC1051 under different concentrations of sodium hypochlorite stress at -20 °C

Sodium hypochlorite stress culture concentration (mmol/L)	Determination coefficient of Weibull model R2	δ(min)	ρ
No sodium hypochlorite stress	0.9957	7.06 ± 0.266b	1.72 ± 0.099a
9	0.9950	7.30 ± 0.284ab	1.73 ± 0.100a
10	0.9976	7.43 ± 0.194ab	1.75 ± 0.075a
11	0.9985	7.58 ± 0.151a	1.77 ± 0.061a

Effect of Sodium Hypochlorite at Different Concentrations on the Acid Resistance of LWCC1051

To determine if sodium hypochlorite stress culture can induce cross-resistance of LWCC1051 to acids, the MIC and MBC values of malic, citric, acetic, lactic, and hydrochloric acid against the control strain and the three sodium hypochlorite resisted strains of LWCC1051 were examined. The results are presented in Table 4. As evident from Table 4, the MIC and MBC values of the three resisted strains exposed to sodium hypochlorite under acetic acid stress are twice that of the original control strain. This suggests that the 9 mmol/L, 10 mmol/L, and 11 mmol/L sodium hypochlorite-resisted strains exhibit cross-resistance to acetic acid. The MIC and MBC values for the other four acids did not significantly differ from those of the original control strain (P > 0.05), indicating that the sodium hypochlorite stress culture did not induce cross-resistance in LWCC1051 to these acids.

Table 4.

Determination of MIC and MBC of different acids against LWCC1051

Sodium hypochlorite stress culture concentration (mmol/L)	Bacteriostatic substances	MIC	MBC
No Sodium hypochlorite stress	Acetic acid	6.25	6.25
9		12.5	12.5
10		12.5	12.5
11		12.5	12.5
No Sodium hypochlorite stress	Malic acid	12.5	12.5
9		12.5	12.5
10		12.5	12.5
11		12.5	12.5
No Sodium hypochlorite stress	Citric acid	12.5	25
9		12.5	25
10		12.5	25
11		12.5	25
No Sodium hypochlorite stress	Lactic acid	12.5	25
9		12.5	25
10		12.5	25
11		12.5	25
No Sodium hypochlorite stress	Hydrochloric acid	3.12	3.12
9		3.12	3.12
10		3.12	3.12
11		3.12	3.12

MIC and MBC units for malic and citric acids are mg/mL; MIC and MBC units for acetic, lactic, and hydrochloric acids are µg/mL

Effect of Sodium Hypochlorite at Various Concentrations on Alkali Resistance of LWCC1051

To determine whether sodium hypochlorite stress culture can induce cross-resistance of LWCC1051 to alkali, the MIC and MBC values of sodium hydroxide and potassium hydroxide against both the control strain and the three sodium hypochlorite-resisted strains of LWCC1051 were assessed. The results are presented in Table 5. The MIC and MBC values for the three resisted strains were 15 mg/mL, consistent with those of the control group. This suggests that LWCC1051 cultured under sodium hypochlorite stress did not develop cross-resistance to sodium hydroxide and potassium hydroxide.

Table 5.

Determination of MIC and MBC of different alkalis against LWCC1051

Sodium hypochlorite stress culture concentration (mmol/L)	Bacteriostatic substances	MIC (mg/mL)	MBC (mg/mL)
No sodium hypochlorite stress	Sodium hydroxide	15	15
9		15	15
10		15	15
11		15	15
No Sodium hypochlorite stress	Potassium hydroxide	15	15
9		15	15
10		15	15
11		15	15

Effect of Sodium Hypochlorite with Different Concentrations on the Salt Resistance of LWCC1051

To determine whether sodium hypochlorite stress culture induces cross-resistance of LWCC1051 to salt, the MIC and MBC values for calcium chloride, sodium chloride, and potassium chloride were assessed for both the control strain and the three sodium hypochlorite-resisted strains of LWCC1051. As illustrated in Table 6, the sodium hypochlorite stress culture did not induce any significant changes in the effects of calcium chloride, sodium chloride, and potassium chloride on LWCC1051. The MIC values for calcium chloride for all four strains were 9.375 mg/mL, and the MBC value was 18.75 mg/mL. Similarly, the MIC values for sodium chloride and potassium chloride for all four strains were 75 mg/mL, with MBC values at 150 mg/mL.

Table 6.

Determination of MIC and MBC of different salts against LWCC1051

Sodium hypochlorite stress culture concentration (mmol/L)	Bacteriostatic substances	MIC (mg/mL)	MBC (mg/mL)
0	Calcium chloride	9.37	18.75
9		9.37	18.75
10		9.37	18.75
11		9.37	18.75
0	Sodium chloride	75	150
9		75	150
10		75	150
11		75	150
0	Potassium chloride	75	150
9		75	150
10		75	150
11		75	150

Discussion

During food production and processing, microorganisms frequently encounter a range of adverse environmental conditions. These challenges induce various self-protection mechanisms, leading to an enhanced resistance to lethal factors [27–29]. Sodium hypochlorite, recognized for its efficiency and broad-spectrum disinfection capabilities, serves as a prevalent preservative in the food industry [30, 31]. A study by Alonso-Calleja et al. [32] revealed that Escherichia coli ATCC 12,806 displayed a heightened tolerance to sodium hypochlorite when continuously cultured under gradient semi-lethal concentrations. This observation aligned with findings by Teixeira et al. [33], who reported that bacterial strains isolated from beef processing plants authorized for export in Brazil also manifested significant tolerance to sodium hypochlorite.

In our study, we noted that S. Enteritidis LWCC1051 exhibited increasing D-values after successive stress cultures with 9 mmol/L, 10 mmol/L, and 11 mmol/L sodium hypochlorite. The bacterium’s resistance to sodium hypochlorite augmented with escalating culture concentrations. In comparison to the original control strain, the 9 mmol/L sodium hypochlorite-resisted strain did not exhibit marked resistance, but its surface texture appeared wrinkled and dry. Conversely, the strains resistant to 10 mmol/L and 11 mmol/L sodium hypochlorite mostly presented enlarged and irregularly jagged colonies. These colonies appeared crumpled and dry, signalling morphological alterations due to sodium hypochlorite stress.

Another study demonstrated that the survival rates of Staphylococcus aureus significantly rose after adaptation to common antibiotics like erythromycin, tetracycline, and gentamicin when subjected to treatments involving hydrochloric acid and temperatures of 63 °C [10]. Dawan and Ahn [34] observed that three Salmonella typhimurium species, when continuously stressed with semi-lethal doses of ceftriaxone, ciprofloxacin, polymyxin B, tetracycline, and other antibiotics, demonstrated cross-adaptation to various antibiotics. This was evidenced by the over-expression of resistance-associated genes and efflux pump genes. Zhang et al. [35] exposed Lactobacillus rhamnosus to varying nitrogen source concentrations and discerned a significant uptick in its heat resistance. Concurrently, heat shock response-related proteases, such as clpL and clpX, were markedly up-regulated.

From our observations, we speculated that S. Enteritidis LWCC1051, during adaptation to sodium hypochlorite stress, elevates the expression of genes related to high-temperature, low-temperature, and acetic acid. This could potentially enable the bacterium to acquire resistance to multiple stress conditions. However, delving deeper to uncover the precise mechanism remains essential.

Conclusion

The results from this study indicated that a specific concentration of sodium hypochlorite in a stress culture promotes the development of cross-resistance in LWCC1051 to treatment conditions such as high temperature, low temperature, and acetic acid. However, it is still necessary to investigate the precise mechanisms through which LWCC1051 develops resistance to these stress conditions after exposure to sodium hypochlorite. Therefore, there is a need to focus on the tolerance of drug-resistant bacteria under environmental stress to prevent its proliferation. Exploring more natural antimicrobial agents, such as tea polyphenols and essential oils from spices, which are non-toxic to humans, may offer alternative solutions for use in food processing.

Author contributions

LZ: Conceptualization, Investigation, Methodology, Data curation, Writing – original draft. DY : Investigation. LL : Conceptualization, Investigation. YG : Funding acquisition, Supervision, Writing – review & editing.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.