γ-Aminobutyric Acid (GABA) Metabolic Bypass Plays a Crucial Role in Stress Tolerance and Biofilm Formation in C. sakazakii ATCC 29544

Abstract

Cronobacter sakazakii is a foodborne pathogen characterized by its robust stress tolerance and ability to form biofilms, which facilitates its survival in powdered infant formula (PIF) processing environments for prolonged periods. Gamma-aminobutyric acid (GABA) is a kind of non-protein amino acid that acts as an osmoprotectant. This study aimed to elucidate the effects of the gabT gene on the survival of C. sakazakii, GABA accumulation, and biofilm formation under desiccation, osmotic stress, and acid exposure. A gabT knockout strain of C. sakazakii was developed using gene recombination techniques. The GABA content and survival rates of both the wild-type and knockout strains were compared under various stress conditions. Scanning electron microscopy (SEM) was used to observe cellular damage and biofilm formation. Statistical analysis was performed using a one-way analysis of variance (ANOVA). The deletion of gabT resulted in enhanced GABA accumulation under different stress conditions, improving the bacterium’s tolerance to desiccation, osmotic pressure, and acid treatment. SEM images revealed that under identical stress conditions, the gabT knockout strain exhibited less cellular damage compared to the wild-type strain. Both strains were capable of biofilm formation under low osmotic pressure stress, but the gabT knockout strain showed higher GABA content, denser biofilm formation, and increased biofilm quantity. Similar trends were observed under acid stress conditions. The gabT gene plays a key role in modulating GABA accumulation, which enhances the stress tolerance and biofilm formation of C. sakazakii. These findings provide new insights into the role of GABA in bacterial survival mechanisms and highlight the potential for targeting GABA pathways to control C. sakazakii in food processing environments.

1. Introduction

Cronobacter sakazakii is a Gram-negative pathogenic bacterium belonging to the Enterobacteriaceae family, posing a significant life-threatening risk to specific populations. C. sakazakii is associated with meningitis, bacteremia, and small intestinal colitis, with mortality rates ranging from 40% to 80% [1]. Although patients infected with C. sakazakii can recover following antibiotic treatment, they often experience neurological sequelae and developmental disorders [2]. Studies have identified contaminated powdered infant formula (PIF) as a major contributor to infections in infants and young children [3]. Furthermore, it has been suggested that the long-term survival of C. sakazakii in PIF can be attributed to two main factors, its strong resistance to adverse conditions and its ability to form biofilms [4].

C. sakazakii demonstrates a remarkable ability to survive under adverse environmental conditions, including desiccation, osmotic pressure, and acidity. It exhibits superior resistance to desiccation stress compared to other members of the Enterobacteriaceae family, which enhances its long-term survival in low-water-activity foods, such as PIF [5]. Research shows that while pathogens such as Salmonella, Escherichia coli, and Klebsiella pneumoniae can survive in PIF for up to 15 months, C. sakazakii can persist for over 2.5 years [6]. Furthermore, this pathogen exhibits significant resistance to osmotic stress. A study investigating the salt stress tolerance of 15 strains of C. sakazakii revealed heterogeneity in survival across different strains in media containing 4–10% NaCl, with clinical isolates demonstrating strong tolerance to osmotic pressure [7]. Further research confirmed that certain strains of C. sakazakii are capable of growth in environments containing 10% NaCl [8]. As for acid tolerance, the pH of adult gastric fluid typically ranges from 1.5 to 3.5; however, neonatal gastric fluid can have a pH as high as 5.17. This higher pH increases the likelihood of survival for pathogens, including C. sakazakii [9]. It has been reported that C. sakazakii is tolerant to acidic conditions, with tolerance levels varying depending on the strain, acid strength, and species involved.

C. sakazakii has been documented to form biofilms on abiotic surfaces, including food processing equipment [10,11] and apparatus involved in the reconstitution of powdered infant formula [12]. An increased synthesis of extracellular polysaccharides during biofilm formation provides physical defense for bacterial cells, enabling them to withstand the stressful conditions encountered during food processing [13]. This characteristic enhances the bacteria’s survival and dissemination, posing a significant challenge for prevention and control in the food industry. Therefore, investigating the resistance of C. sakazakii to adversity and its ability to form biofilms is crucial for developing effective strategies to inactivate this bacterium during food processing.

γ-aminobutyric acid (GABA) is a non-protein amino acid commonly found in microorganisms, animals, and plants. The γ-aminobutyric acid transaminase gene (gabT) plays a critical role in the GABA metabolic bypass (Figure 1). In higher plants, multiple studies have shown that the GABA shunt is an important mechanism for drought tolerance, with stress conditions often leading to significant GABA accumulation [14]. For instance, Bao et al. [15] demonstrated that silencing the gabT gene in tomato resulted in elevated GABA levels, reduced succinic acid content, and enhanced osmotic tolerance. It has been revealed that a flagellin-induced GABA shunt improves drought stress tolerance in Brassica napus L [16]. In addition, the GABA shunt is crucial in microbial responses to environmental stresses, such as acid tolerance [17], general stress response [18], and pathogenic bacterial virulence [19]. Additionally, mutations in gabT have been associated with reduced pathogenicity in Pseudomonas syringae [20].

Proteomic technology using iTRAQ has been conducted to investigate desiccation stress tolerance in C. sakazakii ATCC 29544, and the findings revealed a significant upregulation of enzymes related to putrescine metabolism, specifically PuuR, PuuA, and PuuD, indicating an increase in GABA synthesis under desiccation stress [21]. This suggests that the accumulation of GABA derived from the degradation of putrescine and other polyamines could be a vital mechanism for desiccation tolerance in C. sakazakii. Although the role of GABA in desiccation tolerance is well established, most research has focused on plants rather than bacteria; its specific function in C. sakazakii remains unclear. In particular, it is not well understood whether the gabT gene can influence GABA production through metabolic regulation in this bacterium nor how such regulation might affect its stress resistance and biofilm formation abilities.

In this study, a gabT gene knockout strain of C. sakazakii ATCC 29544 was constructed using gene knockout technology. The survival rates, biofilm formation, and intracellular GABA content of both the WT strain and the knockout strain were analyzed under various stress conditions. These experiments aimed to validate the critical role of the GABA metabolic shunt in stress resistance and biofilm formation in C. sakazakii. This study provides novel evidence regarding the role of GABA in osmotic and acid stress in C. sakazakii, an area in which experimental data are limited in the existing literature. We hypothesize that disrupting the GABA metabolic shunt significantly impairs the ability of C. sakazakii to withstand environmental stress and form biofilms. The findings provide a foundation for developing targeted regulatory strategies based on the GABA shunt, offering new insights for the inactivation of C. sakazakii in the food industry.

2. Materials and Methods

2.1. Construction of Gene Knockout Mutants

The gabT knockout strain of C. sakazakii was prepared following a previously established method [22]. Primers were designed based on the sequences of gabT and the plasmid, with details provided in Table S1. The upstream and downstream fragments of the gabT gene were obtained through amplification, after which the gabT fusion fragment was generated via overlapping PCR. The plasmid was constructed and transformed into E. coli β2163 using the recombinant enzyme Exnase II to ligate the products of the suicide vector pLP12cm and the fusion fragment. Finally, the gabT mutant was screened through two times of homologous recombination, and the deletion mutant was cloned and submitted for the sequencing of the PCR product.

2.2. Culture Conditions

C. sakazakii ATCC 29544 and ΔgabT were stored in tryptic soy broth (TSB) with 30% glycerol at −80 °C. The activation of the strain was achieved by inoculating the culture medium on TSA and cultured at 37 °C for 24 h. Then, a single colony of each strain was transferred into 100 mL TSB and the mixture was incubated at 180 rpm 37 °C for 18 h.

2.3. Growth Curves Assay

Growth curves were measured to verify the effect of gene knockout on the growth of the C. sakazakii strain. After activation, either WT or ΔgabT strains were inoculated into TSB by transferring a single colony from TSA plates using an inoculating loop. The cultures were then incubated at 37 °C in a shaker set to 180 rpm. Optical density at 600 nm (OD₆₀₀) was measured every two hours using a microplate reader (Thermo Fisher, Shanghai, China).

2.4. Stress Tolerance Assay

For the preparation of bacterial suspension, cultures in the logarithmic growth phase were collected and centrifuged at 8000 rpm for 5 min to remove the supernatant. The bacterial pellet was then washed three times with PBS and various concentrations of bacterial suspensions were prepared using a 10-fold serial dilution method. The reconstitution of PIF (RIF) was prepared according to the manufacturer’s instructions.

2.4.1. Desiccation Stress

Ten grams of PIF and 1 mL of bacterial suspension were added to a sterile bag. The mixture was kneaded for 2 min to ensure thorough blending of the bacterial solution with the milk powder. Subsequently, the sample was homogenized using a homogenizing beater to achieve a final contamination level of approximately 6 log₁₀ CFU/g for each strain. After homogenization, the sample was placed in a desiccator containing potassium acetate for equilibration [23]. The sample remained in the desiccator for 5 days to reach a water activity level close to 0.25, during which viable counts were assessed.

2.4.2. Osmotic Stress

The culture of each strain was separately suspended and diluted with RIF containing 9% NaCl (w/v) to obtain an initial population of 6 log₁₀ CFU/mL. Then, the culture was incubated at room temperature (25 °C) for 0, 1, 2, 3, 4, 5, and 6 h [22]. After incubation, those samples were centrifuged and diluted with 0.9% (w/v) saline solution to achieve proper concentrations.

2.4.3. Acid Stress

To simulate the acidic environment of the stomach, simulated gastric fluid (SGF, pH = 3.4) was used to obtain acid cells of C. sakazakii. SGF was prepared as described by Wu [24]. Cells of different strains were subjected in SGF solution to obtain an initial flora of about 6 log₁₀ CFU/mL. Then, the cultures were incubated in a shaker (150 rpm) at 37 °C for different incubation times (0, 1, 2, 3, 4, and 5 h).

After drying, osmotic pressure, or acid stress treatment, the samples were washed three times with PBS, diluted to the appropriate concentration, and then spread onto TSA plates for viable bacteria counting. The survival rate was calculated using the following formula:

Survival rate (%) = (total number of viable bacteria after stress treatment/total number of viable bacteria before stress treatment) × 100%.

2.5. Determination of GABA Content

The GABA content in C. sakazakii WT and ΔgabT strains was measured according to the instructions provided with the GABA assay kit (Merck Biotech, Shanghai, China). The preparation of stress-treated samples for each strain followed the method described in Section 2.4. This kit employs an enzyme-linked immunosorbent assay (ELISA) to quantify GABA content, involving the formation of an antibody–antigen–enzyme complex between the GABA antibody, GABA, and the horseradish peroxidase (HRP)-labeled GABA antibody. After thorough washing, the substrate tetramethylbenzidine (TMB) was added, which is catalyzed by HRP to produce a color change from blue to yellow upon acid addition. The color intensity is directly proportional to the GABA concentration. The optical density at 450 nm (OD₄₅₀) was measured using a microplate reader, and GABA concentration in each sample was calculated based on the standard curve.

2.6. Biofilm Formation Assay

Biofilm formation was assessed using the crystal violet staining method as previously described [25]. The bacterial suspensions of the WT and ΔgabT strains were prepared according to Section 2.2, with the turbidity of the suspensions adjusted to a McFarland standard of 0.5 using sterile physiological saline (0.9% NaCl, w/v). Subsequently, 20 μL of bacterial suspensions were inoculated into 96-well plates made of polystyrene containing 180 μL of TSB medium and incubated at 37 °C for 48 and 72 h. The wells were then gently washed three times with phosphate-buffered saline (PBS) and stained with 0.1% crystal violet for 30 min in the dark. After washing with PBS three times, the stain was solubilized with 95% ethanol, and the optical density at 595 nm (OD₅₉₅) of the supernatant in each well was measured using a microplate reader (Bioteck, Synergy Neo2, Arcugnano, Italy). Each sample was tested in triplicate, and the average value was calculated. TSB without bacteria served as the negative control.

2.7. Determination of Biofilm Formation by SEM

Following the method of Liu et al. [26], scanning electron microscopy (SEM, Hitachi, S-3400N-II, Tokyo, Japan) was used to examine the effects of acid and osmotic stress on the biofilm morphology of C. sakazakii. WT and ΔgabT strains were inoculated onto six-well plates with sterilized coverslips, and the samples were incubated at 37 °C for 48 h. Biofilms formed by WT and ΔgabT strains were fixed onto the coverslips with 2.5% glutaraldehyde for at least 2.5 h. The coverslips were then washed three times with sterile PBS and sequentially dehydrated with ethanol at increasing concentrations (10%, 30%, 50%, 70%, 90%, and 100%). The coverslips were then attached to the SEM-specific specimen holders using specialized conductive adhesive, followed by dehydration and gold sputtering. After metallization, the samples were observed under a Hitachi S-3400N-Ⅱscanning electron microscope to examine the microscopic structural morphology of the biofilms. The magnification used for observing the biofilms was 2500×.

2.8. Statistical Analysis

All experiments were conducted in triplicate with independent biological replicates. Standard deviations are indicated in the plots by error bars. Statistical comparisons were performed using a one-way ANOVA for independent samples in SPSS version 26. Tukey’s post hoc test was used to determine significant differences between groups (p < 0.05).

3. Results

3.1. Growth Curve

The growth dynamics of C. sakazakii ATCC 29544 WT and ΔgabT strains in TSB were analyzed using growth curves. As shown in Figure 2, there was no significant difference in the overall growth between WT and ΔgabT strains. The ΔgabT strain exhibited normal growth and proliferation similar to the WT strain under optimal conditions, with both strains entering the logarithmic and stationary phases simultaneously. This finding suggests that the deletion of the gabT gene does not significantly impact the growth of C. sakazakii in a suitable environment and the metabolic pathways regulated by gabT are not critical for bacterial growth under these conditions.

3.2. Survivability and GABA Content Under Desiccation Stress Conditions

As depicted in Figure 3A, both WT and ΔgabT strains exhibited a significant decline in survival rate following desiccation treatment. After ten days of drying, the survivors of the WT strain decreased from 6 to 3.84 log₁₀ CFU/g, while the viable counts of the ΔgabT strain decreased to 5.52 log₁₀ CFU/g. After twenty days of drying, the survival rate of the WT strain further declined to 3.24 log₁₀ CFU/g, whereas the survivability of the ΔgabT strain decreased to 5.43 log₁₀ CFU/g. Following this period, the survivors of both strains stabilized, with the WT and ΔgabT strains exhibiting a survivability of 3.25 and 5.44 log₁₀ CFU/g, respectively. These findings suggest that the desiccation stress tolerance of C. sakazakii is enhanced upon the knockdown of gabT.

The changing trends in GABA content in C. sakazakii WT and ΔgabT strains is illustrated in Figure 3B. Under desiccation stress, GABA levels increased in both strains. After fifteen days of drying, the GABA content in ΔgabT rose from 6.23 μmol/L to 8.17 μmol/L, whereas the WT strain exhibited an increase from 4.17 μmol/L to 5.42 μmol/L. By thirty days, the ΔgabT strain achieved a GABA content of 9.17 μmol/L, while the GABA content in the WT strain remained stable between the fifteen- and thirty-day marks, reaching approximately 5 μmol/L at the end. These findings indicate that the ΔgabT strain maintained a higher GABA content under drying stress compared to the WT strain.

3.3. Survivability and GABA Content Under Osmotic Stress Conditions

The osmotic stress tolerance of WT and ΔgabT strains in RIF medium demonstrated significant declines in viable bacterial counts (p < 0.05) across treatments (Figure 4A). After 3 h of osmotic stress treatment, the survival count of the WT strain decreased to 4.70 log₁₀ CFU/mL, whereas the ΔgabT strain exhibited minimal change, decreasing from 6.00 to 5.78 log₁₀ CFU/mL. Similarly, after 6 h of incubation in RIF, the survival rates of the ΔgabT and WT strains dropped to 5.68 and 4.23 log₁₀ CFU/mL, respectively. The WT strain exhibited a significantly lower survival rate compared to the ΔgabT strain (p < 0.05).

Under 9% NaCl osmotic stress, the GABA content in the ΔgabT strain increased significantly over time, while the GABA content in the WT strain decreased. After 6 h of treatment, the GABA content in the ΔgabT strain rose from 6.87 to 13.89 μmol/L, nearly doubling, whereas the GABA content in the WT strain declined from 5.08 μmol/L to 2.62 μmol/L (Figure 4B).

3.4. Survivability and GABA Content Under Acid Stress Conditions

In this study, the survival of WT and ΔgabT strains under the acidic stress of simulated gastric fluid at pH 3.4 decreased during the first 3 h (Figure 5A). Both strains experienced a significant decline in survival following acid stress treatment (p < 0.05). Notably, the viable count of ΔgabT strains began to increase after 3 h of acid exposure. After 6 h of treatment, the survivability of ΔgabT decreased to 5.46 log₁₀ CFU/mL, while that of WT dropped to 4.01 log₁₀ CFU/mL. Overall, the survival rate of ΔgabT under acid stress was significantly higher than that of WT (p < 0.05).

Additionally, the changes in GABA content during the growth of WT and ΔgabT under acid stress conditions (pH 3.4) were evaluated (Figure 5B). As the duration of acid stress treatment increased, the GABA content in the WT strain exhibited a decreasing trend, whereas the GABA content in the ΔgabT strain gradually increased. After 6 h of acid stress treatment, the GABA content in the WT strain was 4.74 μmol/L, while in the ΔgabT strain, it reached 11.75 μmol/L, which was significantly higher than that of the WT strain (p < 0.05). The increase in GABA content was consistent with the observed survival trends of the two strains under acid stress conditions.

3.5. Effect of Stress Treatment on Bacterial Cell Morphology

Under normal growth conditions, both the WT and ΔgabT strains exhibited intact and robust cell morphology (Figure 6). However, under desiccation stress, the WT strain displayed noticeable surface holes, while the ΔgabT strain showed signs of cell shriveling without significant damage. In osmotic stress conditions, the WT strain exhibited pronounced cell shrinkage and damage, whereas only slight damage was observed on both sides of the ΔgabT cells. Under acid stress, the WT strain experienced severe cell damage, characterized by a visible leakage of intracellular contents, while the ΔgabT strain underwent shrinkage with holes observed on both sides. These observations suggest that the ΔgabT strain is more capable of maintaining cell morphology under stress conditions, which may contribute to its higher survival rates, consistent with the results from the survival experiments.

3.6. Effects of Osmotic Stress on Biofilm Formation and GABA Content

In this study, crystal violet staining was employed to quantify the formation of biofilms by C. sakazakii. Under normal growth conditions, the ΔgabT strain demonstrated a slightly enhanced capacity for biofilm formation compared to the WT strain (p > 0.05, Figure 7A). In the low-salt-concentration treatment group (3% NaCl), the biofilm formation of the ΔgabT strain remained largely unchanged compared to the control samples. Conversely, the biofilm formation of the WT strain under this osmotic stress condition (OD₅₉₅ = 0.73) was significantly lower than that of the control group (OD₅₉₅ = 1.49). When the NaCl concentration was increased to 5%, biofilm formation decreased in both strains; however, the ΔgabT strain (OD₅₉₅ = 0.89) exhibited significantly greater biofilm formation than the WT strain (OD₅₉₅ = 0.46). As the NaCl concentration was further increased to 9%, the biofilm formation for both strains decreased to approximately 0.18.

As illustrated in Figure 7B, under identical osmotic pressure conditions, the GABA content in the ΔgabT strain consistently surpassed that of the WT strain, with the most significant difference observed at a salt concentration of 7%. At a NaCl concentration of 5%, the GABA content in the WT strain began to decline, while the GABA content in the ΔgabT strain exhibited a reduction only at salt concentrations exceeding 7%.

3.7. Effects of Osmotic Stress on the Biofilm Microstructure

The SEM images in Figure 8 reveal the changes in biofilms formed by C. sakazakii under osmotic stress. After 48 h of incubation, both the ΔgabT and WT strains aggregated into multilayer stacks, demonstrating a distinct three-dimensional structure. Following treatment with 3% NaCl, the ΔgabT strain exhibited a denser biofilm and an increase in extracellular polymer production, which aligns with the findings from the crystal violet assay. As osmotic pressure increased (NaCl concentrations above 5%), biofilm formation decreased for both C. sakazakii strains, resulting in bacterial cells appearing as a sparse monolayer. Notably, the ΔgabT strain maintained higher biofilm formation and greater extracellular polymer content compared to the WT strain. At a NaCl concentration of 9%, fragments of broken cells were observed within the field of view.

3.8. Effects of Acid Stress on Biofilm Formation and GABA Content

With increasing acidity in the stress environment, the biofilm formation ability of C. sakazakii WT gradually declines. For the ΔgabT strain, both biofilm formation and extracellular polymer content under acidic conditions (pH 3.4 and pH 4) were significantly lower than those in untreated samples (p < 0.05). However, as the pH rose to 5.0, biofilm formation by ΔgabT exceeded that of the control group, reaching an OD₅₉₅ value of 2.30 compared to 1.63 in the control. These results suggest that a mildly acidic environment can promote biofilm formation in the ΔgabT strain (Figure 9A).

As illustrated in Figure 9B, GABA content increased in both the WT and ΔgabT strains under acid stress conditions. At a pH of 5, the GABA content in WT and ΔgabT strains was measured at 7.92 and 12.37 μmol/L, respectively, with the ΔgabT strain demonstrating a significantly higher GABA concentration than the WT strain (p < 0.05). As the pH decreased, the accumulation of GABA progressively diminished in comparison to the control group. At a pH of 3.4, the GABA content in the WT and ΔgabT strains was recorded at 3.86 and 5.98 μmol/L, respectively, both of which were lower than the GABA content observed in the control conditions.

3.9. Effects of Acid Stress on the Biofilm Microstructure

Under acid stress, changes in the biofilm morphology of C. sakazakii are evident in SEM images (Figure 10). At pH 3.4, damaged bacteria and lysed cells are visible but no distinct biofilm structure is present. As the pH increases, biofilms appear denser, with richer extracellular polymers. At pH 4.5, notable bacterial aggregation is observed, while at pH 5, extensive biofilm formation and a dense layer of extracellular polymers dominate the field of view. Across all acidic conditions, ΔgabT consistently showed a greater biofilm formation ability than WT, consistent with the crystal violet staining results.

4. Discussion

C. sakazakii is a common opportunistic pathogen found in PIF and exhibits resistance to various environmental pressures. Consequently, a deeper understanding of the potential tolerance mechanisms of C. sakazakii could aid in its activation. This study hypothesized that the GABA metabolic shunt plays a critical role in the stress resistance and biofilm formation of C. sakazakii. Based on the experimental results, this hypothesis was accepted. Specifically, the survival rates, GABA accumulation, and biofilm formation ability of the gabT knockout strain were significantly higher under osmotic, desiccation, and acid stress conditions compared to the WT. These findings provide strong evidence supporting the hypothesis. Specific discussions of these results are described below.

4.1. GABA Accumulation Improve the Ability of Desiccation Tolerance

In this study, desiccation stress significantly increased GABA levels in both ΔgabT and WT strains, with the ΔgabT strain exhibiting a markedly higher accumulation than WT (Figure 3). After ten days of drying, the GABA content in the ΔgabT strain rose from 6.23 μmol/L to 7.93 μmol/L (p < 0.05), while in the WT strain, it increased from 4.17 μmol/L to 5.23 μmol/L (p < 0.05). Desiccation is typically associated with high osmotic stress, and C. sakazakii responds through a phased regulatory mechanism. Initially, cells increase intracellular osmotic pressure by accumulating electrolytes such as potassium ions and glutamate to counteract external osmotic forces. However, prolonged high ion concentrations may lead to ionotoxicity, so a secondary response involves synthesizing or absorbing compatible solutes, like proline and glycine betaine, which stably remain in cells, mitigate ion toxicity, and provide osmotic balance [27,28]. As a small-molecule osmotic regulator, GABA increases cytoplasmic osmotic potential, thereby enhancing cell water retention and reducing desiccation-induced damage [29].

Further analysis revealed that after twenty days of drying, the GABA level in the ΔgabT strain rose to 8.49 μmol/L, while the GABA level in the WT strain remained relatively stable, reaching approximately 5 μmol/L at the end (p < 0.05). This discrepancy suggests that the deletion of the gabT gene in C. sakazakii impairs GABA transaminase activity, hindering further GABA metabolism and facilitating its intracellular accumulation. The elevated GABA accumulation in ΔgabT under desiccation stress aligns with its improved desiccation resistance. Studies indicate that strains with high desiccation tolerance exhibit an increased expression of genes related to the synthesis and transport of compatible solutes (e.g., betAB, proVW) compared to low-tolerance strains, suggesting that such gene expression enhances desiccation resilience [28]. Thus, the substantial GABA accumulation observed in ΔgabT under desiccation in this study not only reflects the effects of the genetic mutation but also enhances the survival rate of ΔgabT in dry conditions. This evidence underscores that GABA accumulation may play a critical protective role under desiccation stress, conferring the ΔgabT strain with significantly greater resistance than WT.

4.2. GABA Accumulation Improves the Ability of Osmotic Tolerance

Osmotic pressure is commonly utilized in the food processing industry as a method to inhibit pathogenic bacteria. The concentration process in PIF production involves osmotic pressure stress. Existing research on the mechanisms by which bacterial cells resist osmotic stress often draws analogies between dry and hypertonic environments [30]. When exposed to hypertonic conditions, C. sakazakii relies on the accumulation of intracellular electrolytes to elevate osmotic pressure and counterbalance the external hypertonic environment [31].

GABA, a four-carbon non-protein amino acid, exists as a zwitterion under normal physiological pH conditions and is readily soluble in water. Its biochemical properties resemble those of small osmotic molecules such as proline and betaine. Consequently, GABA serves as an osmotic adjustment substance that increases osmotic water potential in the cytoplasm, enhances cellular water retention, and mitigates damage caused by dehydration [14]. In this study, the ΔgabT strain exhibited a higher accumulation of GABA under osmotic stress (Figure 4B), correlating with its improved survival rate (Figure 4A). The deletion of the gabT gene in ΔgabT disrupts GABA metabolism, leading to its accumulation, which aids the cells in resisting osmotic stress and results in a higher survival rate compared to WT. Desiccation strategies of C. sakazakii in low-moisture environments have been summarized; the accumulation of osmoprotectants or the production of chaperone proteins that prevent oxidative damages are the main strategies [1]. Therefore, in the ΔgabT strain, GABA accumulation enhances osmotic water potential, ultimately increasing survival rates. The higher survival rate of C. sakazakii in PIF may be attributed to the protective effects of lipids, proteins, and other components present in PIF.

4.3. GABA Accumulation Improves the Ability of Acid Tolerance

Gastric acid serves as the first line of defense against bacterial infections in the host. The survival rate of C. sakazakii during the rehydration of PIF and in simulated gastric fluid was assessed, and the evidence confirms that C. sakazakii can grow under acidic conditions [32,33]. Currently, while the relationship between GABA content and stress tolerance in plants has been extensively studied, research on the role of gabT in bacterial environmental tolerance remains limited. Few studies have reported that GABA enhances the acid resistance of Listeria monocytogenes [17]. In the present study, both the WT and ΔgabT strains exhibited decreased survivors within the first three hours under acid stress of pH 3.4 (Figure 5A); however, the number of ΔgabT strains began to increase after this period. Furthermore, the gabT mutant demonstrated higher viability than the WT throughout the acid tolerance conditions. The glutamate decarboxylase (GAD) system is primarily associated with resistance to acidic conditions in microorganisms. This system produces GABA as a by-product, which serves as a substrate in the GABA shunt pathway [34]. The GAD system contributes to intracellular pH balance by consuming protons during the decarboxylation reaction [35], utilizing glutamate to produce GABA. Consequently, microorganisms exploit this mechanism to remove protons from the intracellular environment under acidic conditions. The authors of [17] reported that GABA accumulation is crucial for the survival of Listeria monocytogenes under acidic stress, with increased GABA levels correlating with enhanced survival at low pH values, a finding consistent with the results of this study. In the ΔgabT strain, the disruption of the downstream metabolic pathway for GABA prevented succinate from being dehydrogenated to produce hydrogen ions, further aiding C. sakazakii in responding to acid stress.

4.4. GABA Accumulation Improves the Ability of Biofilm Formation

C. sakazakii is known to develop biofilms rapidly as a self-protective response to adverse environmental conditions [5]. Research has demonstrated that extracellular polysaccharides can protect cells within biofilms from environmental stressors [36]. In this study, under acid (pH 5) or osmotic conditions (3% NaCl), C. sakazakii exhibited higher extracellular polymer content and a denser tissue structure compared to the untreated group (Figure 8 and Figure 10). The biofilm matrix encapsulating C. sakazakii consists of extracellular polysaccharides, proteins, and extracellular DNA (eDNA), which collectively create mechanical stability and cohesion among the bacteria [21]. This stability enhances the survival and persistence of microorganisms within biofilms [5]. As illustrated in Figure 7A and Figure 9A, C. sakazakii resists environmental stress by forming a biofilm under conditions of weak acid and hypo-osmolality. However, as the pH decreases, the harsh environment negatively impacts C. sakazakii growth, resulting in decreased survival rates and subsequently reduced extracellular polymer production. Notably, regardless of treatment conditions, ΔgabT exhibited greater extracellular polymer content than WT, correlating with its higher survival rate under acid stress. The ΔgabT strain demonstrated not only enhanced survival but also greater viable bacterial counts and increased extracellular polymer secretion, which provide better protection against acidic environments. Similarly, under low osmotic pressure, this strain can resist stress through biofilm formation. However, as osmotic pressure increases, viable bacterial numbers and activity decline, preventing effective biofilm formation, which aligns with survival rate results. Furthermore, measurements of GABA content in the planktonic cells of the biofilm revealed that the deletion of the gabT gene disrupts GABA metabolism, leading to GABA accumulation (Figure 7B and Figure 9B). This accumulation aids in environmental stress resistance, resulting in denser biofilm formation in the ΔgabT strain compared to the WT strain.

In summary, this study confirms for the first time that the GABA metabolic shunt contributes to desiccation, osmotic, and acid stress resistance in C. sakazakii. These findings not only offer new insights into the stress adaptation mechanisms of this pathogen but also establish a foundation for developing targeted strategies to prevent and control C. sakazakii contamination in food processing. Despite these contributions, certain limitations of the study should be acknowledged. For instance, the research focused on a single strain of C. sakazakii, and the broader applicability of these findings across other strains or environmental conditions remains to be explored. Additionally, while this study highlights the role of GABA in stress resistance and biofilm formation, the underlying biochemical mechanisms, particularly at the molecular level, require further investigation. Future studies should aim to elucidate the GABA-mediated regulatory pathways and their interactions with other metabolic processes in C. sakazakii.

5. Conclusions

In this study, the gabT mutant of C. sakazakii was constructed using homologous recombination techniques to investigate its role in environmental stress tolerance and biofilm formation. Comparative analyses of survival rates, biofilm formation, and GABA content between the wild-type strain and the gabT mutant revealed that GABA enhances C. sakazakii’s tolerance to desiccation, osmotic pressure, and acidity while promoting biofilm formation under stress conditions. These findings confirm that gabT plays a critical role in the environmental tolerance and biofilm development of C. sakazakii. This study provides a foundation for developing strategies to reduce the stress resistance and biofilm formation of C. sakazakii in food processing environments. Future research should focus on elucidating the molecular mechanisms underlying GABA-mediated stress responses and exploring potential GABA inhibitors as control measures.