The antibacterial mechanism of the extracellular product of coagulated Heyndrickxia coagulans against Staphylococcus aureus

doi:10.21203/rs.3.rs-7975300/v1

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The antibacterial mechanism of the extracellular product of coagulated Heyndrickxia coagulans against Staphylococcus aureus

https://doi.org/10.21203/rs.3.rs-7975300/v1

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Heyndrickxia coagulans is a probiotic strain combining features of lactic acid bacteria and spore-forming bacilli. Its extracellular metabolites (postbiotics), such as bacteriocins, organic acids, and peptides, have attracted growing interest for skincare due to their antibacterial, anti-inflammatory, and antioxidant activities. In this study, Staphylococcus aureus was selected as a model pathogen to assess the antibacterial effects of H. coagulans metabolites using Oxford cup diffusion and minimum inhibitory concentration assays. Mechanistic investigations included bacterial growth curve analysis, membrane permeability testing, biofilm formation assessment, and evaluation of key metabolic enzyme activities. Results demonstrated that these metabolites disrupted the pathogen’s physiological functions, indicating strong antibacterial potential. This study highlights the promise of H. coagulans metabolites as safe, natural agents for skin health, providing a theoretical foundation for developing functional skincare products and strategies to prevent and control skin infections.

Heyndrickxia coagulans

Staphylococcus aureus

antibacterial activity

biofilm

membrane disruption

Heyndrickxia coagulans (also known as Bacillus coagulans or Weizmannia coagulans) is a spore-forming probiotic bacterium that combines the advantages of lactic acid bacteria and Bacillus species. It has demonstrated significant potential in food safety, medical therapy, and skin health applications^1,2. Its extracellular metabolites—such as organic acids, antimicrobial peptides, short-chain peptides, and bioactive small molecules—show broad-spectrum antibacterial activity with excellent thermal and pH stability³. Genomic studies have identified gene clusters responsible for bacteriocin production, adhesion, and bile acid tolerance, which underpin its probiotic viability and antimicrobial function in both skin and gut environments⁴.

Staphylococcus aureus is a common skin-resident pathogen linked to infections such as folliculitis, impetigo, cellulitis, and skin abscesses⁵. Its colonization increases significantly in patients with chronic skin conditions like atopic dermatitis, where it exacerbates inflammation and disrupts the skin barrier through the secretion of toxins and superantigens^6,7. In addition, S. aureus forms biofilms on the skin surface, enhancing its antibiotic resistance and evading immune responses—factors that contribute to persistent or recurrent infections⁸.

Given the global issue of rising antibiotic resistance, probiotics and their non-viable derivatives, known as postbiotics, have emerged as safe and natural alternatives for antimicrobial therapy^9–11. Postbiotics not only exhibit antimicrobial properties but also provide antioxidant, anti-inflammatory, and skin-barrier-supporting functions^12–14. For instance, LactoSporin—an extracellular metabolite derived from H. coagulans MTCC 5856—has demonstrated inhibitory effects against S. aureus and improvements in skin texture and aging markers¹⁵. The PL-W strain, isolated from traditional cheese, produces a stable bacteriocin that is effective against multiple Gram-positive pathogens³. Additionally, the combination of H. coagulans with prebiotics has been shown to modulate the gut–skin axis, reducing skin roughness and promoting immune balance^16–18. Notably, Zhang Yongtao (2023) revealed that protein components from H. coagulans YTCY fermentation can regulate TLR2/NF-κB and MAPK/ERK signaling pathways, reducing S. aureus-induced inflammation and oxidative stress¹⁹.

Therefore, this study focuses on the extracellular metabolites of Heyndrickxia coagulans to systematically investigate their antibacterial potential against Staphylococcus aureus, a common skin pathogen. The basic antibacterial activity was evaluated using the Oxford cup diffusion method and minimum inhibitory concentration (MIC) tests. Subsequently, the possible mechanisms of action were explored from multiple dimensions, including bacterial growth curves, changes in cell membrane permeability, inhibition of biofilm formation, interference with protein synthesis, and alterations in key metabolic enzyme activities. This research is expected to provide a scientific basis for the development of natural microbe-derived antibacterial agents and expand the potential applications of probiotics in skincare and infection prevention.

2.1 Materials

The experimental materials used in this study included DCFH-DA (Shuangjun Chemical Co., Ltd.), crystal violet (Beijing Solarbio Science & Technology Co., Ltd.), 25% glutaraldehyde (Shanghai Zeye Biotechnology Co., Ltd.), ONPG (Yuan Ye Bio), and a succinate dehydrogenase (SDH) assay kit (Nanjing Jiancheng Bioengineering Institute). In addition, animal-free LB medium (Product No. CM0998B) was obtained from Oxoid Ltd., Wade Road, Basingstoke, Hampshire, United Kingdom.

The main instruments used in the study included a SpectraMax 190 microplate reader (Molecular Devices, Shanghai), a NanoDrop micro-volume spectrophotometer (Thermo Fisher Scientific), a Hitachi SU8200 field emission scanning electron microscope (Hitachi High-Technologies, Shanghai), and an ultrasonic cell disruptor (Shanghai Beyotime Biotechnology Co., Ltd.).

The extracellular products of this experiment were derived from previous experiments and were extracted by ammonium sulfate fractional precipitation. The fermentation supernatant was salted step by step at 25%, 50% and 75% saturation, and the 75% saturated precipitate was collected after centrifugation (8500×g, 4°C, 15 min). The resulting pellet was dissolved in Tris-HCl buffer (pH 7.5) and desalted with a 500 Da dialysis bag after removing impurities from a 0.22 µm filter membrane. The molecular weight distribution of the desalting products was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC), and the main peak was concentrated in 9.3 min, corresponding to a molecular weight of about 1.51–11.44 kDa, accounting for about 80% of the total protein.

2.2 Strain Source

The Weizmannia coagulans YTCY strain used in this study is derived from the laboratory and is the strain used by our team in previous skin-related research. The strain was cultured in LB liquid medium containing tryptone (10 g/L), NaCl (10 g/L) and yeast extract (5 g/L), and fermented at constant temperature at 37°C. After the fermentation broth is centrifuged to remove the bacteria, the supernatant is used as the raw material for extracting extracellular products.

2.3 Pathogenic Bacteria Culture

Frozen Staphylococcus aureus cultures were thawed to room temperature, and 100 µL of the bacterial suspension was inoculated into 100 mL of LB liquid medium and incubated at 37°C with shaking at 200 rpm for 24 hours. Then, 100 µL of the cultured bacterial suspension was evenly spread on an LB agar plate using the four-quadrant streaking method, with sterilization of the inoculation loop after each quadrant. The plates were incubated in an inverted position at 37°C for 18–24 hours until single colonies appeared. A single colony was picked and inoculated into 100 mL of fresh LB liquid medium and cultured at 37°C with shaking at 200 rpm for an additional 5 hours. The optical density (OD) of the bacterial suspension was then measured and adjusted to an OD₆₀₀ range of 0.08–0.12.

2.4 Antibacterial Activity Assessment

2.4.1 Oxford Cup Diffusion Assay

Bacterial suspension was centrifuged at 8000 rpm for 10 minutes at 37°C, and the supernatant was discarded. The bacterial pellet was resuspended in 0.9% sterile saline and adjusted to an OD₆₀₀ value of 0.08–0.12 (corresponding to 1×10⁸ CFU/mL), then diluted to a final concentration of 1×10⁶ CFU/mL. A total of 200 µL of the diluted suspension was evenly spread onto the surface of LB agar medium, and Oxford cups were placed on the plates. Each cup was filled with 200 µL of extracellular product solution at concentrations of 0.5 mg/mL, 1.0 mg/mL, and 2.0 mg/mL, respectively. Plates were incubated at 37°C for 24 hours, and antibacterial activity against Staphylococcus aureus was assessed based on the inhibition zones formed.

2.4.2 Minimum Inhibitory Concentration (MIC) Determination

The MIC value was determined using the broth microdilution method in a 96-well microplate. According to the dilution scheme, 100 µL of the test sample at different concentrations was added to each well. The specific steps are as follows:

First, a high-concentration gradient was prepared: 0.9 mL of the original sample was mixed with 0.1 mL of LB broth in well 1A to obtain a 90% concentration; in well 1B, 0.8 mL of the original sample was mixed with 0.2 mL of LB (80%); 1C contained 0.7 mL + 0.3 mL (70%); 1D, 0.6 mL + 0.4 mL (60%); and 1E, 0.5 mL + 0.5 mL (50%).Subsequently, a two-fold serial dilution was performed starting from the 50% concentration: 0.5 mL from well 1E was mixed with 0.5 mL LB to prepare well 1F (25%), followed by dilutions to obtain 1G (12.5%), 1H (6.25%), 1I (3.125%), and 1J (1.563%).This series of diluted concentrations was used to evaluate the minimum inhibitory concentration (MIC) of the sample.

2.4.3 Bacterial Growth Curve Determination

Sterile nutrient broth (100 mL) was prepared in a 250 mL Erlenmeyer flask. A 1 mL aliquot of S. aureus suspension (1×10⁸ CFU/mL) was inoculated into the flask. The experimental groups received 1 mL of extracellular product at concentrations of 0.5 mg/mL, 1.0 mg/mL, and 2.0 mg/mL, respectively. A solvent control group received 1 mL of absolute ethanol, and a blank control group received 1 mL of sterile saline. All flasks were incubated in a shaking incubator at 37°C, 120 rpm. Samples were taken at various time points (e.g., 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 24 h), and the absorbance at 625 nm (OD₆₂₅) was measured with a spectrophotometer. Growth curves were plotted based on OD₆₂₅ values over time.

2.5 Preliminary Mechanism Analysis

2.5.1 Alkaline Phosphatase (AKP) Activity Assay

A single colony of Staphylococcus aureus was inoculated into sterile nutrient broth and cultured at 37°C, 120 rpm for 12 hours. Cells were harvested by centrifugation at 5000 rpm for 4 minutes and resuspended in saline to a final concentration of 1×10¹⁰ CFU/mL. Samples were then treated with the test agents at final concentrations of 0.5 mg/mL, 1 mg/mL, and 2 mg/mL, respectively. Control groups included 1% (v/v) absolute ethanol (solvent control), 1% saline (blank control), and lysozyme at 10 mg/mL (positive control).After mixing, all samples were incubated in a water bath at 37°C for 3 hours. Afterward, samples were centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatants were collected to determine AKP activity using a commercial AKP assay kit, following the manufacturer’s instructions.

2.5.2 PI Staining for Cell Membrane Permeability

The preparation and standardization of bacterial suspensions were performed as described in Section 2.2.Cells in the logarithmic phase were centrifuged and resuspended in sterile PBS or nutrient broth to an OD₆₂₅ of 0.3.Test samples were added to the suspensions at final concentrations of 1 mg/mL and 2 mg/mL (1% v/v).A blank control group received sterile water instead of antimicrobial agents. All groups were incubated at 37 ± 1°C, 200 rpm for 6 hours. After incubation, PI dye was added to each sample at a final concentration of 1.5 mM and incubated in the dark at room temperature for 30 minutes. PI staining was conducted according to published protocols²⁰.Then, 10 µL of each stained suspension was placed on a microscope slide, covered with a coverslip, and examined under an inverted fluorescence microscope.

2.5.3 Nucleic Acid Leakage Assay

Single colonies of S. aureus were inoculated into nutrient broth and cultured at 37°C, 120 rpm for 12 hours. Cells were harvested by centrifugation at 5000 rpm for 4 minutes and resuspended in saline to an OD₆₂₅ of 0.5.Samples were then treated with the test agents at final concentrations of 1 mg/mL and 2 mg/mL. The solvent control was treated with 1% ethanol, and the blank control with 1% saline. Samples were incubated at 37°C, 120 rpm, and 1 mL aliquots were taken at 2 h, 4 h, 6 h, and 8 h. After centrifugation at 10,000 rpm, 4°C for 5 minutes, the supernatant was collected, and nucleic acid release was measured using a NanoDrop spectrophotometer.

2.5.4 β-Galactosidase Leakage Assay

After 3-hour drug treatment, bacterial suspensions were centrifuged at 12,000 rpm, 4°C for 10 minutes. A 1 mL aliquot of the supernatant was mixed with 0.1 mL of ONPG solution (final concentration 0.75 M) and incubated at 37°C in a water bath for 3 hours. The absorbance at 420 nm was measured to evaluate the effect of antimicrobial agents on β-galactosidase activity.

2.5.5 Cell Membrane Permeability Measurement

Bacterial suspensions (OD₆₂₅ = 0.5) were prepared as described in Section 2.4.3.

Cells were then treated with antimicrobial agents at final concentrations of 0.5, 1, and 2 mg/mL (1% v/v).Control groups included 1% saline (blank) and 1% ethanol (solvent).After incubation at 37°C, 120 rpm for 3 hours, the pH and conductivity of each sample were measured to evaluate the physicochemical changes in the bacterial suspension.

2.5.6 Morphological Observation of Bacterial Cells

A single colony was inoculated into nutrient broth and cultured at 37°C, 120 rpm for 12 hours. Ten milliliters of the culture were harvested by centrifugation at 5000 rpm for 4 minutes. The cells were washed and resuspended in 1 mL sterile saline and then transferred into 100 mL fresh broth. Test samples were added to achieve final concentrations of 1 mg/mL and 2 mg/mL. Ethanol of equal volume was used in the solvent control group. All samples were incubated at 37°C, 120 rpm for another 12 hours. Afterward, cells were harvested and fixed with 1 mL of 4% glutaraldehyde at 4°C overnight. The next day, fixed cells were washed with distilled water three times and resuspended. About 200 µL of suspension was loaded into filter paper envelopes containing glass slides (0.5 mm × 0.7 mm).Samples were dehydrated in graded ethanol (50%, 70%, 90%, 100%), dried by critical-point drying, gold-sputtered, and observed under a Hitachi field-emission SEM to assess morphological changes.

2.6 In-depth Mechanistic Analysis

2.6.1 Reactive Oxygen Species (ROS) Detection

Previous studies have shown²¹ that intracellular ROS levels can be effectively assessed using the DCFH-DA probe, which provides a reliable method for investigating antibacterial mechanisms. In this experiment, bacterial cultures were prepared as described in Section 2.2.Into a 10 mL sterile EP tube, DCFH-DA was added to a final concentration of 2×10⁻⁵ mol/L, followed by the addition of 10 mL of bacterial suspension (final concentration: 1×10⁸ CFU/mL).The mixture was incubated in a 37°C water bath in the dark for 1 hour to allow the probe to enter the cells and be hydrolyzed by intracellular esterases to non-fluorescent DCFH. In the presence of ROS, DCFH is oxidized to fluorescent DCF. After incubation, the cells were centrifuged at 8000 rpm for 5 minutes at 4°C to remove unincorporated DCFH-DA. Then, antimicrobial agents were added to the bacterial pellet to final concentrations of 0.5, 1, and 2 mg/mL. Bacterial suspensions were adjusted to 1×10⁶ CFU/mL and incubated in a 37°C water bath for 12 hours. A saline-treated group served as the blank control. Finally, the intracellular fluorescence intensity of DCF was measured using a microplate reader (excitation wavelength: 488 nm; emission wavelength: 525 nm) to evaluate ROS levels and determine whether oxidative stress contributed to the antibacterial mechanism.

2.6.2 Effect of Extracellular Products on Total Protein Synthesis in Staphylococcus aureus

This assay was based on a modified version of previously published protocols²², which demonstrated that protein levels are a key indicator of antibacterial mechanisms. Bacterial suspensions were harvested by centrifugation at 5000 rpm for 4 minutes. The cell pellet was resuspended in 1 mL of sterile saline and transferred into 100 mL of fresh nutrient broth. Test samples were added to achieve final concentrations of 1 mg/mL and 2 mg/mL, and a blank control was treated with 1% saline. All groups were incubated at 37°C, 120 rpm for 12 hours to ensure sufficient interaction. Cells were harvested by centrifugation again and resuspended in saline to an OD₆₂₅ of 0.3.Cells were disrupted by ultrasonic cell crusher (15 min total time, 15% power, 3 s on / 2 s off).Total protein content in the lysate was quantified using the Coomassie brilliant blue (Bradford) method to evaluate the extent to which the samples affected protein synthesis or release.

2.6.3 Effect of Antimicrobial Agents on Succinate Dehydrogenase (SDH) Activity

This method was adapted from previous studies showing²³ that suppression of key metabolic enzymes such as SDH can interfere with bacterial energy metabolism and contribute to antibacterial effects. Bacterial suspensions were prepared as described in Section 2.4.3 and adjusted to OD₆₂₅ = 0.3.Aliquots were transferred to sterile EP tubes, and antimicrobial agents were added to final concentrations of 1 mg/mL and 2 mg/mL. Control groups included saline (1%, blank) and lysozyme (10 mg/mL, positive control).Samples were incubated at 37°C, 120 rpm for 6 hours to ensure sufficient interaction. SDH activity was then measured using a commercial SDH assay kit, following the manufacturer’s instructions. Absorbance values were measured by microplate reader, and SDH activity levels were calculated to evaluate the impact of the treatment on bacterial energy metabolism.

2.6.4 Effect on Metabolic Activity of Bacterial Cells

This method was based on a modified version of the MTT colorimetric assay used to assess bacterial metabolic activity on surfaces²⁴. Bacterial suspensions were prepared and standardized to OD₆₂₅ = 0.5.Each sample was treated with 1% (v/v) of antimicrobial agent solution, giving final concentrations of 1 mg/mL and 2 mg/mL. A blank control group was treated with 1% saline.

Samples were incubated at 37°C, 150 rpm for 2 hours to allow interaction.

After incubation, the cells were centrifuged at 8000 rpm for 5 minutes at 4°C and resuspended in saline to maintain OD₆₂₅ = 0.5.INT (iodonitrotetrazolium chloride) was then added to a final concentration of 1 mmol/L and incubated in a 37°C water bath for 30 minutes. During incubation, dehydrogenases in metabolically active cells reduce INT to red formazan. Absorbance at 630 nm was measured using a microplate reader to evaluate metabolic activity under each treatment condition.

2.7 Biofilm Inhibition Analysis

Biofilm adhesion assays were conducted to evaluate the effects of test samples on both biofilm formation and removal.

In each well of a 48-well plate, 200 µL of Staphylococcus aureus suspension (1×10⁶ CFU/mL) and 200 µL of test sample at varying concentrations (0.5 mg/mL, 1 mg/mL, and 2 mg/mL) were added. The plate was incubated at 37°C for 48 hours. After incubation, the supernatant was discarded, and each well was gently rinsed twice with sterile water. Subsequently, 500 µL of 25% glutaraldehyde was added to each well and fixed at room temperature for 15 minutes. The fixative was then removed, and the wells were rinsed again with sterile water. Next, 200 µL of 0.1% crystal violet solution was added to each well and stained for 5 minutes. The excess stain was discarded, and the wells were washed three times. After imaging the stained wells using an inverted microscope, absolute ethanol was added to dissolve the bound crystal violet, and absorbance at 590 nm was measured. The biofilm inhibition rate was calculated using the following formula: Biofilm inhibition rate (%)=(1 − C/S)×100%

where C and S represent the average absorbance values of the control group and sample group, respectively.

3.1 Antibacterial Activity Results

As shown in Figure 1, the extracellular product at a concentration of 0.5 mg/mL produced a 12 mm inhibition zone against Staphylococcus aureus. Standard for evaluating antibacterial efficacy based on inhibition zone size, inhibition zone diameters between 10–14 mm indicate a moderate sensitivity of the strain to the treatment. In summary, the extracellular product exhibited inhibitory activity against Gram-positive bacteria, suggesting its potential as a broad-spectrum antibacterial agent.

Staphylococcus aureus was able to fully grow in LB medium within 24 hours. As shown in Figure 2A, antibacterial assays against Staphylococcus aureus were performed using three concentrations of the extracellular product: 0.5 mg/mL, 1 mg/mL, and 2 mg/mL . The goal was to evaluate the antibacterial activity of the sample at gradient concentrations. By comparing the inhibition effects at different concentrations, the most effective antibacterial concentration was identified, and the corresponding inhibition rate was calculated. The results are presented in Table 3.

Table 2. Evaluation of the Inhibition Rate of Extracellular Product Against Staphylococcus aureus

Sample	Optimal Inhibitory Concentration (mg/mL)	Inhibition Rate (%)
Extracellular product 0.5 mg/mL	0.35	53.25%
Extracellular product 1.0 mg/mL	0.80	58.10%
Extracellular product 2.0 mg/mL	1.80	56.98%

As shown in Figure 2B, the control groups (treated with physiological saline and absolute ethanol) exhibited rapid growth, with OD600 values entering a stationary phase around 12 hours, representing a typical bacterial growth curve. After 18 hours, the cells entered the decline phase. In the sample treatment groups, bacterial growth was notably inhibited in a concentration-dependent manner. Among them, the group treated with 2 mg/mL of the sample maintained OD values consistently below 0.5 throughout the incubation period, indicating effective suppression of bacterial proliferation. All three tested concentrations exhibited antibacterial effects, with higher concentrations corresponding to stronger inhibition.

The minimum inhibitory concentration (MIC) of the extracellular product against Staphylococcus aureus was determined to be 400μg/mL.

3.2 Preliminary Mechanism Analysis Results

Propidium iodide (PI) staining further confirmed membrane damage. PI only enters cells with compromised membranes, binds DNA, and emits red fluorescence. As shown in Figures 3A–C, red fluorescence intensified with higher sample concentrations. Figures 3D–F show corresponding dark-field images. The 2 mg/mL group showed the strongest signal, suggesting the highest degree of membrane damage and strongest antibacterial effect.

As shown in Figure 4A, AKP activity rose with increasing concentrations of the extracellular product. Both 0.5 mg/mL and 1 mg/mL treatments significantly increased AKP levels compared to the blank control, while 2 mg/mL approached the level of the lysozyme-positive control, indicating severe membrane disruption and intracellular leakage.

As shown in Figure 4B, the nucleic acid concentration in the physiological saline and ethanol control groups remained nearly unchanged between 2 and 8 hours, indicating that the solvents had no significant effect on bacterial membrane integrity. In contrast, treatment with 1 mg/mL and 2 mg/mL of the extracellular product led to a time- and dose-dependent increase in nucleic acid leakage. The 1 mg/mL group reached 117.2 ng/μL at 4 h, while the 2 mg/mL group rose from 116.9 ng/μL to 145.3 ng/μL between 4 and 8 h, indicating more severe membrane disruption at higher concentration.

As shown in Figure 4C, β-galactosidase activity in the solvent and blank control groups was 0.0816 and 0.0597, respectively. In contrast, the 0.5 mg/mL, 1 mg/mL, and 2 mg/mL treatment groups showed significantly higher activities of 0.094, 0.107, and 0.112. Notably, the 2 mg/mL group approached the activity level of the lysozyme-positive control, further confirming that higher concentrations of the extracellular product cause greater damage to the bacterial cell wall and membrane.

As shown in the figure 5, increasing concentrations of the extracellular product led to a gradual decrease in pH and a clear rise in conductivity, indicating structural damage to Staphylococcus aureus. Membrane disruption allows intracellular ions to leak out, raising electrolyte levels and lowering pH, both markers of compromised membrane integrity.

Further confirmation came from membrane permeability assays. As shown in Figure 5, treatment groups exhibited significantly lower pH values than the blank control, suggesting increased membrane permeability and intracellular leakage.

As shown in Figure 6, bacterial cells in the blank control group were densely arranged with intact, spherical morphology. After treatment with 1 mg/mL of the sample, cell numbers decreased, some cells showed rupture and leakage, and surface deformation appeared. At 2 mg/mL, cells became notably shriveled and collapsed, indicating severe structural damage.

These morphological changes demonstrate that higher concentrations of the extracellular product progressively compromised cell wall and membrane integrity, leading to intracellular leakage. The antimicrobial agent may also penetrate damaged membranes, disrupt metabolism, and ultimately inhibit bacterial growth and viability.

3.3 In-depth Mechanistic Analysis Results

Previous experiments have been determined by LC-MS/MS, and it has been concluded that YTCY-EPs are mainly composed of short peptides, some of which are rich in amino acid residues related to antibacterial and antioxidant activities (such as Val, Leu, Pro, His, Tyr, Trp, and Cys). Based on the sequence characteristics, the research team screened out several representative functional peptides (named YTCY_A–F and YTCY_1–4), the former with the typical cationic characteristics and hydrophobic end structure of antimicrobial peptides, and the latter showing antioxidant potential. It is difficult to determine the key role of a single peptide in overall activity. Combined with the sequence composition and functional characteristics, it is speculated that the biological effects of YTCY-EPs may be due to the structural complementarity and signal regulation synergy between polypeptides, so they are regarded as a functional system with complex activity in subsequent studies, and their antibacterial, antioxidant and anti-inflammatory mechanisms are analyzed from an overall perspective.

As shown in Figures 8A-D , treatment with different concentrations of extracellular product significantly influenced multiple physiological and metabolic parameters in Staphylococcus aureus. Specifically, exposure to 1 mg/mL of the extracellular product resulted in a fluorescence intensity of 7362 AU, indicating a substantial increase in intracellular reactive oxygen species (ROS) levels. Excessive ROS generation can cause irreversible damage to bacterial cell membranes, proteins, and DNA, ultimately leading to bacterial death.

Furthermore, treatment with 1 mg/mL and 2 mg/mL of the extracellular product reduced total protein synthesis in S. aureus to varying extents compared with the saline control group, with the 2 mg/mL group exhibiting the most pronounced inhibition. This suppression of protein synthesis suggests a decrease in bacterial metabolic activity, thereby affecting bacterial growth and viability.

In addition, succinate dehydrogenase (SDH) activity was also markedly reduced by the extracellular product, with enzyme activities of 8.375 U/mL and 7.9 U/mL observed in the 1 mg/mL and 2 mg/mL treatment groups, respectively. These correspond to decreases of 68.69% and 70.47% relative to the saline control, similar to the inhibition observed in the lysozyme-treated positive control group. The reduction in SDH activity was consistent with the observed decline in total protein content, suggesting that the extracellular product may exert its antibacterial effect by disrupting bacterial metabolism, inhibiting SDH activity, and thereby weakening bacterial viability.

Lastly, metabolic activity assays revealed that treatment with 1 mg/mL and 2 mg/mL of the extracellular product led to reductions in bacterial metabolic activity by 6.9% and 13.9%, respectively. This inhibition of metabolic activity may disrupt the normal metabolic cycle of the bacteria, impair energy production and biosynthetic processes, and contribute to growth suppression or bacterial death.

3.4 Biofilm Inhibition Analysis

In this experiment, crystal violet staining was used to assess how different concentrations of extracellular product affect S. aureus biofilm formation and adhesion. As shown in Figure 8, the treated groups had fewer stained biofilm fragments than the blank control, especially at higher concentrations. This suggests that the product can disrupt and inhibit biofilm formation, making S. aureus more vulnerable.
Absorbance data showed that all three concentrations inhibited biofilm formation by over 50%, confirming the extracellular product’s strong anti-biofilm effect.

Imbalance in the skin microbiota is considered a major contributing factor in the development of acne. To address this issue effectively, this study investigates the antibacterial mechanisms of action from multiple perspectives, aiming to identify potential targets and provide a solid data foundation for the development of acne-fighting bioactive ingredients. Staphylococcus aureus, a representative Gram-positive pathogen, was selected as the test organism to evaluate the antimicrobial potential of the extracellular metabolites produced by Condensimonas hendrickxii.

Growth curve analysis revealed that the treated group exhibited minimal changes in OD625 over the 0–22 hour period, lacking a clear logarithmic growth phase, which indicates significant inhibition of bacterial proliferation. Furthermore, treatment with the extracellular metabolites resulted in a marked increase in reactive oxygen species (ROS) levels within the bacterial cells, suggesting interference with the metabolic processes via oxidative stress.

To further validate the mechanism, a series of targeted assays were conducted. The results demonstrated that the extracellular metabolites disrupted the integrity of the bacterial cell membrane in a concentration-dependent manner. Nucleic acid leakage assays showed a time-dependent increase in extracellular DNA concentration; concurrently, a decrease in pH and an increase in electrical conductivity of the culture medium were observed—both indicative of intracellular content leakage. In the biofilm degradation assays, bacterial biofilms were visibly disrupted, fragmenting from their originally intact structure.

Additionally, PI (propidium iodide) fluorescence staining, measurements of alkaline phosphatase (AKP) and β-galactosidase activity, and high-resolution scanning electron microscopy (SEM) were employed to assess changes in membrane permeability and morphology. Under normal conditions, these enzymes and stains should not be detectable in the external environment. However, the treated groups showed increased extracellular enzyme activity and intracellular PI penetration, with SEM images clearly displaying collapsed and deformed cells with substantial leakage of cytoplasmic content—indicating severe membrane damage.

In summary, the extracellular metabolites of Condensimonas hendrickxii exhibit potent antibacterial effects through multiple mechanisms: they inhibit bacterial growth, induce oxidative stress, compromise membrane integrity, and increase permeability, thereby facilitating the penetration of antibacterial agents. Furthermore, these metabolites interfere with intracellular protein synthesis and disrupt metabolic activity, ultimately reducing bacterial viability. These findings provide compelling evidence of both the antimicrobial and anti-inflammatory potential of Condensimonas hendrickxii metabolites, supporting their future application as functional ingredients in acne treatment formulations.

Impact Statement
This study provides new insights into the antibacterial mechanisms of Heyndrickxia coagulans extracellular metabolites against Staphylococcus aureus. The findings not only broaden the understanding of postbiotic applications in dermatology but also offer a scientific basis for developing novel antibacterial and anti-acne agents derived from probiotic metabolites.

Credit Author Statement

Keran Jia: Data curation, Investigation, Writing-original draft. Yaqian Yan: Data curation, Methodology, Validation, Writing-original draft. Ji Yin: Investigation, Validation, Writing-review & editing. Changtao Wang: Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Resources, Supervision. Dongdong Wang: Software, Validation, Writing – review & editing. Dan Zhao: Investigation, Methodology, Writing – review & editing. Jiachan Zhang: Investigation, Project administration, Validation. Meng Li: Data curation, Funding acquisition, Methodology, Project administration, Resources, Software, Writing – review & editing.

Data Availability Statement
The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Funding Statement
This research received no external funding.

Ethical Approval Statement
This study did not involve human participants or animal experiments and therefore did not require ethical approval.

Conflict of Interest
The authors declare no conflict of interest.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

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The antibacterial mechanism of the extracellular product of coagulated Heyndrickxia coagulans against Staphylococcus aureus

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Version 1

Abstract

Figures

1.Introduction

2.Materials and Methods

2.1 Materials

2.2 Strain Source

2.3 Pathogenic Bacteria Culture

2.4 Antibacterial Activity Assessment

2.4.1 Oxford Cup Diffusion Assay

2.4.2 Minimum Inhibitory Concentration (MIC) Determination

2.4.3 Bacterial Growth Curve Determination

2.5 Preliminary Mechanism Analysis

2.5.1 Alkaline Phosphatase (AKP) Activity Assay

2.5.2 PI Staining for Cell Membrane Permeability

2.5.3 Nucleic Acid Leakage Assay

2.5.4 β-Galactosidase Leakage Assay

2.5.5 Cell Membrane Permeability Measurement

2.5.6 Morphological Observation of Bacterial Cells

2.6 In-depth Mechanistic Analysis

2.6.1 Reactive Oxygen Species (ROS) Detection

2.6.2 Effect of Extracellular Products on Total Protein Synthesis in Staphylococcus aureus

2.6.3 Effect of Antimicrobial Agents on Succinate Dehydrogenase (SDH) Activity

2.6.4 Effect on Metabolic Activity of Bacterial Cells

2.7 Biofilm Inhibition Analysis

3.Results

4.Discussion

Declarations

Data Availability Statement

References

Additional Declarations

Status:

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