The phytoconstituents (FCs) represented by gallic acid (GA), Nerolidol (N) and α-Terpineol (αT), were purchased from Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France.

Dimethyl sulfoxide (DMSO) (Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France) was used as a solvent to improve the solubility of GA, N and αT. Solutions were prepared using both the individual phytochemicals and their binary combinations (αT:N, αT:GA and GA:N) in a 1:1 volumetric ratio as well as ternary combination (αT:N:GA) in a 1:1:1 volumetric ratio, all at an initial concentration of 10 mg/mL.

In order to determine the antimicrobial activity of FCs, we utilized an adapted method of disk diffusion assay, following the general guidelines provided in the Clinical Laboratory Standards Institute (CLSI, 2024) (40). A 0.5 McFarland suspension, corresponding to 1.5 × 10⁸ CFU (Colony Forming Units)/mL, prepared in 0.9% NaCl sterile saline solution, was used as a standardized inoculum, which was used to inoculate Petri dishes containing Mueller-Hinton (MH) agar (for bacterial strains) and MRS agar (for LAB strains). LAB strains previously grown in MRS liquid media were centrifuged twice for 3 minutes at 5000 Revolutions per minute (RPM). The sediment was resuspended in 0.9% NaCl sterile saline solution for removing the culture medium (MRS) and to obtain the standardized inoculum with 0.5 McFarland density. Sterile 6 mm diameter absorbent paper discs were disposed on the surface of Petri dishes containing MH agar (for the bacterial strains) and MRS agar (for the LAB strains), which were seeded with microbial inoculums. A volume of 10 μL of each FC solution was placed on a separate sterile disc. The Petri dishes were then incubated to allow microbial growth for 24 h at 37°C. After incubation, the diameter of growth inhibition zone (in mm) around each disc was measured.

The Minimum Inhibitory Concentration (MIC) value of the bioactive compounds was determined using a quantitative procedure based on an adapted serial microdilution standard method which was performed in Nutrient Broth liquid medium (for bacterial strains) and MRS liquid medium (for LAB strains). Sterile broth was added to 96-well microtiter plates and binary dilutions of each bioactive compound were prepared in a volume of 150 μL (with eight different concentrations: 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625 and 0.0078125 mg/mL). After the microdilutions, 15 μL of each microbial suspension with a standard density of 0.5 McFarland was added to each well and the plates were incubated for 24 h at 37°C. After incubation, the MIC values were established by visual analysis and confirmed by spectrophotometric measurement (absorbance at 620 nm) via the Biotek Synergy-HTX ELISA multi-mode reader (VT, USA) (41, 42). Each experiment was conducted in triplicate and repeated on at least three times.

To investigate the effect of the natural bioactive compounds on adhesion and biofilm formation, a semi-quantitative assessment was performed using the crystal violet microdilution method. 96 well microtiter plates were prepared with binary dilutions of the FCs tested in a final volume of 150 μL of sterile broth. Each well was inoculated with 15 μL of each microbial suspension at 1.5 × 10⁸ CFU mL prepared in sterile saline. The plates were incubated for 24 h at 37°C and after incubation, the liquid medium in the wells was carefully removed, and the wells were washed three times with sterile saline to remove unattached cells. The microbial cells adhered to the well surfaces were fixed with cold methyl alcohol/methanol for 5 minutes. After removing the methanol, the plates were stained with 1% crystal violet solution for 20 minutes and the excess dye was washed off with tap water. The appearance of the biofilms developed on the inert substrate was analyzed using an inverted microscope (40X). The stained biofilms were resuspended with a 33% acetic acid solution and the absorbance of the resulting suspensions was measured spectrophotometrically at 492 nm using an ELISA multi-mode reader (41, 43, 44). Each experiment was conducted in triplicate and repeated at least three times.

The THP-1 human leukemia monocytic cell line was purchased from the American Type Culture Collection (ATCC). Cells were maintained in a culture medium (Roswell Park Memorial Institute Medium – RPMI) 1640 supplemented with 10% Fetal Bovine Serum (FBS), antibiotics: 100U/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine) at 37°C in a humidified atmosphere with 5% CO_2, following the supplier’s recommendations. For the assessment of immunomodulatory effects, the cells were seeded into 24-well plates at a concentration of 1 x 10⁶ cells/mL in a volume of 1 mL per well. Differentiation into macrophages was induced by treating the cells with 100 ng/mL of Phorbol 12-Myristate 13-Acetate (PMA) for 72 h (45).

After incubation, the medium containing PMA was removed and cells were washed twice with PBS and the differentiated cells were maintained in RPMI 1640 supplemented with 10% FBS, without PMA. for 48 h, before experimental infection.

THP-1 cells differentiated into macrophages were infected with bacterial suspensions at a standard density of 1–3 × 10⁷ CFU/mL. The infection conditions were as follows: 450 μL RPMI medium and 50 μL microbial suspension. The bacterial strains used included Gram-positive strains: S. aureus MRSA 86, S. aureus ATCC 25923, E. faecalis 188 and E. faecalis ATCC 29212; Gram-negative strains: E. coli 82, E. coli ATCC 25922, K. pneumoniae 102 (representing bacterial strains control) and LAB strains: Lb. paracasei, Lb. rhamnosus and Lc. lactis (representing LAB control).

Decimal dilutions of the selected FCs solutions were prepared in DMSO to obtain subinhibitory concentrations (0.01 mg/mL). Cell cultures were inoculated with the final solutions, which included the following: compound control – 450 μL RPMI medium and 50 μL of the natural bioactive compound (Gallic Acid (GA), Nerolidol (N) or α-Terpineol (αT)) at a concentration of 0.01 mg/mL; the working suspensions (infected with pathogens or exposed to LAB strains and treated with FCs at subinhibitory concentrations) – 400 μL RPMI medium, 50 μL of the natural bioactive compound (G, N or αT) at a 0.01 mg/mL concentration, and 50 μL of the microbial suspension containing the following strains: Gram-positive strains (S. aureus MRSA 86, S. aureus ATCC 25923, E. faecalis 188 and E. faecalis ATCC 29212), Gram-negative strains (E. coli 82, E. coli ATCC 25922, K. pneumoniae 102) and LAB strains (Lb. paracasei, Lb. rhamnosus and Lc. lactis).

We selected a sub-inhibitory concentration of 0.01 mg/mL for the phytoconstituents, to ensure that the observed effects on cytokine production were not confounded with antimicrobial activity. This dose is below the MIC values determined for the tested isolates and is consistent with concentrations reported in the literature (20 – 100 µg/mL) for assessing immune modulation without inducing cytotoxicity (19, 46, 47).

The plates were incubated for 3 h at 37°C in a humidified atmosphere, with 5% CO₂. After incubation, cell supernatants were collected into Eppendorf tubes and stored at −20°C. The cell cultures were then washed twice with PBS to remove non-internalized microorganisms and reinoculated with 500 μL of RPMI medium supplemented with FBS to continue incubation for 24 h and 48 h, ensuring that cell viability was not affected by dehydration.

Throughout the incubation period, cell cultures were analyzed in real time using an inverted microscope.

After the 24 h and 48 h stimulation periods, cell supernatants were collected into Eppendorf tubes and stored at −20°C. The cell cultures were gently washed twice with PBS and fixed with 500 μL of cold methanol for 5 minutes. After removing the methanol, the plates were stained with May-Grünwald Giemsa solution for 20 minutes. Following stain removal, the plates were air-dried at room temperature, and the morphology of macrophages post-infection was analyzed using an inverted microscope.

The production of pro-inflammatory (TNF-α, IL-2, IL-6, IL-12A) and anti-inflammatory (IL-10) interleukins was measured using commercial ELISA kits purchased from EIAab Science INC, Wuhan, China, according to the manufacturer’s instructions.

The experiments were performed on cell supernatants obtained after the experimental infection of differentiated macrophages and treatment with subinhibitory concentrations of FCs: GA, N and αT (0.01 mg/mL), following 24 h and 48 h of stimulation.

Before analysis by ELISA, the cell supernatants were centrifuged at 10–000 RPM for 5 minutes. According to ELISA kit protocols, the results were interpreted after spectrophotometer readings at 450 nm, using the Biotek Synergy-HTX ELISA multi-mode reader (VT, USA).

The qualitative analysis of the tests showed that some of bioactive FCs tested exhibited a selective inhibitory effect, in a strain-dependent manner. In Gram-positive bacterial strains, variation in antimicrobial effect among different strains was observed: a moderate inhibitory effect was generally noted, with a slight enhancement in growth, except for the S. aureus 205 strain which exhibited a significantly stronger inhibitory effect in the presence of αT (Diameter of Growth Inhibition Zone (DGIZ) – 20 mm) ( Figures 1 , 2 ). The behavior of Gram-negative strains was more homogeneous, with most exhibiting inhibition zone with DGIZ ranging between 6 and 9 mm, generally reduced than those of Gram-positive strains ( Figure 3 ). In addition, N exhibited a moderate effect bacterial growth (DGIZ ranging between 6 and 9 mm). The LAB strains exhibited the smallest DGIZ, measuring 6 mm diameters.

In the case of using binary and ternary combinations of the three natural bioactive compounds, the qualitative results did not show significantly larger growth inhibition zones compared to the previously presented qualitative tests for Gram-positive bacterial strains.

The significant inhibitory effect of αT on the S. aureus 205 strain was neutralized when combined with the other two compounds ( Figures 4 , 5 ). For Gram-negative strains, DGIZ ranging between 7 and 11 mm were measured, with slightly larger diameters observed in the αT:N combination (1:1), which was selected for quantitative antimicrobial tests ( Figure 6 ).

In the case of LAB strains, DGIZ was equal to that of the sterile absorbent paper discs (6 mm) in the presence of FCs.

The results of the quantitative assays revealed that tested natural bioactive compounds exhibited different antimicrobial effects depending on the tested strain and concentration.

A noticeable inhibitory effect on the growth and multiplication of bacterial cells in suspension was observed, particularly in Gram-positive species. The minimum inhibitory concentrations (MIC) assay revealed that the most effective compound against Gram-positive strains was N (mean MIC – 0.25 mg/mL) for three out of the seven tested strains: S. aureus MRSA 86, S. aureus 205 and E. faecalis 188 ( Figure 7 ).

However, although less pronounced, the inhibition of planktonic growth in Gram-negative strains remains consistent for all the FCs assessed (mean MIC – 1 mg/mL). The solvent control samples (DMSO) showed no antimicrobial activity against the tested microbial strains (mean MIC values > 1 mg/mL). For LAB strains, the MIC values exceeded 1 mg/ml, confirming the results of the qualitative tests.

Based on the results obtained from the qualitative assay, the αT:N (1:1) combination exhibited notable antimicrobial activity against both Gram-positive and Gram-negative bacterial strains. Therefore, this combination was selected for the quantitative assessment to explore its complementary effects and potential for enhanced efficacy.

αT:N (1:1) combination demonstrated its effectiveness against the multiplication of Gram-positive cells in suspension. Reduced MIC values (0.25–1 mg/mL) were obtained for four out of the seven tested Gram-positive strains, such as S. aureus 216, S. aureus ATCC 25923, E. faecalis ATCC 29212 and E. faecalis 188, showing a significantly improved result compared to the individual testing of the two compounds and the qualitative tests ( Figure 8 ).

On the other hand, the antimicrobial activity against Gram-negative bacterial strains was not enhanced by using the combination of the two compounds, as the MIC values were greater than or equal to 1 mg/mL.

The evaluation of biofilm development on an inert substrate using the crystal violet staining method enabled the determination of the Minimum Biofilm Eradication Concentration (MBEC).

It was observed that monospecific biofilm formation on an inert substrate was altered in a strain-specific and bioactive compound dependent manner. Monospecific biofilm development was only slightly inhibited in the presence of N, specifically in the case of the clinical E. faecalis strain (mean MBEC – 0.25 mg/mL) ( Figure 9 ). In contrast, the tested compounds exhibited low anti-biofilm activity against the other microbial strains (mean MBEC – 1 mg/mL).

On the other hand, the effect of bioactive compounds on biofilm formation was more efficient in the presence of the αT:N combination in a 1:1 volumetric ratio, for most implicated wound biofilm opportunistic pathogens, both standard and clinical resistant strains isolated from HS.

For Gram-positive bacteria, the results indicated that the biofilm development was altered in the presence of the selected combination, with mean MBEC values between 0.0625 and 0.125 mg/mL.

Additionally, the inhibitory effect of this combination was also observed in Gram-negative bacterial strains (mean MBEC values between 7.8125 μg/mL and 0.125 mg/mL), with a significant effect for the clinical E. coli strain (mean MBEC value of 7.8125 μg/mL) ( Figure 10 ). LAB strains didn’t exhibit adhesion capacity to inert substrates.

The results showed that both GA, N and αT individually and in combination did not exhibit antimicrobial activity against LAB strains. This suggests that these compounds do not interfere with the growth of beneficial microorganisms, highlighting their beneficial effects in inhibiting pathogen growth without affecting normal microbiota.

Therefore, these findings support using of natural bioactive compounds as effective agents in HS management, with diverse therapeutic applications. Such compounds could prove to be effective and well-tolerated agents in the treating of chronic wound infections.

The results showed that, following the experimental infection of differentiated THP-1 monocyte-derived macrophages with Gram-negative bacterial strains, significant alterations in cell morphology were observed. Vacuolated cells with rounded or spherical shapes, infiltrated with microbial cells, were detected in both the control group (experimentally infected but untreated cells) and in the presence of FCs. Regarding the experimental infection of differentiated macrophages with Gram-positive bacterial strains, cell viability was strongly affected. This trend was also observed in microscopic evaluations at both 24 and 48 h of incubation, showing dynamic cellular changes. Additionally, cells in various stages of apoptosis, detached from the inert substrate, were identified. On the other hand, co-cultivation of THP-1 cells with LAB strains did not lead to any morphological changes.

After 24 h of macrophage cell line infection with pathogenic bacteria suspensions, the signaling cascades leading to TNF-α secretion were activated, as reflected by the high TNF-α levels ( Figure 11 ). Phytochemical treatment of the cell line interfered with these pathways, resulting in a reduction in TNF-α production, which was more pronounced when the treatment was performed with subinhibitory concentrations (10 μg/mL) of AG, followed by αT, in case of Gram-positive strains infection. Also, TNF-α levels were undetectable after 24 h when the macrophage cell line infected with Gram-negative bacteria was treated with subinhibitory concentrations of N and αT ( Figure 11 ).

TNF-α inhibition could not be correlated with un increasing level of IL-10 after the same experimental treatments ( Figure 12 ). IL-10 secretion is significantly higher in Gram-negative (36 pg/mL) infections compared to Gram-positive (6 pg/mL) across all treatments. But in different treatment conditions the IL-10 production is transient and primarily occurs at the 24 h. For GA, ELISA determinations after 24h of incubation showed an undetectable level of IL-10.

The highest IL-6 concentration (217 pg/mL at 24h) was observed in the control condition, suggesting a strong inflammatory response upon Gram-negative bacterial infection, but not for Gram-positive ( Figure 13 ). By 48 h, IL-6 levels dropped to 0 pg/mL, indicating a transient cytokine response. When the treatment of the infected cell line was performed with GA, the IL-6 levels at 24 h (74 pg/mL) were significantly lower than in the control, suggesting that GA treatment reduced IL-6 production. In αT treatment condition, the IL-6 levels at 24h (117 pg/mL) were lower than the control but higher than GA, suggesting a partial reduction of IL-6 expression. Also, IL-6 expression in N treatment condition was lower than the control (128 pg/mL), but higher than GA and αT, indicating an intermediate effect. By 48 h, IL-6 was completely absent in all conditions, suggesting cytokine clearance or immune regulation over time.

The results suggest that all tested conditions (GA, αT, N) mitigate IL-6 expression compared to the control, with GA showing the strongest suppression. This indicates that these treatments may have anti-inflammatory effects by reducing IL-6 secretion in response to Gram-negative bacterial infection.

In the control condition, both Gram-negative and Gram-positive bacterial infections induced a moderate level of IL-12A expression, with undetectable concentrations after 48 h ( Figure 14 ). When the infected cell line was treated with N and αT, IL-12A expression was significantly higher than in the control, especially in response to Gram-negative bacterial infections (78 pg/ml and 123 pg/ml, respectively). Treatment of Gram-positive infections led to an increased IL-12A level only with αT (61 pg/ml) compared to the control (37 pg/ml). Under the GA treatment condition, IL-12A levels at 24 h were 0 pg/ml for Gram-negative infections and significantly lower (14 pg/ml) than in the control for Gram-positive infections (37 pg/ml) ( Figure 14 ). By 48 h, IL-12A was completely absent in all conditions.

The expression of ILs was also evaluated using a combination of phytocompounds (at subinhibitory concentrations of 10 μg/mL) with LAB probiotic strains. The concentration of 0.01 mg/mL was selected to avoid cytotoxicity while still enabling detection of immune modulatory effects. Published studies report that GA, N, and αT exhibit cytotoxicity in human keratinocytes and fibroblasts at concentrations typically exceeding 0.05–0.1 mg/mL. Therefore, our chosen dose is within a safe, sub-cytotoxic range suitable for assessing immune-related responses without compromising cell viability.

The IL levels were determined after FCs treatment, using a macrophage cell line infected with three different LAB strains as the control. For LAB strains, TNF-α synthesis was inhibited in a strain-dependent manner and influenced by their source of isolation. Specifically, Lc. lactis, isolated from dental plaque, induced the highest level of TNF-α synthesis (1085 pg/mL) at 24h, with a prolonged response at 48h (364 pg/mL). Lb. plantarum, isolated from sheep cheese, exhibited low TNF-α levels (109 pg/mL at 24h and 16 pg/mL at 48h), indicating that the cytokine response was fading. Lb. rhamnosus, isolated from the normal human microbiota (newborn feces), did not stimulate TNF-α synthesis ( Figure 15 ).

Phytochemical treatment of the cell line interfered with these pathways, Lc. lactis infection inducing extremely high TNF-α levels (1135 pg/mL), even higher than the control condition, indicating that GA does not reduce TNF-α secretion but might even enhance the pro-inflammatory response. Lb. paracasei also shows an increased TNF-α level (940 pg/mL) compared to the control, suggesting GA enhances inflammation in this condition. Lc. lactis remains at 0 pg/mL, consistent with the control. At 48h, a complete absence of TNF-α in all bacteria can be observed, showing a rapid decline, which may indicate that GA enhances early cytokine release but then accelerates immune suppression.

Lc. lactis and Lb. paracasei infections show moderate TNF-α levels (341 pg/mL and 258 pg/mL, respectively) after N treatment by 24h, significantly lower than the control condition (1085 pg/mL and 109 pg/mL). This suggests that N treatment effectively reduces the inflammatory response. For Lb. rhamnosus, the level of TNF-α remains 0 pg/mL, like the control. At 48h, TNF-α levels drop to negligible amounts (0–6 pg/mL), showing that N treatment suppresses long-term inflammation.

After αT treatment, Lc. lactis infection shows moderate TNF-α levels (717 pg/mL), significantly lower than the control (1085 pg/mL), indicating, a reduction in inflammation. Lb. paracasei infection shows very low TNF-α levels (22 pg/mL), also suggesting strong immunomodulation at 24 h. Lb. rhamnosus also remains at 0 pg/mL, like the control. At 48 h, TNF-α levels decrease significantly (0–5 pg/mL), reinforcing αT’s anti-inflammatory effects over time.

The IL-10 secretory pattern shows that, at 24h, only Lc. lactis induces IL-10 production (18 pg/mL), Lb. paracasei and Lb. rhamnosus show no detectable IL-10 secretion (0 pg/mL). At 48h, IL-10 levels drop to 0 pg/mL for all three bacterial strains, indicating a transient IL-10 response that is present at 2 4h but disappears by 48 h. IL-10 is an anti-inflammatory cytokine that helps regulate excessive immune responses. The transient production of IL-10 at 24 h in response to Lc. lactis suggests that this strain temporarily activates an anti-inflammatory response, potentially to counterbalance pro-inflammatory cytokines like TNF-α. The level of IL-10 was undetectable (0 pg/ml) after treatment with tested phytoconstituents ( Figure 16 ).

The IL-12A secretory pattern shows that at 24 h, all three LAB strains induce its synthesis at similar concentration levels ( Figure 17 ). By 48h, IL-12A secretion is undetectable (0 pg/mL). When Lb. rhamnosus and Lc. lactis infections were treated with GA at a subinhibitory concentration, IL-12A levels decreased dramatically, whereas no significant reduction was observed for Lb. paracasei infection. N treatment exhibited a similar effect on the infected macrophage cell line, with a higher IL-12A level (136 pg/mL) for Lb. paracasei compared to the control (79 pg/mL).

In contrast, treatment with αT induced higher IL-12A levels in Lb. rhamnosus (117 pg/mL) and Lc. lactis (115 pg/mL) infections than in the control, but a lower IL-12A level for Lb. paracasei infection (45 pg/mL) ( Figure 17 ).

In line with our results, it was reported that a concentration of 5 mg/mL of GA exhibit a strong inhibiting effect on P. aeruginosa monospecific mature biofilms. Additionally, the study showed that GA led to growth inhibition at a MIC of 2.5 mg/mL for both S. aureus and P. aeruginosa (16). In concordance with this study, Tian et al. demonstrated that GA exhibited antimicrobial activity against nineteen clinically E. coli strains and one reference strain, E. coli ATCC 25922, with a MIC value of 5 mg/mL (52).

In evaluating the antibacterial activity of GA on S. aureus strains IS-58 and K2068, which express the TetK and MepA efflux pumps, it was found that GA displayed an MIC ≥ 1 mg/mL, for both strains. However, it was observed that when is combined with antibiotics, it was generated potentiation of antimicrobial activity. Notably, GA enhanced the effects of tetracycline and ciprofloxacin, suggesting its potential to inhibit efflux pumps activity. These findings highlight the potential of GA as an efflux pump inhibitor, which could improve the efficacy of antibiotics against MDR strains and suggesting that it could be possibly use as an adjuvant in the treatment of difficult-to-treat infections (53).

Other authors also reported similar findings regarding the antimicrobial potential of GA, particularly against MRSA and Methicillin Susceptible Staphylococcus aureus (MSSA) strains. The MIC values obtained for GA ranged from 0.2 to 0.4 mg/mL for both MRSA and MSSA. In addition, GA was shown to enhance the effects of β – lactam antibiotics by inducing morphological changes in MRSA strains, generated through cell membrane damage, improving antibiotic’s efficacy against MDR strains and suggesting a potential synergistic effect (54).

Nerolidol has proven their efficiency in the treatment of various bacterial infections including those associated with chronic wounds. Several investigations have been conducted on recognized wound-related strains such as: S. aureus, P. aeruginosa, E. coli, E. faecalis and K. pneumoniae, demonstrating its antimicrobial, antibiofilm and antioxidant properties (55–58).

Previous reports demonstrated that N possesses a strong antibacterial behavior against both Gram-positive and Gram-negative bacteria. A study performed recently reported MIC values of 0.5 mg/mL for P. aeruginosa and K. pneumoniae and 1–4 mg/mL for S. aureus, S. epidermidis, E. faecalis and E. coli. Besides, N showed effective antibiofilm properties, inhibiting adherence and biofilm development in a dose-dependent manner and also exhibited antioxidant effects (55).

Santana et al. explored the antimicrobial properties of N both alone and encapsulated in liposomal nano-formulations, on S. aureus strains that express efflux pumps: NorA, MsrA and Tet(K). The results revealed that N inhibited the growth of MRSA strains, with MIC values ranging from 32 to 1024 µg/mL. Furthermore, N significantly reduced the MICs of antibiotics, suggesting that N could potentiate the efficacy of them by interfering with bacterial efflux pumps mechanisms. By inhibiting efflux pumps, N increases the intracellular accumulation of antibiotics, increasing their antibacterial properties. All these findings position N as a valuable compound and a promising strategy for use as an adjuvant in the treatment of chronic infections, especially when it is combined with conventional antibiotics (56).

In concordance with the results obtained in our study, αT demonstrated superior efficacy against bacterial strains including: E. coli, S. aureus, MRSA, P. aeruginosa, Bacillus cereus, Streptococcus mutans and Salmonella typhimurium (31, 33, 59).

Researchers in biomedical field also revealed that terpenes such as N and αT enhance the absorption of lipophilic molecules and improving permeability in the cutaneous tissue. Their lipophilicity and ability to interact with skin lipids molecules promote drug delivery. These results make terpenes promising candidates for transdermal delivery systems, potentially useful in wound healing formulations which could be able to enhance the efficacy of therapeutic agents (60).

A study conducted by Johansen and Sergere (31) demonstrated that a combination of two terpenoid isomers αT and terpinen-4-ol inhibit microbial growth and proliferation of ESKAPE pathogens, making this combination a strong natural candidate for antimicrobial applications. Additionally, this combination revealed that the doses of these two isomers could potentially be reduced when they are used together, reducing potential side effects such as: toxicity, allergenicity and resistance mechanisms, while maintaining antibacterial behavior. These findings align with our study, where the combination of αT and N, two terpenoids, exhibit promising antimicrobial, anti-adherence and immunomodulatory properties. The cumulative effects of these two compounds could potentially provide an effective therapeutic solution for HS therapy associated with pathogenicity and inflammation, reducing the risks associated with higher concentrations when they are used as individual compounds.

Still, according to our knowledge, there are no references available to compare our results regarding the antimicrobial and antibiofilm activity of αT, N & GA combinations with potential therapeutic applications in the treatment of HS. Additionally, no results have been reported yet on the probiotic – natural bioactive compound combinations effects on the modulation of inflammatory responses.

Interestingly, a study performed on phenolic compounds such as: catechin, gallic acid (GA), vanillic acid, ferulic acid and protocatechuic acid present in fruits and vegetables provides selective growth effects on both beneficial and pathogenic microorganisms. These compounds promoted the growth of beneficial lactobacilli such as Lb. rhamnosus GG and Lactobacillus acidophilus, while inhibiting the growth of E. coli and Salmonella typhimurium. The selective antimicrobial activity holds significant potential for the development of nutraceutical products (61). Moreover, combining natural bioacitive compounds with probiotics, which are known as pathogen antagonists due to their ability to produce antimicrobial molecules such as bacteriocins (62), could amplify their bioactivities. The interaction between LAB probiotic strains and natural bioactive compounds represents a promising strategy in the management of chronic wounds such as HS, not only by inhibiting growth of pathogens, but also promotes the proliferation of beneficial microorganisms that modulate inflammation, offering a promising approach, without affecting normal microbiota (61).

The specific characteristics of the bacteria appear to alter the immune response to Gram-positive bacterial components. The distinct cytokine profiles triggered by Gram-positive and Gram-negative bacteria may help optimize the clearance of bacteria with different cell wall structures (63). The data presented in this study suggests intricate immune modulation of macrophage responses to bacterial infections, particularly in relation to TNF-α, IL-6, IL-10, and IL-12A cytokine profiles. The experiment investigated the effects of different treatments (GA, αT, N) on macrophage responses to Gram-positive and Gram-negative bacterial infections and their interactions with these cytokine pathways.

The pathogenic bacteria infections—both Gram-positive and Gram-negative—activated a strong inflammatory response, as reflected by the elevated TNF-α levels after 24 h. This cytokine was significantly higher in infections involving Gram-negative bacteria compared to Gram-positive bacteria. The treatments with GA, αT and N led to varied effects on TNF-α production. For Gram-negative infections, treatment with subinhibitory concentrations of N and αT led to undetectable TNF-α levels, suggesting a complete suppression of macrophage inflammatory responses in these conditions. The differential effects between Gram-positive and Gram-negative infections suggest that FCs interact with distinct bacterial components (lipoteichoic acid vs. LPS), especially since N and αT, alone or in combination, inhibited the ability of the pathogenic strains to adhere and develop mature biofilms (as suggested in Figures 5 and 6 ). By 48 h, TNF-α levels were undetectable across all treatments, suggesting a time-dependent cytokine clearance or immune regulation.

Pathogenic Gram-negative bacterial infections induced a strong IL-6 response at 24 h, indicating a robust pro-inflammatory reaction. In contrast, IL-6 production following Gram-positive bacterial infections was much lower. The treatments significantly reduced IL-6 levels at 24 h, with gallic acid showing the strongest suppression. This suggests that the tested compounds have anti-inflammatory effects that limit IL-6 secretion, particularly in response to Gram-negative bacterial infections. By 48 h, IL-6 levels dropped to zero in all treatment conditions, supporting the idea that the inflammatory response was transient and under tight immune regulation. The role of GA in reducing inflammation by regulating the expression level of genes involved in neutrophil functionality has been demonstrated. Thus, in milk phagocytes, GA induced a reduction in the expression of proinflammatory genes (IL1B, IL6, TNF) (64) by downregulating nuclear factor-κB (65).

Anti-inflammatory cytokine involved in wound healing and suppressing immune system activators is IL-10 (66). In our experiment, the IL-10 levels were higher in response to Gram-negative bacterial infections compared to Gram-positive ones. However, the secretion of IL-10 was transient, peaking at 24 h and returning to baseline levels by 48 h. IL-10 serves as an anti-inflammatory cytokine that likely functions to counterbalance the pro-inflammatory effects of TNF-α (67). Inhibition of IL-10 secretion was observed under all treatment conditions, with levels undetectable in all tested conditions. This suggests that the phytochemical treatments may suppress the anti-inflammatory responses typically mediated by IL-10, although IL-10 is naturally transient in response to these infections.

IL-12A levels were moderately induced across both Gram-negative and Gram-positive bacterial infections, though it remained at low levels and became undetectable by 48 h. GA treatment reduced IL-12A levels in both Gram-negative and Gram-positive infections, with the most notable suppression seen in Gram-negative infections, where IL-12A levels dropped to zero. This suggests that GA might not only suppress pro-inflammatory cytokines like TNF-α but could also dampen IL-12A, which is typically involved in promoting Th1 immune responses (68).

The study evaluated the immune response modulation by different LAB probiotic strains in combination with FCs (GA, N, and αT) by measuring TNF-α, IL-10, and IL-12A levels in a macrophage cell line. The findings provide insight into how probiotics and phytoconstituents influence pro-inflammatory and anti-inflammatory pathways, which has implications for their potential therapeutic role in Hidradenitis Suppurativa (HS) treatment.

LAB strain-dependent TNF-α synthesis was observed, with the highest levels seen in Lc. lactis (from dental plaque), followed by Lb. plantarum (from sheep cheese), while Lb. rhamnosus (from newborn feces) did not induce TNF-α secretion. The source of isolation appears to influence the strain’s immunogenicity, as Lc. lactis triggered strong inflammatory responses compared to other strains.

Phytochemical treatment with GA significantly enhanced TNF-α levels in Lc. lactis and Lb. paracasei infections, suggesting that this compound may exacerbate inflammation rather than reducing it in some contexts. However, by 48 h, TNF-α levels dropped to negligible amounts for all strains, indicating that FC may trigger an early inflammatory response but then accelerate immune suppression over time.

N treatment significantly reduced TNF-α levels, especially in Lc. lactis and Lb. paracasei infections, suggesting a strong anti-inflammatory effect. αT also reduced TNF-α expression, with Lb. paracasei showing the most significant reduction, indicating potential immunomodulatory properties that may be beneficial for HS treatment.

Regarding the anti-inflammatory response, only Lc. lactis induced IL-10 production at 24h, while Lb. paracasei and Lb. rhamnosus did not. After 48h, IL-10 levels dropped to 0 pg/mL in all conditions, suggesting a transient anti-inflammatory response at 24h that is not sustained. Future studies could explore combining LAB strains with other anti-inflammatory phytochemicals that enhance IL-10 secretion, such as resveratrol or quercetin (69, 70). Some studies on mast cells (MC), as innate immune skin-resident cells, strongly support the role of resveratrol (a phenolic compound also) on local MC stabilization, accompanied by decreased levels of chemokines and its infiltration in otherwise inflamed skin (70).

IL-12A is involved in Th1 immune responses, which contribute to chronic inflammation in HS (68). The transient nature of IL-12A response suggests that LAB strains activate immune pathways early but do not sustain inflammation over time.

These findings suggest that the interplay between probiotics and phytochemical treatments is strain-dependent, with certain strains being more responsive to immunomodulation than others. The variation in cytokine responses to different LAB strains further underscores the importance of strain-specific interactions in modulating the immune system. This could have implications for designing targeted therapies using probiotics or FCs for inflammatory conditions. A possible application for HS treatment may consider the combination of Lb. paracasei with N or αT that could provide balanced immune modulation, reducing excess inflammation while maintaining protective immune responses. Future therapies must focus on personalized probiotic treatments based on individual microbiome profiles and immune response patterns.