Exploring the Antibacterial Potential of Lamiaceae Plant Extracts: Inhibition of Bacterial Growth, Adhesion, Invasion, and Biofilm Formation and Degradation in Pseudomonas aeruginosa PAO1

In response to the global rise in antibiotic resistance and the prevalence of bacterial biofilm-related infections, the antibacterial efficacy of methanolic, ethanolic, and aqueous extracts of 18 Lamiaceae plants from Serbia was evaluated. The total coumarins and triterpenes were detected spectrophotometrically, while a microdilution assay measured their effects on bacterial growth. Additionally, the impact of these extracts was assessed on Pseudomonas aeruginosa PAO1 adhesion and invasion in human fibroblasts and biofilm formation and degradation. The alcoholic extracts had the highest phytochemical content, with Teucrium montanum and Lavandula angustifolia being the richest in coumarins and triterpenes, respectively. Gram-positive bacteria, particularly Bacillus subtilis, were more susceptible to the extracts. Hyssopus officinalis ethanolic and Sideritis scardica methanolic extracts inhibited bacterial growth the most efficiently. Although the extracts did not inhibit bacterial adhesion, most ethanolic extracts significantly reduced bacterial invasion. Origanum vulgare and H. officinalis ethanolic extracts significantly inhibited biofilm formation, while Teucrium chamaedrys extract was the most active in biofilm degradation. This study significantly contributes to the literature by examining the antibacterial activity of Lamiaceae extracts, addressing major literature gaps, and underscoring their antibacterial potential, particularly Satureja montana and O. vulgare ethanolic extracts, linking their efficacy to coumarins and triterpenes.


Introduction
Infectious diseases are considered responsible for significant health loss, annually claiming the lives of millions of people worldwide (13.7 million infection-related deaths, of which 7.7 million deaths were caused by 33 bacterial pathogens in 2019) [1].With the clinical use of antibiotics, the development of bacterial infections has significantly slowed down [2].However, in recent decades, and especially in the post-COVID-19 era, their efficacy has diminished due to both their overuse and misuse, as well as the ability of bacteria to develop mechanisms of tolerance or resistance to antibiotics [3][4][5].It is estimated that in developed countries, over 60% of human bacterial infections are caused by bacterial biofilms, while the removal of biofilms represents a serious health, social, and economic difficulty that people face nowadays [5,6].The reason for this is that bacterial cells within the biofilm are more resistant to antimicrobial agents than planktonic bacteria, which is a major challenge in the therapy of certain infections [7].Particularly, due to their antibiotic resistance, multi-resistant strains, including Enterococcus faecium, Staphylococcus aureus, Aqueous extracts of H. officinalis, L. angustifolia, M. officinalis, O. majorana, R. officinalis, S. montana, T. chamaedrys, Th. serpyllum, and Th.vulgaris had a higher yield than their methanolic and ethanolic extracts.Methanolic extracts of G. hederacea, M. vulgare, M. piperita, O. basilicum, O. vulgare, S. scardica, and T. montanum had higher yields compared to other solvents.Ethanolic extracts of L. cardiaca and S. officinalis had higher yields compared to their methanolic and aqueous extracts.M. vulgare ethanolic extract had the highest yield of all the tested plants (34.1%), while the lowest yield was obtained for the aqueous extract of G. hederacea (9.07%) (Table 1).

Total Coumarin and Triterpene Contents in Lamiaceae Extracts
Since the total coumarin and triterpene contents were not detected at 100 and 250 µg/mL of extracts, only the results using the highest tested concentration of extracts (500 µg/mL) are shown in Figures 1 and 2.

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methanolic extracts of H. officinalis, L. angustifolia, and M. vulgare, as well as aqueous extracts of G. hederacea, H. officinalis, and M. vulgare.As was the case with total coumarins, in certain extracts, triterpenes were not detected even in the highest tested concentration.Overall, the highest amounts of triterpenes were detected in methanolic extracts (range from <1 to 210.42 mg UAE/g), while the aqueous extracts contained the lowest amounts of triterpenes (<1-52.12mg UAE/g).All the ethanolic extracts had a measurable amount of triterpenes in the range of 1.61-109.4mg UAE/g.The most triterpenes were found in L. angustifolia extracts, especially in the methanolic one (210.42mg UAE/g), which contained almost two times more triterpenes than the ethanolic extract and four times more triterpenes than the aqueous extract.Finally, the lowest content of triterpenes was found in O. basilicum extracts (Figure 2).

Inhibition of Bacterial Growth by Lamiaceae Extracts
The MIC and MBC of methanolic and ethanolic extracts of the examined Lamiaceae representatives are presented in Table 2.The results for the aqueous extracts are omitted due to their lack of antibacterial activity.Additionally, results for the inhibition of E. coli and S. typhimurium growth are not provided in Table 2, as their MICs and MBCs exceeded the highest tested concentration of extracts, 1000 µg/mL.In general, the antibacterial effect of the extracts was more pronounced against Gram-positive compared to Gram-negative bacteria, except for E. faecalis.Of all the tested bacteria, B. subtilis showed the highest sensitivity; H. officinalis ethanolic extract and S. scardica methanolic extract inhibited B. subtilis at the same MIC value, 31.25 µg/mL, while the MIC of S. officinalis ethanolic extract was The majority of the examined members from the Lamiaceae family have significant amounts of total coumarins.The highest level of coumarins was detected in alcoholic extracts-methanolic <1 to 161.22 mg CE/g, ethanolic 11.22 to 126.53 mg EC/g-while aqueous extracts had the lowest amount of coumarins (<1-97.96mg CE/g) (Figure 1).The methanolic extract of T. montanum contained the highest amount of total coumarins (161.22 mg CE/g), while among the ethanolic extracts, the G. hederacea, H. officinalis, and M. vulgare ones had significantly more of these compounds in comparison to the methanolic and aqueous extracts.The aqueous extracts of L. angustifolia and O. vulgare contained significantly more coumarins compared to the extracts prepared using the other two solvents.It should be noted that in certain extracts, coumarins were not detected, such as the methanolic extracts of H. officinalis, L. angustifolia, and M. vulgare, as well as aqueous extracts of G. hederacea, H. officinalis, and M. vulgare.
As was the case with total coumarins, in certain extracts, triterpenes were not detected even in the highest tested concentration.Overall, the highest amounts of triterpenes were detected in methanolic extracts (range from <1 to 210.42 mg UAE/g), while the aqueous extracts contained the lowest amounts of triterpenes (<1-52.12mg UAE/g).All the ethanolic extracts had a measurable amount of triterpenes in the range of 1.61-109.4mg UAE/g.The most triterpenes were found in L. angustifolia extracts, especially in the methanolic one (210.42mg UAE/g), which contained almost two times more triterpenes than the ethanolic extract and four times more triterpenes than the aqueous extract.Finally, the lowest content of triterpenes was found in O. basilicum extracts (Figure 2).

Inhibition of Bacterial Growth by Lamiaceae Extracts
The MIC and MBC of methanolic and ethanolic extracts of the examined Lamiaceae representatives are presented in Table 2.The results for the aqueous extracts are omitted due to their lack of antibacterial activity.Additionally, results for the inhibition of E. coli and S. typhimurium growth are not provided in Table 2, as their MICs and MBCs exceeded the highest tested concentration of extracts, 1000 µg/mL.In general, the antibacterial effect of the extracts was more pronounced against Gram-positive compared to Gramnegative bacteria, except for E. faecalis.Of all the tested bacteria, B. subtilis showed the highest sensitivity; H. officinalis ethanolic extract and S. scardica methanolic extract inhibited B. subtilis at the same MIC value, 31.25 µg/mL, while the MIC of S. officinalis ethanolic extract was 62.5 µg/mL.In contrast to E. coli and S. typhimurium, where the MIC for all tested extracts was higher than 1000 µg/mL, the MIC values of the ethanolic extracts for P. aeruginosa were in the range of 250-500 µg/mL (Table 2).The positive control, streptomycin, showed the strongest antibacterial activity (B.subtilis 3.125 µg/mL, E. coli Plants 2024, 13, 1616 5 of 22 12.5 µg/mL, and S. typhimurium 3.125 µg/mL), while the negative controls, ethanol and methanol, in the tested concentrations, did not affect the growth of the tested bacteria.

Inhibition of Adhesion and Invasion of P. aeruginosa PAO1 on Human Fibroblasts by Lamiaceae Extracts
In the adhesion assay, the tested ethanolic extracts did not inhibit the adhesion of P. aeruginosa PAO1 to the MRC-5 cells.
On the other hand, all the tested ethanolic extracts, except H. officinalis and M. officinalis, significantly reduced the number of bacteria capable of invading MRC-5 cells (Figure 3).

Inhibition of Adhesion and Invasion of P. aeruginosa PAO1 on Human Fibroblasts by Lamiaceae Extracts
In the adhesion assay, the tested ethanolic extracts did not inhibit the adhesion of P. aeruginosa PAO1 to the MRC-5 cells.
On the other hand, all the tested ethanolic extracts, except H. officinalis and M. officinalis, significantly reduced the number of bacteria capable of invading MRC-5 cells (Figure 3).

Inhibition of P. aeruginosa PAO1 Biofilm Formation and Degradation by Lamiaceae Extracts
P. aeruginosa PAO1 was used to monitor the inhibitory effect of ethanolic extracts on the formation and degradation of biofilm.The selection of extracts for these assays was based on the obtained MIC values for P. aeruginosa ATCC 15442.All extracts with an MIC of 250 µg/mL (Table 2) were tested, except for S. scardica, due to its low solubility.
In the biofilm formation inhibition assay, ethanolic extracts were tested in the range from 156 to 1250 µg/mL (⅛MIC-MIC), and the results are presented in Figure 4.All tested extracts showed an inhibitory effect on biofilm formation.The extracts of O. vulgare and H. officinalis demonstrated the highest overall level of biofilm formation inhibition, with significant inhibition rates of 45.43% and 49.1%, respectively, both at a concentration of 625 µg/mL.Moreover, M. officinalis, M. piperita, R. officinalis, S. montana, T. chamaedrys, T. montanum, Th. serpyllum, and Th.vulgaris extracts at certain concentrations also exhibited significant inhibition of the formation of P. aeruginosa PAO1 biofilm.It is important to note that, except for M. piperita, O. basilicum, and T. montanum, the highest concentrations of extracts did not result in the strongest inhibition.This indicates that there was no concentration-dependent inhibition observed.The negative control, ethanol (tested in the range of 1.092-8.75%),did not affect the viability of bacteria during biofilm formation.Hence, the statistical analysis was performed to assess the significance of differences between the extracts and ethanol to determine the extent to which the extracts themselves contribute to biofilm inhibition.Also, streptomycin (tested in the range of 0.781 to 6.25 µg/mL) did not cause a reduction in bacterial viability (Figure 4).2) were tested, except for S. scardica, due to its low solubility.
In the biofilm formation inhibition assay, ethanolic extracts were tested in the range from 156 to 1250 µg/mL ( Plants 2024, 13, x FOR PEER REVIEW 6 of 22

Inhibition of Adhesion and Invasion of P. aeruginosa PAO1 on Human Fibroblasts by Lamiaceae Extracts
In the adhesion assay, the tested ethanolic extracts did not inhibit the adhesion of P. aeruginosa PAO1 to the MRC-5 cells.
On the other hand, all the tested ethanolic extracts, except H. officinalis and M. officinalis, significantly reduced the number of bacteria capable of invading MRC-5 cells (Figure 3).

Inhibition of P. aeruginosa PAO1 Biofilm Formation and Degradation by Lamiaceae Extracts
P. aeruginosa PAO1 was used to monitor the inhibitory effect of ethanolic extracts on the formation and degradation of biofilm.The selection of extracts for these assays was based on the obtained MIC values for P. aeruginosa ATCC 15442.All extracts with an MIC of 250 µg/mL (Table 2) were tested, except for S. scardica, due to its low solubility.
In the biofilm formation inhibition assay, ethanolic extracts were tested in the range from 156 to 1250 µg/mL (⅛MIC-MIC), and the results are presented in Figure 4.All tested extracts showed an inhibitory effect on biofilm formation.The extracts of O. vulgare and H. officinalis demonstrated the highest overall level of biofilm formation inhibition, with significant inhibition rates of 45.43% and 49.1%, respectively, both at a concentration of 625 µg/mL.Moreover, M. officinalis, M. piperita, R. officinalis, S. montana, T. chamaedrys, T. montanum, Th. serpyllum, and Th.vulgaris extracts at certain concentrations also exhibited significant inhibition of the formation of P. aeruginosa PAO1 biofilm.It is important to note that, except for M. piperita, O. basilicum, and T. montanum, the highest concentrations of extracts did not result in the strongest inhibition.This indicates that there was no concentration-dependent inhibition observed.The negative control, ethanol (tested in the range of 1.092-8.75%),did not affect the viability of bacteria during biofilm formation.Hence, the statistical analysis was performed to assess the significance of differences between the extracts and ethanol to determine the extent to which the extracts themselves contribute to biofilm inhibition.Also, streptomycin (tested in the range of 0.781 to 6.25 µg/mL) did not cause a reduction in bacterial viability (Figure 4).MIC-MIC), and the results are presented in Figure 4.All tested extracts showed an inhibitory effect on biofilm formation.The extracts of O. vulgare and H. officinalis demonstrated the highest overall level of biofilm formation inhibition, with significant inhibition rates of 45.43% and 49.1%, respectively, both at a concentration of 625 µg/mL.Moreover, M. officinalis, M. piperita, R. officinalis, S. montana, T. chamaedrys, T. montanum, Th. serpyllum, and Th.vulgaris extracts at certain concentrations also exhibited significant inhibition of the formation of P. aeruginosa PAO1 biofilm.It is important to note that, except for M. piperita, O. basilicum, and T. montanum, the highest concentrations of extracts did not result in the strongest inhibition.This indicates that there was no concentration-dependent inhibition observed.The negative control, ethanol (tested in the range of 1.092-8.75%),did not affect the viability of bacteria during biofilm formation.Hence, the statistical analysis was performed to assess the significance of differences between the extracts and ethanol to determine the extent to which the extracts themselves contribute to biofilm inhibition.Also, streptomycin (tested in the range of 0.781 to 6.25 µg/mL) did not cause a reduction in bacterial viability (Figure 4).The effects of the selected extracts on the P. aeruginosa PAO1 biofilm degradation are shown in Figure 5.The extracts were tested at twice the concentration used in the inhibition of biofilm when performing the biofilm formation assay (from 312 to 2500 µg/mL, ⅟4MIC-2 × MIC), since the degradation of already-formed biofilm requires agents of stronger activity compared to the ones used for the inhibition of its formation.Among the tested extracts, T. chamaedrys extract displayed the best activity, degrading 42.26% of the pre-existing biofilm at 625 µg/mL.Similar to the assessment of the Lamiaceae extracts effects on biofilm formation, several extracts showed statistically significant differences in biofilm degradation compared to ethanol, namely, H. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, S. montana, T. montanum, Th.Serpyllum, and Th.vulgaris extracts, which at lower concentrations showed a significant potential for biofilm degradation.Ethanol was tested in the range of 2.184-17.5% and streptomycin in the range of 1.562-12.5 µg/mL.Ethanol degraded 22.48% of the pre-existing biofilm at the highest tested concentration, and streptomycin acted in a dose-dependent manner, leaving only 28.3% of bacteria viable at the highest tested concentration (Figure 5).The effects of the selected extracts on the P. aeruginosa PAO1 biofilm degradation are shown in Figure 5.The extracts were tested at twice the concentration used in the inhibition of biofilm when performing the biofilm formation assay (from 312 to 2500 µg/mL, ⅟4MIC-2 × MIC), since the degradation of already-formed biofilm requires agents of stronger activity compared to the ones used for the inhibition of its formation.Among the tested extracts, T. chamaedrys extract displayed the best activity, degrading 42.26% of the pre-existing biofilm at 625 µg/mL.Similar to the assessment of the Lamiaceae extracts effects on biofilm formation, several extracts showed statistically significant differences in biofilm degradation compared to ethanol, namely, H. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, S. montana, T. montanum, Th.Serpyllum, and Th.vulgaris extracts, which at lower concentrations showed a significant potential for biofilm degradation.Ethanol was tested in the range of 2.184-17.5% and streptomycin in the range of 1.562-12.5 µg/mL.Ethanol degraded 22.48% of the pre-existing biofilm at the highest tested concentration, and streptomycin acted in a dose-dependent manner, leaving only 28.3% of bacteria viable at the highest tested concentration (Figure 5).MIC-2 × MIC), since the degradation of already-formed biofilm requires agents of stronger activity compared to the ones used for the inhibition of its formation.Among the tested extracts, T. chamaedrys extract displayed the best activity, degrading 42.26% of the pre-existing biofilm at 625 µg/mL.Similar to the assessment of the Lamiaceae extracts' effects on biofilm formation, several extracts showed statistically significant differences in biofilm degradation compared to ethanol, namely, H. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, S. montana, T. montanum, Th.Serpyllum, and Th.vulgaris extracts, which at lower concentrations showed a significant potential for biofilm degradation.Ethanol was tested in the range of 2.184-17.5% and streptomycin in the range of 1.562-12.5 µg/mL.Ethanol degraded 22.48% of the pre-existing biofilm at the highest tested concentration, and streptomycin acted in a dose-dependent manner, leaving only 28.3% of bacteria viable at the highest tested concentration (Figure 5).The effects of the selected extracts on the P. aeruginosa PAO1 biofilm degradation are shown in Figure 5.The extracts were tested at twice the concentration used in the inhibition of biofilm when performing the biofilm formation assay (from 312 to 2500 µg/mL, ⅟4MIC-2 × MIC), since the degradation of already-formed biofilm requires agents of stronger activity compared to the ones used for the inhibition of its formation.Among the tested extracts, T. chamaedrys extract displayed the best activity, degrading 42.26% of the pre-existing biofilm at 625 µg/mL.Similar to the assessment of the Lamiaceae extracts effects on biofilm formation, several extracts showed statistically significant differences in biofilm degradation compared to ethanol, namely, H. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, S. montana, T. montanum, Th.Serpyllum, and Th.vulgaris extracts, which at lower concentrations showed a significant potential for biofilm degradation.Ethanol was tested in the range of 2.184-17.5% and streptomycin in the range of 1.562-12.5 µg/mL.Ethanol degraded 22.48% of the pre-existing biofilm at the highest tested concentration, and streptomycin acted in a dose-dependent manner, leaving only 28.3% of bacteria viable at the highest tested concentration (Figure 5).

Correlation Analysis across Assays: Phytochemical Content, Bacterial Growth, Invasion, Biofilm Formation, and Degradation
The correlations between the total coumarin and triterpene contents of the extracts and the results of the antibacterial assays, as well as the inter-correlation of these results, were defined by Pearson correlation coefficients (r).The calculated individual r values in Table 3 depict only the results of ethanolic extracts, either because only these were examined or due to their higher antibacterial activity compared to that of the methanolic and aqueous extracts.[24].The correlations highlighted in bold are significant at p < 0.05.
The coumarin content correlated moderately with the inhibition of B. subtilis, E. faecalis, and P. aeruginosa growth, as well as with the degradation of pre-existing P. aeruginosa PAO1 biofilm.However, only the correlations between coumarin content and the inhibition of B. subtilis and P. aeruginosa growth were statistically significant (Table 3).
The results of the MIC assay obtained for the different bacteria exhibited variations, with a weak correlation observed between the MIC assay results of Gram-positive and Gram-negative bacteria.Notably, a significant, strong correlation was identified only between the inhibition of B. subtilis and S. aureus growth.Furthermore, the correlation found between the inhibition of E. faecalis and L. innocua was identified as significant and moderate.Moderate correlations were also found among the inhibition of S. aureus, E. faecalis, and L. innocua growth (Table 3).
The findings from the P. aeruginosa PAO1 invasion assay exhibited moderate correlation with the inhibition of L. innocua growth, while only their moderate correlation with the inhibition of P. aeruginosa PAO1 biofilm formation was identified as statistically significant (Table 3).
The assays employed to assess the extracts' capacity to inhibit the formation and promote the degradation of P. aeruginosa PAO1 biofilm exhibited a moderate inter-correlation.Additionally, a moderate correlation was identified between the ability of the extracts to Plants 2024, 13, 1616 9 of 22 inhibit the formation of P. aeruginosa PAO1 biofilm and the growth of E. faecalis.The correlation between the ability of the extracts to inhibit the formation of P. aeruginosa PAO1 biofilm and the growth of P. aeruginosa was identified as moderately and statistically significant.On the other hand, the ability of extracts to degrade P. aeruginosa PAO1 biofilm had a moderate and significant correlation with the growth inhibition of B. subtilis and S. aureus (Table 3).

Integrated Biomarker Response Analysis
Finally, the acquired results underwent standardization using the Integrated Biomarker Response (IBR) analysis technique.The IBR analysis included the following: The IBR analysis of antibacterial activity included the standardization and graphical presentation of results obtained for the ethanolic extracts of the following Lamiaceae representatives: H. officinalis, M. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, R. officinalis, S. montana, T. chamaedrys, T. montanum, Th. serpyllum, and Th.vulgaris (Figures 6 and 7).The MIC assay on E. coli and S. typhimurium and the P. aeruginosa PAO1 adhesion assay in lung fibroblast infection were excluded from this analysis as none of the ethanolic extracts exhibited activity in these assays.
Additionally, a moderate correlation was identified between the ability of the extracts to inhibit the formation of P. aeruginosa PAO1 biofilm and the growth of E. faecalis.The correlation between the ability of the extracts to inhibit the formation of P. aeruginosa PAO1 biofilm and the growth of P. aeruginosa was identified as moderately and statistically significant.On the other hand, the ability of extracts to degrade P. aeruginosa PAO1 biofilm had a moderate and significant correlation with the growth inhibition of B. subtilis and S. aureus (Table 3).

Integrated Biomarker Response Analysis
Finally, the acquired results underwent standardization using the Integrated Biomarker Response (IBR) analysis technique.The IBR analysis included the following: The IBR analysis of antibacterial activity included the standardization and graphical presentation of results obtained for the ethanolic extracts of the following Lamiaceae representatives: H. officinalis, M. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, R. officinalis, S. montana, T. chamaedrys, T. montanum, Th. serpyllum, and Th.vulgaris (Figures 6  and 7).The MIC assay on E. coli and S. typhimurium and the P. aeruginosa PAO1 adhesion assay in lung fibroblast infection were excluded from this analysis as none of the ethanolic extracts exhibited activity in these assays.Results from the IBR analysis revealed that the ethanolic extract of S. montana displayed the highest antibacterial potential, followed by O. vulgare and T. chamaedrys extracts.In contrast, the ethanolic extract of T. montanum demonstrated the weakest antibacterial potential (Figures 6 and 7).

Discussion
Plants with antibacterial potential are increasingly being researched on, as it was demonstrated that their metabolites, including phenolic compounds and terpenes, have better bioavailability compared to synthetically derived drugs.Current studies show that plant extracts, essential oils, and their isolated phytoconstituents affect the growth of both Gram-positive and Gram-negative bacteria, influencing adhesion and invasion during infections in various cells and tissues, as well as the dynamics of bacterial biofilm formation [25].
While many literature references align with our results of the yield of extraction [26][27][28][29], certain studies indicate variations in the extraction yield for the examined extracts [30][31][32][33].Research has demonstrated that differences in extraction yield values are significantly influenced by factors such as the chosen extraction protocol, extraction time, solvent polarity, and, most importantly, the plant species [34,35].Additionally, one of the most critical factors is the temperature applied in the extraction procedure, specifically the solvent temperature.Kivilompolo and Hyötyläinen [36] validated that a higher temperature enhances the extraction yield.Hence, the higher yield observed in our study for aqueous extracts can be attributed to the use of boiling, rather than cold, distilled water in the extraction procedure.
Given the low extraction yields for both methanol and ethanol in our study, and the use of ethanol in developing various plant-based products, optimizing the extraction protocol is essential for future applications.Enhancing extraction parameters and incorporating advanced techniques, such as ultrasound-assisted extraction and binary solvent systems, can significantly improve the extraction yield.Combining methanol or ethanol with water in optimized ratios enhances solvent polarity and improves the solubility and extraction efficiency of phytochemicals.Even small amounts of water have been reported to significantly enhance the extraction outcome [34,35].Moreover, ultrasound-assisted extraction disrupts cell walls and facilitates the release of bioactive compounds [37], while increasing the solid/solvent ratio prevents saturation of the extraction medium, resulting in improved extraction yield [38]. Reducing solvent volumes makes the process more sustainable and cost-effective, especially with environmentally friendly solvents like ethanol.Comparative studies of methanol, ethanol, and their binary mixtures can identify the most Results from the IBR analysis revealed that the ethanolic extract of S. montana displayed the highest antibacterial potential, followed by O. vulgare and T. chamaedrys extracts.In contrast, the ethanolic extract of T. montanum demonstrated the weakest antibacterial potential (Figures 6 and 7).

Discussion
Plants with antibacterial potential are increasingly being researched on, as it was demonstrated that their metabolites, including phenolic compounds and terpenes, have better bioavailability compared to synthetically derived drugs.Current studies show that plant extracts, essential oils, and their isolated phytoconstituents affect the growth of both Grampositive and Gram-negative bacteria, influencing adhesion and invasion during infections in various cells and tissues, as well as the dynamics of bacterial biofilm formation [25].
While many literature references align with our results of the yield of extraction [26][27][28][29], certain studies indicate variations in the extraction yield for the examined extracts [30][31][32][33].Research has demonstrated that differences in extraction yield values are significantly influenced by factors such as the chosen extraction protocol, extraction time, solvent polarity, and, most importantly, the plant species [34,35].Additionally, one of the most critical factors is the temperature applied in the extraction procedure, specifically the solvent temperature.Kivilompolo and Hyötyläinen [36] validated that a higher temperature enhances the extraction yield.Hence, the higher yield observed in our study for aqueous extracts can be attributed to the use of boiling, rather than cold, distilled water in the extraction procedure.
Given the low extraction yields for both methanol and ethanol in our study, and the use of ethanol in developing various plant-based products, optimizing the extraction protocol is essential for future applications.Enhancing extraction parameters and incorporating advanced techniques, such as ultrasound-assisted extraction and binary solvent systems, can significantly improve the extraction yield.Combining methanol or ethanol with water in optimized ratios enhances solvent polarity and improves the solubility and extraction efficiency of phytochemicals.Even small amounts of water have been reported to significantly enhance the extraction outcome [34,35].Moreover, ultrasound-assisted extraction disrupts cell walls and facilitates the release of bioactive compounds [37], while increasing the solid/solvent ratio prevents saturation of the extraction medium, resulting in improved extraction yield [38]. Reducing solvent volumes makes the process more sustainable and cost-effective, especially with environmentally friendly solvents like ethanol.Comparative studies of methanol, ethanol, and their binary mixtures can identify the most efficient solvent systems for specific bioactive compounds.By integrating these advanced techniques and optimizing parameters, future research can significantly improve extraction yields, enhancing practical applications.
Coumarins (2H-1-benzopyran-2-ones) are structurally diverse phenolic compounds that exhibit a myriad of biological activities, such as antioxidant, antibacterial, antifungal, antiviral, cytotoxic, and antitumor activities [39].In our study, among the methanolic extracts, the T. montanum one had the highest coumarin content.Among the ethanolic extracts, the M. piperita one was the richest in coumarins, while among the aqueous extracts, the S. officinalis one had the highest coumarin content.Our review of the literature indicated that the analysis of coumarin content in the 18 Lamiaceae representatives examined in this study has not been frequently conducted up to the present day.Nevertheless, Patil et al. [40] demonstrated that the aqueous extract of M. piperita from India had a higher coumarin content compared to the ethanolic extract, in contrast to our findings, which revealed a higher coumarin content specifically in the ethanolic extract.Moreover, Mahdi et al. [41] demonstrated the presence of coumarins and terpenes in the ethanolic extract of S. officinalis.Although the specific composition of coumarins in Lamiaceae representatives is infrequently analyzed; it was documented that O. basilicum and S. officinalis contain esculetin (6,7-dihydroxycoumarin), with O. basilicum also containing esculin (6,7-dihydroxycoumarin-6-glucoside), while L. angustifolia possesses coumarin, herniairin (7-methoxycoumarin), santonin, and umbelliferone, along with lavnadupyrone A and B [42][43][44].Despite previous reports, the extracts of L. angustifolia examined in our studies did not contain a significant amount of coumarins.
Triterpenes are a class of secondary metabolites belonging to the terpene family, characterized by a structural motif composed of six isoprene units [45].Triterpenes are commonly present in plants, and they contribute to the pharmacological and therapeutic potential of these plants, playing a crucial role in the development of new bioactive products [46,47].While the antibacterial activity of terpenes from essential oils is well-established [12], their exploration and characterization in Lamiaceae family plant extracts have been relatively limited.This is partly due to the unsaturated carbon bonds in terpenes, making them susceptible to reactions with atmospheric oxidants such as OH − , O 3 , and NO 3 .As a result, terpenes have a short lifespan (typically ranging from a few minutes to a few hours), making their identification and accurate quantification in plant material challenging [48].In our study, similar to coumarins, the concentration of triterpenes in the extracts showed statistically significant variations depending on the extraction solvent.L. angustifolia extracts, particularly the methanolic one, exhibited the highest triterpene content.Lavandula species are renowned for having a high terpene content, making the identification of L. angustifolia extracts as the richest in triterpenes among all tested extracts unsurprising.Héral et al. [44] reported in their study that plants from the Lavandula genus contain over 30 different triterpenes, including tetracyclic, pentacyclic, and steroid derivatives.Notable triterpenes include betulin, 2α-hydroxyursolic, 2,3-hydroxytormentic, 3-epiursolic, betulinic, micromeric, oleanolic, and ursolic acids, as well as α-amyrin, β-amyrin, and uvaol.Moreover, previous research indicates that extracts of M. vulgare contain high amounts of triterpenes [32], a finding not consistent with our results.In our study, O. basilicum extracts displayed the lowest amount of triterpenes.Although the examination of total triterpene content in O. basilicum extracts was not conducted previously, it was found that its ethyl acetate extract exhibited a high content of total terpenes [48].The existing literature data diverge from our study results, likely due to the authors quantifying total terpenes, which encompass various classes such as mono-, di-, and triterpenes.Additionally, it is important to note that the choice of solvent for plant material extraction can significantly influence the content of these secondary metabolites in the sample.To the best of our knowledge, there are no previous reports on the total triterpene content for the rest of the Lamiaceae representatives that were investigated in our study.
In our investigation, the MIC assay was employed to assess the antibacterial activity of extracts from 18 Lamiaceae representatives.Additionally, all extracts underwent further examination in the P. aeruginosa PAO1 adhesion and invasion assays.Moreover, selected ethanolic extracts were subjected to assays evaluating their ability to inhibit the formation and degradation of P. aeruginosa PAO1 biofilm.The Pearson's correlation results indicated a probable association between coumarins and triterpenes with the displayed antibacterial potential of Lamiaceae extracts.Particularly, coumarins showed a significant correlation with the growth inhibition of B. subtilis and P. aeruginosa ATCC 15442, as well as with the degradation of P. aeruginosa PAO1 ATCC 15692 biofilm.Indeed, previous studies indicate that the multifaceted nature of coumarins allows them to interfere with various bacterial processes, which makes plants containing them potential candidates for combating bacterial infections [49].
In the MIC assay, the results revealed that the ethanolic extracts exhibited the highest activity.Unlike their alcoholic counterparts, aqueous extracts displayed no activity.The lack of antibacterial activity observed in aqueous extracts of Lamiaceae representatives can be attributed to the testing of relatively low extract concentrations (the highest tested concentration was 1000 µg/mL), as well as the insufficient solubilization of hydrophobic bioactive compounds from these plants [34,35].These hydrophobic compounds, which often contribute significantly to the antibacterial properties, may not be sufficiently extracted in water-based solutions, leading to lower efficacy against bacterial growth compared to extracts obtained with other organic solvents.Additionally, antibacterial activity is frequently associated with lipophilic phytocomponents with physicochemical properties that influence their ability to diffuse and dissolve within bacterial membranes, leading to various antibacterial effects.Nonetheless, they are more effectively extracted in non-polar solvents such as ethanol or methanol, as opposed to water [50].
Previous studies have explored the impact of extracts of Lamiaceae representatives on bacterial growth activity [40,[51][52][53][54][55][56][57][58][59]; however, our literature review revealed that current research mainly focuses on the antibacterial properties of essential oils derived from Lamiaceae plants [12,50,[60][61][62].In our study, B. subtilis showed the highest susceptibility to the effects of Lamiaceae extracts, with H. officinalis and S. officinalis ethanolic and S. scardica methanolic extracts demonstrating the most potent growth-inhibitory activity.Our literature survey uncovers that Lamiaceae extracts typically exhibit inhibition of bacterial growth at concentrations surpassing those examined in our study, reaching as high as 40 mg/mL, which was specifically found for M. officinalis ethanolic extract and the inhibition of E. coli and P. aeruginosa growth [63].Despite the previous study, it is noteworthy that the S. officinalis extracts investigated by Mocan et al. [64] exhibited high inhibition against Gram-negative bacteria growth (E. coli at 45 µg/mL and P. aeruginosa and S. typhimurium at 90 µg/mL) in contrast to our results.Additionally, the results of our MIC assay for the different bacteria exhibited variations, indicating a weak correlation between the MIC assay results for Gram-positive and Gram-negative bacteria.This outcome was expected due to the presence of an outer membrane with a hydrophilic polysaccharide chain in Gram-negative bacteria, acting as a hydrophobic barrier against many plant secondary metabolites [25].Therefore, similar to essential oils [62], plant extracts may encounter difficulties in efficiently targeting the phospholipid layers of Gram-negative bacterial cells, potentially compromising their permeability and structural integrity.
The infection of invasive bacteria, exemplified by P. aeruginosa, is marked by three stages: (i) adhesion and colonization, (ii) local infection through tissue penetration and internalization, and (iii) dissemination through the bloodstream [65].Initial tissue penetration stages (extracellular matrix protein and tight junction cleavage) and host cell invasion are pivotal for bacterial survival and infection initiation.In P. aeruginosa PAO1, cell invasion, facilitated by the secretome (comprising toxins, proteases, lipases, and lysines), occurs independently of lipopolysaccharide production or cytotoxicity.Notably, these bacteria exhibit a higher binding affinity for inflamed or compromised cells, emphasizing the importance of employing natural antioxidants and anti-inflammatory agents, such as plant extracts, particularly in pathological conditions [9,66,67].
While our study indicated that methanolic, ethanolic, and aqueous extracts of Lamiaceae species did not affect P. aeruginosa PAO1 adhesion to MRC-5 cells, earlier investiga-tions [15,[67][68][69][70] demonstrated inhibition of bacterial adhesion to human cells for extracts of L. cardiaca, M. vulgare, M. piperita, R. officinalis, S. montana, and Th.vulgaris.Furthermore, our findings indicated that all ethanolic extracts of the evaluated Lamiaceae representatives, except for H. officinalis and M. officinalis, significantly reduced the number of bacteria capable of invading MRC-5 cells.It is important to emphasize that there are no previous reports on the bacterial invasion of human cells by these plants, except for the study of Šimunović et al. [70], who demonstrated that the S. montana ethanolic extract (62.5 µg/mL) inhibited the invasion of Campylobacter jejuni 11168 into INT407 epithelial cells by up to 81%, a percentage higher than the one observed in our results.
Additionally, P. aeruginosa, a prominent biofilm former, serves as a valuable model for studying the dynamics of biofilm formation and degradation.Gaining insight into biofilm composition, structure, and the molecular mechanisms contributing to antibacterial tolerance is essential for developing strategies to not only manage and prevent but also eradicate biofilm-associated infections.However, the treatment of P. aeruginosa displays distinctive challenges in utilizing most of the available antibiotics since it showcases multi-drug resistance mechanisms [71].Research on the dynamics of P. aeruginosa biofilm, especially its degradation, has been scarce for Lamiaceae plant extracts.In addition to studies that have researched the dynamics of P. aeruginosa biofilm formation and/or degradation [54,[72][73][74][75][76], there are several studies focused on the effect of these extracts on the biofilms of Bacillus cereus, C. jejuni, S. aureus, E. coli, Acinetobacter baumannii, Klebsiella pneumoniae, and MRSA [15,54,68,73,74,77,78].Our findings indicate that ethanolic extracts of O. vulgare and H. officinalis significantly inhibited P. aeruginosa PAO1 biofilm formation by up to 50%.Additionally, at certain concentrations, extracts from M. officinalis, M. piperita, R. officinalis, S. montana, T. chamaedrys, T. montanum, Th. serpyllum, and Th.vulgaris significantly inhibited the formation of P. aeruginosa PAO1 biofilm.Moreover, T. chamaedrys showed the best activity, degrading 42.26% of pre-existing biofilm at 625 µg/mL.Additionally, H. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, S. montana, T. montanum, Th. serpyllum, and Th.vulgaris extracts demonstrated significant biofilm degradation at lower concentrations.Interestingly, the extracts that exhibited the highest inhibition of biofilm formation were not as effective in degrading a pre-existing biofilm.However, this outcome is anticipated as plant extracts and their phytoconstituents may employ various mechanisms for their anti-biofilm activities: (i) plant extracts, containing compounds like phenolics, can modify the microenvironment around bacterial cells and disrupt bacterial communication systems, specifically quorum sensing, crucial for biofilm formation, thereby impeding the coordination of bacterial cells in biofilm development [79][80][81]; (ii) plant extracts and phenolic compounds, specifically flavonoids, directly inhibit the growth of P. aeruginosa cells and their adherence to surfaces, preventing the initial stages of biofilm formation [82]; and (iii) phytoconstituents may disrupt the biofilm matrix by enzymatically breaking down the extracellular polymeric substances, making the biofilm structure more susceptible to degradation [10].The precise mechanisms rely on the composition of the plant extract and their bioactive compounds.Altogether, it was proven that various plant species and their extracts may exhibit unique properties that contribute to their effectiveness in both inhibiting biofilm formation and degrading existing biofilms [83].
Last but not least, our IBR analysis identified the ethanolic extracts of S. montana and O. vulgare as highly promising antibacterial agents.This observation is noteworthy as prior studies predominantly emphasized the antibacterial activity of their essential oils [50,61,84].Our findings further reveal the promising antibacterial efficacy of their extracts, shedding light on their potential as a powerful natural defense against various aspects of bacterial infections.

Plant Material
The experimental plant material, commercially available and sourced from the Institute for Medicinal Plant Research "Dr.Josif Pančić" (IMPR) in Belgrade, Serbia, comprises 18 medicinal, aromatic, and spice Lamiaceae species from Serbia.Collected during the spring of 2018, vouchers for each plant species used in this study have been cataloged in the IMPR's herbarium (Table 4).

Preparation of Lamiaceae Extracts
Pre-ground plant material (10 g) underwent extraction using the classic maceration method (10% w/v, 24 h at 25 • C) [33].Three solvents-70% methanol, 70% ethanol, and boiling distilled water (100 • C)-were employed for the extraction process.Ultrasonic treatment was applied for a total of two hours (one hour before and one hour after maceration, 30 • C).The resulting mixture underwent double filtration with Whatman No. 1 filter paper, followed by the removal of excess solvent using a Büchi rotavapor R-114 evaporator under reduced pressure.The resulting crude extracts were stored in glass vials at 4 • C until subsequent experimentation.

Yield of Extracts
The dry extract's mass was determined for each sample, and the yield was calculated using the following equation: where m 0 represents the mass of the empty vial; m 1 is the mass of the vial with the extract; (m 1 − m 0 ) is the mass of the dry extract; and M is the mass of the dry plant material used for extraction.The results are expressed as percentages.

Total Coumarin Content
The method for determining the total coumarin content followed the procedure by de Amorim et al. [85] with some modifications, conducted in 96-well microtiter plates.An amount of 2 µL of extract at concentrations of 100, 250, and 500 µg/mL were dispensed into the wells, followed by the addition of 8 µL distilled water and 2 µL lead acetate solution (5% w/v).Additionally, 28 µL distilled water and 160 µL of 0.1 M hydrochloric acid were added to each well.A blank was prepared with all components, substituting the sample with an appropriate solvent (70% methanol, 70% ethanol, or distilled water).The microtiter plate with the reaction mixtures and the blank was incubated for 30 min at room temperature, and absorbances were measured at 320 nm using a Multiskan Sky Thermo Scientific microtiter plate reader, Vantaa, Finland.
To generate the calibration curve, coumarin (C) dissolved in methanol was utilized in concentrations ranging from 5 to 1000 µg/mL instead of the extract.All the samples were tested in triplicate.The total coumarin content in the samples was calculated using the calibration curve equation (y = 0.2440x + 0.0093; R 2 = 0.9983) and expressed in coumarin equivalents as mg CE/g of dry extract.The results are presented as the mean of three replicates ± standard error.

Total Triterpene Content
The method for determining total triterpene content followed the procedure by Chang et al. [86], with adjustments, using 96-well microtiter plates.In brief, 10 µL of extract at concentrations of 100, 250, and 500 µg/mL were dispensed into the well, followed by the addition of 15 µL vanillin-glacial acid solution (5% w/v) and 50 µL perchloric acid.The microtiter plate with the reaction mixtures was incubated at 60 • C for 45 min, then cooled to 25 • C on ice, and afterward, 225 µL of glacial acetic acid was added to each well.A blank, containing all the components except the sample, substituted with an appropriate solvent (70% methanol, 70% ethanol, or distilled water), was prepared.Absorbances were measured at 548 nm using a Multiskan Sky Thermo Scientific microtiter plate reader, Finland.For the calibration curve, ursolic acid (UA) dissolved in 100% methanol (concentration of 5 to 1000 µg/mL) was used instead of the extract.Each sample was tested in triplicate.
The total triterpene content of the samples was calculated from the calibration curve equation (y = 0.0008x + 0.0082, R 2 = 0.995) and expressed in UA equivalents as mg UAE/g of dry extract.The results are presented as the mean of three replicates ± standard error.

Preparation of Bacterial Strains
The Gram-positive and Gram-negative bacterial strains employed in the experimental work are part of the ATCC collection (Table 5).A colony of the respective bacteria was transferred to a test tube containing 5 mL of MHB medium, pre-warmed to 37 • C, and allowed to cultivate overnight at 37 • C.
To ascertain the minimum inhibitory concentration (MIC) of the extracts, the bacteria listed in Table 5 were cultured to the exponential growth phase, providing the required bacterial density per milliliter for each strain [87].Subsequently, a dilution of 0.01 M magnesium sulfate was made for each strain to achieve a final bacterial concentration in the inoculum of 10 5 CFU (colony forming unit)/mL which was used to determine the MIC.
The impact on both biofilm formation and the degradation of existing biofilm was examined in P. aeruginosa PAO1 ATCC 15692.The optical density (OD 600 ) was adjusted to 0.4 using an MHB medium, resulting in a bacterial concentration of 10 8 CFU/mL.4.7.Cell Culture Preparation for Assessing the Impact of Lamiaceae Extracts on Adhesion and Invasion of P. aeruginosa PAO1 ATCC 15692 MRC-5 cells (ECACC no.84101801) were cultivated in 75 cm 2 flasks, and 1 × 10 5 cells were subsequently transferred to 12-well microtiter plates.The cells were then incubated in DMEM complete (with fetal bovine serum) nutrient medium at 37 • C with 5% CO 2 for 48 h to achieve a confluent monolayer.

Microdilution Assay
The MICs of the methanolic, ethanolic, and aqueous extracts were determined by the microdilution method in 96-well plates, using a series of double dilutions.Resazurin (final concentration 675 µg/mL) served as a bacterial growth indicator.Sample stocks containing 10 mg/mL extracts were used to prepare two-fold gradient dilutions in 96-well plates with the highest tested concentration of 1000 µg/mL, 200 µL total volume.Each well contained 20 µL of pre-prepared bacterial inoculum (10 5 CFU/mL).Resazurin (22 µL) was added the same day.
Microtiter plates were incubated at 37 • C for 24 h.To assess the bacteriostatic or bactericidal effect, solid nutrient media (MHA) screening was conducted, followed by a 24 h incubation at 37 • C. Bacterial growth presence or absence determined the substance's bacteriostatic or bactericidal effect, with the lowest concentration at which there was no bacterial growth being referred to as minimum bactericidal concentration (MBC) and the concentration at which bacterial growth occurred being referred to as minimum bacteriostatic concentration (MIC).The following controls were included in the experiment: (i) medium sterility; (ii) solvents (for methanol and ethanol); (iii) bacterial growth (negative control); and (iv) relevant antibiotics (positive controls).4.9.Assessment of Lamiaceae Extracts Impact on P. aeruginosa PAO1 Adhesion and Invasion in Lung Fibroblast Cell Infection 4.9.1.Adhesion Assay Confluent MRC-5 cell monolayers in a 24-well microtiter plate were washed with buffered PBS, and 1 mL of fresh DMEM complete nutrient medium (supplemented with 10% FBS, antibiotic-free) containing the plant extracts was added to each well.Bacterial cultures were introduced to achieve a multiplicity of infection (MOI) of a 5:1 bacteriato-cell ratio, followed by a three-hour incubation at 37 • C. Post-incubation, the medium was removed, and the cells were washed thrice with tempered PBS.For cell lysis and detachment of adhered bacteria, 100 µL of 1% Triton X-100 was added, and after a 10 min incubation at room temperature, 900 µL of LB medium was added.The well contents were homogenized with a micropipette.Dilutions of adherent bacteria and bacterial inoculum were seeded on an LA medium, and after overnight incubation at 37 • C, grown colonies were counted.The negative control contained ethanol instead of the sample.

Invasion Assay
After achieving the MOI and incubating the plates (3 h, 37 • C, as explained in Section 4.9.1), the adhered cells were washed with a medium containing 300 µg/mL gentamicin, followed by additional incubation (1 h, 37 • C).Post-medium removal, the cells were washed with PBS to eliminate bacteria and lysed with 1% Triton X-100.The collected lysate was centrifuged at 4000× g rpm (5 min), and the cells were rinsed with 1 × PBS to remove bacteria.Additionally, 10 µL of bacterial dilution (1 × 10 −1 , 1 × 10 −2 , 1 × 10 −3 , and 1 × 10 −4 in 1 × PBS) was seeded on a TSA medium and incubated for 24 h at 37 • C. Colony counts were conducted, and the number of bacteria per milliliter was calculated.The negative control contained ethanol instead of the sample.4.10.Assessment of Lamiaceae Extracts' Impact on P. aeruginosa PAO1 Biofilm Formation Selected Lamiaceae extracts' impact on P. aeruginosa PAO1 biofilm formation was assessed by the crystal-violet method [88].In the first microtiter plate wells, 200 µL of MIC concentration of extracts (¼MIC for H. officinalis and O. basilicum, Table 6) was added, after which 100 µL of MHB medium was added to the remaining wells.A concentration gradient was created by transferring 100 µL from row to row, testing MIC and sub-MIC concentrations.An amount of 100 µL of P. aeruginosa PAO1 culture, diluted to an OD 600 of 0.05 (10 7 CFU/mL), was added to all wells, resulting in double dilutions of the extracts.After a 24 h incubation at 37 • C, the medium was removed, and the biofilm remained.To remove residual planktonic bacteria, the biofilm was washed with sterile distilled water twice, followed by drying (15 min, 25 • C).Biofilm cells were fixed with methanol (10 min) and dried, and each well was stained with 125 µL of 0.1% crystal violet (15 min) for biofilm mass determination.Post-staining, the plates were washed with distilled water twice and left to dry for 10 min.To dissolve residual dye, 200 µL of absolute ethanol was added to each well.After mixing, absorbance was read at 570 nm on a Multiskan FC microtiter plate reader (Thermo Scientific, Waltham, MA, USA).The values were compared with streptomycin (positive control), and the results were presented as the percentage of viable bacteria in the newly formed biofilm.The results are presented as µg/mL.

Assessment of Lamiaceae Extracts Impact on Pre-Existing P. aeruginosa PAO1 Biofilm
The impact of the extract on pre-existing P. aeruginosa PAO1 biofilm was assessed following the protocol by Merritt et al. [89].Overnight culture of P. aeruginosa PAO1 was diluted to OD 600 = 0.01 in an MHB nutrient medium, and 200 µL of suspension was added to each well and incubated for 6 h at 37 • C.After incubation, the microtiter plate was washed twice with sterile PBS buffer to eliminate planktonic bacteria.After drying, 100 µL of MHB nutrient medium containing plant extracts (2 × MIC, except for H. officinalis and O. basilicum, where ½MIC was used; Table 6) was added, as prepared in the same concentration as described earlier (Section 4.10).Following a 24 h incubation at 37 • C, plate contents were removed, and each well was stained with 125 µL of 0.1% crystal violet for 15 min.The results were evaluated using the same procedure as described earlier (Section 4.10).

Statistical Analyses
For the effects of solvents on total coumarin and triterpene contents, one-way ANOVA and Tukey's post hoc tests were used, while the t-test assessed the significance of results in the adhesion and invasion assays.A statistical analysis was performed with the computer software IBM SPSS v29.The distribution between samples in the assays applied for the determination of biofilm formation and degradation was examined by the Shapiro-Wilk test, and the t-test was used to determine statistical significance between the samples.A significance level of p < 0.05 was set for all relationships.Pearson's correlation coefficients (r) represented correlations among total coumarin, triterpene contents, and antibacterial assays, interpreted following Taylor [24].Integrated Biomarker Response (IBR) values were calculated only for the ethanolic extracts and the results were standardized according to Beliaeff and Burgeot [90].The results were standardized for the extracts that underwent testing in all experiments (12 ethanol extracts in total).The results of the biofilm formation assay were standardized for the concentration of extracts of 625 µg/mL, while the results of the biofilm degradation were standardized for the concentration of extracts of 312 µg/mL.

Conclusions
Amidst the consistent increase in bacterial resistance to existing antibiotics and the declining effectiveness of these antibiotics against common infections, our study delved into the antibacterial potential of methanolic, ethanolic, and aqueous extracts derived from 18 aromatic and medicinal Lamiaceae species from Serbia.The effects of these extracts on the growth of Gram-positive and Gram-negative bacteria, P. aeruginosa PAO1 invasion of human cells and P. aeruginosa PAO1 biofilm formation and degradation were investigated.The results showed that the aqueous extracts had the highest extraction yield.A Pearson's correlation analysis revealed significant correlations between the phytochemical content of the extracts, especially coumarins, and the inhibition of B. subtilis and P. aeruginosa growth, suggesting that coumarins may be key contributors to the antibacterial activity of the Lamiaceae extracts.Although none of the extracts inhibited the adhesion of P. aeruginosa PAO1, all ethanolic extracts, except those of H. officinalis and M. officinalis, significantly reduced the number of bacteria capable of invading MRC-5 cells.Furthermore, ethanolic extracts of O. vulgare, H. officinalis, M. officinalis, M. piperita, R. officinalis, S. montana, T. chamaedrys, T. montanum, Th. serpyllum, and Th.vulgaris significantly inhibited biofilm formation at certain concentrations.For pre-existing biofilm degradation, T. chamaedrys was the most effective, while H. officinalis, M. piperita, O. basilicum, O. majorana, O. vulgare, S. montana, T. montanum, Th. serpyllum, and Th.vulgaris showed significant degradation at lower concentrations.The IBR analysis identified S. montana and O. vulgare ethanolic extracts as the most promising antibacterial agents, highlighting their potential in defending against bacterial infections.Our findings demonstrate the potent antibacterial efficacy of Lamiaceae extracts and, for the first time, their ability to inhibit P. aeruginosa PAO1 invasion of MRC-5 cells, a critical event for bacterial survival and infection initiation.With previous studies primarily focusing on the antibacterial activity of Lamiaceae essential oils, our research underscores the potential of Lamiaceae extracts as powerful natural defenses against multiple aspects of bacterial infections.Further research is needed to identify the specific compounds responsible for the antibacterial activity and to elucidate their mechanisms of action.

Figure 1 .
Figure 1.Total coumarin content of methanolic, ethanolic, and aqueous extracts of Lamiaceae representatives.Mean values from different extracts of a plant species, denoted by distinct superscript letters (a-c), show significant differences: a vs. methanolic, b vs. ethanolic, and c vs. aqueous extract (one-way ANOVA, Tukey s post hoc test, p < 0.05).

Figure 1 .
Figure 1.Total coumarin content of methanolic, ethanolic, and aqueous extracts of Lamiaceae representatives.Mean values from different extracts of a plant species, denoted by distinct superscript letters (a-c), show significant differences: a vs. methanolic, b vs. ethanolic, and c vs. aqueous extract (one-way ANOVA, Tukey's post hoc test, p < 0.05).

Figure 1 .
Figure 1.Total coumarin content of methanolic, ethanolic, and aqueous extracts of Lamiaceae representatives.Mean values from different extracts of a plant species, denoted by distinct superscript letters (a-c), show significant differences: a vs. methanolic, b vs. ethanolic, and c vs. aqueous extract (one-way ANOVA, Tukey s post hoc test, p < 0.05).

Figure 2 .
Figure 2. Total triterpene content of methanolic, ethanolic, and aqueous extracts of Lamiaceae representatives.Mean values from different extracts of a plant species, denoted by distinct superscript letters (a-c), show significant differences: a vs. methanolic, b vs. ethanolic, and c vs. aqueous extract (one-way ANOVA, Tukey s post hoc test, p < 0.05).

Figure 2 .
Figure 2. Total triterpene content of methanolic, ethanolic, and aqueous extracts of Lamiaceae representatives.Mean values from different extracts of a plant species, denoted by distinct superscript letters (a-c), show significant differences: a vs. methanolic, b vs. ethanolic, and c vs. aqueous extract (one-way ANOVA, Tukey's post hoc test, p < 0.05).

Figure 3 .
Figure 3.The effect of ethanolic extracts on the invasion of Pseudomonas aeruginosa PAO1 in the infection of lung fibroblasts (MRC-5 cells), presented by mean ± standard deviation of log CFU/mL.The values marked with * differ significantly from those of the control (t-test, p < 0.05).

Figure 3 .
Figure 3.The effect of ethanolic extracts on the invasion of Pseudomonas aeruginosa PAO1 in the infection of lung fibroblasts (MRC-5 cells), presented by mean ± standard deviation of log CFU/mL.The values marked with * differ significantly from those of the control (t-test, p < 0.05).

2. 5 .
Inhibition of P. aeruginosa PAO1 Biofilm Formation and Degradation by Lamiaceae Extracts P. aeruginosa PAO1 was used to monitor the inhibitory effect of ethanolic extracts on the formation and degradation of biofilm.The selection of extracts for these assays was based on the obtained MIC values for P. aeruginosa ATCC 15442.All extracts with an MIC of 250 µg/mL (Table

Figure 3 .
Figure 3.The effect of ethanolic extracts on the invasion of Pseudomonas aeruginosa PAO1 in the infection of lung fibroblasts (MRC-5 cells), presented by mean ± standard deviation of log CFU/mL.The values marked with * differ significantly from those of the control (t-test, p < 0.05).

Figure 4 .
Figure 4.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm formation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.

Figure 5 .
Figure 5.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm degradation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.

Figure 4 .
Figure 4.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm formation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.The effects of the selected extracts on the P. aeruginosa PAO1 biofilm degradation are shown in Figure5.The extracts were tested at twice the concentration used in the inhibition of biofilm when performing the biofilm formation assay (from 312 to 2500 µg/mL,

Figure 4 .
Figure 4.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm formation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.

Figure 5 .
Figure 5.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm degradation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.
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Figure 4 .
Figure 4.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm formation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.

Figure 5 .
Figure 5.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm degradation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.

Figure 5 .
Figure 5.The effect of ethanolic extracts on Pseudomonas aeruginosa PAO1 biofilm degradation.Statistically significant differences (p < 0.05) between the samples and the negative control (ethanol) are represented by the letters a, b, c, and d for each concentration of extract and ethanol.

Figure 6 .
Figure 6.Radial diagrams from IBR analysis: antibacterial activity of ethanolic extracts from selected Lamiaceae representatives.

Figure 7 .
Figure 7. Summarized area of radial diagrams in Figure 6: A larger area indicates a higher antibacterial potential of the tested ethanolic extracts.

Table 2 .
Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of extracts of Lamiaceae representatives.

Table 3 .
The inter-correlation of assays for total coumarin and triterpene contents with antibacterial activity.

Table 4 .
Plant species used in this study and their respective voucher numbers.

Table 5 .
Bacterial strains used in the experimental work.

Table 6 .
Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of ethanolic extracts and positive control against Pseudomonas aeruginosa PAO1.