1. 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,
Klebsiella pneumoniae,
Acinetobacter baumannii,
Pseudomonas aeruginosa, and
Enterobacter spp., collectively referred to as “ESKAPE” pathogens, pose a serious and significant obstacle in treating nosocomial infections [
5,
8].
Persisters, a subset of bacterial cells, exhibit transient tolerance to antibiotics. They typically exhibit slow or halted growth in the presence of antibiotics but can resume growth after exposure to lethal stress. This formation of persistent cells introduces phenotypic heterogeneity within the bacterial population, a critical mechanism for adapting to environmental changes [
9]. Moreover, since biofilms can form on abiotic surfaces, there is an increased interest in finding ways to suppress them, especially from medical devices and equipment such as catheters. Additionally, the release of microorganisms from biofilms can often cause life-threatening infections [
5,
8,
10,
11]. Commonly used antibiotics impose increased selective pressure for multi-drug resistance and often lack selectivity, affecting both pathogenic and commensal bacteria’s biochemical and physiological functions [
5]. Hence, there is an urgent demand for safe and effective alternatives that can either substitute or complement existing antibiotics while remaining unchallenged by bacterial resistance [
4,
5]. Given that more than 70 years ago, before the advent of antibiotics, 22% of medicinal drugs originated from herbs, nowadays there is a renewed interest in exploring the antibacterial activity of plants, their essential oils, extracts, and secondary metabolites [
2]. It has been reported that phytoconstituents such as phenolics, coumarins, terpenoids, and alkaloids have various mechanisms of action, among which are the disturbance of the integrity of the bacterial membrane; inactivation of bacterial proteins, adhesins, and other enzymes; blocking of cell-to-cell signalization; and also the inhibition of biofilm formation and the promotion of biofilm degradation [
9,
12,
13].
Essential oils from Lamiaceae representatives have previously been studied for their antibacterial potential. It has been demonstrated that they can, independently or in combination with existing antibiotic drugs, inhibit bacterial cell growth, diminish the virulence of highly resistant strains such as MRSA (Methicillin-resistant
Staphylococcus aureus), and both hinder the formation and induce the degradation of pre-existing biofilms in specific bacteria [
14,
15,
16,
17,
18,
19,
20,
21]. However, to this day, there is a limited amount of research available on the antibacterial mechanisms of extracts derived from these plants. Hence, this study aimed to determine the antibacterial potential of methanolic, ethanolic, and aqueous extracts derived from 18 aromatic and medicinal Lamiaceae plant species traditionally used in Serbia by employing a multi-tier study design. Firstly, the total contents of coumarins and triterpenes were determined. Afterwards, the lowest concentration of an extract that inhibited the bacterial growth (minimum inhibitory concentration—MIC) was assessed on four Gram-positive (
Bacillus subtilis,
Enterococcus faecalis,
Listeria innocua, and
Staphylococcus aureus) and three Gram-negative bacteria (
Escherichia coli,
Pseudomonas aeruginosa, and
Salmonella enterica subsp.
enterica serovar Typhimurium—
Salmonella typhimurium). Additionally, the effects of the ethanolic extracts on adhesion and invasion of
P. aeruginosa PAO1 during lung fibroblast infection (MRC-5 cells) were examined.
P. aeruginosa PAO1 was chosen as a model in this study, as it is the most commonly used strain for research, being highly resistant to existing antibiotics and disinfectants and particularly responsible for severe hospital infections, which makes its removal from hospital environments and the treatment of infected patients particularly difficult [
22,
23]. Subsequently, the efficacy of selected ethanolic extracts on bacterial biofilm was studied in terms of their impact on
P. aeruginosa PAO1 biofilm formation and degradation. The results were standardized using the Integrated Biomarker Response (IBR) technique to reveal the extract with the highest antibacterial potential.
3. 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 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 investigations [
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.
4. Materials and Methods
4.1. 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).
4.2. Chemicals and Reagents
Ethanol, glacial acetic acid, hydrochloric acid, and methanol were bought from Zorka Pharma, Šabac, Serbia. Rifampicin was obtained from Hemofarm, Belgrade, Serbia; lead acetate trihydrate was obtained from Superlab, Belgrade, Serbia; while streptomycin and gentamicin were obtained from Galenika, Belgrade, Serbia. Coumarin, crystal violet, DMSO (dimethyl sulfoxide), glucose, magnesium sulfate, sodium chloride, Triton X-100, trypan blue, tryptone, ursolic acid, and vanillin were purchased from Merck, Rahway, NJ, USA. DMEM 5523 (Dulbecco’s Modified Eagle Medium), FBS (Fetal Bovine Serum), and PBS (Phosphate-Buffered Saline) were purchased from Gibco, Invitrogen, Waltham, MA, USA, while perchloric acid was obtained from VWR, Radnor, PA, USA. Agar, Brain-HeartInfusion (BHI), and yeast extract were obtained from Lab M Ltd., Neogen, Heywood, UK. Resazurin sodium salt (>90% (LC)) was bought from SERVA Electrophoresis GmbH, Heidelberg, Germany, while penicillin–streptomycin solution was purchased from PAA Laboratories GmbH, Pasching, Austria. Muller–Hinton Broth (Himedia, MHB, Maharashtra, India) was obtained from Biomedics, Málaga, Spain.
4.3. 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.
4.4. 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; R2 = 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.
4.5. 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, R2 = 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.
4.6. 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 (OD600) was adjusted to 0.4 using an MHB medium, resulting in a bacterial concentration of 108 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 cm2 flasks, and 1 × 105 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% CO2 for 48 h to achieve a confluent monolayer.
4.8. 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 (105 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 bacteria-to-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.
4.9.2. 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.
4.11. 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).
4.12. 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.