Synergistic Activity of Thymol with Commercial Antibiotics against Critical and High WHO Priority Pathogenic Bacteria

Citation: Gan, C.; Langa, E.; Valenzuela, A.; Ballestero, D.; Pino-Otín, M.R. Synergistic Activity of Thymol with Commercial Antibiotics against Critical and High WHO Priority Pathogenic Bacteria. Plants 2023,12, 1868. https:// doi.org/10.3390/plants12091868 Academic Editor: Despina Vokou Received: 9 March 2023 Revised: 28 April 2023 Accepted: 29 April 2023 Published: 2 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Synergistic Activity of Thymol with Commercial Antibiotics against Critical and High WHO Priority Pathogenic Bacteria Cristina Gan , Elisa Langa, Antonio Valenzuela, Diego Ballestero and M. Rosa Pino-Otín * Faculty of Health Sciences, Universidad San Jorge, 50830 Villanueva de Gállego, Zaragoza, Spain; [email protected] (C.G.); [email protected] (E.L.) *Correspondence: [email protected] Abstract: The use of synergistic combinations between natural compounds and commercial antibiotics may be a good strategy to fight against microbial resistance, with fewer side effects on human, animal and environmental, health. The antimicrobial capacity of four compounds of plant origin (thymol and gallic, salicylic and gentisic acids) was analysed against 14 pathogenic bacteria. Thymol showed the best antimicrobial activity, with MICs ranging from 125 µ g/mL (for Acinetobacter baumannii,Pasteurella aerogenes, and Salmonella typhimurium) to 250 µ g/mL (for Bacillus subtilis,Klebsiella aerogenes,Klebsiella pneumoniae,Serratia marcescens,Staphylococcus aureus, and Streptococcus agalactiae). Combinations of thymol with eight widely used antibiotics were studied to identify combinations with synergistic effects. Thymol showed synergistic activity with chloramphenicol against A. baumannii (critical priority by the WHO), with streptomycin and gentamicin against Staphylococcus aureus (high priority by the WHO), and with streptomycin against Streptococcus agalactiae, decreasing the MICs of these antibiotics by 75% to 87.5%. The kinetics of these synergies indicated that thymol alone at the synergy concentration had almost no effect on the maximum achievable population density and very little effect on the growth rate. However, in combination with antibiotics at the same concentration, it completely inhibited growth, confirming its role in facilitating the action of the antibiotic. The time–kill curves indicated that all the combinations with synergistic effects were mainly bactericidal. Keywords: thymol; antibiotics; synergy; Staphylococcus aureus;Streptococcus agalactiae;Acinetobacter baumannii; natural product 1. Introduction The discovery of antibiotics (ABXs) was a true revolution for public health, and has saved millions of lives. However, their excessive consumption and irrational use have led to their dispersion in the environment and the emergence of ABX-resistant bacteria [ 1 ]. The World Health Organization (WHO) has declared that the emergence of multidrug-resistant (MDR) pathogens is one of the greatest threats to global health, food security, and development [ 2 ]. In recent decades, the consumption of ABXs has continued to grow. Between 2000 and 2010, ABX drug consumption increased by 36% (from 54,083,964,813 standard units in 2000 to 73,620,748,816 standard units in 2010) worldwide, with aminoglycosides as one of the most used [ 3 ]. This enormous quantity of ABXs, once consumed, passes into the wastewater where, in the best of cases, it reaches sewage treatment plants that do not eliminate these residues [ 4 ], and discharge them into watercourses. Levels in the ng/L range have been detected in effluents containing high concentrations of most ABXs that we study here, e.g., chloramphenicol (CHL) [ 5 ]. Some of these wastes become part of the sludge from wastewater treatment plants and end up being applied to soils as fertilizers [ 6 ]. ABXs have been detected in soils at different concentrations ranging from ng/kg to mg/kg [ 7 ]. For example, a concentration of 5.6 ng/kg of streptomycin (STM) was reported in US sandy loam soil after the addition of manure [ 8 ]. All this leads to a large dissemination of ABX residues in the environment, which will facilitate the selective pressure and the spread of Plants 2023,12, 1868. https://doi.org/10.3390/plants12091868 https://www.mdpi.com/journal/plants Plants 2023,12, 1868 2 of 22 resistance genes [ 9 ]. Resistance can arise from mutations that alter the bacterial molecular targets of the ABX. The difficulty in treating infections due to MDR pathogens makes it urgent to search for new antimicrobial substances with different mechanisms of action capable of producing less resistance and, if possible, with fewer side effects on human, animal, and environmental health, in line with the “One Health” strategy. The WHO has also developed an action plan to combat MDR strains, and one of the key points is the development of new antimicrobial products [ 10 ]. Therefore, many studies have focused on prospecting natural products to find new potential antimicrobial agents [11]. Many natural products from plants, especially essential oils (EOs), have been explored for the treatment and prevention of MDR bacteria [ 12 , 13 ]. Unfortunately, natural products usually have weaker antibiotic activity than common ABXs; therefore, it is difficult for them to effectively replace current ABXs in clinical practice. However, some plant-derived antimicrobial compounds have been shown to synergistically enhance antibiotic activity [ 14 ]. The synergistic interaction of natural compounds with already available ABXs may allow for the combination to be as effective as the ABX alone, and while maintaining the use of commercial ABXs, it lowers the minimum inhibitory concentration (MIC) of both the ABX and the natural product [15]. The use of lower concentrations of both agents offers important opportunities in the search for alternatives to the treatment of infectious diseases, as combinations with synergistic effects may reduce the probability of the emergence of bacterial resistance [16] while having effective pharmacological results [ 17 ]. Moreover, it may involve a reduction in ABX toxicity [ 18 ] with fewer side effects compared to those derived from high doses of synthetic drugs [19]. Thymol (2-isopropyl-5-methylphenol, THY) is one of the main phenolic monoterpenes found in EOs extracted from plants belonging to the Lamiaceae family, such as those of the genera Thymus,Ocimum,Origanum,Satureja,Thymbra, and Monarda [ 20 – 23 ]. It has a molecular weight of 150.22 g/mol and a solubility of 900 mg/L [ 24 ] and logP (o/w) = 3.3 [25] , which indicates that it is a slightly water-soluble compound. Moreover, its pKa = 10.6 [ 26 ], which indicates that it is a molecule that at physiological pH 7.4 will be non-ionized. Essential oils of these plants have demonstrated antimicrobial properties primarily attributed to their main components, THY [ 20 , 21 ] among them. THY exhibits broad bioactivity [ 27 ]; especially, its antimicrobial activity has been quantitatively assessed on Escherichia coli, Staphylococcus aureus,Listeria monocytogenes, and Bacillus subtilis [ 28 – 31 ]. However, the antimicrobial effect of THY in combination with commercial ABXs has been much less explored. Other authors [ 14 ] have studied the interaction of THY with ampicillin, bacitracin, erythromycin, and penicillin in four ABX-resistant bacteria, finding synergistic effects in several cases, as with Salmonella typhimurium combined with ampicillin, tetracycline, penicillin, or erythromycin. Other authors have found synergistic activity between THY and other ABXs, such as vancomycin against E. coli [ 31 ], and antibiofilm activity in combinations of THY with three aminoglycosides against Klebsiella pneumoniae [ 32 ]. These studies indicate that THY presents favourable characteristics to be used in combination with ABXs in the treatment of infectious diseases, but their interaction with most of the ABXs used, as well as the synergistic effects on the numerous pathogenic bacteria of major clinical interest, have not yet been studied. The European Commission considers THY a low-risk product in consumption, and it is tested for use as a food flavouring. The Food and Drug Administration (FDA) has further classified THY as “generally safe” [27]. Another interesting group of plant secondary metabolites are the hydroxybenzoic acids, which are phenolic compounds characterized by an aromatic ring with an acid group and one or more hydroxyl groups. Among the representatives of this chemical family are salicylic acid (2-hydroxybenzoic acid, SA), gentisic acid (2,5-dihydroxybenzoic acid, GEA), and gallic acid (3,4,5-trihydroxybenzoic acid, GA). SA is a natural product that is frequently used in cosmetics because of its ability to promote exfoliation and of its anti-inflammatory and topical antibacterial activity [ 33 ]. Antibacterial activity against various bacterial strains, such as E. coli and S. aureus [ 34 ], has also been demonstrated. Similar to SA, GEA also Plants 2023,12, 1868 3 of 22 exhibits antimicrobial activity against both Gram-positive and negative bacteria [ 35 ], and has antiarrhythmic, antirheumatic, analgesic, and anti-inflammatory properties [ 33 ]. GA has antioxidant, antimelanogenic [ 36 ], and antimicrobial properties, with demonstrated activity against Enterococcus faecalis,S. aureus,E. coli, and Pseudomonas aeruginosa among others [37]. The aim of this study is to explore combinations of natural products of plant origin with commercial ABXs in search of the ones with synergistic effects and with lower required doses of the ABX. For this purpose: (1) the MIC of four natural products and eight widely consumed ABXs are studied against 14 microbial strains responsible for numerous human and veterinary diseases and food spoilage; (2) from the natural products with the lowest MIC, combinations with ABXs are studied to identify synergistic combinations. For this purpose, bactericidal and bacteriostatic synergistic effects are quantified and the growth kinetics and time–kill curves of bacteria exposed to the most promising natural product/ABX combinations are analysed. Bacterial types were selected based on their clinical interest, as they cause some of the most common infections today [ 38 , 39 ], and on their potential severity and ability to generate resistance, according to the WHO’s list of priority pathogens [40]. 2. Results 2.1. Antimicrobial Properties of Natural Products The antibacterial activity of THY, GA, SA, and GEA against 14 microorganisms is shown in Table 1. THY had strong antimicrobial effects (See Material and Methods for the qualitative evaluation of the antimicrobial activity of the natural products tested) against seven out of the nine Gram-negative bacteria tested and against three out of the five Gram-positive bacteria, at concentrations below or equal to 500 µ g/mL. The lowest MICs were 125 µ g/mL for the Gram-negative Acinetobacter baumannii,Pasteurella aerogenes, and S. typhimurium. The values of the ratio between the minimum bactericidal concentration (MBC) and the MIC of THY showed that the activity was bactericidal in all cases (MBC/MIC ≤ 4) [ 41 – 43 ]. THY had higher MBC/MIC ratios for the Gram-positive cocci. SA was the second most bioactive natural product, showing weak antibacterial activity against 12 out of the 14 bacteria tested, with MIC values between 1000 and 5000 µ g/mL. P. aerogenes was the most sensitive strain to this compound (MIC = 625 µ g/mL); the same strain was also the most sensitive with THY and GA. GEA and GA exhibited low to no antibacterial activity, with MIC values ranging between 1250 and 5000 µ g/mL, and between 2500 and 5000 µ g/mL, respectively. According to the MBC/MIC index, all three acids exhibited bactericidal activity. The MICs of the ABXs are given in Table 2. These concentrations will be used to calculate the fractional inhibitory concentration index (FIC I ) in the combinations with synergistic effects. 2.2. Synergies between Thymol and Antibiotics The FIC I s of the combinations of THY with the ABXs from the checkerboard test are shown in Table 3. The corresponding isobolograms of the combinations that showed one or more interactions with a FIC I≤ 0.5 are shown in Figure 1. Among the 30 combinations of THY with the ABXs, four showed synergism (FIC I≤ 0.5), 14 showed additivity (0.5 < FIC I≤ 1), and 12 showed no interaction (1 < FIC I < 2). None of the combinations showed antagonistic effects (FICI≥2). Two of the most pronounced results were obtained with the combination of THY and STM against S. aureus, and THY and CHL against A. baumannii, both showing a significant synergistic effect (FIC I = 0.375) and achieving an ABX dose reduction from 62.5 to 7.8 µg/mL (ABX dose reduction of 87.5%). Two other very promising results, with a four-fold dose reduction of ABX (75% dose reduction), were observed with THY and gentamycin (GTM) against S. aureus (FIC I = 0.375), and THY and STM against S. agalactiae (FIC I = 0.5). For all the other combinations tested, there were either additive effects or no interaction of the compounds (Table 3). Plants 2023,12, 1868 4 of 22 Table 1. Sensitivity of the microorganisms to the natural products examined. Microorganism Thymol Gallic Acid Salicylic Acid Gentisic Acid MIC MBC MBC/MIC MIC MBC MBC/MIC MIC MBC MBC/MIC MIC MBC MBC/MIC Acinetobacter baumannii ATCC 19606 125 250 2 5000 5000 1 1250 1250 1 2500 2500 1 Bacillus subtilis ATCC 6633 250 500 2 5000 5000 1 1250 1250 1 5000 5000 1 Enterococcus faecalis ATCC 19433 >1000 >1000 - >5000 >5000 - 1250 1250 1 2500 2500 1 Escherichia coli ATCC 25922 500 500 1 >5000 >5000 - 1250 >1250 - 5000 >5000 - Klebsiella aerogenes ATCC 13048 250 250 1 5000 5000 1 1250 1250 1 5000 5000 1 Klebsiella pneumoniae C6 250 250 1 >5000 >5000 - 1250 1250 1 5000 5000 1 Listeria monocytogenes ATCC 7644 >1000 >1000 - >5000 >5000 - 1250 1250 1 2500 2500 1 Pasteurella aerogenes ATCC 27883 125 125 1 5000 5000 1 625 >1250 - 1250 1250 1 Proteus mirabilis ATCC 35659 - - - 5000 5000 1 >1250 >1250 - 5000 5000 1 Pseudomona aeruginosa ATCC 27853 >1000 >1000 - 5000 >5000 - >1250 >1250 - 5000 >5000 - Salmonella typhimurium ATCC 13311 125 125 1 5000 5000 1 1250 1250 1 2500 2500 1 Serratia marcescens ATCC 13880 250 250 1 5000 5000 1 1250 1250 1 2500 2500 1 Staphylococcus aureus ATCC 9144 250 1000 4 >5000 >5000 - 1250 >1250 - 5000 >5000 - Streptococcus agalactiae ATCC 12386 250 1000 4 2500 2500 1 1250 1250 1 1250 1250 1 Concentration is given in µg/mL; -: insufficient data or test not carried out due to incompatibility with solvents. Table 2. MIC values (µg/mL) of the commercial antibiotics that were examined. Microorganism Amoxicillin Ampicillin Chloramphenicol Erythromycin Gentamycin Penicillin G Streptomycin Tetracycline A. baumannii 250 250 62.5 15.6 15.6 500 250 0.8 B. subtilis 0.3 0.3 1.9 0.5 7.8 1.3 15.6 1.6 E. coli 7.8 7.8 7.8 250 31.3 - 125 0.8 K. aerogenes >500 >500 31.3 62.5 0.8 - 3.9 2 K. pneumoniae 250 125 7.8 62.5 3.1 - 7.8 0.5 P. aerogenes >500 >500 7.8 >500 6.3 - 7.8 7.8 S. agalactiae 0.2 0.2 15.6 0.5 7.8 0.2 62.5 0.2 S. aureus 0.6 0.2 31.3 0.6 15.6 1.3 62.5 62.5 S. marcescens 125 125 125 250 6.3 - 0.5 125 S. typhimurium 3.9 3.9 15.6 31.3 0.8 - 31.3 0.5 -: not tested. Plants 2023,12, 1868 5 of 22 Table 3. FICIvalues of thymol—antibiotics combinations. Microorganism Commercial ABX MIC THY in Combination MIC ABX in Combination FICI* Interpretation A. baumannii AMO 62.5 125 1 Additivity AMP 62.5 62.5 0.75 Additivity CHL 31.3 7.8 0.375 Synergy ERY 125 15.6 2 No interaction GTM 62.5 1 0.56 Additivity PEN 125 500 2 No interaction STM 62.5 125 1 Additivity B. subtilis STM 125 7.8 1 Additivity E. coli ERY 250 7.8 0.53 Additivity GTM 250 1.9 0.56 Additivity STM 250 7.8 0.56 Additivity K. aerogenes CHL 15.6 15.6 0.56 Additivity ERY 250 62.5 2 No interaction K. pneumoniae AMO 250 250 2 No interaction AMP 250 125 2 No interaction ERY 250 62.5 2 No interaction S. agalactiae CHL 250 15.6 2 No interaction STM 62.5 15.6 0.5 Synergy S. aureus CHL 250 31.3 2 No interaction GTM 31.3 3.9 0.375 Synergy STM 62.5 7.8 0.375 Synergy TC 250 62.5 2 No interaction S. marcescens AMO 250 125 2 No interaction AMP 250 125 2 No interaction CHL 125 62.5 1 Additivity ERY 125 125 1 Additivity TC 250 125 2 No interaction S. typhimurium CHL 62.5 3.9 0.75 Additivity ERY 62.5 15.6 1 Additivity STM 62.5 15.6 1 Additivity Concentration is given in µg/mL; * FICIvalues are calculated according Equation (1). As shown in Figure 1a, the synergy of THY and GTM against S. aureus presented two points of synergistic interaction, with FIC I values of 0.375 and 0.5 (in both cases, the ABX concentration was reduced to 3.9 µ g/mL). The combination of THY with STM (Figure 1b,c) showed only one point of synergistic interaction when tested against both S. aureus and S. agalactiae (points above or below the lower dotted line). The reduction of STM concentration was greater in the case of S. aureus. Figure 1d shows how the combination of THY and CHL produced two interaction points with FIC I = 0.375, one with a reduction of CHL to 7.8 µ g/mL and the other to 15.6 µ g/mL. In the cases where two combinations had the same FICI, the one with the highest ABX reduction in its MIC was chosen for the kinetic tests. Plants 2023,12, 1868 6 of 22 Plants 2023, 12, x FOR PEER REVIEW 5 of 23 FICI ≤ 1), and 12 showed no interaction (1 > FICI < 2). None of the combinations showed antagonistic effects (FICI ≥ 2). Figure 1. Isobolograms (blue solid line) of THY interactions with (a) GTM; (b) STM; (c) STM; (d) CHL that include synergistic effects. The THY concentration is represented on the x-axis and the different ABX concentrations on the y-axis. The MIC values are located on the respective axes (points where the isobologram intersects the coordinate axes). The straight “addition line” (upper dashed line), allows for the distinction of additive effects (above the straight line or in its immediate vicinity) from synergistic effects (concave isoboles below the line). It also has a line representing the synergy edge (lower doed line). The points above or below the laer line represent synergistic combinations. Table 3. FICI values of thymol—antibiotics combinations. Microorganism Commercial ABX MIC THY in Combination MIC ABX in Combination FICI * Interpretation A. baumannii AMO 62.5 125 1 Additivity AMP 62.5 62.5 0.75 Additivity CHL 31.3 7.8 0.375 Synergy ERY 125 15.6 2 No interaction GTM 62.5 1 0.56 Additivity PEN 125 500 2 No interaction STM 62.5 125 1 Additivity B. subtilis STM 125 7.8 1 Additivity E. coli ERY 250 7.8 0.53 Additivity GTM 250 1.9 0.56 Additivity STM 250 7.8 0.56 Additivity K. aerogenes CHL 15.6 15.6 0.56 Additivity ERY 250 62.5 2 No interaction K. pneumoniae AMO 250 250 2 No interaction AMP 250 125 2 No interaction ERY 250 62.5 2 No interaction Figure 1. Isobolograms (blue solid line) of THY interactions with ( a ) GTM; ( b ) STM; ( c ) STM; ( d ) CHL that include synergistic effects. The THY concentration is represented on the x-axis and the different ABX concentrations on the y-axis. The MIC values are located on the respective axes (points where the isobologram intersects the coordinate axes). The straight “addition line” (upper dashed line), allows for the distinction of additive effects (above the straight line or in its immediate vicinity) from synergistic effects (concave isoboles below the line). It also has a line representing the synergy edge (lower dotted line). The points above or below the latter line represent synergistic combinations. 2.3. Synergy Kinetics Study and Time–Kill Curves Figures 2a, 3a, 4a and 5a illustrate the growth kinetics of the synergistic combinations (blue line). The growth kinetics of ABXs alone (red lines) and THY alone (green lines), at different concentrations, are also shown. The curves have a greater colour intensity at higher concentrations (the darkest curve is the MIC concentration and the lightest one represents the synergistic concentration) for both ABX and THY. The control is represented by a black line. Cmax, r, and Tm50 values are included in a table below the graphs to better characterize the growth kinetics curves. Figures 2b, 3b, 4b and 5b show the time–kill curves that present the mortality of the bacteria along the growth kinetics. As can be seen in all of the growth kinetics curves of the synergies (as well as those of the respective MICs of ABXs and THY), there was complete growth inhibition, so these curves are plotted horizontally on the x-axis. The kinetic study of THY and GTM synergy against S. aureus is shown in Figure 2. Treatment with GTM at the synergistic concentration caused a decrease in the growth rate of S. aureus (Figure 2a), causing a delay in the exponential growth phase. Although exposure to THY alone at the synergistic concentration had little effect on the growth rate (r, Tm50) or on the maximum growth (Cmax) of the bacteria, it contributed to enhancing the effect of the ABX when combined, as the synergistic combination produced a total inhibition of growth over the 24 h studied. Figure 2b shows how at 6 h, the combination (blue line) had already killed a large part of the bacterial population, resulting in a reduction in the bacterial population of approximately 5 log 10 CFU/mL compared to the control, and 2.9 log 10 CFU/mL compared to GTM. This confirms the bactericidal effect of the combination and its synergistic effect. Plants 2023,12, 1868 7 of 22 Plants 2023, 12, x FOR PEER REVIEW 7 of 23 effect of the ABX when combined, as the synergistic combination produced a total inhibition of growth over the 24 h studied. Figure 2b shows how at 6 h, the combination (blue line) had already killed a large part of the bacterial population, resulting in a reduction in the bacterial population of approximately 5 log10 CFU/mL compared to the control, and 2.9 log10 CFU/mL compared to GTM. This confirms the bactericidal effect of the combination and its synergistic effect. Figure 2. Kinetic assays and Cmax, r, and Tm50 values of THY (greenish curves) and GTM (reddish curves) alone and in combination (blue curves) against S. aureus; the darker the colour of the curve, the higher the concentration applied for the two compounds when tested alone. Black curves correspond to the control. (a) Growth kinetics assay. -: values achieved outside the studied range. Error bars are standard deviations (n = 4). (b) Time–kill curves. Error bars are standard deviations (n = 3). The kinetics of THY and STM against S. aureus (Figure 3a) indicates that both products at the synergistic concentration decrease the bacterial growth but they have lile effect on bacterial Cmax values. At half the MIC, both products markedly slowed the growth rate of the bacteria, and neither product reached the stationary phase after 24 h. In Figure 3b, we can see that at 24 h, the combination produced a decrease in survivors of 8.29 log10 Figure 2. Kinetic assays and Cmax, r, and Tm50 values of THY (greenish curves) and GTM (reddish curves) alone and in combination (blue curves) against S. aureus; the darker the colour of the curve, the higher the concentration applied for the two compounds when tested alone. Black curves correspond to the control. ( a ) Growth kinetics assay. -: values achieved outside the studied range. Error bars are standard deviations (n = 4). (b) Time–kill curves. Error bars are standard deviations (n = 3). Plants 2023,12, 1868 8 of 22 Plants 2023, 12, x FOR PEER REVIEW 8 of 23 CFU/mL compared to the control, and 8.14 log10 CFU/mL compared to STM, thus demonstrating the bactericidal and synergistic effects, respectively. Figure 3. Kinetic assays and Cmax, r, and Tm50 values of THY (greenish curves) and STM (reddish curves) alone and in combination (blue curves) against S. aureus; the darker the colour of the curve, the higher the concentration applied for the two compounds when tested alone. Black curves correspond to the control. (a) Growth kinetics assay. -: values achieved outside the studied range. Error bars are standard deviations (n = 4). (b) Time–kill curves. Error bars are standard deviations (n = 3). Figure 4 shows the kinetics of THY and STM against S. agalactiae. The synergistic combination produced a total inhibition of growth throughout the 24 h studied (Figure 4a), whereas both products alone only slightly affected the Cmax (curves very similar to the control). If we look at the synergy curve (blue) in Figure 4b, it can be seen that the combination was able to kill bacteria very quickly (4 h), with a reduction in the bacterial population of 3.6 log10 CFU/mL compared to the control, and 3.17 log10 CFU/mL compared to STM. Figure 3. Kinetic assays and Cmax, r, and Tm50 values of THY (greenish curves) and STM (reddish curves) alone and in combination (blue curves) against S. aureus; the darker the colour of the curve, the higher the concentration applied for the two compounds when tested alone. Black curves correspond to the control. ( a ) Growth kinetics assay. -: values achieved outside the studied range. Error bars are standard deviations (n = 4). (b) Time–kill curves. Error bars are standard deviations (n = 3). Plants 2023,12, 1868 9 of 22 Plants 2023, 12, x FOR PEER REVIEW 9 of 23 Figure 4. Kinetic assays and Cmax, r, and Tm50 values of THY (greenish curves) and STM (reddish curves) alone and in combination (blue curves) against S. agalactiae; the darker the colour of the curve, the higher the concentration applied for the two compounds when tested alone. Black curves correspond to the control. (a) Growth kinetics assay. -: values achieved outside the studied range. Error bars are standard deviations (n = 4). (b) Time–kill curves. Error bars are standard deviations (n = 3). The growth curves of A. baumannii are shown in Figure 5. The results show that both compounds at sub-MIC concentrations affected the Cmax of the bacteria in a concentration-dependent manner (Figure 5a). The time–kill curve of the synergy (Figure 5b) revealed a reduction in the bacterial population of 8.15 log10 CFU/mL in comparison to the control, and 7.42 log10 CFU/mL in comparison to CHL at 24 h. Figure 4. Kinetic assays and Cmax, r, and Tm50 values of THY (greenish curves) and STM (reddish curves) alone and in combination (blue curves) against S. agalactiae; the darker the colour of the curve, the higher the concentration applied for the two compounds when tested alone. Black curves correspond to the control. ( a ) Growth kinetics assay. Error bars are standard deviations (n = 4). (b) Time–kill curves. Error bars are standard deviations (n = 3). The kinetics of THY and STM against S. aureus (Figure 3a) indicates that both products at the synergistic concentration decrease the bacterial growth but they have little effect on bacterial Cmax values. At half the MIC, both products markedly slowed the growth rate of the bacteria, and neither product reached the stationary phase after 24 h. In Figure 3b, we can see that at 24 h, the combination produced a decrease in survivors of 8.29 log 10 CFU/mL compared to the control, and 8.14 log 10 CFU/mL compared to STM, thus demonstrating the bactericidal and synergistic effects, respectively. Figure 4shows the kinetics of THY and STM against S. agalactiae. The synergistic combination produced a total inhibition of growth throughout the 24 h studied (Figure 4a), whereas both products alone only slightly affected the Cmax (curves very similar to the control). If we look at the synergy curve (blue) in Figure 4b, it can be seen that the combination was able to kill bacteria very quickly (4 h), with a reduction in the bacterial Plants 2023,12, 1868 16 of 22 M07-A9 (2018). In order to achieve a more accurate measurement of microbial growth, the absorbance of each well was also measured at 625 nm using a microplate reader. Natural product activity was classified as strong (<400 µ g/mL), moderate (400–800 µ g/mL), or weak (>800 µ g/mL) [ 44 ]. In addition, for natural products, the MBC was also studied; this is defined as the lowest concentration at which all bacteria are killed. For its determination, a 10 µ L aliquot was taken from each non-growth column of the incubated 96-well plates and inoculated onto an agar plate. The plates were subsequently cultured for 24 h at the optimal growth temperature for each bacterial strain (Table S1) and monitored for any growth. The MBC/MIC ratio determines the bactericidal or bacteriostatic effect of the product on a bacterium. Antimicrobial substances are considered to have bactericidal activity when MBC/MIC ≤4 [41–43]; therefore, in this study the same criterion was followed for THY. 4.4. Determination of the Product Combination Behaviour 4.4.1. Checkerboard Assays and Fractional Inhibitory Concentration Index Selection of the combinations to be examined (among all possible ones) was made according to the following criteria: (1) the natural product should have the strongest antimicrobial activity among the four tested; (2) the ABX should have a MIC > 10 µ g/mL (this increases the importance of reducing its effective dose than if it was already low). The checkerboard method was used to measure potential synergies [ 14 , 122 , 123 ] between THY (drug A) and the tested ABXs (drug B). For the microdilution checkerboard test, THY was serially diluted vertically from columns 1 to 7 of 96-well microtiter plates. The corresponding ABX was then serially diluted horizontally from rows A to G of the plate, both products starting with a stock dilution corresponding to four times the MIC obtained for that product against a specific bacterium. Next, the plates were inoculated with bacterial suspension adjusted to the McFarland standard prepared as discussed in Section 4.3. The plates were incubated at the optimal temperature for each strain (Table S1) for 24 h and then the absorbance (625 nm) was measured to evaluate the bacterial growth in the same way as described in Section 4.3. To test the type of interaction between the drug combinations, the FIC I was calculated for each combination, as follows [123,124]: FICI=FICA+FICB=MICA+B MICA +MICB+A MICB (1) where FIC A is the MIC of drug A (natural product) in the presence of the commercial ABX (drug B) (MIC A+B ) divided by the MIC of drug A alone (MIC A ). FIC B is the MIC of drug B in the presence of drug A (MIC B+A ) divided by the MIC of the drug B alone (MIC B ). According to the European Committee on antimicrobial susceptibility testing guidance [ 125 ], a FIC I value ≤ 0.5 indicates synergy; between 0.5 and 1 indicates additivity, whereas from >1 to 2, there is “no interaction” between the agents; FIC I values ≥ 2 imply antagonistic effects [126,127]. 4.4.2. Isobolograms An isobologram (Figure 1) is a representation of the interaction between two substances. Isobolograms have been used to display the results of the checkerboard tests [ 128 ]. Unlike the growth kinetics or time–kill curves, this representation allows for the study of the interaction of ABXs and the natural product at several tested concentrations. Only isobolograms that showed in the checkerboard test one or more interactions with a FICI≤0.5 have been plotted. 4.4.3. Growth Kinetics Tests For a better interpretation of the bacteriostatic effects of synergistic combinations (those with a FIC I≤ 0.5), growth kinetics tests were carried out. Bacterial cultures were adjusted to the McFarland standard, as previously described (Section 4.3). They were then exposed to different concentrations (MIC and sublethal concentrations) of natural Plants 2023,12, 1868 17 of 22 products, commercial ABX, and a combination of both (according to the results obtained in the checkerboard test) in a 96-well microplate. They were then incubated at the corresponding temperature for each bacterium and absorbance measurements were taken every hour for 24 h. The results were plotted as absorbance vs. time to obtain growth curves (Figures 2a, 3a, 4a and 5a). All experiments were performed in quadruplicate. Kinetic curves were fitted to a logistic model (Equation (2)) for sigmoid microbial growth [ 129 ] with the Excel Solver add-in (Microsoft 365): Absorbance =Cmax 1+eb−rt (2) where Cmax is the carrying capacity, meaning the maximum achievable population density, ris the intrinsic rate of the population increase, and bis a fitting parameter. Cmax,r, and Tm50 (time in which half of the carrying capacity is reached) were calculated to characterize the kinetics of the different curves (see Figures 2a, 3a, 4a and 5a). 4.4.4. Time–Kill Curves To study the bactericidal properties of the combinations, time–kill curves were obtained according to Hu et al. [ 126 ] (Figures 2b, 3b, 4b and 5b). To this end, bacterial cultures (adjusted to the McFarland standard, as previously described) were exposed to THY and ABXs (alone and in combination) to a final volume of 10 mL, at concentrations of the selected synergistic combinations. Control tubes without antimicrobial agents were also included. Bacterial cultures exposed to the different concentrations were incubated at 37 ◦ C for 24 h. Samples (100 µ L) were collected at 0, 2, 4, 6, and 24 h. Serial dilutions of each sample were then prepared from 10 −1 to 10 −7 , and 10 µ L of each dilution was seeded on agar plates in triplicate. Following overnight incubation at 37 ◦ C, the colonies were counted. The results were plotted as log 10 CFU (y-axis) vs. time (x-axis) to obtain the time–kill curves. A product was considered bactericidal when the decrease in the number of survivors was greater than 3 log 10 CFU/mL-fold compared to the control. In addition, synergy was defined as a ≥ 2 log 10 CFU/mL-fold decrease by the combination compared to the most active single agent [130]. 5. Conclusions In this study, the antimicrobial properties of THY and three other products of natural origin (GA, SA, and GEA) were assessed on 14 Gram-positive and Gram-negative pathogenic bacteria. THY proved active against 10 of them. THY also showed synergistic effects when combined with GTM, STM, and CHL, and the reaction of some of these against bacteria, were considered as critical (A. baumannii) and of high priority (S. aureus) by the WHO, reducing the MIC of these ABXs by 75% to 87.5%. The study of the growth kinetics together with the time–kill curves seems to indicate that the possible role of THY in the synergies is to facilitate the access of the ABX, probably by altering the bacterial envelope. The results presented in this work show that THY as a product to be explored as an RMA, which may allow for a reduction in the consumption of ABXs in clinical and veterinary settings. This could contribute to reducing their impact on the environment and the generation of resistance, in line with the One Health strategy. The identification and characterization of these synergies is the first step in a series to be made towards a healthier life and a safer environment. Although the ABXs are already marketed and THY is considered by the EPA and FDA as a safe product, the mode of application that would be the safest and most effective for treating human and veterinarian infections of these three bacteria is still a challenge. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12091868/s1, Table S1: Microorganisms’ reference and culture conditions according to ATCC datasheets for each microorganism. Figure S1: Chemical structures of the tested natural products. Plants 2023,12, 1868 18 of 22 Author Contributions: Conceptualization, M.R.P.-O.; methodology, C.G. and M.R.P.-O.; validation, C.G., E.L. and M.R.P.-O.; formal analysis, C.G.; investigation, C.G., A.V. and M.R.P.-O.; resources, M.R.P.-O.; data curation, C.G. and A.V.; writing—original draft preparation, C.G. and M.R.P.-O.; writing—review and editing, E.L., D.B. and M.R.P.-O.; visualization, C.G. and A.V.; supervision, E.L. and M.R.P.-O.; project administration, M.R.P.-O.; funding acquisition, M.R.P.-O. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Cátedra Novaltia, Universidad San Jorge and Foundation Sabadell. Data Availability Statement: Data is contained within the article or supplementary material. Conflicts of Interest: The authors declare no conflict of interest. References 1. Markowicz, A.; Bondarczuk, K.; Cycon, M.; Sulowicz, S. 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