Studies Regarding the Antimicrobial Behavior of Clotrimazole and Limonene

The paper presents the results of the studies performed to establish the effect of the mixtures between limonene and clotrimazole against microbial pathogens involved in dermatological diseases, such as Candida albicans, Staphyloccocus aureus, and Escherichia coli. Preliminary data obtained from the studies performed in microplates revealed a possible synergism between the mixture of clotrimazole and limonene for Staphylococcus aureus. Studies performed “in silico” with programs such as CLC Drug Discovery Workbench and MOLEGRO Virtual Docker, gave favorable scores for docking each compound on a specific binding site for each microorganism. The tests performed for validation, with the clotrimazole (0.1%) and different sources of limonene (1.9% citrus essential oils), showed a synergistic effect on Staphylococcus aureus in the case of the mixtures between clotrimazole and the essential oils of Citrus reticulata or Citrus paradisi. The studies performed on Staphylococcus aureus MRSA showed a synergistic effect between clotrimazole and the essential oils obtained from Citrus bergamia, Citrus aurantium, or Citrus paradisi. In the case of Pseudomonas aeruginosa, essential oils and clotrimazole used alone did not exhibit antimicrobial activities, but the mixtures between clotrimazole and the essential oils of Citrus bergamia or Citrus sinensis exhibited a synergistic antimicrobial effect.


Introduction
Citrus fruits generate large amounts of residues, generally made up of peels, seeds, and membranes. The amount of this waste exceeds 120 million tons annually worldwide. The recovery of these residues can be completed to obtain raw materials for the pharmaceutical, food, or biofuel industry [1]. Citrus peels contain essential oils, polyphenolic compounds, and carotenoids, the use of which has been recognized as safe by the U.S. Food and Drug Administration [1,2]. The most important products obtained from citrus peels are essential oils (EO). The EO content in limonene (1-methyl-4-(1-methyl ethenyl)-cyclohexene) can reach 97-98%. Limonene can be separated from citrus peels by microwave-assisted extraction (in the absence of solvents) or by extraction with supercritical fluids [2]. In medicine, citrus oil is important in treating bacterial infections with E. coli melanoma) [28]. Proapoptotic effects on melanoma tumor cell lines were and continue to be also reported by other researchers [29][30][31].
Despite the biological effects reported by the literature, clotrimazole's main disadvantages are its low solubility in aqueous media, and the adverse effects it can generate. Thus, the dermal administration may cause itching, hives, and even sensitivity to this antibiotic. When administered orally, this compound may disturb the synthesis of mesenchymal liver enzymes; in the case of intraperitoneal (i.p.) administration, it may cause a heart attack. Regarding the toxicity, the tests conducted on lab animals (rats and mice) indicate for the case of a single ingestion, a value for DL50 = 708 mg/kg, and, respectively, of 761 mg/kg. In the case of i.p. administration, the DL50 = 445 mg/kg and, respectively, 108 mg/kg. For these reasons, the use of clotrimazole is limited at topical administration [32,33].
Taking into account the results reported in the literature, it is estimated that the essential oils obtained from citrus residues (containing mainly limonene) and the antibiotic reagent named CT present interest in the management of dermatological diseases, due to the broad spectrum of action of the two components and to the possibility of these to act synergistically, thus being important in the actual context of increased resistance to antibiotics of pathogenic microorganisms.
The main objective of the studies performed was to establish if the mixture between limonene (and different natural sources which contain limonene) and clotrimazole can exhibit a different behavior against the main microbial pathogens involved in dermatological diseases. This main objective was accomplished during three types of investigations, made in vitro, on microplates; in silico with the help of docking programs such as CLC Drug Discovery Workbench and MOLEGRO Virtual Docker; and finally again in vitro, on Petri plates, with natural sources of limonene such as the essential oils from different citrus species.
In the experiments performed in vitro, on microplates, the main purpose was to evaluate the antimicrobial susceptibility of limonene, clotrimazole, and the mixtures between them, for three microorganisms: Candida albicans, Escherichia coli, and Staphylococcus aureus. The aim of the tests performed "in silico" was to establish the chemical interactions between limonene and/or clotrimazole with each specific binding site from Candida albicans, Escherichia coli, and Staphylococcus aureus, using two programs of docking studies: CLC Drug Discovery Workbench and MOLEGRO Virtual Docker. In the third experimental part, we aimed to test "in vitro" the antimicrobial action of some natural sources of limonene (different essential oils from citrus species) and mixtures of each one with clotrimazole for Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus aureus MRSA, and Pseudomonas aeruginosa. These tests have been performed in order to validate "the proof of concept" resulting from studies made on microplates and from docking studies performed "in silico". Finally, based on pieces of information obtained in the three types of studies, we will select the best natural sources of limonene, which can be used in pharmaceutical formulations with clotrimazole, with improved antimicrobial properties.

Reagents
Limonene (97%) was purchased from the local Merck Bucharest, Romania. Clotrimazole was used as a liquid solution with 10.87 mg CT/mL in polyethylene glycol (Biofarm, Bucharest, Romania).
Essential oils of Citrus sinensis, Citrus limon, Citrus aurantium, Citrus paradisi, Citrus reticulata (green and red), and Citrus bergamia were purchased from the Romanian market, and characterized by gas chromatography coupled with a mass spectrometer (GC-MS Table 1).

Microorganisms and Culture Media
Mueller Hinton broth (Oxoid) was used as a culture medium for Escherichia coli ATTC 11303, Staphylococcus aureus ATTC 25923, Staphylococcus aureus MRSA ATTC 33592, and Pseudomonas aeruginosa ATTC 1338. Potato dextrose broth (BD Difco Sigma Aldrich, Bucharest, Romania) was used as a liquid culture medium for Candida albicans ATTC 10231.
Experiments on Petri plates were conducted using fresh microorganisms (growth during 24 h for bacteria and 48 h in the case of C. albicans, growth on TSA (Tryptic soy agar, Sigma Aldrich. Bucharest, Romania), and PDA (potato dextrose agar, Sigma Aldrich, Bucharest, Romania)).

Devices
Optical density measurements were made on microplate reader type Dynatex at 600 nm (Dynex Technologies-MRS, Chantilly, VA, USA). The microplate was incubated at 37 • C (incubator type Cole Palmer H2200 Vernon Hills, IL, USA) in the case of bacteria, and, respectively, at 25 • C in the case of C. albicans. All experiments were made in laminar hood type Faster Bio 48, (Cornaredo, Italy).
A GC-MS/MS TRIPLE QUAD Agilent 7890 A (Santa Clara CA, USA) used in the analysis of the essential oil was equipped with a DB-WAX capillary column (30 m length, 0.25 mm internal diameter, 0.25 mm film thickness) and helium as the carrier gas at 1 mL/min. For the essential oils analysis, the oven temperature was initially set at 100 • C, held for 2 min, then increased gradually to 280 • C, with 4 • C per minute and 6 min holding time. The GC injector and MS ion source temperatures were 250 • C and 150 • C, respectively. The transfer line temperature was 280 • C. The MS detector was operated in EI mode at 70 eV, with an m/z scanning range of 50-450.

Experimental Design on Microplates
Solutions of limonene, CT, and a mixture of them, with concentrations in the range of (5000 ÷ 2.37) µg/mL, were made in Mueller Hinton broth and in potato dextrose broth, respectively, in 96-well plates. Each inoculum was made in a sterile physiological serum containing suspensions of S. aureus, E. coli, or C. albicans, prepared according to McFarland standard 0.5. The inoculation rate in each well was made using a volumetric report of 1:10. The optical density of each plate was measured by a plate reader device, without microorganism inoculum, and with microorganism inoculum, respectively, at the time 0. The plate was incubated for 24 h at 37 • C in the case of bacterial strains, and at 25 • C for 48 h in the case of fungal strains. After incubation, each plate's optical density (OD) was measured. Optical density at 24 h was assigned by the difference between OD at 24 h and 0 h and culture media without microbial inoculants. Each experimental variant was made in 3 repetitions.
The results are presented as an average value, with standard deviations. The total viable cells (UFC) after 24 h and, respectively, after 48 h were determined by taking out 100 µL from each well, diluting it properly, and spreading it out on the Petri plates; with a solid specific culture media (Mueller Hinton agar for bacteria and, respectively, potato dextrose agar for fungi); incubating at 37 • C in the case of bacteria and, respectively, at 24 • C for 48 h for C. albicans; counting the total number of colonies formed and reporting for each case. Results were reported as a log of colonies forming units (logCFU).

Docking Studies
Predictions regarding potential interactions between the selected active molecules and selected targets were achieved by molecular docking studies using the CLC Drug Discovery Workbench ( QIAGEN, Aarhus, Denmark) and MOLEGRO Virtual Docker programs (Molexus IVS, Odder, Denmark), respectively. Both approaches considered the occurring interactions between the native (co-crystallized) ligands, as references, specific to each studied microorganism, in complex with the selected receptor protein, imported from the International Protein Database (PDB Bank, PDB consortium). The studies performed with CLC Drug Discovery Workbench (QIAGEN Aarhus, Silkeborgvej 2, Prismet 8000, Aarhus C, Denmark) have been accomplished according to the following docking protocol [34]: ligand and protein preparation, setup binding site, docking simulations for the co-crystallized ligand, validation, docking of the investigation ligands, and results validation.
The results were given in terms of the docking score and hydrogen bonds created with the amino acids' residues from the amino acid group interaction within the active catalytic site. Thus, the obtained data have been used to predict the binding modes, the binding affinities, and the best orientation of the docked compounds in the active site of the protein receptor. Additionally, the molecular properties of the small molecules, such as parameters of Lipinski's rule of five [35], i.e., the molecular weight, number of hydrogen bond donors, number of hydrogen bond acceptors, and Log P (octanol-water partition coefficient), have been calculated. The studies performed with MOLEGRO have been accomplished according to the following docking protocol: importing the protein receptor from the PDB bank, preparation of the protein receptor, detection of cavities, search space setup, and docking simulation. The docking score and hydrogen bonds created with the amino acid residues from the group of interactions have been used to assess the binding modes and the orientation of the docked compounds in the active site of the protein receptor. In both approaches, the protein-ligand complex has been realized based on the Xray structure of Candida albicans Dihydrofolate Reductase, downloaded from the Protein Data Bank PDB ID: 1AI9 [36,37]; in the case of S. aureus, the protein-ligand complex has been realized based on the X-ray structure of Wild-type Staphylococcus aureus DHFR in complex with trimethoprim, downloaded from the Protein Data Bank (PDB ID: 2W9H) [36,38]. For Escherichia coli, the protein-ligand complex has been realized based on the X-ray structure of Escherichia coli K12 (protein Alpha-ketoglutarate-dependent dioxygenase AlkB), downloaded from the Protein Data Bank, PDB ID: 4JHT [36][37][38][39]. The two simulation programs considered the same co-crystallized (native ligand) as a reference, respectively: • in the case of C. albicans, the follow was considered as a ligand (co-crystallized): NADPH Dihydro-Nicotinamide-Adenine-Dinucleotide Phosphate (NDP); • in the case of S. aureus, the following was considered as a ligand: Trimethoprim (TOP); • in the case of E. coli, the following was considered as a ligand: 8-hydroxyquinoline-5-carboxylic acid (8XQ). The co-crystallized ligands introduced in the protein fragments chosen from the PDB bank also guided the docking simulations for the studied ligands (clotrimazole; limonene), in the same binding site. In the case of the selected protein fragments, the two programs validated the docking protocol, both for each co-crystallized (the reference ligands), and the selected ligands (limonene and clotrimazole).
During the simulations, the co-crystallized ligand was removed from the protein complex; in its place, the studied molecule was re-docked. Each program calculated the length of the detected hydrogen bonds and the docking score. The predictions were favorable to the initially assumed docking mechanism if values close to those of the natural ligand were obtained. Both programs detected the binding site and the binding pocket, complementary to a specific ligand.

Results Validation
Results validations were made on the Petri plates with Mueller Hinton culture media in the case of bacteria and, respectively, with PDA (potato dextrose agar) culture media in the case of fungi, using five microorganisms (Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus aureus MRSA, and Pseudomonas aeruginosa). The inoculum of each microorganism was prepared as a suspension in sterile saline solution (0.9% NaCl) using a fresh culture of each microorganism (fresh culture: 24-h-old bacteria and 48-h-old fungi), according to standard McFarland 0.5. The sterile inoculum of each microorganism was spread on the surface of each Petri plate with a cotton swab. After 20 min, in each Petri plate the cellulose discs with 0.6 mm diameter (3 or 5 cellulose discs for each treatment), impregnated with a solution that contained clotrimazole (0.1%, w/w), essential oils of citrus (1.9%), or a mixture of them at the same concentrations were added. All solutions were made with propylene glycol as a solvent. The Petri plates were incubated at 37 • C for 24 h (in the case of bacteria) and, respectively, for 48 h at 25 • C for Candida albicans.
The results obtained are presented as the average diameter of inhibition with a standard deviation. The methodology used in the study is presented in Figure 1.

Studies on Microplates Performed on E. coli
Studies performed on E. coli exposed to limonene reveal that this compound inhibits cell growth at concentrations in the range of (5000 ÷ 39) μg/mL (Tables 2 and 3). Due to this fact, at these concentrations, we considered that the studied microorganism is susceptible (S). At concentrations of (9.5 ÷ 2.37) μg/mL, the values of the optical densities (OD) are in the range of (0.05 ÷ 0.01) units, and we considered that, at these levels of limonene, the microorganism is intermediate (I) (or susceptible to increased exposure, according to EUCAST modification established in 2022 [40]). These observations are in agreement with results obtained by other researchers [3,8,9] regarding limonene activity against E. coli. In the case of exposure to E. coli at CT (Tables 2 and 3), the measurements made after 24 h showed that, at concentrations of CT in the range of (5000 ÷ 19) μg/mL, the development of cells is inhibited, and the microorganisms are considered to be susceptible. For CT concentrations in the range of (9.5 ÷ 4.75) μg/mL, the OD range is (0.08 ÷ 0.06) units, and we considered that, at these CT levels, the microorganism has intermediate susceptibility at concentrations of CT less than 4.75 μg/mL and the microorganisms are considered resistant (R).  Studies performed on E. coli exposed to limonene reveal that this compound inhibits cell growth at concentrations in the range of (5000 ÷ 39) µg/mL (Tables 2 and 3). Due to this fact, at these concentrations, we considered that the studied microorganism is susceptible (S). At concentrations of (9.5 ÷ 2.37) µg/mL, the values of the optical densities (OD) are in the range of (0.05 ÷ 0.01) units, and we considered that, at these levels of limonene, the microorganism is intermediate (I) (or susceptible to increased exposure, according to EUCAST modification established in 2022 [40]). These observations are in agreement with results obtained by other researchers [3,8,9] regarding limonene activity against E. coli. In the case of exposure to E. coli at CT (Tables 2 and 3), the measurements made after 24 h showed that, at concentrations of CT in the range of (5000 ÷ 19) µg/mL, the development of cells is inhibited, and the microorganisms are considered to be susceptible. For CT concentrations in the range of (9.5 ÷ 4.75) µg/mL, the OD range is (0.08 ÷ 0.06) units, and we considered that, at these CT levels, the microorganism has intermediate susceptibility at concentrations of CT less than 4.75 µg/mL and the microorganisms are considered resistant (R).   The results obtained are in agreement with those obtained by other scientists [41][42][43] who have reported similar biological activities of CT against E. coli. Under the influence of a mixture between limonene and CT (Tables 1 and 2), the concentration at which E. coli is susceptible ranges from 5000 µg/mL to 9.5. µg/mL; at concentrations less than/below 9.5. µg/mL, microorganisms are resistant. In this case, the concentration range for which E. coli is susceptible is larger than the case of using only limonene or CT. These results permit us to affirm that by using a mixture between limonene and CT in equal parts, the antibiotic reagent concentration at which the microorganism is susceptible can be lowered to 4.75 µg/mL.

Studies on Microplates Performed on S. aureus
Studies performed on S. aureus revealed that under the influence of limonene, the growth of the cells is inhibited at a concentration of 5000 µg/mL (Table 4, Figure 2), with the studied microorganism being susceptible at this value (Table 5).  When using 2500 µg/mL limonene, the OD value is 0.007, and at this value of concentration, the microorganism is considered with intermediate susceptibility. If the concentration of limonene in the culture medium is less than 2500 µg/mL, then the S. aureus cells are resistant. Regarding the number of CFU, its value decreases with one logarithmic unit to the concentration of 1250 µgL/mL ( Figure 2); at the level of 2500 µg/mL, the CFU decreases with 1.5 log units, and at 5000 µgL/mL all cells are inactivated. Regarding the mechanism of action, studies performed with limonene on S. aureus revealed that this compound inhibits bacterial growth by the destruction of cell wall integrity [9].
Following the exposure of S. aureus at CT the results showed that, at concentrations of CT in the range of (5000 ÷ 312) µg/mL, the development of cells is inhibited; hence, the microorganisms are susceptible. When concentrations of CT less than 312 µg/mL are used, the microorganism is resistant. Regarding the number of the CFU, its value decreases with one logarithmic unit to the concentration of 156 µgL/mL ( Figure 3); at concentrations greater than 156 µgL/mL, the CFU decreases with nine logarithmic units. When using 2500 μg/mL limonene, the OD value is 0.007, and at this value of concentration, the microorganism is considered with intermediate susceptibility. If the concentration of limonene in the culture medium is less than 2500 μg/mL, then the S. aureus cells are resistant. Regarding the number of CFU, its value decreases with one logarithmic unit to the concentration of 1250 μgL/mL ( Figure 2); at the level of 2500 μg/mL, the CFU decreases with 1.5 log units, and at 5000 μgL/mL all cells are inactivated. Regarding the mechanism of action, studies performed with limonene on S. aureus revealed that this compound inhibits bacterial growth by the destruction of cell wall integrity [9]. Following the exposure of S. aureus at CT the results showed that, at concentrations of CT in the range of (5000 ÷ 312) μg/mL, the development of cells is inhibited; hence, the microorganisms are susceptible. When concentrations of CT less than 312 μg/mL are used, the microorganism is resistant. Regarding the number of the CFU, its value decreases with one logarithmic unit to the concentration of 156 μgL/mL ( Figure 3); at concentrations greater than 156 μgL/mL, the CFU decreases with nine logarithmic units. Other scientists obtained similar results on commercial CT formulation [22,43,44]. Kalhapure et al. have reported that after 18 h of exposure to CT, a MIC = 31.25 µgL/mL is obtained for both S. aureus and S. aureus MRSA [22]. In experiments performed by the Kirby Bauer method, El-Halim et al. reported the antibacterial activity of CT solutions with concentrations in the range of (1000 ÷ 5000) µg/mL [41]. Analyzing the influence of the mixture between limonene and CT, the interval of concentration at which S. aureus is susceptible, is situated in the range of (5000 ÷ 625) µg/mL (Tables 4 and 5, Figure 4).
When concentrations of a mixture of (312 ÷ 78) µg/mL (Table 4) are used, OD ranges between 0.03 and 0.08 units, and we have considered that, at these concentrations, the microorganisms are intermediate (Table 4, Figure 4). If the concentration of the mixture is less than 78 µg/mL, the microorganism is resistant. Regarding the number of the CFU, its value decreases with one logarithmic unit to the concentration of 312 µg/mL ( Figure 4). If the mixture concentrations are greater than 312 µg/mL, the CFU decreases with 7 log units, and the cell growth is inhibited. Interestingly, under the influence of the mixture between limonene and CT, the CFU is lower compared to experimental variants in which each reagent is used alone. The mixture concentrations of (156 ÷ 78) µg/mL are interesting because if the reagents are used individually, then the microorganism is resistant (  Other scientists obtained similar results on commercial CT formulation [22,43,44]. Kalhapure et al. have reported that after 18 h of exposure to CT, a MIC = 31.25 μgL/mL is obtained for both S. aureus and S. aureus MRSA [22]. In experiments performed by the Kirby Bauer method, El-Halim et al. reported the antibacterial activity of CT solutions with concentrations in the range of (1000 ÷ 5000) μg/mL [41]. Analyzing the influence of the mixture between limonene and CT, the interval of concentration at which S. aureus is susceptible, is situated in the range of (5000 ÷ 625) μg/mL (Tables 4 and 5, Figure 4).
When concentrations of a mixture of (312 ÷ 78) μg/mL (Table 4) are used, OD ranges between 0.03 and 0.08 units, and we have considered that, at these concentrations, the microorganisms are intermediate (Table 4, Figure 4). If the concentration of the mixture is less than 78 μg/mL, the microorganism is resistant. Regarding the number of the CFU, its value decreases with one logarithmic unit to the concentration of 312 μg/mL ( Figure 4). If the mixture concentrations are greater than 312 μg/mL, the CFU decreases with 7 log units, and the cell growth is inhibited. Interestingly, under the influence of the mixture between limonene and CT, the CFU is lower compared to experimental variants in which each reagent is used alone. The mixture concentrations of (156 ÷ 78) μg/mL are interesting because if the reagents are used individually, then the microorganism is resistant (Table  5); however, in the presence of both reagents, the microorganism has intermediate susceptibility (I).  In the case of C. albicans, the obtained results revealed that, under the influence of limonene, the cell growth is inhibited at concentrations of 5000 ÷ 2500 μg/mL (Tables 6  and 7, Figures 5 and 6). For these concentrations, in our experimental model, the microor-

Studies on Microplates Performed on C. albicans
In the case of C. albicans, the obtained results revealed that, under the influence of limonene, the cell growth is inhibited at concentrations of 5000 ÷ 2500 µg/mL (Tables 6  and 7, Figures 5 and 6). For these concentrations, in our experimental model, the microorganism is susceptible (Table 7). At concentrations of limonene less than 2500 µg/mL, C. albicans is resistant. In our experimental model, the obtained values showed that the reduction of the CFU at limonene concentrations of (0 ÷ 1250) µg/mL is made slowly ( Figure 5).   [45]. Authors reported that at a concentration of 20 mM limonene in a culture medium, the microorganism growth, the biofilm development, and the biofilm maturation can be reduced substantially up to 90%. Nidhi and collab. have reported that, when using essential oils with 28% limonene obtained from Citrus aurantium (bitter orange), on two types of C. albicans, they obtained a MIC value of 0,15% and 0.31%, respectively [11]. Regarding the clotrimazole effect on C. albicans, in our experimental model, at concentrations in the range of (5000 ÷ 625) µg/mL, the microorganism is susceptible. At a CT level of 312 µg/mL, the microorganism has intermediate susceptibility (I) ( Tables 6 and 7). At this level of CT, in the system, the number of the CFU decreases with two logarithmic units ( Figure 6, Table 7).
For CT concentrations situated below 312 µg/mL, the C. albicans cells are resistant. Similarly, in experiments performed on C. albicans, other scientists have indicated MIC and MLC (minimum lethal concentration) values greater than 128 µg/mL [10,46]. Other researchers in studies performed on microorganisms implied in skin disease, with different antibiotic formulations from the market, revealed that the products with 1% CT are effective against infection with C. albicans [44,47]. In experiments performed with a mixture of (limonene + CT) on C. albicans, the results revealed that the microorganism is susceptible only at concentrations in the range of (5000 ÷ 2500) µg/mL (Tables 4 and 5). At concentrations situated below these values, C. albicans is resistant. Regarding the number of the CFU in the system, these values are relatively close in the range of CT concentrations ranging between 0 and 1250 µg/mL (Figure 7).     ent antibiotic formulations from the market, revealed that the products with 1% CT are effective against infection with C. albicans [44,47]. In experiments performed with a mixture of (limonene + CT) on C. albicans, the results revealed that the microorganism is susceptible only at concentrations in the range of (5000 ÷ 2500) μg/mL (Tables 4 and 5). At concentrations situated below these values, C. albicans is resistant. Regarding the number of the CFU in the system, these values are relatively close in the range of CT concentrations ranging between 0 and 1250 μg/mL (Figure 7).

Figure 7.
Influence of (CT + limonene) exposure on C. albicans growth, after 48 h. At a concentration of limonene + clotrimazole in the culture medium higher than 1250 μg/mL, C. albicans is inhibited.

Docking Studies Performed on C. albicans
The two programs validate a protein fragment of C. albicans for which the reference ligand is possible to be fitted, in the active binding site of 1AI9 (Figure 8a1,a2,b1,b2). In both prediction models, the docking score of the two ligands (clotrimazole; limonene) is lower (in absolute value) than the docking score of the co-crystallized (Table 8). In the Figure 7. Influence of (CT + limonene) exposure on C. albicans growth, after 48 h. At a concentration of limonene + clotrimazole in the culture medium higher than 1250 µg/mL, C. albicans is inhibited.

Docking Studies Performed on C. albicans
The two programs validate a protein fragment of C. albicans for which the reference ligand is possible to be fitted, in the active binding site of 1AI9 (Figure 8(a1,a2,b1,b2)). In both prediction models, the docking score of the two ligands (clotrimazole; limonene) is lower (in absolute value) than the docking score of the co-crystallized (Table 8). In the CLC model, the co-crystallized (NDP) creates a total of 16 hydrogen bonds as well as other interactions with amino acids of the binding site (Figure 8(a2,a3)), from which there are three bonds with ARG79, three bonds with ALA115, two bonds with THR 58, two hydrogen bonds with GLU 116, and two bonds with LYS 57. In the case of the studied ligands (clotrimazole, and limonene), the two prediction models do not detect the presence of hydrogen bonds (Table S1 in Supplementary), but they predict interactions with amino acids from the binding site (Figure 8(a4,a5)). The docking score of the studied compounds generated by the CLC prediction model in the absolute value decreases in the following order: limonene (docking score −41.94; RMSD: 0.01 Å) < clotrimazole (docking score: −57.87; RMSD: 0.10 Å) < co-crystallized NDP (docking score: −79.35; RMSD: 2.86 Å) ( Table 8).
In the case of clotrimazole, the model identifies a single hydrogen bond with THR ( Figure 8(b3)) and interactions between about 16 amino acids (Figure 8(b3-b5)) (details presented in the table from the Annex). For limonene, the predictive model does not identify hydrogen bonds but indicates the existence of interactions between 12 amino acids (Figure 8(b5)). The docking score obtained in this model, in absolute value, decreases in the following order: limonene (docking score −41.94; RMSD: 0.00 Å) < clotrimazole (docking score −77.75; RMSD: 0.00 Å) < co-crystallized NDP (docking score −155.24; RMSD: 0.00 Å).   Negative docking scores obtained in both models show the following aspects: 1.
The two ligands (limonene; clotrimazole) are fitting in the binding site predicted by the two models; 2.
Biological activities of the two ligands (clotrimazole; limonene) are similar but smaller than co-crystallized NDP; 3.
According to Lipinski's rule of five [35], the values generated by the CLC model for the three ligands studied (clotrimazole, limonene, and the co-crystallized) are more favorable for delivery into the cell for limonene and clotrimazole (molecular mass less than 500) in comparison with co-crystallized (molecular mass 739. 37 Da). In addition, the value obtained for the partition coefficient in octanol (logP) is more favorable for clotrimazole (logP = 3.36) and the co-crystallized (log P = −4.72) ( Table 9). Analyzing the value of the partition coefficient (logP) in the two ligands (Table 9), it can be appreciated that in the case of a mixture at low concentrations between the two compounds (i.e., clotrimazole; limonene), the presence of clotrimazole is determinant in transdermal delivery, due to the absence of the hydrogen bond [48][49][50][51], and more likely will be docked first.

Docking Studies Performed on S. aureus
In the case of studies performed in silico on S. aureus, the programs validate the binding site named 2W9H, in which the ligands' co-crystallized TOP, clotrimazole, or limonene are possible to be fitted (Figure 9(a1,b1)). In the CLC model (Table S2 in Supplementary, Figure 9(a1-a5)), the co-crystallized TOP creates a total of six hydrogen bonds, from which two bonds are with ASP27, two bonds with SER49, one bond with PHE 92, one bond with LEU5, and two bonds with LYS 57 (Table S2 in Supplementary, Figure 9(a2)). Here are the possible the interactions between the co-crystallized and the rest of the 18 amino acids from the binding site. Regarding clotrimazole and limonene, in the CLC model, clotrimazole generates one hydrogen bond with SER 49 (Figure 9(a3)) and 13 interactions with amino acids from the binding site (Figure 9(a4)); limonene can generate interactions with 15 amino acids from the binding site (Figure 9(a5)). The docking score of the studied compounds decreases in the following order: limonene (docking score −39,63; RMSD: 0.02 Å) > clotrimazole (docking score: −47.56.87; RMSD: 0.13 Å) > co-crystallized TOP (docking score: −53.12; RMSD: 0.35 Å). In the CLC model, clotrimazole can interact with 13 groups of amino acids, and limonene with 15 groups. In the Molegro model (Figure 9(b1-b5)), the co-crystallized generates interactions with the rest of the 18 amino acids of the binding site (Figure 9(b2)) and 8 hydrogen bonds, from which 4 bonds are with H 2 O, and 4 bonds with 4 amino acids. In the case of clotrimazole, the model identifies a single hydrogen bond with H 2 O 2092, and interactions between about 13 amino acids (tables from the Annex; Figure 9(b3,b4)). For limonene, the predictive model does not identify hydrogen bonds, but indicates the possibility of interactions between 11 amino acids (Figure 9(b5)). The docking score obtained in this model decreases in the following order: limonene (docking score −50.58; RMSD: 0.00 Å) > clotrimazole (docking score −59.82; RMSD: 0.00 Å) > cocrystallized TOP (docking score −100,53; RMSD: 0.00 Å). Negative docking scores obtained in both models suggest the following:

•
Both ligands (limonene, clotrimazole) can be fitted in the binding site predicted by the two models; • The partition coefficient is comparable with the co-crystallized (logP= 3.46) in the case of clotrimazole (logP = 3.36) (table from the Annex), but greater than TOP in the case of limonene (logP = 5.41); • It is not clear if limonene and clotrimazole can act synergically.

Docking Studies Performed on E. coli
In the case of E. coli, the two programs validate the binding site named 4JHT (Figure 10(a1,b1)).
In the CLC Drug Discovery Workbench model, the co-crystallized 8XQ creates a total of five hydrogen bonds from which three bonds are with ASP, one bond with TYR, one bond with TRP, and fifteen possible interactions with amino acids from the binding site (Table S3 in Supplementary, Figure 10(a2)). Regarding clotrimazole and limonene, in the CLC model ( Figure 10(a1-a5)) clotrimazole generates 1 hydrogen bond with GLY188 and 27 other possible interactions with amino acids from the binding site ( Figure 10(a3,a4)). Limonene does not generate hydrogen bonds but can interact with 18 groups of amino acids (Figure 10(a5)). The docking score of the studied compounds decreases in the following order: clotrimazole (docking score −10,06; RMSD: 0.01 Å) > 8XQ (docking score: −37,48; RMSD: 0.03 Å) > limonene (docking score: −45.14; RMSD: 0.12 Å) ( Table S3 in Supplementary). In the Molegro model (Table S3 in Supplementary, Figure 10(b1-b5)), the co-crystallized generates 18 interactions with the rest of the amino acids from the binding site and 6 amino acids (Figure 10(b1,b2)). In the case of clotrimazole, the model identifies 6 hydrogen bonds and 24 interactions between amino acids (Table S3 in Supplementary, Figure 10(b3,b4)). For limonene, the predictive model does not identify hydrogen bonds but indicates the possibility of interactions with 17 amino acids (Figure 10(b5)).
Due to the similar docking score obtained in both models, the great number of interactions with amino acids from the binding site, and the low number of Lipinski's violations for clotrimazole, this ligand can be docked first in the binding site. The synergy between the two ligands (limonene; clotrimazole) in the case of E. coli is less probable.

a.
Validating the Antimicrobial Activities by Tests Performed "In Vitro" In the case of C. albicans, the best results are obtained for the mixture between the essential oil from Citrus sinensis (NJoy) and clotrimazole, and, respectively, in the case of the mixture between the essential oil from Citrus aurantium and clotrimazole (diameter of inhibition obtained is 35 mm), followed by the mixture between Citrus limone and clotrimazole (diameter of inhibition obtained is 34.33 mm) (Figure 11). The docking score obtained in this model decreases in the following order: clotrimazole (docking score −64.85; RMSD: 0.00 Å) > limonene (docking score −65,79; RMSD: 0.00 Å) > co-crystalized 8XQ (docking score −79,94; RMSD: 0.00 Å).
Negative docking scores obtained in both models suggest the following: • The ligands can be fitted in the binding site predicted by the two models; • The partition coefficients of the co-crystallized (logP = 2.44) are smaller than clotrimazole (logP = 3.36) ( Table 9) and limonene (logP = 5.41).
Due to the similar docking score obtained in both models, the great number of interactions with amino acids from the binding site, and the low number of Lipinski's violations for clotrimazole, this ligand can be docked first in the binding site. The synergy between the two ligands (limonene; clotrimazole) in the case of E. coli is less probable.

a. Validating the Antimicrobial Activities by Tests Performed ''In Vitro''
In the case of C. albicans, the best results are obtained for the mixture between the essential oil from Citrus sinensis (NJoy) and clotrimazole, and, respectively, in the case of the mixture between the essential oil from Citrus aurantium and clotrimazole (diameter of inhibition obtained is 35 mm), followed by the mixture between Citrus limone and clotrimazole (diameter of inhibition obtained is 34.33 mm) (Figure 11). Figure 11. Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w), on C. albicans. No synergism between clotrimazole and the different sources of limonene was observed.
Regarding E. coli, an easily increased antimicrobial activity appears in the case of the mixture between the essential oil from Citrus reticulata (red mandarin) and clotrimazole (diameter of inhibition obtained is 10.83 mm), followed by the mixture between clotrimazole and the essential oil from Citrus bergamia (supplied by Life and Mayam) when an inhibition diameter of 10 mm is obtained (Figure 12). Regarding E. coli, an easily increased antimicrobial activity appears in the case of the mixture between the essential oil from Citrus reticulata (red mandarin) and clotrimazole (diameter of inhibition obtained is 10.83 mm), followed by the mixture between clotrimazole and the essential oil from Citrus bergamia (supplied by Life and Mayam) when an inhibition diameter of 10 mm is obtained (Figure 12).
A good performance is obtained in the case of S. aureus when the antimicrobial activity increases slightly in the case of the mixture between clotrimazole and the essential oil from Citrus reticulata (red mandarin) when an inhibition diameter of 18 mm is obtained (Figure 13). In second place is the mixture between clotrimazole and the essential oil from Citrus bergamia (Mayam) (diameter of inhibition is 17.92 mm); in third place is the mixture between clotrimazole and the essential oil from Citrus paradisi (17.33 mm inhibition diameter), and, finally, the mixture between clotrimazole and the essential oil from Citrus reticulata (green mandarin) with an inhibition diameter of 16.67 mm. A good performance is obtained in the case of S. aureus when the antimicrobial activity increases slightly in the case of the mixture between clotrimazole and the essential oil from Citrus reticulata (red mandarin) when an inhibition diameter of 18 mm is obtained ( Figure 13). In second place is the mixture between clotrimazole and the essential oil from Citrus bergamia (Mayam) (diameter of inhibition is 17.92 mm); in third place is the mixture between clotrimazole and the essential oil from Citrus paradisi (17.33 mm inhibition diameter), and, finally, the mixture between clotrimazole and the essential oil from Citrus reticulata (green mandarin) with an inhibition diameter of 16.67 mm.  A good performance is obtained in the case of S. aureus when the antimicrobial activity increases slightly in the case of the mixture between clotrimazole and the essential oil from Citrus reticulata (red mandarin) when an inhibition diameter of 18 mm is obtained ( Figure 13). In second place is the mixture between clotrimazole and the essential oil from Citrus bergamia (Mayam) (diameter of inhibition is 17.92 mm); in third place is the mixture between clotrimazole and the essential oil from Citrus paradisi (17.33 mm inhibition diameter), and, finally, the mixture between clotrimazole and the essential oil from Citrus reticulata (green mandarin) with an inhibition diameter of 16.67 mm. Figure 13. Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w) on S. aureus. The synergistic effect is evident in the case of the mixtures between clotrimazole and the essential oils of mandarin (red mandarin or green mandarin), and, respectively, between clotrimazole and the essential oil of grapefruit.
Good results are obtained in the case of S. aureus MRSA (Figure 14), for which generally the citrus essential oils in the concentration of 1.9% (w/w) do not have any antimicrobial effect, except the essential oil from Citrus sinensis (orange of Valencia), for which an inhi-bition diameter of 9.13 mm is obtained. In comparison with the solution of clotrimazole 0.1% that gives an inhibition diameter of 16.4 mm, the mixture between clotrimazole and the essential oils of Citrus bergamia, Citrus aurantium, Citrus paradisi, and Citrus limone give the inhibition diameters greater than each essential oil or CT. The best antimicrobial activities against S. aureus MRSA are obtained for a mixture of clotrimazole and the two essential oils of Citrus bergamia, (supplied by Life and Mayam), when the average inhibition diameters obtained are situated between 32.33 mm and 38.33 mm. Figure 13. Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w) on S. aureus. The synergistic effect is evident in the case of the mixtures between clotrimazole and the essential oils of mandarin (red mandarin or green mandarin), and, respectively, between clotrimazole and the essential oil of grapefruit.
Good results are obtained in the case of S. aureus MRSA (Figure 14), for which generally the citrus essential oils in the concentration of 1.9% (w/w) do not have any antimicrobial effect, except the essential oil from Citrus sinensis (orange of Valencia), for which an inhibition diameter of 9.13 mm is obtained. In comparison with the solution of clotrimazole 0.1% that gives an inhibition diameter of 16.4 mm, the mixture between clotrimazole and the essential oils of Citrus bergamia, Citrus aurantium, Citrus paradisi, and Citrus limone give the inhibition diameters greater than each essential oil or CT. The best antimicrobial activities against S. aureus MRSA are obtained for a mixture of clotrimazole and the two essential oils of Citrus bergamia, (supplied by Life and Mayam), when the average inhibition diameters obtained are situated between 32.33 mm and 38.33 mm. In the case of P. aeruginosa, the results obtained are more spectacular. The clotrimazole and the citrus essential oil used alone do not have any antimicrobial effect on this microorganism. However, in the case of the mixture of clotrimazole and the essential oil from Citrus bergamia (Mayam), an average inhibition diameter of 46.125 mm is obtained ( Figure 15). In the case of P. aeruginosa, good performances are obtained in the case of the mixtures between clotrimazole and the essential oils of Citrus sinensis (Life, nJoy), which give the inhibition diameters of 12.52 mm and, respectively, 21 mm. Local antimicrobial activities are registered in the case of the mixture between clotrimazole and the essential oil from Citrus reticulata (green mandarin) or Citrus paradisi, when the inhibition diameters obtained are 8 mm and 7 mm, respectively. In the case of P. aeruginosa, the results obtained are more spectacular. The clotrimazole and the citrus essential oil used alone do not have any antimicrobial effect on this microorganism. However, in the case of the mixture of clotrimazole and the essential oil from Citrus bergamia (Mayam), an average inhibition diameter of 46.125 mm is obtained ( Figure 15). In the case of P. aeruginosa, good performances are obtained in the case of the mixtures between clotrimazole and the essential oils of Citrus sinensis (Life, nJoy), which give the inhibition diameters of 12.52 mm and, respectively, 21 mm. Local antimicrobial activities are registered in the case of the mixture between clotrimazole and the essential oil from Citrus reticulata (green mandarin) or Citrus paradisi, when the inhibition diameters obtained are 8 mm and 7 mm, respectively.

Discussion
If we analyze the results obtained from the studies performed on microplates in terms of interaction types between clotrimazole and each target (microorganism), taking into account the values obtained after calculating the fractional inhibitory concentration index (FICI), we obtain an indifference effect in the case of C. albicans and, respectively, a synergism effect in the case of S. aureus (Table 10). In the case of E. coli, the effect can be due to the processes of addition and/or synergy, due to the close value obtained for FICI (0.4926) [52]. Regarding docking studies, in the case of C. albicans, both models give a negative score of docking for limonene and CT; however, these values are less than the co-crystallized (NDP) in both models. The number of hydrogen bonds formed between studied molecules in both models is less than 1. Both models identified more interactions between analyzed molecules and the rest of the amino acids from the binding site (1AI9). Due to the near value of the docking score for clotrimazole and the co-crystallized, we can conclude that in the case of a mixture between limonene and clotrimazole, the antibiotic reagent will be docked preferentially. This supposition is confirmed by studies performed on Petri plates, where in the case of C. albicans, all inhibition diameters obtained in the case of a mixture between different sources of limonene and clotrimazole are closer as a mathematical value to clotrimazole, without a synergistic effect.
In the case of E. coli, both docking models give negative docking scores for limonene and clotrimazole, which means that both molecules can interact with the binding site (8XQ). The CLC model indicates the formation of one hydrogen bond between clotrimazole and the binding site, whereas the MOLEGRO model indicates the formation of six hydrogen bonds between the antibiotic reagent and the binding site. Regarding the interactions with the rest of the amino acids from the binding site, both models indicate a great number of interactions between clotrimazole and the rest of the amino acids from the binding site (24 possible interactions identified by Molegro models, and 27 interactions identified by the CLC model).

Discussion
If we analyze the results obtained from the studies performed on microplates in terms of interaction types between clotrimazole and each target (microorganism), taking into account the values obtained after calculating the fractional inhibitory concentration index (FICI), we obtain an indifference effect in the case of C. albicans and, respectively, a synergism effect in the case of S. aureus (Table 10). In the case of E. coli, the effect can be due to the processes of addition and/or synergy, due to the close value obtained for FICI (0.4926) [52]. Regarding docking studies, in the case of C. albicans, both models give a negative score of docking for limonene and CT; however, these values are less than the co-crystallized (NDP) in both models. The number of hydrogen bonds formed between studied molecules in both models is less than 1. Both models identified more interactions between analyzed molecules and the rest of the amino acids from the binding site (1AI9). Due to the near value of the docking score for clotrimazole and the co-crystallized, we can conclude that in the case of a mixture between limonene and clotrimazole, the antibiotic reagent will be docked preferentially. This supposition is confirmed by studies performed on Petri plates, where in the case of C. albicans, all inhibition diameters obtained in the case of a mixture between different sources of limonene and clotrimazole are closer as a mathematical value to clotrimazole, without a synergistic effect.
In the case of E. coli, both docking models give negative docking scores for limonene and clotrimazole, which means that both molecules can interact with the binding site (8XQ). The CLC model indicates the formation of one hydrogen bond between clotrimazole and the binding site, whereas the MOLEGRO model indicates the formation of six hydrogen bonds between the antibiotic reagent and the binding site. Regarding the interactions with the rest of the amino acids from the binding site, both models indicate a great number of interactions between clotrimazole and the rest of the amino acids from the binding site (24 possible interactions identified by Molegro models, and 27 interactions identified by the CLC model). If we take into account only this information, it suggests that the antibiotic reagent will be docked first. A careful analysis of the values of the docking scores shows that both models give a value close to the co-crystallized for limonene; the CLC model gives a docking score of −45.14 (the co-crystallized docking score in this model is −34.48), whereas the Molegro model gives a docking score of −65.79 (the co-crystallized docking score in the Molegro model is −79.94). Taking into account only the values of the docking score, the molecule of limonene will be docked first. Applying Lipinski's rule of five in this case indicates that limonene is docked first because its molecular mass is less than that of clotrimazole and of the co-crystallized.
In this case, we suppose that in the case of E. coli, a competition for the binding site between limonene and clotrimazole exists, without a clear conclusion. The studies performed on Petri plates with clotrimazole (0.1%) and different sources which contain limonene reveal that the inhibition diameters are closer to clotrimazole, without a synergistic effect. The value of inhibition diameter obtained in the case of both essential oils from Citrus bergamia (inhibition diameter of 10 mm) is probably due to the linalool content (CBM: 20.83%; CBC 23.31%) [53][54][55].
In conclusion, in the case of E. coli, the effect of the clotrimazole and limonene is most probably obtained by "Addition" and not by "Synergy".
In the case of S. aureus, studies performed in microplates indicate synergism in the case of a mixture between clotrimazole and limonene (FICI = 0.0159). Docking studies performed on the two molecules with both programs (CLC and Molegro) reveal that docking scores are favorable for the two molecules. Studies performed on this microorganism with a mixture of clotrimazole (0.1%) and various sources of citrus essential oils (1.9%) reveal a clear synergistic effect in the case of different sources of limonene, such as essential oils from Citrus reticulata (red), Citrus reticulata (green), and Citrus paradisi, probably because these essential oils contain limonene and or/linalool (Citrus paradisi essential oil) [56,57]. In agreement with the results obtained by Guimarães and collab., beta-pinene and gammaterpinene do not exhibit antimicrobial activity on S. aureus [56].
In the validation studies, noticeable results are obtained in the case of S. aureus MRSA, for which clearer synergistic effects are obtained in the case of a mixture between clotrimazole and the following citrus essential oils: Citrus bergamia (Life and Mayam supplier). Citrus aurantium, Citrus limone, and Citrus paradisi. Spectacular results are obtained with the essential oils of Citrus bergamia (two sources), which give the inhibition diameters of 32.33 mm and 38.33 mm.
These spectacular results are due to favorable reports between linalool and linalyl acetate (bergamole) from the bioproducts which are responsible for synergism in antimicrobial activity for S. aureus MRSA [58][59][60]. A synergistic effect from linalool and antibiotic reagent gentamicin is found in the case of S aureus, S. aureus MRSA. The synergistic effect is observed also in the study performed on P. aeruginosa for which a clear synergism is obtained in the case of the mixture between clotrimazole (0.1%) and essential oils from Citrus sinensis (supplier by Life or NJoy), and Citrus bergamia, for the last one with an exceptional result (inhibition diameter of 41.25 mm).
In the case of the mixture between the essential oil of Citrus bergamia and clotrimazole, the results are due to the presence of linalool and linalyl acetate, which destroy cell membrane, change the nucleotides' integrity, and interfere with cellular respiration [61]. In the case of P. aeruginosa, an additive interaction was reported by Adaszyńska-Skwirzyńska and collab. in the case of a mixture between linalool and gentamicin [61].
In fact, these studies represent just a proof of concept that the mixture of different sources of limonene and clotrimazole can enhance antimicrobial activity. In neither case, will the mixture of essential oils and clotrimazole in propylene glycol represent the end pharmaceutical formulation. Studies are still in progress regarding the formulations which contain limonene and clotrimazole. In one of these studies, we developed the solid formulations with clotrimazole [62] as a sponge, with collagen, chitosan, and clotrimazole. Here, the studies performed in vitro on normal cell lines confirm that the matrix used to obtain end products diminishes the toxicity of the formulations with clotrimazole.

Conclusions
Studies performed in vitro on microplates in an experimental model made with limonene, clotrimazole, and a mixture of them on three types of microorganisms involved in skin diseases revealed the following: in the case of E.coli, the mixture between CT and limonene decreases the range of concentration at which this microorganism is susceptible, at levels in the range of (19 ÷ 9.5) µg/mL; in the case of S. aureus, results showed that it is susceptible to increased exposure at levels of concentrations in the range of 156 ÷ 78 µg/mL; the mixture between CT and limonene has no influence on C. albicans' growth.
Docking studies performed using CLC Drug Discovery Workbench and MOLEGRO Virtual Docker showed the following:

•
Regarding C. albicans, data obtained suggest that, in the case of a mixture between limonene and clotrimazole, in small quantities, clotrimazole is docked first; • Data for S. aureus show a similar docking score using both models and suggest that the limonene will be docked first in the binding site. Synergism between the two ligands (limonene; clotrimazole) in the case of S. aureus is more probable; • In the case of E. coli, taking into account the similar docking data obtained in both models, the great numbers of interactions with amino acids from the binding site, and zero Lipinski's violations for clotrimazole, it can be assumed that this ligand will be docked first in the binding site. The synergism between the two ligands (i.e., limonene; clotrimazole) in the case of E. coli is less probable. For E. coli, the effect of the mixture between clotrimazole and limonene is probably made by addition; • Results obtained in validation studies indicate a clear synergism between the mixture of clotrimazole and different sources of limonene (citrus essential oils) against S. aureus, S aureus MRSA, and P. aeruginosa, with the best results for the essential oil of Citrus bergamia for S aureus MRSA and P. aeruginosa. The studies performed are important because they: -Indicate the synergism between clotrimazole and limonene against S. aureus, in the case of sources with limonene with the content of limonene and/or linalool; -Show what the best natural resources for limonene are, in order to obtain a synergistic effect; -Reveal the importance of lab studies (performed "in vitro"), which must be in agreement with the results obtained "in silico".
The future developments of this study consist of: 1.
Obtaining pharmaceutical products with clotrimazole, limonene, and different sources of limonene that exhibit the best antimicrobial activity and low toxicity; 2.
Testing these pharmaceutical products on pathogenic microorganisms involved in skin injury.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
• Susceptible (S) (or sensitive) The term is used in two senses, one microbiological and the other clinical. Microbiological Susceptibility: Susceptible (sensitive) bacteria are those that belong to the most susceptible sub-populations and lack mechanisms of resistance.
Clinical Susceptibility: Susceptible (sensitive) bacteria are those that ideally have been shown to respond to a standardized therapeutic regimen when causing infection.

•
Intermediate = a bacterium is classified with intermediate susceptibility if it belongs to the group of strains that lies between the clinically susceptible and the clinically resistant. Infections caused by such strains have variable (or indeterminate) responses to chemotherapy, but they may be eliminated if the antibiotic is concentrated at the site of the infection, or the dosage is increased. • Resistance = this term is used in two senses, microbiological and clinical.
Microbiological resistance organisms are those that possess any resistance mechanism. The term may be qualified, as in "moderately or highly resistant" or "low-level, or high-level resistance".
Clinical resistance occurs when the infection is highly unlikely to respond even to maximum doses of a given antibiotic.
• Score = the docking score used in the Drug Discovery Workbench is the PLANTSPLP score [63]. This score has a good balance between accuracy and evaluation time. The score mimics the potential energy change when the protein and ligand come together. This means that a very negative score corresponds to a strong binding; a less negative or even positive score corresponds to a weak or non-existent binding. (Score = S target-ligand + S ligand). The S target-ligand term is a sum of contributions from all heavy atom contacts between the ligand and the molecules included in the binding site setup. It scores the complementarity between the binding site and ligand by rewarding and punishing different types of heavy atom contacts (inter-atom distance below~5.5 Å). Five different types of contacts are defined: