Plant Monoterpenes Geraniol, Eugenol and Carvacrol Against Multidrug-Resistant ESKAPE Isolates from Surgical Wounds

1. Introduction

In 2017, WHO released for the first time a list of bacterial pathogens presenting a serious threat to human health which is updating constantly [1,2]. Among other pathogens, subgroup marked with the ESKAPE acronym contain the ones with “priority status”, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. All specified are commonly isolated as multidrug-resistant pathogens (MDR), i.e. pathogens resistant to at least one agent belonging to three or more antibiotic classes [3]. Furthermore, subclasters of MDR pathogens were described in order to better estimate AR grade: (1) extensively drug-resistant bacteria (XDR) including the strains featured with nonsusceptibility to almost all tested agents belonging to different antimicrobial classes, except only one or two agents belonging to the same or different categories and (2) pandrug-resistant bacteria (PDR) involving nonsusceptible strains to all tested agents belonging to all tested antimicrobial categories [4].

It has been suggested that the improper use of antibiotics has contributed to the emergence of MDR pathogens, which resulted in a phenomenon that infections, being previously regarded as routine, are now becoming crucial threats [5]. Accordingly, surgical site infections (SSIs) are considered to be one of the most prevalent causes of hospital infections. In 70% to 95%, constituents of endogenous patient’s microbiome are causative agents of SSIs. It is commonly suggested that Escherichia coli and Enterococcus species contribute to 9.5% and 5.1% of all SSIs, respectively [6]. Moreover, S. aureus accounts for 24% of surgical site infections, and among them methicillin-resistant S. aureus (MRSA) is associated with poorer clinical outcome and extended hospital stay [6]. Further, contribution of all ESKAPE pathogens to SSIs and especially to mortality resulted from them, has also been recorded [7].

Taking into account aforementioned, usage of antiseptics is crucial for prevention of the infections in postoperative period. The most commonly used povidone-iodine can inhibit fibroblast proliferation and cellular viability even in diluted forms, therefore negatively affecting wound healing [8]. Moreover, its frequent utilization could be associated with various health problems, such as acute renal failure, seizures and hyperchloremic acidosis, thus suggesting a need for alternative but efficient antimicrobial agents [8].

Promising alternative could be seen in essential oils (EOs) and their constituents, since they are embedded with strong antibacterial activity and especially antibiofilm properties [9]. This investigation has been focused on selected monoterpenoid constituents of EOs, namely geraniol (G), carvacrol (C) and eugenol (E), due to their strong and proven antibacterial potential. Although G, C and E have already been proved as antibacterials against various bacterial species including some of the members of the ESKAPE group, there is limited information regarding their efficacy against MDR ESKAPE strains, especially in the form of biofilms. Moreover, there is no published data about the activity of the binary monoterpenes mixtures of aforementioned monoterpenes prepared in clearly defined ratio, against MDR ESKAPE strains’ biofilms. Taking this into account, the aims of the study were (1) to collect surgical wounds isolates of the ESKAPE group, determine their antibiotic resistance pattern and classify them as MDR, XDR or PDR, (2) to evaluate the antibacterial and antibiofilm potential of G, C and E applied individually and in the binary mixtures, against selected MDR isolates belonging to ESKAPE, and (3) to screen for the cytotoxicity of the monoterpenes and their selected binary mixtures in order to provide preliminary risk assessment.

2. Results

Screening of the antibiotic resistance pattern pointed out that all tested isolates belonging to the ESKAPE group were MDR. However, A. baumannii, P. aeruginosa, and K. pneumoniae belong to the XDR subgroup, while Enterobacter sp. isolate could be denoted as PDR (Table 1).

2.1. Antibacterial Potential of Monoterpenes and Their Interactions

Comparison of the inhibitory potential among monoterpenes (Table 2) revealed that C (average MIC value 0.32 ± 0.24 mg mL^-1) has achieved higher potential in comparison to E and G (p=0.00544 and p=0.02912, respectively). Activity of the E and G were notable (MICs 2.69±2.10 and 3.63±3.08 mg mL^-1, respectively), and similar (p =0.340749). In addition, inhibitory potential of all tested monoterpenes was significantly higher (p<0.00001) in comparison to positive control (povidone-iodine MIC 41.67±16.70 mg mL^-1).

Concerning strain sensitivity, it can be suggested that C seemed to be similarly efficient against all tested isolates. For both E and G, A. baumannii and P. aeruginosa were the most sensitive isolates. The least sensitive isolates were E. faecium, S. aureus MRSA and K. pneumoniae for E, and E. faecium, K. pneumoniae and Enterobacter sp. for G. Bactericidal effect of C was achieved on the lower tested concentrations (range 0.26±0.00-1.06±0.00 mg mL^-1) in comparison to two other tested monoterpenes (0.68±0.00-13.5±0.00 mg mL^-1, for E and 0.67±0.58 to >10 mg mL^-1 for G) and positive control (31.25±0.00 to >125±0.00 mg mL^-1).

Analysis of the interaction among the monoterpenes obtained in checkerboard assay (Table 3) showed synergism among all three monoterpenes.

2.2. Antibiofilm Potential

At higher concentrations (MIC/4-MIC) within the tested range MIC/32-MIC of C (Figure 1a) had inhibitory effect on E. faecium biomass biofilm formation (range 18.86-39.14%). In the case of K. pneumoniae C induced prevention of the biofilm formation at the lowest (MIC/32 and MIC/16 concentrations, 42.08% and 33.14%, respectively) and the highest tested concentration (34.52% of inhibition). Concerning A. baumannii inhibition of the biofilm formation was observed at all tested concentrations except at MIC, i.e., the range was from 39.68% (MIC/8) to 56.17% (MIC/2) inhibition. In the case of P. aeruginosa and Enterobacter sp. inhibition was observed only at MIC concentrations, 66.35% and 39.65%, respectively. Only in the case of S. aureus MRSA, C had no inhibitory effect or even induced biofilm production at tested higher concentrations (MIC/8-MIC).

Concerning E (Figure 1b), the only inhibitory potential was observed in the case of A. baumannii and K. pneumoniae, at MIC/32-MIC/8 and MIC/32 (35.38-47.14% and 18.85% inhibitions), respectively. At all other concentrations and species, the increase of biofilm biomass production was observed.

G (Figure 1c) had significant inhibitory effect on K. pneumoniae at all tested concentrations (MIC/32-MIC, within the range 59.63-84.72% of inhibition). Similar was observed in the case of A. baumannii, and Enterobacter sp. with the inhibitory ranges 38.78-76.39% and 39.49%-72.93%, respectively. G had inhibitory effect on the P. aeruginosa biofilm formation at MIC/4 (63.37%) and MIC (70.71%). In the case of S. aureus MRSA, G induced prevention of the biofilm formation at almost all tested concentrations (53.49-67.34% of inhibition) except MIC/8 where it caused biofilm production. G at all tested concentrations showed upgrading effect on the E. faecium biofilm.

In order to valorize the monoterpenes potential to inhibit biofilm formation, positive control i.e., solution of 10% povidone-iodine used for the wound’s disinfection, was also screened. It has also significantly prevented the biofilm formation of all isolates in all tested concentrations: the inhibitions’ ranges were in the case of MIC/32 54.32% (E. faecium) – 93.91% (Enterobacter sp.); MIC/16 29.27% (E. faecium) to 93.30% (P. aeruginosa); MIC/8 52.27% (E. faecium) to 92.36% (P. aeruginosa); MIC/4 22.81% (E. faecium) to 92.38% (P. aeruginosa) (Figure 1d).

In the further work screening of disruptive potential of the monoterpenes (MIC-8MIC) against pre-formed biofilm was performed. The activity of C tested on 48h old biofilm (Figure 2a) was heterogeneous. The highest susceptibility was observed in the case of Enterobacter sp., where significant biofilm reduction was detected at 4MIC (61.34%) and MIC (46.26%). A comparable disruption was recorded for A. baumannii at 2MIC (56.34%) and 4MIC (47.12%), as well as for K. pneumoniae at 4MIC (48.56%). Concerning P. aeruginosa, disruptive range was 29.63-41.77%, while the highest tested concentration increased overall biofilm biomass. In S. aureus MRSA, disruption was detected only at the highest concentrations tested (4MIC 28.42% and 8MIC 25.21%), but the overall effect remained low. Similarly, in E. faecium, biofilm eradication was observed exclusively at 2MIC.

E demonstrated the strongest antibiofilm activity against already formed biofilm of S. aureus MRSA across all tested concentrations, in the range of 34.41% (2MIC) to 52.83% (MIC) (Figure 2b). In contrast, eradicative effects against E. faecium (21.74%) and A. baumannii (44.32%) were detected only at 4MIC. No disruptive potential of E against pre-formed biofilms was observed in remaining isolates.

G (Figure 2c) displayed pronounced antibiofilm activity against E. faecium, with the strongest inhibition recorded at 8MIC (55.26%) and 2MIC (51.49%), followed by MIC (33.52%). Slightly lower effect was observed in the case of S. aureus MRSA where G had a consistent diminishing effect at all tested concentrations within the range of 38.98% to 44.09%, while the most effective was the lowest concentration. For P. aeruginosa, the lowest concentration tested resulted in the disruption of 28.83%, whereas the highest concentration induced biofilm upgrading. In A. baumannii, both the lowest and the highest tested concentrations stimulated further biofilm formation.

The activity of the positive control (10% povidone-iodine solution) against pre-formed biofilms was also evaluated (Figure 2d). Disruptive potential was observed only against K. pneumoniae and A. baumannii, with the most pronounced reduction detected at 2MIC (47.21%) and at 8MIC (57.12%), respectively. In all other cases, exposure to the positive control resulted in biofilm proliferation.

Concerning the observed potential of singular monoterpenes, whereas E induced increased biofilm formation, monitoring of combined effect of two other monoterpenes was interesting. In accordance with the results given in Table 4, mixtures of the monoterpenes G and C providing synergistic antibacterial effect, i.e. (MIC/32+MIC/16, MIC/16+MIC/32, MIC/8+MIC/32 and MIC/4+MIC/32, respectively) were further tested and their potential to prevent formation of biofilms and to eradicate already formed biofilms was shown in Figure 3. Results suggested that all four mixtures significantly prevented the biofilm formation of all isolates, with the only exception of the P. aeruginosa biofilm, whose production was even stimulated when it was exposed to the mixtures with the lowest concentrations of the G and C. The mixture MIC/32(G)+MIC/16(C) prevented biofilm formation of the isolates for 25.27% (A. baumannii) and 66.77% (E. faecium) (Figure 3a). Similar was observed with the mixture MIC/16(G)+MIC/32(C) where inhibition range was from 31.40% (Enterobacter sp.) to 65.32% (E. faecium). In the case of the mixture containing MIC/8(G)+ MIC/32(C), inhibitory potential was from 28.34% (Enterobacter sp.) to 81.02% (S. aureus MRSA). The mixture with the highest concentration of the G (MIC/4) was the most efficient i.e., it inhibited biofilm formation in the range 39.82% - 84.77%, for K. pneumoniae and S. aureus MRSA, respectively.

The same G-C mixtures were also screened for the disruptive potential on pre-formed 48h old biofilms. Results obtained (Figure 3b) pointed out that all tested combinations significantly reduced biofilm biomass of P. aeruginosa only. The eradicative antibiofilm effect of all tested concentrations was relatively uniform (38.84-44.67%), while the most effective was the mixture MIC/32(G)+MIC/16(C). Biofilm biomass decrease was also observed in S. aureus MRSA on MIC/32(G)+MIC/16(C) (27.81%) and MIC/16(G)+MIC/32(C) (35.78%) concentrations. No disruptive effects were detected in the remaining strains.

Finally, in order to evaluate and visually compare the potential of sole monoterpenes and their mixtures to prevent biofilm formation, and consequently estimate if the sole monoterpene’s activity was amplified, Table 4 summarizing the overall activity profiles was prepared. As shown, the combinations exhibited superior antibiofilm activity, compared to the single compounds, against E. faecium, S. aureus MRSA, A. baumannii and Enterobacter sp.

2.3. Cytotoxicity Assessment

In order to provide the risk assessment of usage of the monoterpenes and their selected mixtures inducing promising antibiofilm potential, the cytotoxicity assay was applied. Tested monoterpenes have not impacted the cell survival at any tested concentration being active in antibiofilm assay (Figure 4). Although selected mixtures were cytotoxic, the cell survival was significantly higher compared to commonly used disinfectant (povidone-iodine). Moreover, the mixture with the lowest abundance of monoterpenes (MIC/32(G) + MIC/16(C)) was not cytotoxic.

3. Discussion

In this study, the antimicrobial and antibiofilm activity of G, E and C against MDR isolates belonging to all members of the ESKAPE group was demonstrated. Further on, the interactions among the monoterpenes were explored and synergistic effect of binary combinations of all tested monoterpenes was observed. Furthermore, searching for the antibiofilm effect of sole monoterpenes pointed out the combination of G and C as the most promising for further research. According to that, for the first time, their mixtures, prepared in appropriate ratios providing synergism in antibacterial activity, were investigated for the antibiofilm potential, as well as the cytotoxicity in order to provide preliminary safety assessment of their potential use.

In respect to selected test organisms, it is worth noting that the literature data confirmed ESKAPE group as the most commonly isolated from SSIs suggesting also that 63% of recognized ESKAPE pathogens exhibited resistance to more than three antibiotic classes [10]. The isolates tested in this study i.e., E. faecium 5573-DM, S. aureus MRSA 3073-DM, K. pneumoniae 3625-DM, A. baumannii 3066-DM, P. aeruginosa 3082-DM, Enterobacter sp. 3071-DM were also derived from surgical site wounds. Majority of selected isolates were XDR, namely A. baumannii, P. aeruginosa and K. pneumoniae being sensitive to colistin only and E. faecium strain which was sensitive to linezolid only. On the other hand, S. aureus MRSA isolate was denoted as MDR, while Enterobacter sp. 3071-DM was resistant to all tested antibiotics (PDR).

Compared to the positive control (10% povidone-iodine as a routine wound antiseptic), all tested monoterpenes showed significantly higher antimicrobial effect (p˂0.05). Moreover, approximately 12, 16 and even 134 times higher antibacterial activity of G, E and C, respectively, was observed in comparison to that of povidone-iodine. Up to our best knowledge, for the first time these monoterpenes were screened against multidrug-resistant strains in the following manner: G against E. faecium, P. aeruginosa and K. pneumoniae, and E and C against Enterobacter sp. Concerning the activity of G against S. aureus MRSA and A. baumannii, the results obtained in this study were comparable with presented by Sroiphetcharat et al. [11], and Choudhary et al. [12], respectively. In addition, sensitivity of A. baumannii against E in our study also closely align with the one reported by Montagu et al. [13], while activity of E against K. pneumoniae and E. faecium was also comparable to the results of de Souza et al. [14], and Owen et al. [15], respectively. Marchese et al. [16], and Da Silva et al. [17] presented similar activity of C against S. aureus MRSA and P. aeruginosa as the one in this study. However, in other studies activity of G, E and C against multidrug-resistant strains isolates of ESKAPE group differed in efficiency from the results presented in our study [18,19,20,21,22,23,24]. Actually, literature data pointed out higher activity which may be attributed to the variety in isolate origin and consequent genetic background of each. Considering a decade has passed between some research available in the literature and this resent study, bacterial species had time to additionally mutate and/or to get certain resistance genes by horizontal genetic transfer and consequently develop new resistance, which contributes to a growing nonsusceptibility to different antibiotics.

G is acyclic monoterpene and its antibacterial mechanism involves an increase in membrane permeability, attributed to its interaction with lipids and other cellular membrane components [25,26]. In addition, Lin et al [27] showed that G interact with cell wall of Gram-positive bacteria and induce its rupture. On the other hand, E and C are aromatic phenolic compounds and their activity is attributed to hydrophobic properties and hydroxyl group. Hydroxyl groups of E and C could interact with proteins and prevent enzymatic reactions, while high lipophilicity is responsible for the action on cellular membrane. However, although E is also phenolic compound, its activity compared to C was rather lower, indicating that additional structural features determine activity of each sole compound. In addition, C is more lipophilic than E, owing its higher lipophilicity due to the absence of methoxy group. This increased lipophilicity enhances its ability to bind to cell membrane and increase its fluidity. Furthermore, the hydroxyl group of C is directly attached to the phenolic ring, facilitating effective interactions with phospholipids and proteins. In contrast, it could probably be suggested that the presence of a methoxy group in E reduces its hydrogen bonding potential, slightly diminishing its antimicrobial activity [28].

Taking into account the structural differences among monoterpenes, the idea of the study was that the monoterpenes may be used in combination, in order to get higher antibacterial efficiency in comparison to their solely use. To investigate this, antibacterial checkerboard assay was utilized to identify the most effective monoterpene combinations with the lowest concentrations, thereby minimizing the risk of their potential cytotoxicity.

The third part of the study was dedicated to investigate antibiofilm potential of the sole monoterpenes and their mixtures since SSI are biofilm associated infections. Monoterpenes, involving G, C and E, exert their antibiofilm activity by reducing bacterial adhesion and synthesis of extracellular polymeric substance, disrupting hydrophobic interactions inside the exopolysaccharide matrix, and suppressing quorum sensing pathways [29,30,31]. Concerning the range of antibiofilm activity observed in this study, it is worth nothing that there is available data concerning individual effect of G, C and E on ESKAPE pathogens’ biofilms inhibition, but not always on the biofilms of the multidrug-resistant isolates, especially of surgical wounds origin. Regarding the inhibitory potential against biofilm formation, available literature data pointed out that G is effective against S. aureus MRSA [32] and MDR A. baumannii [33]. In addition, biofilm formation was prevented with C in the case of MDR A. baumannii and K. pneumoniae [34,35] and with E treatment of A. baumannii [36], K. pneumoniae [37], MRSA [24], and P. aeruginosa [38]. As far as we know, antibiofilm potential, i.e., inhibition of formation, against (1) G against K. pneumoniae, P. aeruginosa, E. faecium and Enterobacter sp., (2) C against S. aureus MRSA, E. faecium, P. aeruginosa and Enterobacter sp. and (3) E against the E. faecium and Enterobacter sp., all referring to the multidrug-resistant strains, was evaluated for the first time in this study.

Regarding the disruption of pre-formed biofilms, available data suggest that up to the best of our knowledge, this study is the first to evaluate the effect on multidrug-resistant strains, namely of (1) G against of E. faecium, K. pneumoniae, P. aeruginosa and Enterobacter sp., (2) C against E. faecium and Enterobacter sp. and (3) E against E. faecium, P. aeruginosa and Enterobacter sp. Our results are in line with Sreepian et al. [39] who reported efficacy of G in disrupting S. aureus MRSA biofilms, but opposite to Aslan & Alim [40] reporting effect on MDR A. baumannii (no inhibition was detected in our study). Kashi et al. [41,42] also showed effect of C on MDR A. baumannii and K. pneumoniae, while Nikolic et al. [43] reported even higher disruptive effect of C on pre-formed S. aureus MRSA biofilms. Regarding E, Yadav et al. [24] and Kashi et al. [41] showed eradication of S. aureus MRSA and MDR A. baumannii biofilms, respectively, being consistent with this study. Although E did not reduce pre-formed biofilms of K. pneumoniae in this study, Kashi et al. [42] reported opposite result.

Concerning the observed pattern of all monoterpenes activity, it is interesting to note that the curves of both antibiofilm effect, i.e., formation inhibition and eradication of already formed biofilm were rather (inverted) U-shaped than a linear (dose-dependent). Actually, in the cases of several pathogens and treatments, such as in the inhibition of biofilm formation of E. faecium, K. pneumoniae and P. aeruginosa with G, P. aeruginosa and Enterobacter sp. with E, and A. baumannii and Enterobacter sp. with C, medium applied concentrations induced the most pronounced effect. Such hormetic response could be explained as follows: lower tested concentrations actually acts as mild stressors triggering adaptive response, i.e. induction of biofilm formation as an attempt of bacteria to protect themselves from antibacterial substances [44,45]. While medium concentrations induce the highest antibiofilm activity, the highest ones result in decrease of activity mainly due to possible aggregation of test substances which consequently reduce bioavailability and possibly block penetration through the biofilm matrix [46]. Similar response has already been established for different antimicrobial substances including antibiotics, antimicrobial peptides and natural products of plant origin [47,48,49,50,51].

Given that E was found to induce biofilm formation of the majority of tested strains, only the combinations of G and C, determined to be synergistic in antibacterial checkerboard assay (MIC/32(G)+MIC/16(C), MIC/16(G)+MIC/32(C), MIC/8(G)+MIC/32(C) and MIC/4(G)+MIC/32(C)), was tested for the antibiofilm activity. According to our knowledge, mixture of G and C was for the first time tested in our study on ESKAPE pathogens. Concerning the mixtures’ antibiofilm potential, it is worth noting that the most common manner to analyze type of interaction between test substances is by calculation of the minimum biofilm inhibition/eradication concentration 50% used for FICI determination [52,53]. However, dose dependent response of test strains to the sole test substances is a necessity to quantify FICI values and clearly determine type of interaction between the substances. Taking into account that the obtained response in the antibiofilm assay performed with sole monoterpenes was dominantly U-shaped, this methodology could not be applied. However, thorough comparative analysis of the biofilm biomass reduction values obtained in the experiments determining biofilm formation inhibition (Table 4) could provide some information concerning types of interaction. Indeed, in the case of E. faecium, only G and C mixture induced notable antibiofilm activity, while sole monoterpenes stimulate biofilm formation (G) or had no effect (C), indicating some affirmative interaction between the substances. Similar was obtained in the case of combinations containing MIC/8 and MIC/4 of G influencing S. aureus MRSA, A. baumannii and Enterobacter sp. biofilms. However, in the case of all combination treatments of K. pneumoniae and P. aeruginosa, as well as in the case of combinations with lower G concentrations (MIC/32 and MIC/16) affecting biofilm of A. baumannii and Enterobacter sp., antagonistic activity between monoterpenes was observed. Such response being species- and concentration-dependent indicates complex type of interactions between G and C and should be further investigated.

The main form of prevention of SSIs is the use of antiseptics and the most common antiseptic is 10% povidone-iodine. This widely used antiseptic exhibits rapid antimicrobial activity by targeting multiple cellular sites, distinguishing it from antibiotics [8]. However, iodine may induce acute pain, irritation and staining at the site of use [8]. Additionally, iodine has potential to form complexes with organic matter, thereby reducing its bactericidal efficacy [54]. In our study, povidone-iodine was used as a commonly used disinfectant in the antibacterial and antibiofilm assays and even though it was effective, the effective concentrations were much higher compared to tested mixtures of G and C.

Following the evaluation of the antibiofilm activity of the monoterpenes and their mixtures, the final objective of this study was to assess their safety. To achieve this, a cytotoxicity assay was conducted, solely for monoterpenes, but also for the mixtures. It was previously suggested that all three monoterpenes possess some cytotoxic potential [55], however, our results suggested that monoterpenes at concentrations tested in this study did not affect cell viability. Similar was suggested by Rodenak-Kladniew et al. [56] who investigated the cytotoxic effects of G at concentrations ranging from 0.04 to 0.19 mg mL^-1 and concluded that G did not significantly inhibit cell viability, exhibiting only low cytotoxicity. The possible explanation could be seen in a fact that cytotoxicity of aforementioned monoterpenes depends on the applied concentrations as well as the specific cell line used in the assay. In addition, in order to screen for the effect of possible interactions which could affect human cells’ viability, cytotoxicity assay was also applied with the most efficient antibiofilm combinations of the G and C. Obtained results demonstrated that the used mixtures, especially the one containing the lowest tested concentrations of monoterpenes (MIC/32(G)+MIC/16 (C)) carry a lower risk of injury to human cells compared to the conventional povidone-iodine. Based on these findings, this mixture could be considered as the valuable one to enhance efficacy at lower concentrations of the monoterpenes, attributed to the synergistic interaction between G and C.

4. Materials and Methods

4.1. Bacterial Strains and Ethical Approval

This study was conducted in accordance with the Declaration of Helsinki, with approval from the Local Ethical Committee of Clinical Hospital Center “Dr. Dragiša Mišović-Dedinje” (No 15341/3-2025). Clinical isolates tested in the study (Table 5), originated from the patients of the Surgical Department of University Hospital Center “Dr. Dragiša Mišović – Dedinje”, however, the study was not a clinical trial and bacteria were collected as a part of the standard procedure in hospital (they were identified as a common procedure in treatment of the patients within the Hospital). No personal data or other data from the patients were collected, neither informed consent was required.

Identification and determination of susceptibility to antibiotics for all isolates were performed using the Vitek 2 system (bioMerieux, Marcy I’Etoile, France). Breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used for determining bacterial resistance.

4.2. Bacterial Cultivation

The bacterial cultivation was carried out by streaking frozen stock cultures onto Mueller Hinton Agar plates (MHA, Torlak, Belgrade, Serbia) followed by 48 h incubation at 37 °C.

The bacterial inoculum used for all further tests were adjusted to be 0.5 Mc Farland, corresponding to 1x10⁸ CFU mL^-1. All microbial procedures were carried out in a sterile environment.

4.3. The Assessment of the Antibacterial Potential

Antibacterial potential of the monoterpenes was examined by the use of the broth-microdilution assay. Minimal inhibitory concentrations (MICs) as well as minimal bactericidal concentrations (MBCs) were determined according to the method previously outlined by Vasilijević et al. [57] with slight modifications. Briefly, monoterpenes were prepared by dissolving them in Tween 80 (G: Tween 80 1:1, E: Tween 80 1:1, C: Tween 80 1:2). Concentration ranges were 0.21 – 13.50 mg mL⁻¹, 0.08 - 10.80 mg mL⁻¹, 0.06 – 8.40 mg mL⁻¹ for E, G and C, respectively. The negative control was the solvent Tween 80 (1.66 -106 mg mL^-1), while a positive control was commercial solution of 10% povidone-iodine (tested concentration range 0.98-125 mg mL^-1). MIC values were determined in accordance with the color changes of growth indicator resazurin. MBCs were determined by plating from the wells with no noticeable growth onto Mueller Hinton Agar plates. The experiment was performed in triplicate and repeated two times.

4.4. Antibacterial Checkerboard Assay

Checkerboard assay was performed as previously described in Vasilijević et al. [57]. Briefly, tested concentration range of the substances was (1/32)×MIC – 2×MIC. All combinations that did not induce color change of resazurin (lack of growth) were used for the calculation of the fractional inhibitory concentration index (FICI) for two antimicrobials in combination. The FICI was calculated according to equation (1),

$$FICI={\displaystyle \frac{\mathrm{M}\mathrm{I}\mathrm{C}\mathrm{A} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{m}\mathrm{b}.}{\mathrm{M}\mathrm{I}\mathrm{C}\mathrm{A} \mathrm{a}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{e}}}+{\displaystyle \frac{\mathrm{M}\mathrm{I}\mathrm{C}\mathrm{B} \mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{m}\mathrm{b}.}{\mathrm{M}\mathrm{I}\mathrm{C}\mathrm{B} \mathrm{a}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{e}}}$$

(1)

where substances A and B were monoterpenes.

FICI was used to distinguish between the mode of interactions as follows: FICI≤0.5 – synergistic; 0.5<FICI≤1 – additive; 1<FICI≤4 – indifferent; FICI>4 – antagonistic effect. The checkerboard assay was performed in triplicate in two individual experiments.

4.5. The Assessment of the Antibiofilm Activity of the Monoterpenes and Selected Mixtures

The impact of antibacterial agents on biofilm formation was determined in 96-well microtiter plates according to the previously described in Marinkovic et al. [58]. The biofilms were formed during 24h at 37°C from the bacterial inoculums of 10⁵ CFU/well, with or without sole monoterpenes (tested concentration ranges MIC/32 –MIC), or G-C mixtures. Selection of these two monoterpenes, as well as of their concentrations tested for the combination activity, was done in respect with the antibiofilm potential of individual monoterpenes and the results of the antibacterial checkerboard assay.

On the other hand, in the biofilm disruption assay the bacterial cell suspensions (2×10⁵ CFU/well) were first incubated for 48h in order to allow biofilm formation. Pre-formed biofilms were exposed for 24h to the tested monoterpenes (tested concentrations range MIC-8MIC). In addition, eradication potential of the monoterpene’s mixtures (the same concentrations were combined as for the biofilm formation inhibition experiments) was also determined.

Biofilm biomass quantification was provided for both the inhibition of biofilm formation and pre-formed biofilm eradication using crystal violet (0.1%). The percentage of the biofilm biomass reduction (R), obtained as a result of inhibition of biofilm formation or pre-formed biofilm eradication was calculated according to the equation (2):

$$R=\left(1-{\displaystyle \frac{At}{Ac}}\right)\times 100\%$$

(2)

Ac and At are absorbances at 570 nm of control and treatment, respectively. The experiment was performed in five replicas and repeated two times.

4.6. Cytotoxicity Assessment

4.6.1. Cell Culture and Treatment Within XTT Assay

Cytotoxicity testing of G and C mixtures was performed on normal human lung fibroblast cell line MRC-5 (ATCC, Manassas, VA, USA ECACC 84101801), according to the standard procedures [59]. Ratios of the monoterpenes in the tested mixtures were determined in the accordance with the checkerboard assay, as the most effective synergistic mixtures against tested bacteria.

The cell cultures were treated with each monoterpene and the selected mixtures for 24 hours. Untreated cells were used as a negative control, while 10% povidone-iodine was used to compare cytotoxicity of tested monoterpenes mixtures with the same effect of already approved conventional disinfectant. For the positive control, doxorubicin (2 µg mL-1) was used. All treatments were set up in triplicate and repeated twice.

4.7. Statistical Analysis

Results are presented as mean value ± standard error of the mean (SEM). Statistical analysis was performed using One-way ANOVA statistical test from SPSS 10 statistical package for Windows (IBM, Armonk, NY, USA). P values < 0.05 were accepted as the level of significance.

5. Conclusions

This work includes investigation of antibacterial and antibiofilm effect of selected monoterpenes G, C and E on clinical isolates of ESKAPE group of pathogens. All tested strains originated from infected surgical sites. Isolate of S. aureus MRSA was MDR, of E. faecium, A. baumannii, P. aeruginosa, and K. pneumoniae were XDR, and Enterobacter spp. was PDR. All monoterpenes induced notable antibacterial effect, with C being the most potent. Some binary combinations of all tested monoterpenes showed synergistic antibacterial activity. Prevention of biofilm formation of all ESKAPE isolates was observed for G and C, while their mixtures could be effective as antibiofilm agents against E. faecium, S. aureus MRSA, A. baumannii and Enterobacter sp. Cytotoxicity screening pointed out lower risk from the use of monoterpenes and the mixtures compared to the conventional povidone-iodine. Accordingly to all abovementioned, obtained results could be considered as an important step forward with potentially high clinical implications. For that reason, further investigation is strongly encouraged, especially in order to determine the mechanism underlying enhancement of antibacterial and antibiofilm activities of the monoterpenes’ mixtures.