Elucidation of the synergistic action of Mentha Piperita essential oil with common antimicrobials

Mentha piperita L. essential oil (EO) is employed for external use as antipruritic, astringent, rubefacient and antiseptic. Several studies demonstrated its significant antiviral, antifungal and antibacterial properties. The aim of this work is the study of the synergistic effects of M. piperita EO with antibacterials and antifungals that are widely available and currently prescribed in therapies against infections. The observed strong synergy may constitute a potential new approach to counter the increasing phenomenon of multidrug resistant bacteria and fungi. In vitro efficacy of the association M. piperita EO/drugs was evaluated against a large panel of Gram-positive and Gram-negative bacteria and yeast strains. The antimicrobial effects were studied by checkerboard microdilution method. The synergistic effect of M. piperita EO with gentamicin resulted in a strong growth inhibition for all the bacterial species under study. The synergistic effect observed for M. piperita EO and antifungals was less pronounced.


Introduction
Bacterial resistance to antibiotic therapy is a growing emergency [1]. In the last years, several research programs have focused on designing new compounds possessing potential antimicrobial activity in order to avoid this problem [2][3][4][5][6]. New sources, especially plant-derived antimicrobial compounds, have been extensively studied in recent years [7,8].
Plant essential oils (EOs) have been examined in detail for their pharmacological properties and may constitute a promising source for new natural drugs [9][10][11][12][13][14]. Currently, approximately 3000 EOs are known of which 300 are commercially available in the agronomic, alimentary, sanitary and pharmaceutical fields [15][16][17][18][19]. Plant-derived EOs are natural mixtures of a certain complexity. At times, they may contain more than 100 components at quite different concentrations. These components encompass two groups of distinct biosynthetic origin: the main group includes terpenes and terpenoids, while the other one includes aromatic and aliphatic PLOS

Chemical composition and potential therapeutic applications of M. piperita L. EO
The content of EOs can be detected by hyphenated gas chromatography with mass spectrometry (GC/MS) technique [24,25]. M. piperita EO is composed by monoterpenes, menthone, menthol and their derivatives. Several authors have underlined the role of EOs in the management of several therapeutic conditions, as inflammation of the oral mucosa, irritable colon, spastic discomfort of the upper gastrointestinal tract and bile ducts, catarrhs of the respiratory tract. A therapeutic approach based on a combination of drugs could contribute to overcoming antibiotic resistance [26][27][28].
Recently, we assessed and reported the positive synergism against Candida spp. of the echinocandin anidulafungin combined with aspirin or with other NSAIDs [29,30]. All these observations prompted us to investigate the combination of commercially available M. piperita EO with well-known synthetic antimicrobials, with the aim of providing a greater effectiveness to combat infections and overcome the phenomenon of drug resistance [31][32][33][34][35].

Aims of research
Gentamicin and ampicillin were chosen as antibacterial agents, while amphotericin B was chosen as antifungal. Gentamicin is an aminoglycoside antibiotic largely used for the treatment and prevention of severe Gram-negative bacterial infections. However, its severe side effects (oto-and nephrotoxicity) limit its use. In addition, psychiatric symptoms related to gentamicin (confusion, anorexia, depression, disorientation and visual hallucinations) may occur.
Ampicillin is a β-lactam antibiotic that showed several side effects. Recently, an increased resistance to this antibiotic has been reported [36].
Amphotericin B is a polyene antifungal drug which is considered the drug of choice for the treatment of mycosis; it is often combined with azoles. However, several authors have observed that in the last decades Candida species have become resistant to treatment to azoles alone and to azoles in association with amphotericin B [37].
In the class of azoles, fluconazole and miconazole were chosen for the association with M. piperita EO. Regarding all this, the aim of the present study was to examine the possible synergistic effect of M. piperita EO with common antimicrobials.
We recently reported the in vitro synergistic activity of certain combinations of essential oils with antimicrobials [38][39][40]. The antimicrobial activity of the M. piperita EO against different Gram-positive and Gram-negative bacteria and fungi, along with its synergistic effects when combined with antimicrobial drugs (gentamicin, ampicillin, amphotericin B, miconazole and fluconazole), has been studied by following the microdilution checkerboard method. The composition of commercially available M. piperita EO used in our experiments has been confirmed by GC/MS analyses [41][42][43][44][45].

Microbial and yeast cultures
The bacterial species were cultured on Mueller Hinton agar (MHA, Oxoid) and each bacterial suspension was composed of 2-3 colonies for each strain taken from an MHA plate and dissolved in 2 mL of MHB (Mueller Hinton Broth). The resulting suspensions were diluted with 0.85% NaCl solution and then adjusted to 1x10 8 CFU/mL (0.5 McFarland).
The fungal strains were subcultured twice on Sabouraud dextrose agar before being tested. Yeast cells were washed four times in sterile saline. Each fungal suspension was taken from its frozen stock at -70˚C. The strains were inoculated in 5 mL of Sabouraud dextrose broth, and then incubated under stirring at 35˚C for 48 h.

MIC evaluation protocols
MIC values were determined by broth microdilution method, in accordance with CLSI (Clinical and Laboratory Standards Institute) Protocol M07-A9 guidelines for bacteria and Protocol M27-A3 guidelines for yeasts [46,47].

Antimicrobial activity
For antibacterial tests, a stock solution (EO/Ethanol 1:2.5, 40% v/v with Tween 80, 0.1%) was diluted 1:20 in MHB to obtain a 2% (v/v) final solution. Doubling dilutions of the EO from 2% to 0.015% for EO were prepared directly in 96 well microtiter trays in MHB. After the addition of 0.02 mL of the inoculum, the microtiter trays were incubated at 36˚C for 24 h. The final concentration of ethanol was 1.5% (v/v). The MHB medium 0.1% (v/v) Tween 80 and ethanol 1.5%, (without EO) was used as a positive growth control.
Preparation of the EO for the antifungal tests followed the same procedure as the one for the antibacterial tests. A small quantity of inoculum was dissolved in RPMI 2% glucose and then spectrophotometrically adjusted to 0.5 x 10 3 to 2.5 x 10 3 CFU/mL (McFarland, turbidity standard). The initial inocula were confirmed by plating serial dilutions and determining the colony counts. A total of 0.1 mL of each yeast suspension was dispensed into serially diluted wells containing the drugs or the EO, achieving final drug concentration. After the addition of 0.1 mL of inoculum, the plates were incubated at 36˚C for 48 h. MIC was defined as the lowest concentration of the mixtures at which no visible growth of the fungal strains could be detected compared to their growth in the negative control well. MIC values are given in mg/ mL and μg/mL for M. piperita EO and antimicrobial drugs, respectively.
MIC determinations were realized in triplicate in three independent assays. . MIC values for the EO-drugs associations were defined as the lowest concentration at which no visible growth of the microbial strains could be detected compared to their growth in the control well, as described in Eucast document [48].

Microdilution checkerboard method
In the combination assays, the checkerboard procedure described by White et al. [49] was followed to evaluate the synergistic action of the EO with selected drugs. Twelve double serial dilutions of the EO were prepared following the same method used to evaluate the MIC. Dilutions of the EO were prepared together with a series of double dilutions of the antimicrobial drugs: for antifungal drugs in the range of 32.0-0.66 μg/mL, for antibacterial in the range of 64.0-0.125 μg/mL and for M. piperita EO in the range of 18.2-0.09 mg/mL. This method was used to mix each antimicrobial compounds dilution with the appropriate concentrations of EO in order to obtain a series of concentration combinations of the EO with each particular drug. In our experimental protocol, the substance combinations were analysed by calculating the FIC index (FICI) as follows: FIC of the EO plus FIC of the drug. Generally, the FICI value was interpreted as: i) a synergistic effect when 0.5; ii) an additive or indifferent effect when > 0.5 and <1; iii) an antagonistic effect when > 1 [49]. The concentrations prepared accounted for 40%, 20%, 10%, and 5% of the MIC value for the EO, and 25%, 12.5%, 6.25%, 3.12% of the MIC value for the antibiotic. Also, the combination of two components is shown graphically in a Cartesian diagram by applying the isobole method. The non-interaction of the two components results in a straight line, whereas the occurrence of an interaction is shown by a concave isobole [50][51][52][53].

Gas chromatography-mass spectrometry equipment
The gas chromatographic analyses have been performed on HP GC/MS 6890N-5973N MSD HP ChemStation equipped with autosampler and HP-5MS column (crosslinked 5% PH ME siloxane) 30 m x 0.25 mm x 0.25 μm Film Thickness. The following temperature program was applied: 40˚C (4 min), 4˚C per minute heating up to 280˚C (30 min). The mass spectrometer was operated in EI mode at 70 eV; the ion source temperature was 220˚C. The mass spectra were measured in the range of 35-360 amu.

Compound identification
For chemical characterization, a standard solution of 100 μL of the pure EO in 1 mL of ethyl acetate was prepared. The solution was filtered and 1 μL was analyzed by GC/MS. The sample was analyzed in triplicate. Qualitative analysis was executed comparing the calculated Linear Retention Indices (LRI) and Similarity Index Mass Spectra (SI/MS) for the obtained peaks with the analogous data from NIST2011 and Adams 4th ed. (2007) databases. LRI of each compound was obtained by temperature programming analysis and was determined in relation to an homologous series of n-alkanes (C7-C30) under the same operating conditions. LRI was calculated following the Van den Dool and Kratz equation [24,25,45,54] and compared with the Arithmetic Index (AI) from NIST2011 database and Adams, 4th ed. (Adams 2007). SI/MS were determined as reported by Koo et al. [55]. Component relative percentages were calculated on the basis of GC peak areas without using correction factors.

Results and discussion
The present research has tested M. piperita EO in association with several antifungal and antibacterial drugs. The effects have been evaluated on ten strains of Gram-positive and Gramnegative bacteria and seven strains of Candida spp. FICI values for M. piperita EO in combination with antibacterial agents as gentamicin and ampicillin are reported in Table 1.
FICI values for the association with gentamicin and ampicillin were, respectively, in the range of 0.07-0.46 and 0.13-0.65 against all tested bacterial strains. It is also interesting to underline that the MIC value for gentamicin is markedly reduced when combined with M. piperita EO, as the MIC value for this particular association was found to be more than 30-fold lower against six out of the ten bacteria strains considered. The most interesting result was obtained for Bacillus subtilis ATCC 6633 for which the MIC value of gentamicin was found to have decreased from 0.5 to 0.01 μg/mL (FICI = 0.07). Moreover, a promising result was obtained also against Acinetobacter baumannii ATCC 19606, a Gram-negative bacillus resistant to treatment with gentamicin alone. In this case, a 16-fold reduction of gentamicin MIC (FICI = 0.46) was observed when used with M. piperita EO. The strong synergy observed between gentamicin and the EO against the Gram-negative Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC 19833 is worthy of note. In particular, the MICc value for gentamicin is much lower than that normally required to achieve the direct inhibition of bacterial growth (MICc: 0.06 μg/mL vs MICo: 2 μg/mL, MICc: 1 μg/mL vs MICo: 32 μg/mL). Associations of ampicillin with M. piperita EO showed a marked synergistic effect against Escherichia coli ATCC 25922 (FICI = 0.08) and Bacillus subtilis ATCC 6633 (FICI = 0.13). Results for the associations with antifungals are reported in Table 2.
A synergistic antifungal action was observed when M. piperita EO was combined with fluconazole, amphotericin B or miconazole against yeast strains under study. FICI values close to 0.4 were found for amphotericin B and fluconazole, whereas they ranged between 0.23 and 0.46 for miconazole. It is interesting to note that this association had a strong synergistic effect against the C. albicans and C. guilliermondii spp.. On the whole, the synergistic antimicrobial action demonstrated by combining M. piperita EO with antifungals was less pronounced against yeast strains than that of antibacterial agents combined with the same EO against bacteria strains. The synergistic interaction between M. piperita EO and the most promising antimicrobials, gentamicin and miconazole, is shown in Figs 1-3.
The combination of the two components is shown by an isobole method [50]. An isobole is an "iso-effect" curve, in which a combination of constituents is represented on a graph, whose axises represent the inhibitory doses of the individual agents. If the agents do not interact, the isobole (the line joining the points representing the combination to the points on the dose axises representing the individual doses with the same effect as the combination) will be a straight line. If the effect is additive, the curve of the isobole will be a "concave" line, thus indicating that the agents in the mixture are synergic. When the opposite occurs, a "convex" line will result, showing antagonism. In other words, the same biological effects of the isolated agents are obtained at lower (or higher) doses than those observed for the mixture. Our graphs indicate, indeed, a high synergism against all the bacteria and yeast strains examined. The synergistic or antagonistic relationship between antimicrobials may result from competition for a possible primary target [56]. Conversely, a synergistic multi-target effect may occur, involving Table 1 enzymes, substrates, metabolites and proteins, receptors, ion channels, transport proteins, ribosomes, DNA/RNA and physicochemical mechanisms [40]. An alternative explanation may be that the interaction between different compounds may lead to changes in the structural conformation, and it may result in the reduction of the inhibitory activity. However, it is difficult to elucidate the exact mechanism of the synergistic effect without further investigation. In order to assess the impact of M. piperita EO in association with antimicrobials we evaluated the chemical composition of this EO by GC/MS analysis. For the chemical characterization of the commercially available EO used for the biological assay, GC/MS analysis were performed [42,43]. 27 components have been identified in the pure EO, 20 of which corresponded to 97.64% of the mixture. Menthol was found to be the major component, amounting to 68% of the mixture. The composition of the EO has been summarized in Table 3. Several constituents fell within the terpene fraction: isomenthone 9.48%; menthone 8.36%; limonene 1.85%. These results are in agreement with those previously reported for EOs of different M. piperita species [37]. Conceivably, the antibacterial activity, as well as the synergistic effect of EO, may be attributed to the high percentage of oxygenated monoterpenes, which Gram-negative outer membrane has a strong negative charge conferred to it by the lipopolysaccharide, which is connected to lipid composition and to the net surface charge of the microbial membrane. The lower synergistic effect observed on yeasts may be attributed to a negative interaction with the antifungal drugs.

Conclusions
This paper describes a study regarding the association of M. piperita EO with several antimicrobial agents against a large panel of bacteria and fungi strains. Gentamicin and ampicillin were chosen as antibacterial agents, whereas amphotericin B, fluconazole and miconazole were chosen as antifungals. On the whole, a synergism between M. piperita EO and antimicrobials was found. The FIC indices for the association of gentamicin and M. piperita EO indicate, indeed, a very strong synergistic mode of action for all tested Gram-positive and Gram-negative strains. As a consequence, the combination of these two compounds allows for a significant reduction of the amount of gentamicin needed to inhibit bacteria strains. bacteria are difficult to treat with commonly employed antibacterial drugs. Generally, a synergistic effect was also observed against yeast strains, although it was less evident than against bacteria. This result may conceivably depend on the poor interaction between the EO with azoles and amphotericin B. Further investigation would allow a more complete understanding of the antimicrobial potential of this association and may be useful for the preparation of new agents for the cure of infections caused by these important pathogens.