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Chemical Characterization and in Vitro Antibacterial Activity of Myrcianthes hallii (O. Berg) McVaugh (Myrtaceae), a Traditional Plant Growing in Ecuador

Myrcianthes hallii (O. Berg) McVaugh (Myrtaceae) is a plant native to Ecuador, traditionally used for its antiseptic properties. The composition of the hydro-methanolic extract of this plant was determined by submitting it to ultra-high performance liquid chromatography (UHPLC) hyphenated to heated-electrospray ionization mass spectrometry and UV detection. The presence of antimicrobial components prompted us to test the extract against methicillin-resistant and methicillin-susceptible Staphylococcus aureus, multidrug-resistant and susceptible Escherichia coli, Pseudomonas aeruginosa, Enterococcus spp. and Streptococcus pyogenes strains. The chromatographic analysis led to the identification of 38 compounds, including polyphenols and organic acids, and represents the first chemical characterization of this plant. The extract showed modest antibacterial activity against all tested bacteria, with the exception of E. coli which was found to be less sensitive. Whilst methicillin-resistant strains usually display resistance to several drugs, no relevant differences were observed between methicillin-susceptible and resistant strains. Considering its long-standing use in folk medicine, which suggests the relative safety of the plant, and the presence of many known antibacterial polyphenolic compounds responsible for its antibacterial activity, the results show that M. hallii extract could be used as a potential new antiseptic agent. Moreover, new anti-infective biomaterials and nanomaterials could be designed through the incorporation of M. hallii polyphenols. This prospective biomedical application is also discussed.


Introduction
The Myrtaceae family includes about 30 genera and 1500 species found in the Neotropics. In Ecuador there are 15 native genera and about 200 species. Native species are mainly grown for their edible fruits and wood and some are used as medicinal plants due to their biological properties [1]. In recent years, increasing interest in herbal medicine has been registered in developed and developing countries, and much attention has been paid to natural antibacterial substances for use in alternative therapies against conventionally resistant infections or as new antiseptic agents. In the last decade, many species of Myrtaceae (including those from the Myrtus, Eucalyptus, Psidium, and Syzygium genera) have been studied for their antimicrobial properties [2][3][4][5]. Myrcianthes is a genus of Myrtaceae that includes shrubs and small trees. At present, 38 species of Myrcianthes are known to be distributed across several countries of Central and South America, from Mexico to Chile, including Ecuador [6]. Although Myrcianthes is closely related to the large genus Eugenia L. recent studies have confirmed its individuality and role as a sister group to the rest of the Eugenia clades [7]. Thanks to recent taxonomic research [8,9], new species belonging to the Myrcianthes genus have been described. Myrcianthes hallii (O. Berg) McVaugh is a medicinal and aromatic species commonly known in Ecuador as "arrayán" [10,11]. It grows as a shrub or a tree up to 8 m high. Leaves and branchlets are nearly glabrous, flowers are tetramerous and the hypanthium is characteristically densely pale and strigose. This species is recorded in Peru, Ecuador, Venezuela and possibly in Colombia [12,13]. In Ecuador M. hallii is found both native and cultivated, growing in the Andean region from 2500 to 3000 metres above mean sea level (MAMSL), and mainly in the provinces of Azuay, Bolivar, Carchi, Chimborazo, Imbabura, Loja and Pichincha [11,13]. Its leaves and berries are used in both traditional medicine and in cosmetics and foods, as a culinary herb or spice. In traditional medicine, arrayán is consumed as an infusion and a decoction for its antiseptic, haemostatic, and balsamic properties. Moreover, dried ground leaves applied to wounds aid healing and are used in baths, vapors and massages for their stimulating and tonic properties. Green leaves are used to clean teeth and gums, with the added effect of whitening teeth naturally. Finally, fresh leaves macerated in olive oil seem to prevent hair loss [10, 11,14]. The chemical compositions of plants belonging to the Myrtaceae family (such as Eucalyptus and Myrtus) have been widely studied. Eucalyptus species are rich sources of biologically active terpenoids, tannins, flavonoids and phloroglucinol derivatives [3,15,16]. In Myrtus communis L. (myrtle) the presence of different polyphenolic classes is reported, including phenolic acids, flavonoids, galloyl derivatives and hydrolysable tannins [2,17,18]. Some of the main polyphenols found in myrtle are also present in green tea (catechin and gallocatechin derivatives) which is widely recognized as an excellent source of powerful nutraceuticals [17,18]. A recent study on leaf extract obtained from Myrcianthes cisplatensis (Cambess.) O. Berg, reported the presence of α-methyl-1-(2 1 ,4 1 ,6 1 -trimethoxyphenyl)-1-propanone, known as conglomerone, which shows antibacterial activity against methicillin-sensitive and resistant Staphylococcus aureus strains [19].
Although studies reporting the composition of essential oils or the biological activity of extracts do exist for several species of Myrcianthes  [19][20][21][22][23][24][25][26], the available information concerning M. hallii is particularly limited. Therefore, in view of the fact that M. hallii is commonly employed as an antibacterial agent in Ecuadorian folk medicine, and therefore is likely safe and effective, and the importance of finding new anti-infective extracts to be loaded into or coated onto biomaterials, the aims of this study were to investigate the chemical composition of M. hallii and its antibacterial activity against Gram positive and Gram negative multidrug-resistant strains.

UHPLC-PDA-hESI-MSn Analysis of LMW Fraction of MHE
The acidic hydro-methanolic leaf extract of M. hallii (MHE) was submitted to dialysis, using a membrane with a nominal molecular weight cut-off (MWCO) of 3500 Da. A low molecular weight (LMW) fraction, suitable for UHPLC-PDA-hESI-MSn analysis, was obtained. The chromatographic profile, acquired at 280 nm, is reported in Figure 1. Thirty-eight compounds were identified based on their chromatographic behaviour and mass spectra, in comparison with the literature. Table 1 summarizes the identified compounds, their retention times and m/z values for the parent ion and fragment ions.
Twelve flavonols were identified in MHE: four myricetin derivatives, five quercetin derivatives, a quercetin aglycone, and two kaempferol derivatives. Four myricetin derivatives were identified in MHE due to their MS and MS/MS spectra. Peaks 21, 23, 24, 32 were assigned to myricetin derivatives due to the presence of the aglycone at m/z 317, in their MS 2 spectra. Peak 21 was identified as myricetin glucoside or galactoside (MW 480) due to the parental ion at m/z 479 producing the MS 2 fragment [M-162]´; peak 24 was assigned to myricetin rhamnoside (MW 464) due to the loss of 146 Da from the molecular ion at m/z 463, according to Faria et al. [28]. Peak 23 was identified as myricetin arabinoside (MW 450), since it produced the MS 2 ion at m/z 317 from the molecular ion at m/z 449, due to the loss of [M-132]´corresponding to the arabinoil moiety. Peak 32 was assigned to acylated myricitrin (MW 506). Its molecular ion at m/z 505 produced a base peak at m/z 316, corresponding to the myricetin aglycone, and a fragment at m/z 463, due to the loss of the acylic moiety. In MHE five quercetin derivatives were identified, because all peaks presented the characteristic fragment ions of quercetin aglycone (e.g., MS n data at m/z 300, 301, 179).  [15]. Peak 34 may correspond to Cypellogin A or B (MW 630), which is a quercetin glucoside or galactoside acylated with an oleuropeic acid residue [29]; in fact, the compound yielded a base peak at m/z 301 corresponding to the aglycone. Quercetin (MW 302) was identified as peak 35 due to the presence of the molecular ion [M-H]´at m/z 301. With regards to kaempferol derivatives, peak 28 was assigned to kaempferol 3-O-glucoside (MW 448) by observing the MS 2 data closely related to the glycosylation position. According to Ablajan et al. [30], peak 28 showed a MS 2 spectrum typical of a 3-O-glucosyl derivative. It produced a base peak at m/z 284, related to the homolytic cleavage of deprotonated flavonoid glycosides, a fragment at m/z 255 more abundant than that registered at m/z 257 and a fragment at m/z 327 that is not present in 7-O-glucosyl derivatives. Regarding flavanone and flavone derivatives, peak 38 was assigned to pinobanksin 3-O-butyrate, the parent ion (m/z 343) producing fragment ions as reported by Chua et al. [31]. Peak 37 was identified as apigenin-hexoside, since it produced a molecular ion [M-H]´at m/z 431, and yielded a fragment ion at m/z 269 corresponding to apigenin aglycone due to the loss of a hexosyl group. Three anthocyanidin derivatives were identified in MHE. Peaks 8, 10, 29 showed the presence of the cyanidin aglycone at m/z 287 in their MS 2 spectra. Peak 8 was assigned to cyanidin-dihexoside (MW 610), since the parent ion (m/z 611) produced fragment ions at m/z 449 and m/z 287, corresponding to the loss of the sugar molecules linked to the aglycone. Peak 10 was identified as cyanidin-3-rutinoside thanks to the pseudomolecular ion [M + H] + at m/z 595 and its MS 2 fragments at m/z 433, caused by the loss of one of the sugar molecules linked to the aglycone, and m/z 287, corresponding to the cyaniding aglycone (MW 594). Peak 29, which produced a molecular ion [M + H] + at m/z 449 and yielded a fragment ion at m/z 287 corresponding to the loss of the hexosyl moiety on MS 2 , may be assigned to the glucoside or galactoside derivative of cyanidin (MW 448).
With regards to benzoic acids, gallic acid (MW 170) was identified as peak 4 in MHE, since it produced a base peak at m/z 125 corresponding to the loss of a carboxyl group [M-H-CO 2 ]´.
As far as tannins are concerned, condensed tannins and hydrolizable tannins (gallotannins and ellagitanninis) were detected. Seven condensed tannin compounds were detected in MHE. Three peaks (12,14,16) showed molecular ions at m/z 577 with the same MS 2 fragmentation pattern (m/z 425, 407, 289), but different retention times: consequently they can be considered to be three isomers of procyanidin (MW 578) [32]. Peak 20 produced a molecular ion [M-H]´at m/z 729 and the most significant MS 2 fragments at m/z 577, corresponding to the loss of a galloyl group, and at m/z 289, corresponding to the monomer: for these reasons peak 20 was assigned to a monogalloyl procyanidin dimer. Peaks 7, 11, 17 produced a molecular ion [M-H]´at m/z 881, which could correspond to procyanidin digallate, and the same MS 2 fragmentation spectra. The main fragments produced were at m/z 729 (corresponding to the loss of a galloyl group), at m/z 711 (corresponding to the loss of a molecule of gallic acid), at m/z 577 (corresponding to procyanidin), and at m/z 289 (corresponding to the monomer (´)-epicatechin or (´)-catechin). Consequently, peaks 7, 11, and 17 were assigned to three different isomers of procyanidin digallate. With regards to hydrolizable tannins, peaks 3 and 5 were assigned to hesahydroxydiphenoyl-glucose and hesahydroxydiphenoyl-galloylglucose, respectively. Peak 3 was identified as hesahydroxydiphenoyl-glucose (MW 480), since it produced a molecular ion at m/z 481 and a MS 2 fragment ion at m/z 301, corresponding to the loss of a glucose unit as reported in the literature [33]. Peak 5 produced a molecular ion at m/z 633, an intense fragment ion at m/z 301 suggesting the loss of a galloylglucose unit, and a fragment ion at m/z 481, corresponding to the molecular ion of hesahydroxydiphenoyl-glucose, due to the loss of a galloyl unit. Through comparison with data from the literature [33], the fragmentation pattern of this molecule suggests that the galloyl moiety is directly linked to the glucose unit, so the compound was identified as hesahydroxydiphenoyl-galloylglucose (MW 634).

Antibacterial Activity of MHE
The antibacterial activity of MHE obtained from M. hallii dried leaves was tested against ten MRSA and MSSA, ten E. coli, ten P. aeruginosa, ten Enterococcus spp, and ten S. pyogenes strains. MHE showed antibacterial activity against all the tested S. aureus (MIC range 0.007-0.0019 gr/mL), P. aeruginosa (MIC range 0.125-0.062 gr/mL), S. pyogenes (MIC range 0.007-0.0039 gr/mL) and Enterococcus spp. (MIC range 0.0039-0.0019 gr/mL). MHE was found to be less active against E. coli strains (MIC range 0.25 > 0.5 gr/mL) (Tables 2 and 3 and Figure 2). Our data showed that MHE extract can inhibit bacterial strains irrespectively of their mechanisms of resistance. No significant differences were observed between strains carrying well-known mechanisms of resistance and susceptible ones, because one-dilution differences in this kind of analysis are taken for granted.

Discussion
This research represents the first report on the isolation and identification of polyphenols and organic acids from M. hallii to date. UHPLC-PDA-hESI-MSn analysis revealed the presence of thirty-eight compounds, belonging to flavonoids, phenolic acids, tannins and organic acids. Some of them have been previously identified in extracts obtained from plants and fruits belonging to the Myrtaceae family, whilst for others, this is the first report of their identification in a Myrtaceae species.
In MHE extract, 29 flavonoids were identified, consisting of flavan-3-ols, condensed tannin derivatives, flavonols, a flavanone and a flavone derivative and anthocyanin derivatives. Flavan-3-ols were also detected in the methanolic extract of the bark of Tristaniopsis callobuxus Brongn. & Gris, by Bellosta et al. [35], which showed the presence of gallocatechin and

Table 2.
In vitro antibacterial cumulative activity of MHE against ten S. aureus, ten E. coli, ten P. aeruginosa, ten Enterococcus spp., and ten S. pyogenes strains.

Discussion
This research represents the first report on the isolation and identification of polyphenols and organic acids from M. hallii to date. UHPLC-PDA-hESI-MSn analysis revealed the presence of thirty-eight compounds, belonging to flavonoids, phenolic acids, tannins and organic acids. Some of them have been previously identified in extracts obtained from plants and fruits belonging to the Myrtaceae family, whilst for others, this is the first report of their identification in a Myrtaceae species.
In MHE extract, 29 flavonoids were identified, consisting of flavan-3-ols, condensed tannin derivatives, flavonols, a flavanone and a flavone derivative and anthocyanin derivatives. Flavan-3-ols were also detected in the methanolic extract of the bark of Tristaniopsis callobuxus Brongn. & Gris, by Bellosta et al. [35], which showed the presence of gallocatechin and epigallocatechin. Catechin was also detected in the methanolic extracts obtained from the air-dried leaves of Myrtus communis L. (myrtle) and Eucalyptus globulus Labill [16,18]. Amongst Myrtaceae, flavonol derivatives were also detected in the methanolic extracts of Eucalyptus globulus. For myrtle, infusions and leaf methanolic extracts were both shown to contain myricetin and quercetin derivatives [2,18] [24]. Regarding anthocyanins, in the fruits of Myrciaria dubia (Kunth) McVaugh, a plant native of Amazonian rainforest, cyanidin-3-glucoside was identified as the main pigment [37]. The other two cyanidin-derivatives identified in MHE have never been reported in plants belonging to Myrtaceae family. Condensed tannins, such as procyanidin, were detected in many species belonging to Eucalyptus and Eugenia genera [38]. Ellagitannis have only been found in dicotyledoneous angiosperms and Myrtaceae are indicated as being rich in ellagitannins. In support of this fact, ellagitannis were detected in Callistemon lanceolatus Sweet, Eucalyptus alba Reinw. Ex Blume, Eugenia grandis Wight, Kunzea ambigua (Sm.) Druce, Melaleuca squarrosa Sm., Pimenta dioica (L.) Merr., Siphoneugena densiflora, Syzygium aqueum (Burm. f.) Alston, and Syzygium aromaticum (L.) Merr & L.M. Perry [39]. In MHE, gallic acid, hydrolysable tannins and organic acids have been detected. Gallic acid is a secondary metabolite of plants, mainly formed from 3-dehydroshikimic acid through the shikimic acid pathway [40]. It is widely distributed in many different families of higher plants, both in the free state and as a part of more complex molecules such as ester derivatives or polymers. Amongst Myrtaceae, gallic acid was detected in the methanolic extract obtained from the leaves of Eucalyptus globulus [16], the fruits of Rhodomyrtus tomentosa (Aiton) Hassk. [41], the methanolic extract of the bark of Tristaniopsis callobuxus [35], leaf infusions or methanolic extract obtained from Myrtus communis [2,18] and the seed and fruit extracts of Syzygium cumini (L.) Skeels [42]. Quinic acid has been identified in many plants in the Myrtaceae family, such as Syzygium cumini and Myrtus communis [17,43].
Amongst the thirty-eight compounds occurring in M. hallii leaf extract, many of them possess well-documented antibacterial activity [44]. For example, the in vitro antibacterial activity of catechin derivatives has been known since the 1990s and has been demonstrated against different strains, such as Streptococcus mutans, E. coli, Clostridium perfringes and Bacillus cereus [45][46][47]. Moreover, flavonols, especially myricetin, quercetin and kaempferol derivatives, are characterized by a remarkable antibacterial activity against both Gram-positive and Gram-negative bacteria, such as S. aureus, Lactobacillus acidophilus, Porphyromonas gingivalis and Prevotella melaninogenica [48]. Other categories of compounds with known antibacterial activity are gallotannins and procyanidins. The former showed antibacterial activity against Gram-positive food-borne bacteria (i.e., Clostridium botulinum, Bacillus subtilis, B. cereus) [49]; the latter, especially those derived from berries, were found to be active against E. coli, S. mutans and oxacillin-resistant S. aureus [50]. On this basis, we evaluated MHE activity against different strains of S. aureus, P. aeruginosa, S. pyogenes, Enterococcus spp. and E. coli. To the best of our knowledge, no data are available on the antibacterial activity of M. hallii against drug resistant bacteria. Although MHE was found to be as rich in polyphenolic components that could exert antibacterial activity, our results showed that the antibacterial activity of MHE against S. aureus, P. aeruginosa, S. pyogenes and Enterococcus spp. strains is modest but appreciable, especially against Enterococcus spp. MHE was shown to be much less active against E. coli strains. Moreover, our data shows that, whilst methicillin-resistant strains usually display resistance to several drugs, no relevant differences were observed between methicillin-susceptible and resistant strains. Our results agree with earlier studies carried out on other species belonging to the Myrcianthes genus. The ethanol extract obtained from Myrcianthes discolor (Kunth) McVaugh dried leaves showed antibacterial activity against a S. aureus strain isolated from laryngitis samples, but did not show any activity against two E. coli strains isolated from urinary tract infection samples [51,52]. Moreover, M. cisplatensis showed antibacterial activity against methicillin-sensitive and resistant S. aureus strains [19]. According to the results obtained from UHPLC-PDA-hESI-MSn analysis, the appreciable antibacterial activity of MHE against S. aureus, P. aeruginosa, S. pyogenes, and especially Enterococcus spp., could be explained by the wide spectrum of polyphenols identified in the extract, which act as antimicrobial substances via different mechanisms of action (MOA). In fact, many MOA are ascribed to polyphenols, such as cytoplasmatic membrane damage, inhibition of nucleic acids, cell walls and cell membrane synthesis. Moreover, in addition to their direct antibacterial activity, a growing body of evidence suggests that polyphenols may interfere with some bacterial virulence factors such as enzymes, toxins and signal receptors [44].
The search for natural antimicrobial compounds is incited by the need to thwart the increasing bacterial resistance to antibiotics. In the biomedical field, this microbial antibiotic-resistance leads to a growing need for new, effective anti-infective materials for the prevention and delay of implantand device-associated infections [53]. Anti-infective biomaterials have become a primary strategy to achieve this end. Natural polyphenols are interesting candidates for new antimicrobial agents to be loaded into or coated onto biomaterials. The development of nanoparticles carrying bioactive compounds with antimicrobial activity has been the target of investigations over the past years. In particular, several technologies have been developed at the nanoscale, such as nanoparticles, nanofibers, and nanocapsules, providing targeted delivery of polyphenols for therapeutic uses [54]. Recently, polyphenol delivery systems with antimicrobial activity have been described, focusing on nanoparticles based on chitosan as the main structural and functional material [55]. Polyphenols, ubiquitously expressed in plants have been shown to exert anticancer and immunomodulatory properties along with their anti-inflammatory and antimicrobial activities. However, some issues have been raised regarding the use of free polyphenols as medical drugs due to their fast metabolism and excretion in the human body. Indeed, this behavior might restrict or hamper their in vivo bioactive effects [56]. Therefore, a successful strategy for the use of M. hallii polyphenols as antimicrobial agents in the biomedical field may be require their administration in a form linked to or incorporated with nanomaterials able to support their controlled and prolonged release.

Extraction and Dialysis
Two 10 g aliquots of MHE were separately taken from the dried and finely ground leaves of M. hallii in 100 mL of H 2 O/methanol solution (70:30; % v/v) containing formic acid (0.1%, v/v), prepared in the dark, under constant stirring at room temperature for 24 h. Both extracts were filtered on a paper filter, gathered and freeze-dried. The residue was weighted and then reconstituted to 20 mL with Millipore-grade water and subdivided into two 10 mL aliquots. The first one was subjected to microbiological assays, the second was submitted to dialysis. Dialysis was performed using a Spectra/Por ® Biotech Regenerated Cellulose membrane (Spectrum Europe B.V., Breda, The Netherlands) with a nominal molecular weight cut-off (MWCO) of 3500 Da. An aliquot (10 mL) of MHE was submitted to dialysis in 1000 mL of Millipore-grade water for 24 h at 4˝C under constant stirring. The dialysate (low molecular weight fraction, LMW) was freeze-dried and the dry residue was assessed and dissolved in 10 mL of Millipore-grade water and subjected to microbiological assays and to UHPLC-PDA-hESI-MSn analysis, following filtration through 0.45 and 0.22 µm filters.

UHPLC-PDA-hESI-MSn Analysis
The experiment was performed using a Jasco X-LC system (Jasco, Easton, MD, USA) equipped with a quaternary pump, an UHPLC photodiode array detector (PDA) and a linear ion trap mass spectrometer LTQ-XL (Thermo Scientific, Waltham, MA, USA) through an h-ESI source. Separation was achieved on a Purospher ® STAR RP-18e (5 µm) LiChroCART ® 250-4 (250ˆ4 mm 2 i.d., 5 µm) with its corresponding guard column (both from Merck KGaA, Darmstadt, Germany). The mobile phase consisted of A (0.1% formic acid in water) and B (acetonitrile) at a flow rate of 1 mL/min with an injection volume of 5 µL. Gradient elution was carried out using the following timetable: 98% A/2% B 0-5 min, 60% A/40% B 5-40 min, 0% A/100% B 40-45 min, 0% A/100% B 45-47 min, 98% A/2% B 47-52 min, 98% A/2% B 52-57 min. The resulting total run time was 57 min, including column reconditioning. The sample tray was set at 4˝C and the column oven temperature was set at 24˝C. The chromatograms were recorded at λ 280, 220, 366 and 520 nm); spectral data were acquired in the range of 200-650 nm for all peaks. The ion trap operated in data dependent, full scan (80-1500 m/z), zoom scan and MS n mode. To obtain the fragment ions a collision energy of 35% and an isolation of 2 m/z were applied; the voltage was kept at 3 kV for negative ionization and 5 kV for the positive one, the temperature of the capillary tube was 275˝C with a sheath gas flow rate of 45 arbitrary units and an auxiliary gas flow rate of 20 arbitrary units, while the ionization chamber was maintained at 100˝C. ThermoFisher Scientific Excalibur 2.0 software (SR2, Thermo Electron Corporation 1998-2006, Waltham, MA, USA) was used for data acquisition and processing. Three independent assays were performed to analyze the sample (filtered through a cellulose acetate/cellulose nitrate mixed esters membrane, 0.22 µm) and no relevant variations attributable to the nature of the detected fragments or their relative intensities were observed.

Bacterial Strains and Growth Conditions
The bacteria were recent clinical isolates, belonging to the Institute of Microbiology (University of Genova) collection. They comprised of: (i) ten S. aureus strains, including five methicillin-resistant (MRSA) and five methicillin-susceptible (MSSA) strains. Of the MRSA strains, three were multi-resistant (resistant to at least three classes of antibiotics); (ii) ten multi-resistant Escherichia coli strains; (iii) ten Pseudomonas aeruginosa strains; (iv) ten vancomycin-resistant and susceptible Enterococcus faecalis and Enterococcus faecium strains; and (v) ten group A streptococci (Streptococcus pyogenes) strains, which remain universally susceptible to penicillin. All isolates were identified at the species level using clinical methods and an API STAPH, API20E, API NE and API STREP system (bioMèrieux, Marcy l'Etoile, France) for S. aureus, E. coli, P. aeruginosa, Enterococcus spp. and S. pyogenes respectively. The antibiotype was determined using the disk diffusion test, as according to the latest Clinical and Laboratory Standards Institute (CLSI) guidelines [57]. Strains were cultured in Mueller-Hinton Broth, Mueller-Hinton agar, MacConkey agar and Columbia blood agar (Biolife, Milan, Italy) at 37˝C.

Susceptibility (MIC Determination)
The Minimum Inhibitory Concentration (MIC) of plant extract was determined using the broth microdilution method, following the CLSI guidelines [58]. In brief, exponentially growing bacteria (5ˆ10 5 cells per mL, final inoculum) were added to the various concentrations of plant extract, 2-fold serially diluted in 96-well microtitre plates of Mueller-Hinton broth or cation-adjusted Mueller Hinton broth with 5% of lysed horse blood for S. pyogenes. The following concentrations (gr/mL) of extracts were used: 0.5, 0.25, 0.125, 0.006, 0.031, 0.015, 0.007, 0.0039, 0.019, 0.00097, 0.00048. After 18-24 h of incubation at 37˝C, the concentration at which the plant extract prevented visible bacterial growth was identified as the MIC. All tests were performed in triplicate and executed three times. S. aureus ATCC 29213, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were added as control strains.

Conclusions
In conclusion, this study represents the first attempt to phytochemically characterize of M. hallii leaf extract and demonstrate its antimicrobial activity against drug resistant bacteria. The extract exhibits antimicrobial activity against S. aureus, P. aeruginosa, S. pyogenes, and Enterococcus spp. strains due to the presence of many different classes of polyphenolic compounds that possess antibacterial activity. The data reported in this paper reveal that M. hallii is a potential source of polyphenols with antimicrobial properties and shows the great potential of this species, not just for pharmaceutical applications, but also for biomedical, food and cosmetic applications.

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

Abbreviations
The following abbreviations are used in this manuscript: