**3. Discussion**

For more than half a century, humans have relied primarily on antibiotics and vaccines to treat and prevent microbial infections. In recent decades, despite the grea<sup>t</sup> progress in the medical and pharmaceutical fields, the traditional treatment of infectious diseases is often ineffective due to the increased resistance of microbial strains to antibiotics. To date, one fifth of global deaths is due to infectious diseases [17], as the uncontrolled use of antibiotics in the clinical, veterinary, and agricultural fields has led to the spread of multidrugresistant microbial strains. While the pharmaceutical industry has addressed this problem by modifying existing antibiotics and developing new ones, microbial strains respond to the pharmaceutical industry by inactivating these new strategies with the development of antibiotic resistance. This scenario clearly highlights the need for new antimicrobial agents with different modes of action than those of traditional antibiotics.

Natural products are among the most promising candidates because they have low toxicity, low environmental impact, and a broad spectrum of action when compared to synthetic antimicrobial substances.

Many studies have shown the antimicrobial activity of various EOs [18,19] also regarding muti-drug resistant bacteria and fungi, due to a broad spectrum of cytocidal activity [20,21]. For example, the EO of *S. montana,* in addition to anti-oxidant activity, proved effective against bacteria and dermatophytes; especially *T. violaceum*, *T. rubrum*, *T. tonsurans*, *T. mentagrophytes* and *P. oryzae* [22,23], while the EO obtained from *O. hirtum* showed antimicrobial activity against both Gram+ and Gram- strains [24,25]. The EOs belonging to the *Lavandula* genus, in addition to having an antimicrobial activity against a broad spectrum of microorganisms [26–28], show sedative properties on the central nervous system, as well as anti-inflammatory and re-epithelializing properties [29–31]. Furthermore, EOs and Hys derived from non-native plants belonging to the *Monarda* genus grown in Italy, have shown interesting antimicrobial activities towards Gram+, Gram- yeasts and environmental fungi [32–34].

The effectiveness of active ingredients was also studied. β-Linalool is a non-toxic alcohol most common in nature. It is present in the phytocomplexes of lavender EOs but also of many other EOs. In the EO of *Cinnamomum camphora* (Ho wood) it can reach concentrations higher than 90%. Literature data show its comprehensive range of bioactive properties including antimicrobial activity [35]. The main component of both EO and Hy of *O. hirtum* is the thymol, a phenol monoterpene isomer of carvacrol, particularly present in EOs obtained from species belonging to the *Thymus* genus. This natural compound has an antimicrobial spectrum wider than that of β-linalool, including Gram-positive, Gram-negative bacteria (especially pathogens of the airways), and fungi. Finally, it shows the ability to interfere with the fungal transformation process from the cellular form to the hyphal form [36]. The antimicrobial activity of carvacrol, main component of both *S. montana* and *Monarda* spp. natural products, is higher than that of the other volatile compounds due to the free hydroxyl group, hydrophobicity, and the phenol moiety. In particular, it shows a grea<sup>t</sup> activity against Gram- food-borne pathogens [37].

Among the main active compounds analyzed, it is possible to identify an activity gradient (linalool < thymol < carvacrol). This gradient is consistent with the data of antimicrobial efficacy actually observed, as the least active natural compounds are those obtained from the *Lavandula* genus, while the others show stronger antimicrobial activities.

Moreover, several EOs have been shown to interfere with the ability of microorganisms to form biofilm, which is often linked to chronic, difficult-to-treat infections such as skin and wound infections [38,39]. *S. montana* EO was shown to be able to inhibit biofilm formation and interfere with preformed biofilms of Gram+ bacteria, including *S. aureus* [23].

Despite the high antimicrobial activity of EOs, use as such is not recommended due to their high concentration of hydrophobic active ingredients with a toxic potential. Therefore, to avoid toxic effects, EOs need to be used in low concentrations by diluting them in an appropriate vehicle before use.

On the contrary, Hys are hydrophilic solutions containing up to a maximum 1g/L of the EOs active compounds. Although more perishable than EOs, they are generally safe and do not need to be diluted in a vehicle before use. This feature of Hys makes them interesting both for oral intake and skin applications. The latter use becomes especially important in the presence of skin infections.

However, the antimicrobial activity of Hys would certainly appear to be milder than that of the corresponding EOs. In fact, the simple comparison of MIC values obtained from the antimicrobial analysis of the EOs and Hys used in this study evidence that the first are more effective at a lower concentration. Tables 1 and 2 show that the EOs active on at least the 50% of the strains have inhibitory and cytocidal actions at concentrations ranging between 0.125% *v*/*v* and 2% *<sup>v</sup>*/*<sup>v</sup>*. Whereas, the Hys must be used at concentrations between 25% *v*/*v* and 50% *v*/*v* to reach the same antimicrobial activity, i.e., they need to be from 25 to 200 times more concentrated than EOs.

However, if we consider the relative concentration of active chemicals, can we say that Hys really have milder antimicrobial actions than the corresponding EOs? Tables 5 and 6 show that this cannot be said. In fact, the calculated IR50Hy/CF is lower than the IR50EO, as well as the IC50Hy/CF calculated for each microbial strain is lower than the IC50EO. This means that, to obtain the inhibition of 50% of growth of both the initial inoculum of each strain and total microbial strains, a concentration of EOs' volatiles greater than that of the corresponding Hys is required. It results, therefore, in the Hys' volatiles being relatively more effective than those of EOs. This activity could be due to the hydrophilic environment of Hy, which provides a greater bioavailability of volatiles for the interaction with bacteria and fungi [40], or to the antagonistic action present among chemical components of the EO phytocomplex.

These data are interesting because they show the antimicrobial activity of Hys from another point of view, especially as it concerns potential clinical applications for the treatment of skin infections. In fact, in these pathologies, local applications that are simultaneously effective for the patient and safe for intact or damaged skin are indispensable.

Potential applications encompass all small skin infections that need daily local treatments with antimicrobial creams and ointments, but also of more serious pathologies such as *Tinea capitis* generated by dermatophytes that essentially afflicts children, or antibiotic resistant/sensitive infections of sores or wounds whose treatment becomes important for skin re-epithelialization, or chronic vaginal infections induced by yeasts in which the topical use of concentrated EOs is absolutely contraindicated due to their toxicity.

In all cases, the use of Hys with antimicrobial activity compatible with a cutaneous or mucosal treatment would be of grea<sup>t</sup> interest. In fact, Hys are already on the market, and they can be used on the skin of non-allergic subjects without inducing adverse effects. Currently, Hys in Italy are used in formulations of cosmetic products for body care, or they are sold pure for cosmetic and food use. As is well known, the Italian market is a famous perfume and fragrance hub that is constantly looking for new products and is able to influence the Hys production of primary producers. Globally, the Hys market in Europe has been growing for several years, attaining, in 2018, a 40% share of the world market [41]. From 2019 to 2024, this share is set to increase by an additional 5.2% [42]. Owing to these reasons and in light of our preliminary data, it becomes more and more interesting to deepen the studies on Hys.

#### **4. Materials and Methods**

#### *4.1. Clinical Strains*

Fifteen clinical strains (six Gram-positive bacterial strains and nine fungal strains), which are potential skin pathogens provided by the UOC of Microbiology of Policlinico Universitario A. Gemelli of Rome, Italy, were used. Two of the six bacterial strains were resistant (R) to antibiotics. Bacterial strains were: *Staphylococcus aureus* MRSA (0.1R), *Streptococcus pyogenes* (0.2), *Enterococcus faecalis* VRE (0.3R), *Enterococcus faecium* (0.4), *Staphylococcus aureus* MSSA (0.5), *Enterococcus faecalis* (0.6). Whereas, four of the nine fungal strains were yeasts (*Candida albicans* (3.1), *Candida parapsilosis* (0.1R), *Candida glabrata* (0.2R), and *Candida tropicalis* (0.3R)), three of which were resistant to common antifungals, and five dermatophytes (*Trichophyton rubrum*, *Trichophyton tonsurans*, *Trichophyton soudanense*, *Trichophyton violaceum*, and *Microsporum canis*). Mueller Hinton medium (Becton Dickinson and Company, Cockeysville, MD, USA) was used to grow bacterial strains at 37 ◦C for 24 h, while fungal strains were grown on RPMI broth and Sabouraud agar medium (Oxoid, Wade Road, Basingstoke, Hants, UK). In particular, yeasts were grown at 37 ◦C for 24 h, and dermatophytes at 30 ◦C for 7 days.

#### *4.2. Essential Oils and Related Hydrolates*

EOs and Hy from six aromatic plants grown and processed in Italy were studied (*S. montana*, *L. angustifolia*, *L. intermedia*, *O. hirtum*, *M. didyma*, and *M. fistulosa*). All EOs and Hys were kindly granted by FX Laboratorio Benessere srl (Arzignano, Vicenza, Italy), except for those isolated from *M. didyma* and *M. fistulosa* species, which were provided by DISTAL, University of Bologna.

#### *4.3. Gas Chromatography Mass Spectrometry Analysis*

Analyses were performed on a Bruker ScionSQ gas chromatograph, coupled with a single quadrupole mass-spectrometer (GC-MS) (Bruker, Milan, Italy). Compounds were separated BD-5 a semi-standard non-polar column (30 m × 0.25 mm, i.d. 0.25 μm) (Phenomenex, Bologna, Italy). EOs were diluted 1:1000 (*v*/*v*) in ethyl acetate, and 1 μL of this dilution was injected into GC-MS. Samples of hydrolate were diluted 1:5 (*v*/*v*) in ethanol (99.8%), and 1 μL of this dilution was injected into GC-MS. The percentage (*w*/*w*) of the amount of the compounds of EO present in Hy was carried out gravimetrically. Peaks were identified by comparing the retention times with those of authentic standard MS fragmentation patterns and final confirmation by matching with the components of the commercial library NIST mass spectral database (vers. 6.41). The percentage composition of the oils was computed by the normalization method from the GC peak areas. R.I. were generated by using a series of n-alkanes from C7 to C40 (Sigma-Aldrich, Milan, Italy) and compared with data reported in the literature [43–46]. All analyses were repeated in triplicate.

#### *4.4. Gravimetric Analysis*

Five mL of each Hy were subjected to liquid/liquid isolation with 5 mL of CH2Cl2 (*n* = 3). The organic phases were pooled, and the solvent evaporated by means of a rotary evaporator at reduced pressure. The residue obtained was weighed and the percentage (*w*/*v*) content of volatiles in the hydrolate evaluated.

#### *4.5. Broth Microdilution Susceptibility Test*

The broth microdilution (BMD) susceptibility test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) international guidelines were performed. The BMD test was performed on a 96-well plate by adding 100 μL of a cell

suspension equal to 5 × 10<sup>5</sup> CFU/mL to a final volume of 200 μL. Scalar dilutions, between 50% *v*/*v* (500 μL/mL) and 3.125% *v*/*v* (31.25 μL/mL) of Hy and between 2% (20 μL /mL) and 0.06% (0.6 μL/mL) of EO were tested. EOs and Hys were dissolved in a suitable nutrient agar (as specified in paragraph 4.1) and 0.5% *v*/*v* of Tween 80 was used to deliver the EOs into the hydrophilic medium. Plates were incubated overnight at 37 ◦C. After this period, MIC values were determined by spectrophotometric reading at 450 nm (EL808, Biotek, Winooski, VT, USA), except for MICs values of the dermatophytes, which were assessed by visual reading. To evaluate the MLC, 5 μL of the content of each well was seeded on Muller Hilton or Sabouraud agar plates, which were incubated for 24 h at 37 ◦C. The MIC is defined as the lowest concentration that completely inhibits the organism's growth when compared to the growth of control. Whereas, the MLC is defined as the lowest concentration corresponding to the death of 99.9% or more of the initial inoculum. Each test was performed in triple, and both negative and positive controls were included. Values corresponding to the IR or LR of 50% and 90% of all strains were calculated. As discussed in the "Data management" paragraph, the value corresponding to a concentration of EOs or Hys necessary to obtain the inhibition of 50% of the initial inoculum was extrapolated for each strain analyzed.

#### *4.6. Comparison Between EO and Hy*

Hy and EO comparison was made, as described in Di Vito M et al. [16]. Comparison was based on comparing the total volatiles content of EO with that of the corresponding Hy. Briefly, the Essential Oil Total volatiles Area (EOTA) and the Hydrolate Total volatiles Area (HYTA) were calculated by evaluating areas covered by the total volatiles in the chromatograms multiplied by EO and Hy respective dilutions prior to GC–MS (1000 and 5, respectively). The semi-quantitative volatiles' Conversion Factor (CF) between the EO and the Hy was assumed to be the EOTA/HYTA ratio. Comparison between an EO and its corresponding Hy was made by dividing the IC50 or IR50 of each Hy by its CF. If the value of this ratio corresponds to the value of IC50 or IR50 of the EO, it means that the two natural products are equivalent in terms of relative antimicrobial activity, as the same amount of volatiles is needed in both EO and Hy to inhibit the growth of 50% of the initial inoculum. Whereas, values of this ratio lower or higher than the IC50 or IR50 of the OE show a relative antimicrobial activity of volatiles contained in the Hy higher or lower than that of the EO, respectively.

#### *4.7. Data Management*

The IC50 value of each natural substance (*O. hirtum, S. montana, M. didyma* and *M. fistulosa*) and distillation product (EO and Hy) vs. each microbial strain was obtained by interpolating the OD450 values corresponding to the tested dilutions with a regression line, and calculating the dilution value (% *v*/*v*) corresponding to half of the OD450 value of the positive control. All the values obtained from both the microbiological and chemical analyzes were processed obtaining mean and standard deviation values.
