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Article

Original Contributions to the Chemical Composition, Microbicidal, Virulence-Arresting and Antibiotic-Enhancing Activity of Essential Oils from Four Coniferous Species

by
Diana-Carolina Visan
1,
Eliza Oprea
2,*,
Valeria Radulescu
1,
Ion Voiculescu
3,
Iovu-Adrian Biris
3,4,
Ani Ioana Cotar
5,
Crina Saviuc
6,7,
Mariana Carmen Chifiriuc
6,7,8 and
Ioana Cristina Marinas
6,7
1
Department of Organic Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy “Carol Davila”, 6 Traian Vuia Street, 020956 Bucharest, Romania
2
Department of Organic Chemistry, Biochemistry and Catalysis, Faculty of Chemistry, University of Bucharest, 4-12 Regina Elisabeta, 030018 Bucharest, Romania
3
“Marin Drăcea” National Institute for Forestry Research and Development, 128 Eroilor, 077190 Voluntari, Romania
4
Faculty of Agriculture, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Mărăşti, 011464 Bucharest, Romania
5
Cantacuzino National Medico-Military Institute for Research and Development, 103 Splaiul Independenței, 050096 Bucharest, Romania
6
Research Institute of the University of Bucharest—ICUB, 91-95 Spl. Independentei, 050657 Bucharest, Romania
7
Microbiology Department, Faculty of Biology, University of Bucharest, 1-3 Portocalilor Way, 060101 Bucharest, Romania
8
Academy of Romanian Scientists, 3 Ilfov Street, 50044 Bucharest, Romania
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2021, 14(11), 1159; https://doi.org/10.3390/ph14111159
Submission received: 11 October 2021 / Revised: 7 November 2021 / Accepted: 10 November 2021 / Published: 13 November 2021
(This article belongs to the Special Issue Bioactive Compounds from Plants and Foods with Pharmaceutical Interest 2022)
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Versions Notes

Abstract

:
This study aimed to establish the essential oil (EO) composition from young shoots of Picea abies, Larix decidua, Pseudotsuga menziesii, and Pinus nigra harvested from Romania and evaluate their antimicrobial and anti-virulence activity, as well as potential synergies with currently used antibiotics. The samples’ EO average content varied between 0.62% and 1.02% (mL/100 g plant). The mono- and sesquiterpene hydrocarbons were dominant in the composition of the studied EOs. The antimicrobial activity revealed that the minimum inhibitory concentration (MIC) values for the tested EOs and some pure compounds known for their antimicrobial activity ranged from 6.25 to 100 µL/mL. The most intensive antimicrobial effect was obtained for the Pinus nigra EO, which exhibited the best synergistic effect with some antibiotics against Staphylococcus aureus strains (i.e., oxacillin, tetracycline, erythromycin and gentamycin). The subinhibitory concentrations (sMIC) of the coniferous EOs inhibited the expression of soluble virulence factors (DN-ase, lipase, lecithinase, hemolysins, caseinase and siderophore-like), their efficiency being similar to that of the tested pure compounds, and inhibited the rhl gene expression in Pseudomonas aeruginosa, suggesting their virulence-arresting drug potential.

1. Introduction

Plant essential oils (EOs) are complex mixtures of volatile natural compounds. EOs are formed in aromatic plants as secondary metabolites (terpenes, terpenoids, phenylpropenes and “others”) [1], which play an important role in plant defense [2] and have been used since ancient times as natural remedies for fighting infectious diseases caused by different microbial and viral pathogens [3,4,5]. They are relatively easy to obtain, have low mammalian toxicity, and degrade quickly in water and soil, making them relatively environmentally friendly [1].
It has been shown that plant extracts have pronounced antimicrobial activities, even when used in subinhibitory concentrations, which do not interfere with bacteria growth but only with their behavior [6], leading to a reduced risk of developing resistance to that compound and to a lower risk of dysbiosis [7]. Moreover, these low concentrations will have minimal or no effect against host cells. Taking into account these aspects, the most recent anti-infective approaches, also called anti-pathogenic or anti-virulence strategies, propose targeting virulence factors expression and biofilm development rather than inhibition of microbial growth or killing the pathogens [6,8]. Despite their superior resistance to antibiotics, biofilm-embedded bacteria seem to be more susceptible than their planktonic counterparts to some EOs, probably because (i) the extracellular matrix of the biofilm adsorbs the active phytocomponents and increases their local concentration; and (ii) the cellular envelope (capsule, cellular wall and membrane) in biofilm cells is different from that of free cells due to differential gene expression in the two growth states and more susceptible to EOs [9].
For this study we have chosen to evaluate the EOs of four coniferous species, being known that coniferous forests are a renewable source of EOs that are distributed in various organs of these plants: needle/leaves, roots, cones/seeds, wood/stem/twigs, bark and berries [8].
The EOs main compounds of coniferous species are monoterpenes, monoterpenoides, sesquiterpenes, sesquiterpenoides and diterpenes [10,11]; however, the chemical composition of EOs could be variable, depending on the anatomical part of the tree, the genetic factors [12], the health condition of plant and also on the geographic and environmental conditions: soil and water composition, humidity and air pollution [13,14,15]. Research conducted in recent decades have highlighted the antibacterial [16,17], antifungal [17,18,19,20] and antioxidant [21,22] properties of EOs isolated from different coniferous species [15,19,22,23,24,25,26,27,28,29,30] but there is scarce information concerning the biologically active principles isolated from populations of coniferous species on the Romanian territory. This type of research is very important if we consider that Romania is the first place in Europe regarding the rates of antimicrobial resistance [11,31]. We have previously shown that the Abies alba EO inhibits agrI gene expression in Staphylococcus aureus, suggesting an inhibitory effect on the quorum sensing (QS) genes expression and indirectly on the strain virulence, and therefore their anti-pathogenic potential [32].
The objectives of this study were to investigate the composition of the EOs from young shoots of four coniferous species and to evaluate their antimicrobial activity. The biological material consisted of three Eurasian species native to the Carpathian area—spruce (Picea abies), larch (Larix decidua) and black pine (Pinus nigra)—and a species of North American origin—the Douglas fir (Pseudotsuga menziesii)—naturalized and often used in forest plantations in Europe.

2. Results and Discussion

2.1. Essential Oil Content and Composition

The average EO content of the P. abies, L. decidua, P. menziesii and P. nigra samples (five determinations for each sample) was 1.02 ± 0.19, 0.62 ± 0.13, 0.87 ± 0.04 and 0.82 ± 0.14% (mL essential oil/100 g dried plant), respectively. Figure 1 shows the chromatogram of the P. abies EO and in Table 1 are listed the identified compounds for all four EO samples. The other chromatograms of the EOs are included in Supplementary Materials (Figures S1–S3). The numbers from the peak of the compounds on the four chromatograms correspond to the numbers listed in Table 1.
The main compound classes from the studied EOs were mono- and sesquiterpene hydrocarbons, representing 69.20% (P. abies), 84.53% (L. decidua), 93.72% (P. menziesii) and 94.39% (P. nigra) from each EO.
Camphene, α-pinene, β-pinene and limonene are prevalent among the monoterpene hydrocarbons. The EO compositions isolated from P. abies [11], L. decidua and P. menziesii [34] are similar to the previously published papers. For the EO extracted from P. abies, the content in compounds with oxygen is significantly higher than in other oils, i.e., 11.08% for bornyl acetate, 9.40% for manool, 3.87% for α-cadinol, and 2.15% for α-muurolol. Germacrene D is the major compound (19.80%) in the EO of Larix decidua, while pinene (α and β) is one of the most important components of the EO extracted from P. abies, these data being in accordance with those reported by Mofikoya (2020) [35].

2.2. Antimicrobial Activity

2.2.1. Qualitative and Quantitative Analysis

The qualitative screening of the EOs and the pure compounds revealed the occurrence of a growth inhibition zone in the area where the EOs: DMSO stock solution was spotted. We started by assessing the efficiency of the tested EOs against a larger batch of microbial strains, but only those strains for which a growth inhibition zone was observed were further tested by quantitative assay. Thus, in Table 2 are presented only the strains that proved to be sensitive to the EOs and pure compounds when tested by the qualitative assay.
The quantitative assay revealed that the MIC values for the tested EOs as well as for some pure compounds known for their antimicrobial activity ranged from 6.25 to 50 µL/mL, the most intensive effect being obtained for P. nigra, exhibiting the lowest MIC values against all tested strains (Table 2).
Concerning the antimicrobial activity of the pure compounds, the most active proved to be α-pinene, S. aureus strains being more susceptible than the Gram-negative ones. Phellandrene, borneol and camphor had the same effect as nerolidol. The EOs proved also to be more effective against S. aureus strains, as well as towards Bacillus subtilis and Candida albicans strains, as compared to the Gram-negative species. It is to be noticed that in many cases, the MIC values were lower for the EOs than those obtained for the pure compounds, demonstrating the synergistic effect of the active compounds found in the EOs. The antimicrobial activity of P. abies EO could mainly be due to bornyl acetate because this compound has shown a good activity on S. aureus (MIC 1.95 mg/mL), P aeruginosa (MIC 2.30 mg/mL) and Escherichia coli (MIC 4.88 mg/mL) [36]. Some studies showed that α-pinene and β-pinene are able to destroy the cellular integrity by inhibition of respiration and ion transport processes [37,38]. Helander et al. [39] showed that the low molecular mass lipophilic compounds are responsible for the toxicity of EO components on Gram-negative bacteria because these compounds are able to penetrate the bacterial membrane and may thus be able to influence the proliferation of certain pathogenic bacteria.
By comparing the effect of the P. abies EO on Gram-positive and Gram-negative strains, a statistically significant activity was observed for Gram-positive bacteria (p < 0.001) (Figure 2a). For L. decidua, P. menziesii and P. nigra, the difference between the antimicrobial activity on Gram-positive and Gram-negative strains was not significant (p > 0.05), despite the fact that a better effect was observed on Gram-positive bacteria (Figure 2b–d).
According to Magwa et al. [40], Sesuvium portulacastrum exhibited an antibacterial activity against S. aureus, which may be due to the camphene found in its EO. This compound, identified in the analyzed conifer EOs, seems to be responsible for their antibacterial activity, especially on Gram-positive bacteria, because P. abies has the highest concentration of camphene and the best effect on S. aureus and B. subtilis. The presence of trans-caryophyllene and camphene, known to possess antifungal activity [40,41], in the studied EO composition explain the effect on the Candida strain. The P. abies EO had the biggest percent of these compounds (camphene: 10.7% and trans-caryophyllene: 1.17%) and the best antifungal activity, followed by P. nigra (with camphene: 1.24% and trans-caryophyllene: 1.99%).

2.2.2. The Adherence Capacity to the Inert Substrate

In the natural environment, but also in the infected host, microorganisms usually produce extracellular capsular polymers, mostly polysaccharides, known as a capsule, slime, or glycocalyx, which, in the case of pathogenic strains, are an important virulence factor, being involved in adhesion and colonization of inert substrata, such as medical devices [42].
The adherence capacity to the inert substrate of the reference and clinical strains was inhibited by all the tested EOs and pure compounds at subinhibitory concentrations, respectively, MIC/2. The results are represented as the minimum biofilm eradication concentration (MBEC) values in Table 2. These results show the promising potential of these EOs to appropriately address the challenges of biofilm-associated infections diagnosis and treatment, often remaining unresolved with the present approaches [43,44].

2.2.3. The Synergistic Activity with Antibiotics

The tested EOs potentiated the currently used antibiotics against S. aureus and P. aeruginosa strains, the most intensive effect being observed in case of P. nigra. The S. aureus 12 H strain, in the presence of EOs obtained from P. abies and P. menziesii, switched from resistant to susceptible to oxacillin and tetracycline, and in the presence of the P. nigra EO, to erythromycin, while S. aureus 35 PL became susceptible to gentamycin (Table 3). In case of the P. aeruginosa strains, the growth inhibition diameters for piperacillin, ticarcillin-clavulanic acid, imipenem, aztreonam, ceftazidime, ciprofloxacin, colistin and gentamycin were not modified by the EOs, probably due to the multi-drug resistance phenotype of these strains, which is often mediated by efflux pumps that are not substrate-specific, thus being able to provide cross-resistance to the EOs [45].
The synergic effects could be produced by α-pinene, according to Kovač, who reported that the MICs reduced from 32 to over 512-fold when (−)-α-pinene was applied in combination with erythromycin, ciprofloxacin or triclosan [46].
Other recent studies revealed that numerous plant-derived compounds and EOs, by interfering with adherence, biofilm formation and motility, are also affecting antibiotic susceptibility [47,48,49].

2.2.4. The Influence of EOs on the QS Genes Expression

Taking into account the involvement of P. aeruginosa strains in the etiology of opportunistic and nosocomial infections, as well as their high resistance rates to the current antibiotics, a significant number of the respective strains isolated from clinical specimens have been tested in order to establish the modulatory effect of the EOs on the expression of QS genes. A relatively new strategy for combating bacterial infections and resistance to antibiotics is represented by QS inhibitors (QSI). Some EOs [50,51,52,53] or their components [32,54,55] were already reported to inhibit QS genes expression. P. aeruginosa, a critical opportunistic nosocomial pathogen, produces different virulence factors synthesized under the control of QS systems las and rhl. The first one consists of LasI, which modulates the synthesis of the autoinducer N-(3-oxododecanoyl) homoserine lactone, and a transcriptional activator (lasR). The second is composed of a putative transcriptional activator, rhlR and rhlI, which manage the synthesis of N-butyryl homoserine lactone. An interconnecting role between these two systems in the QS hierarchy of P. aeruginosa is held by the PQS signaling system (which produces 2-heptyl-3-hydroxy-4-quinolone) [56].
The rhl and lasR genes expression was significantly downregulated by the coniferous EOs, while lasI expression was upregulated (Figure 3).
According to Kostylev (2019), the RhlIR QS system requires induction by LasR, and as a consequence, a decreasing in the expression of QS-activated genes could be possible by lasR or lasI deletion in strain PAO1 (a laboratory model P. aeruginosa strain) [57].
As a result, these findings suggest that EOs interfere with the QS pathways in P. aeruginosa and could be a promising lead for the development of virulence-arresting drugs. These findings indicate that EOs may have an inhibitory effect on rhamnolipid production, which is regulated by the P. aeruginosa QS regulator rhlR, while an inhibitory effect on elastase and protease activities are regulated by the rhlI-rhlR system [58].

2.3. Influence of EO on the Expression of Soluble Enzymatic Virulence Factors

The tested strains have been previously analyzed for their virulence potential and selected as positive for producing the investigated virulence determinants, i.e., toxins forming pores in the membrane of eukaryotic cells (lecithinase, hemolysins and lipase), proteases (caseinase) and DN-ase [32].
Taking into account the rapid emergence of resistance to classical antimicrobial drugs, a new but expanding class, the so-called virulence-arresting drugs, targeting the inhibition of virulence factor production rather than kill pathogens, has emerged. These drugs can restore or augment the antibiotics’ effect in a pathogen-specific manner, thus decreasing the risk of resistance and side effects [59,60,61,62].
We have previously demonstrated the effects of some natural pure compounds or products (probiotic fractions, essential oils, bacteriophages, etc.) on the phenotypic or genotypic expression of virulence factors in opportunistic pathogens [32,63,64,65].
In the present study, the tested EOs inhibited the expression of the analyzed soluble virulence factors by different degrees, their efficiency being similar to that of the pure compounds. The most inhibited virulence factors in S. aureus were haemolysins, followed by siderophore-like compounds and lecithinase, while in P. aeruginosa, DN-ase, siderophore-like and haemolysins (Table 4).

3. Materials and Methods

3.1. Reagents and Solvents

SupraSolv dichloromethane was used for the gas chromatography, anhydrous Na2SO4 granulated for organic trace analysis, and the pure compounds (Ph. Eur.)—α-pinene, (+)-limonene, phellandrene, eucalyptol, borneol, camphor and nerolidol—were purchased from Merck, Darmstadt, Germany. The n-alkanes C8–C24 used for the determination of the Kovats retention indices were from Fluka, Switzerland.

3.2. Plant Material

The samples of young shoots with needles (around 1000 g) of Douglas fir (P. menziesii), European larch (L. decidua ssp. Carpathica), Norway spruce (P. abies) and black pine (P. nigra ssp. nigra) were collected from an intensive plantation located in the tree nursery of the “Marin Drăcea” National Institute for Forestry Research and Development (Voluntari, Romania). The plants grew in natural vegetation conditions, without the use of chemical fertilizers or pesticides to control weeds, diseases and pests. For each species, the samples were harvested from ten individual 6–8-year-old trees. The samples were dried, separated from branches and manually grounded.

3.3. Essential Oil Extraction

The needles (50 g) were hydro-distilled in a Clevenger-type apparatus for 4 h [66]. The EOs was dried over anhydrous Na2SO4, stored in a dark glass bottle and kept at 4 °C until analysis. The oil samples were diluted in dichloromethane (1/200) and 1 μL was injected for GC analysis.

3.4. Gas Chromatography–Mass Spectrometry

GC-MS analysis of the EOs was carried out using a Fisons Instruments GC 8000 with an electron impact quadrupole, MD 800 mass spectrometer detector.
The electron ionization energy was 70 eV. A fused silica column of 5% phenylpoly (dimethylsiloxane) (SLB–5 ms, 30 m × 0.32 mm i.d., film thickness = 0.25 μm) was employed. The operating conditions were as follows: a split-splitless injector (split ratio, 1:30) at 280 °C, ion-source temperature 200 °C and the interface temperature 280 °C; initial column temperature, 40 °C for 3 min, raised at 4 °C/min to 280 °C and finally held isothermally for 20 min; the carrier gas (helium) flow rate was 2 mL/min; and sample volume injected, 1 μL. Data acquisition was performed with MassLab 3.4 Software for the mass range 30–600 u with a scan speed of 1 scan/s. The identity of the EO components was established from their GC Kovats retention indices and from mass spectra by computer matching with a mass spectra library (NIST, Wiley and a personal library of 600 spectra). The Kovats retention indices were determined in relation to a homologous series of n-alkanes (C8–C24) and compared with those reported in the literature [67,68,69]. The components’ relative concentrations were calculated from the GC peaks without using correction factors.

3.5. Antimicrobial Activity

3.5.1. Microbial Strains

The antimicrobial and anti-biofilm activity was tested on Gram-positive (Staphylococcus aureus ATCC 25923, Bacillus subtilis 6633, Enterococcus faecalis ATCC 29212) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) bacterial as well as fungal (Candida albicans ATCC 10231) reference strains, but also on S. aureus and P. aeruginosa strains isolated from hospitalized patients. S. aureus 19 F, S. aureus 8 V and S. aureus 35 PL were isolated from pharyngeal exudates, vaginal swabs and wound secretions, respectively; S. aureus 12 H and P. aeruginosa 1 H from blood cultures; and P. aeruginosa 61/2, P. aeruginosa 399 and P. aeruginosa 261/1 from urine cultures.

3.5.2. Qualitative Assessment

The antimicrobial activity was determined by an adapted diffusion method. Briefly, the microbial inoculum with a density corresponding to 0.5/1 McFarland standard for bacterial/fungal strains was evenly swabbed on the agar surface in three directions, and thereafter, 10 μL of the stock solution of EO: DMSO was spotted on the seeded medium.

3.5.3. Quantitative Analysis

Serial microdilution method in liquid medium using 96-well plates was performed, the intensity of bacterial growth being appreciated by the absorbance value read spectrophotometrically at 620 nm; the MIC (μL/mL) was determined as the last concentration at which no microbial growth was observed [70].

3.5.4. The Microbial Adherence Capacity to the Inert Substratum

The slime test was used to highlight the EOs influence on the microbial adherence capacity to the inert substratum represented by the polymeric material of the 96 multi-well plates. Following the quantitative analysis of the antimicrobial effect, the adhered biomass was fixed with methanol, stained with violet crystal, resuspended in 33% acetic acid solution and assessed spectrophotometrically at 490 nm [71].

3.5.5. The Influence of EOs on the QS Genes Expression

The effects of the EOs and limonene (EO or limonene: DMSO, 1:1, v/v) on QS gene expression in P. aeruginosa were investigated by real-time reverse transcriptase quantitative PCR (RT–qPCR), using a commercial kit (GeneJet RNA Purification Kit Fermentas), following the manufacturer’s indications. Total RNA was extracted overnight from P. aeruginosa bacterial cultures treated and untreated with EOs. All the details about this experimental part were previously published [32,63].

3.5.6. The Synergistic Activity with Antibiotics

The antibiotic susceptibility of the S. aureus and P. aeruginosa strains was tested by the disk-diffusion method (Kirby–Bauer), according to the CLSI recommendations. The standardized bacterial suspensions were seeded onto a solid medium (Mueller–Hinton agar), as described for the qualitative screening. Two replicate plates were prepared for each strain [72]. For establishing the EOs’ influence on the antibiotic susceptibility, 10 μL of each EO stock solution (essential oil: DMSO 1:1, v/v) were placed on each antibiotic disk, with one replicate per strain. Plates were incubated for 16–18 h at 35 ± 2 °C. The results were read by measuring the diameter of the inhibition zones by using a hand-held caliper with a ruler, as generated by the different antibiotics comparatively to the antibiotics–EOs solution combination.

3.6. The Soluble Enzymatic Virulence Factors

The microbial strains were cultivated in liquid medium (nutrient broth) with and without the addition of subinhibitory concentrations of the tested EOs stock solution. The obtained overnight bacterial cultures were spotted onto special media for assessing the following virulence factors production [70,73].
Plate haemolysis: the strains were streaked on blood Sabouraud agar plates containing 5% (v/v) sheep blood in order to obtain isolated colonies. After incubation at 37 °C for 24 h the clear zone (total lysis of the red blood cells) around the colonies was registered as positive reaction.
Gelatinase activity: determined by using 3% gelatine agar as substrate medium. After incubation at 37 °C up to 48 h, a clear zone surrounding the growth area indicated gelatine proteolysis (gelatinase presence).
Caseinase activity: determined using 15% soluble casein agar as substrate. The strains were spotted and after incubation at 37 °C for 24 h, a precipitation zone surrounding the bacteria growth indicated the casein production.
DNA-se production: studied using DNA agar medium. The strains were spotted and after incubation at 37 °C for 24 h, a drop of HCl 1N solution was added upon the spotted cultures; a clearing zone around the culture was interpreted as positive reaction.
Lipase production: the cultures were spotted on Tween 80 agar with a substrate at a final concentration of 1% and were incubated at 37 °C up to 7 days. An opaque (precipitation) zone around the spot was registered as positive reaction.
Lecithinase production: the cultures were spotted into 2.5% yolk agar and were incubated at 37 °C for 7 days. An opaque (precipitation) zone around the spot indicated the lecithinase production.

3.7. Statistical Analysis

All experiments were done in triplicate, but the MIC and MBEC had the same values, so they were not expressed as the mean ± SD. The results obtained were represented by the last concentration at which no microbial growth was observed. The statistical impact of the EOs on microbial type (Gram-positive and Gram-negative) highlighted whether the antimicrobial effect is significant on the microorganism classes. The statistical analysis was performed using GraphPad Prism v9 (paired t-test). A p value < 0.05 was considered statistically significant. Significance values for antimicrobial activity against Gram-positive and Gram-negative strains are shown as *** p < 0.001.

4. Conclusions

Our study revealed that the EOs extracted from the coniferous species L. decidua, P. nigra, P. abies and P. menziesii exhibited significant antimicrobial features, in many cases equal or superior to those obtained for the major compounds, demonstrating the synergistic effect of the active compounds found in the EOs. They inhibited the microbial growth of a large number of reference and clinical and resistant P. aeruginosa and S. aureus strains as well as of the reference C. albicans strain, with MIC values varying from 6.25 to 100 µL/mL and the most susceptible strains being the Gram-positive and fungal ones. The most intensive and broad-spectrum microbicidal effect was exhibited by the P. nigra EOs. A synergistic effect with some antibiotics recommended to be tested against S. aureus strains (i.e., oxacillin, tetracycline, erythromycin and gentamycin) was also observed.
The subinhibitory concentrations of the tested EOs inhibited the adherence and the expression of the soluble virulence factors and modulated the QS genes’ expression in P. aeruginosa. All these features make the tested EOs promising leads for the development of novel antimicrobial strategies.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ph14111159/s1, Figure S1. Chromatogram of the Larix decidua essential oil. Figure S2. Chromatogram of the Pseudotsuga menziesii essential oil. Figure S3. Chromatogram of the Pinus nigra volatile essential oil.

Author Contributions

Conceptualization, V.R., methodology, V.R., E.O. and C.S., formal analysis, I.C.M.; investigation, D.-C.V., I.V., I.-A.B., V.R., E.O., C.S., A.I.C. and I.C.M.; resources, I.V. and I.-A.B.; data curation, D.-C.V. and E.O.; writing—original draft preparation, V.R., D.-C.V. and E.O.; writing—review and editing, E.O., I.C.M. and M.C.C.; visualization, E.O., I.C.M., C.S. and M.C.C.; supervision, E.O. and M.C.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by O.I.M., research project ICUB 20968/30.12.2020 and CNFIS-FDI-2021-0405.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or supplementary material.

Conflicts of Interest

The authors declare no conflict of interest.

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Pharmaceuticals 14 01159 g001 550
Figure 1. Chromatogram of the P. abies essential oil.
Figure 1. Chromatogram of the P. abies essential oil.
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Figure 2. Estimation plots of the antibacterial activity of the P. abies (a), L. decidua (b), P. menziesii (c) and P. nigra (d) EOs. The Gram-negative–Gram-positive effect size is generated by the difference between means. The precision of the calculated effect size was at a 95% confidence interval.
Figure 2. Estimation plots of the antibacterial activity of the P. abies (a), L. decidua (b), P. menziesii (c) and P. nigra (d) EOs. The Gram-negative–Gram-positive effect size is generated by the difference between means. The precision of the calculated effect size was at a 95% confidence interval.
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Figure 3. The QS genes expression levels in P. aeruginosa strains cultivated in the presence of limonene and EOs.
Figure 3. The QS genes expression levels in P. aeruginosa strains cultivated in the presence of limonene and EOs.
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Table
Table 1. The chemical composition of P. abies, L. decidua, P. menziesii and P. nigra EOs obtained by gas chromatography coupled with mass spectrometry.
Table 1. The chemical composition of P. abies, L. decidua, P. menziesii and P. nigra EOs obtained by gas chromatography coupled with mass spectrometry.
No.Compound NameRI
Exp		RI a
Lit		Relative Area (%)
Pa bLd bPm bPn b
14-hexen-1-ol872879Tr0.42 ± 0.030.10 ± 0.01
2santene8878883.83 ± 1.10
3tricyclene 9219261.23 ± 0.230.10 ± 0.010.09 ± 0.030.11 ± 0.01
4α-thujene928931 Tr0.27 ± 0.010.08 ± 0.04
5α-pinene93493911.64 ± 1.3426.99 ± 2.5718.42 ± 2.2974.27 ± 2.73
6camphene95095210.70 ± 0.250.52 ± 0.150.62 ± 0.101.24 ± 0.12
7sabinene973973 0.15 ± 0.071.67 ± 0.590.02 ± 0.01
8β-pinene9769804.62 ± 1.348.20 ± 0.8349.84 ± 3.574.33 ± 0.55
9β-myrcene9919912.26 ± 0.492.05 ± 0.521.17 ± 0.120.70 ± 0.18
10α-phellandrene100310050.13 ± 0.070.32 ± 0.180.15 ± 0.010.05 ± 0.01
11δ-3-carene100610090.89 ± 0.215.97 ± 1.190.66 ± 0.10Tr
12α-terpinene101610170.08 ± 0.030.38 ± 0.100.87 ± 0.190.05 ± 0.01
13p-cymene102410260.05 ± 0.010.06 ± 0.010.36 ± 0.140.04 ± 0.01
14limonene1029103121.14 ± 2.276.69 ± 0.933.58 ± 0.267.06 ± 0.78
151,8-cineole103210330.18 ± 0.110.22 ± 0.01
16(Z)-β-ocimene10401040Tr TrTr
17(E)-β-ocimene105010500.05 ± 0.01 0.50 ± 0.21
18γ-terpinene106010620.07 ± 0.020.48 ± 0.231.38 ± 0.310.08 ± 0.06
19α-terpinolene108510840.64 ± 0.231.59 ± 0.543.71 ± 1.020.22 ± 0.07
20linalool11001100Tr0.07 ± 0.04 0.04 ± 0.03
21fenchol111711170.07 ± 0.010.14 ± 0.10
22α-campholenal112610270.11 ± 0.03 0.10 ± 0.00
23camphor114511450.06 ± 0.03
24ethyl benzoate11691170 0.49 ± 0.01
25β-terpineol115411590.07 ± 0.02
26borneol117111710.78 ± 0.06 0.09 ± 0.010.12 ± 0.06
27terpinen-4-ol118011790.08 ± 0.000.60 ± 0.332.24 ± 0.180.04 ± 0.01
28α-terpineol119311970.57 ± 0.042.27 ± 0.621.86 ± 0.440.35 ± 0.14
29estragole119611950.27 ± 0.01
30fenchyl acetate121612200.05 ± 0.01
31methyl thymyl ether12311235 TrTr0.04 ± 0.01
32bornyl acetate1283128511.08 ± 1.801.26 ± 0.040.20 ± 0.041.21 ± 0.17
33sabinyl acetate129312930.16 ± 0.01
34δ-elemene13341337 0.6 ± 0.43
35terpenyl acetate13471351 0.18 ± 0.01
36α-longipinene134913510.34 ± 0.03
37citronellyl acetate135113540.04 ± 0.010.06 ± 0.01Tr
38longicyclene137113730.05 ± 0.03
39α-copaene137513760.06 ± 0.030.08 ± 0.03 0.03 ± 0.03
40geranyl acetate138013820.04 ± 0.03 0.12 ± 0.01
41β-elemene138713910.15 ± 0.030.27 ± 0.030.42 ± 0.26
42longifolene140414080.50 ± 0.09
43trans-caryophyllene141614151.17 ± 0.132.68 ± 0.180.34 ± 0.161.99 ± 0.30
44γ-elemene14281430 0.13 ± 0.01
45trans-α-bergamotene143214360.04 ± 0.01 0.48 ± 0.04
46α-humulene145314521.23 ± 0.041.12 ± 0.131.07 ± 0.540.27 ± 0.03
47ethyl cinnamate14641460 0.11 ± 0.01
48β-cadinene147014720.15 ± 0.060.16 ± 0.07
49γ-muurolene147314770.24 ± 0.030.76 ± 0.060.20 ± 0.040.16 ± 0.04
50germacrene D147814800.76 ± 0.0719.80 ± 4.405.47 ± 2.702.74 ± 1.07
51ledene14851487 0.08 ± 0.01
52phenylethyl isovalerate14891489 0.03 ± 0.01
53valencene149114900.10 ± 0.01 0.06 ± 0.04
54β-selinene148514850.05 ± 0.04 0.38 ± 0.18
55α-selinene149314940.19 ± 0.08
56α-muurolene149614990.75 ± 0.131.01 ± 0.180.09 ± 0.010.13 ± 0.06
57α-farnesene150415080.25 ± 0.01
58γ-cadinene151015140.86 ± 0.310.72 ± 0.040.20 ± 0.010.11 ± 0.03
59δ-cadinene151715234.21 ± 0.694.52 ± 0.660.74 ± 0.010.34 ± 0.06
60zonarene152115260.15 ± 0.070.16 ± 0.06
61cadina-1,4-diene153115320.07 ± 0.040.10 ± 0.00
62α-cadinene153615380.14 ± 0.070.17 ± 0.04
63trans-α-bisabolene15411544 0.95 ± 0.24
64germacrene B15531560 0.10 ± 0.040.30 ± 0.00
65nerolidol 156015650.06 ± 0.000.04 ± 0.01
66γ-eudesmol16311630 0.11 ± 0.01
67α-muurolol164316452.15 ± 0.282.55 ± 0.54
68δ-cadinol164616460.32 ± 0.05
69α-cadinol165516563.78 ± 0.764.11 ± 0.880.36 ± 0.21
70manool205320569.40 ± 1.85
71verticillol21022106 c 2.14 ± 1.60
TOTAL98.06 ± 0.9797.75 ± 2.2399.13 ± 0.0498.77 ± 1.68
Monoterpene hydrocarbons57.77 ± 3.8552.90 ± 0.5382.50 ± 6.6888.56 ± 4.83
Sesquiterpene hydrocarbons11.43 ± 1.7831.63 ± 6.5311.22 ± 4.355.83 ± 2.09
Monoterpene alcohols and esters12.90 ± 2.974.01 ± 1.854.39 ± 0.311.86 ± 0.32
Sesquiterpene alcohols6.28 ± 0.486.69 ± 1.430.39 ± 0.310.00
Diterpene alcohols9.40 ± 1.85002.14 ± 1.60
a RI, the retention index relative to C8–C24 n-alkanes on a DB-5MS column. b Pa: P. abies; Ld: L. decidua; Pm: P. menziesii; Pn: P. nigra. c [33].
Table
Table 2. The MIC (μL/mL) a and MBEC (μL/mL) b values for P. abies, L. decidua, P. menziesii and P. nigra essential oils and for some pure compounds against Gram-positive bacteria, Gram-negative bacteria and C. albicans.
Table 2. The MIC (μL/mL) a and MBEC (μL/mL) b values for P. abies, L. decidua, P. menziesii and P. nigra essential oils and for some pure compounds against Gram-positive bacteria, Gram-negative bacteria and C. albicans.
StrainaPinLimPhelBorCamCinNerPaLdPmPnGen
b
S. aureus ATCC 25923a502550505025502512.56.2512.50.48
b2512.525252512.52512.56.253.136.25n.t.
S. aureus
19 F		a505050505050506.255025250.48
b252525252525253.132512.512.5n.t.
S. aureus 8 Va25505050505050202525250.96
b12.52525252525251012.512.512.5n.t.
S. aureus 12 Ha12.55050505050506.2550256.250.96
b6.252525252525253.132512.53.13n.t.
S. aureus 35 PLa12.55050505050506.2525256.252.88
b6.252525252525253.1312.512.53.13n.t.
Pseudomonas aeruginosa ATCC 27853a25505050505050505025251.96
b12.525252525252525252512.512.5n.t.
P. aeruginosa 1 Ha25255050505050505025257.84
b12.512.52525252525252512.512.5n.t.
P. aeruginosa 61/2a505050505050505050252515.68
b25252525252525252512.512.5n.t.
P. aeruginosa 399a505025252525255050502515.68
b252512.512.512.512.512.525252512.5n.t.
P. aeruginosa 261/1a50502525252525505050257.84
b252512.512.512.512.512.525252512.5n.t.
B. subtilis 6633an.t.n.t.n.t.n.t.n.t.n.t.n.t.6.2550256.250.24
bn.t.n.t.n.t.n.t.n.t.n.t.n.t.3.132512.53.13n.t.
E. faecalis ATCC 29212an.t.n.t.n.t.n.t.n.t.n.t.n.t.255050257.68
bn.t.n.t.n.t.n.t.n.t.n.t.n.t.12.5252512.5n.t.
E. coli ATCC 25922an.t.n.t.n.t.n.t.n.t.n.t.n.t.505050250.48
bn.t.n.t.n.t.n.t.n.t.n.t.n.t.25252512.5n.t.
C. albicans ATCC 10231an.t.n.t.n.t.n.t.n.t.n.t.n.t.6.2525256.25n.t.
bn.t. n.t.n.t.n.t.n.t.n.t.n.t.3.1312.512.53.13n.t.
a: MIC (μL/mL): minimum inhibitory concentration; b: MBEC (μL/mL): minimum biofilm eradication concentration on an inert substrate; Pin: α-pinene; Lim: (+)-limonene; Phel: phellandrene; Bor: borneol; Cam: camphor; Cin: 1,8-cineole; Ner: nerolidol; Pa: P. abies; Ld: L. decidua; Pm: P. menziesii; Pn: P. nigra, Gen: gentamycin (μg/mL), n.t. = not tested.
Table
Table 3. The synergistic activity of the tested EOs with different antibiotics.
Table 3. The synergistic activity of the tested EOs with different antibiotics.
StrainsSampleThe Diameter of the Inhibition Zones (mm)
oxacliciptetgenpenery
S. aureus ATCC 25923control18322224222626
P. abies18402623232630
L. decidua18302422222622
P.menziesii18262423212626
P. nigra18382722242623
S. aureus 19 Fcontrol1032282219022
P. abies1028332121822
L. decidua10403220251327
P.menziesii10362822231825
P. nigra1034292223727
S. aureus 8 Vcontrol1138252019010
P. abies113427212109
L. decidua1133272121011
P.menziesii1132272121011
P. nigra1136272121012
S. aureus 12 Hcontrol10342420201511
P. abies10362726211912
L. decidua10382822191512
P.menziesii10362520191511
P. nigra10443030232424
S. aureus 35 PLcontrol14252301800
P. abies14262501800
L. decidua14262302000
P.menziesii14282401980
P. nigra14292492000
oxa = oxacillin; cli = clindamycin; cip = ciprofloxacin; tet = tetracycline; gen = gentamycin; pen = penicillin; ery = erythromycin.
Table
Table 4. The number of strains in which the inhibition of at least one virulence factor expression was noticed in the presence of the EOs and of their major compounds.
Table 4. The number of strains in which the inhibition of at least one virulence factor expression was noticed in the presence of the EOs and of their major compounds.
BorneolCamfor1,8-CineoleLimoneneα-PinenePaaLdaPmaPna
S. aureus
Total no. of strains101010101010101010
DN-ase111111111
Lipase112121111
Lecithinase334335535
Haemolysins889999999
Caseinase333345333
Siderophore-like557787656
P. aeruginosa
Total no. of strains101010101010101010
DN-ase109109101010109
Lipase566556566
Lecithinase455566655
Haemolysins676776666
Caseinase998900000
Siderophore-like10710101010101010
a Pa: P. abies; Ld: L. decidua; Pm: P. menziesii; Pn: P. nigra.
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Visan, D.-C.; Oprea, E.; Radulescu, V.; Voiculescu, I.; Biris, I.-A.; Cotar, A.I.; Saviuc, C.; Chifiriuc, M.C.; Marinas, I.C. Original Contributions to the Chemical Composition, Microbicidal, Virulence-Arresting and Antibiotic-Enhancing Activity of Essential Oils from Four Coniferous Species. Pharmaceuticals 2021, 14, 1159. https://doi.org/10.3390/ph14111159

AMA Style

Visan D-C, Oprea E, Radulescu V, Voiculescu I, Biris I-A, Cotar AI, Saviuc C, Chifiriuc MC, Marinas IC. Original Contributions to the Chemical Composition, Microbicidal, Virulence-Arresting and Antibiotic-Enhancing Activity of Essential Oils from Four Coniferous Species. Pharmaceuticals. 2021; 14(11):1159. https://doi.org/10.3390/ph14111159

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Visan, Diana-Carolina, Eliza Oprea, Valeria Radulescu, Ion Voiculescu, Iovu-Adrian Biris, Ani Ioana Cotar, Crina Saviuc, Mariana Carmen Chifiriuc, and Ioana Cristina Marinas. 2021. "Original Contributions to the Chemical Composition, Microbicidal, Virulence-Arresting and Antibiotic-Enhancing Activity of Essential Oils from Four Coniferous Species" Pharmaceuticals 14, no. 11: 1159. https://doi.org/10.3390/ph14111159

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