Next Article in Journal
One-Step Multiplex RT-PCR Method for Detection of Melon Viruses
Previous Article in Journal
Regulation of the Rhizosphere Microenvironment by Arbuscular Mycorrhizal Fungi to Mitigate the Effects of Cadmium Contamination on Perennial Ryegrass (Lolium perenne L.)
Previous Article in Special Issue
Isolation, Antibacterial Activity and Molecular Identification of Avocado Rhizosphere Actinobacteria as Potential Biocontrol Agents of Xanthomonas sp.
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 12
Issue 11
10.3390/microorganisms12112336
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

In Vitro Antimicrobial Activity of Volatile Compounds from the Lichen Pseudevernia furfuracea (L.) Zopf. Against Multidrug-Resistant Bacteria and Fish Pathogens

by
Yasser Essadki
1,
Adel Hilmi
2,
Antonio Cascajosa-Lira
3,
Mariana Girão
4,
El Mehdi Darrag
5,
Rosário Martins
4,6,
Abderrahmane Romane
5,
Soukaina El Amrani Zerrifi
1,7,
Richard Mugani
1,
Zakaria Tazart
8,
El Mahdi Redouane
9,
Angeles Jos
3,
Ana M. Cameán
3,
Vitor Vasconcelos
4,10,
Alexandre Campos
4,
Fatima El Khalloufi
11,
Brahim Oudra
1,
Mustapha Barakate
2,8 and
Maria de Fátima Carvalho
4,12,*
1
Water, Biodiversity and Climate Change Laboratory, Phycology, Biotechnology and Environmental Toxicology Research Unit, Faculty of Sciences Semlalia Marrakech, Cadi Ayyad University, Bd Prince Moulay Abdellah, Marrakech 40000, Morocco
2
Laboratory of Microbial Biotechnology, AgroSciences and Environment, CNRST Labeled Research Unit N◦4, Faculty of Sciences Semlalia, Cadi Ayyad University, Bd Prince Moulay Abdellah, Marrakech 40000, Morocco
3
Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla, C. Tramontana, 2, 41012 Sevilla, Spain
4
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, 4450-208 Matosinhos, Portugal
5
Laboratory of Applied Chemistry and Biomass, Faculty of Sciences Semlalia Marrakech, Cadi Ayyad University, Bd Prince Moulay Abdellah, Marrakech 40000, Morocco
6
Health and Environment Research Centre, School of Health, Polytechnic Institute of Porto (E2S/P.PORTO), R. Dr. António Bernardino de Almeida 400, 4200-072 Porto, Portugal
7
Higher Institute of Nurses Professions and Health Techniques of Guelmim, Guelmim 81000, Morocco
8
AgroBioSciences Department, College of Agriculture and Environmental Sciences, Mohammed VI Polytechnic University, Ben Guerir 43150, Morocco
9
UMR-I 02 INERIS-URCA-ULH SEBIO, Stress Environnementaux et BIOsurveillance des Milieux Aquatiques, Université de Reims Champagne-Ardenne, Campus du Moulin de la Housse, BP 1039, 51687 Reims, CEDEX, France
10
Department of Biology, Faculty of Sciences, University of Porto (FCUP), Rua Campo Alegre s/n, 4169-007 Porto, Portugal
11
Natural Resources Engineering and Environmental Impacts Team, Multidisciplinary Research and Innovation Laboratory, Polydisciplinary Faculty of Khouribga, Sultan Moulay Slimane University of Beni Mellal, Bd 2 Mars, Khouribga 25000, Morocco
12
ICBAS—School of Medicine and Biomedical Sciences, University of Porto, R. Jorge de Viterbo Ferreira 228, 4050-313 Porto, Portugal
 
add Show full affiliation list
 
remove Hide full affiliation list
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(11), 2336; https://doi.org/10.3390/microorganisms12112336
Submission received: 23 September 2024 / Revised: 11 November 2024 / Accepted: 13 November 2024 / Published: 15 November 2024
(This article belongs to the Special Issue Research on Antimicrobial Activity of Natural Products, Second Edition)
Browse Figures
Versions Notes

Abstract

:
Lichens are symbiotic organisms with unique secondary metabolism. Various metabolites from lichens have shown antimicrobial activity. Nevertheless, very few studies have investigated the antimicrobial potential of the volatile compounds they produce. This study investigates the chemical composition and antimicrobial properties of volatile compounds from Pseudevernia furfuracea collected in two regions of Morocco. Hydrodistillation was used to obtain volatile compounds from samples collected in the High Atlas and Middle Atlas. Gas chromatography–mass spectrometry (GC-MS) analysis identified phenolic cyclic compounds as the primary constituents, with atraric acid and chloroatranol being the most abundant. Additionally, eight compounds were detected in lichens for the first time. The antimicrobial activity of these compounds was assessed using disc diffusion and broth microdilution methods. Both samples demonstrated significant antimicrobial effects against multidrug-resistant human bacteria, reference microorganisms, fish pathogens, and Candida albicans, with minimum inhibitory concentrations (MICs) ranging from 1000 µg/mL to 31.25 µg/mL. This study provides the first report on the volatile compounds from Pseudevernia furfuracea and their antimicrobial effects, particularly against fish pathogens, suggesting their potential as novel antimicrobial agents for human and veterinary use. Further research is warranted to explore these findings in more detail.

Graphical Abstract

1. Introduction

Lichenization is a nutritional specialization of lichen-forming fungi [1], wherein the exhabitant fungus and inhabitant autotrophic algae or cyanobacteria interact for mutual benefit, forming a symbiotic association [2]. The morphology of the lichen thallus is highly diverse, ranging from leprose to fruticose [3]. Approximately 20,000 species of lichens have been described worldwide [4]. They produce around one thousand secondary metabolites, primarily through the shikimic acid, mevalonic acid, and polymalonate pathways [5]. These metabolites have demonstrated antioxidant, anticancer, and antimicrobial potential [6]. For instance, usnic acid, one of the most studied lichen metabolites, has exhibited antibacterial activity against Staphylococcus aureus and Bacillus subtilis, inhibiting DNA and RNA synthesis [7].
In Morocco, a catalogue updated in 2013 stated the presence of 1169 lichen species, distributed in 21 orders, 75 families, and 241 genera [8]. A more recent revision was provided based on the changes in nomenclature and indicated that Moroccan national biodiversity includes 1237 lichen species [9]. Amidst this broad national diversity, Pseudevernia furfuracea (L.) Zopf, a lichen of the Parmeliaceae family, is found in large populations in Morocco’s Middle and High Atlas regions [10], and in North Africa in general [11]. This lichen species was widely used in the fragrance industry under the appellation ‘cedarmoss’ when growing on Cedrus atlantica, or ‘treemoss’ when growing on other trees [12]. In 1997, 1900 tons of this lichen, harvested globally, including in Morocco, were processed in France [12]. In Morocco, it has been traditionally used as a decoction believed to improve blood volume and aid in liver detoxification [13]. While the antimicrobial potential of extracts from this lichen has been investigated worldwide and in Morocco [14,15,16], to our knowledge, no research has been conducted on the antimicrobial potential of its volatile compounds.
With an extensive diversity of secondary metabolites exhibiting antimicrobial properties, lichens, such as Pseudevernia furfuracea, present promising natural alternatives to synthetic antibiotics. These compounds could play an important role in addressing antimicrobial resistance (AMR), a challenge of growing concern to human health. Indeed, the World Health Organization (WHO) identifies AMR as one of the top three threats to public health [17]. It occurs when pathogens—bacteria, fungi, parasites, and viruses—stop responding to compounds that previously inhibited them [18]. This resistance complicates the treatment of infections or even renders it ineffective. In 2019 alone, AMR was estimated to contribute to 1.27 million deaths, and predictive models suggest that this number could reach 10 million by 2050 [19]. Among the most concerning “superbugs” are methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii, carbapenem-resistant Klebsiella pneumoniae, and strains of Escherichia coli [19,20]. Additionally, Mycobacterium tuberculosis remains a global health threat, especially in developing countries [19]. Beyond the death toll, AMR also poses a significant economic burden, potentially costing trillions of dollars worldwide [17]. The One Health approach, which emphasizes the connections between human, animal, and environmental health [18], highlights the link between human health and aquaculture. Indeed, the field of aquaculture has experienced a significant rise over the past 40 years, driven by global demographic growth and the need to provide a sustainable protein source [21,22,23]. By 2017, the annual production of fish and shellfish reached 80 million tons, with freshwater species accounting for approximately 45 million tons and Asia contributing over 97% of global production [21]. Commonly farmed freshwater species include grass carp, silver carp, Nile tilapia, and common carp [21]. However, the rapid expansion of aquaculture has introduced serious challenges, such as the proliferation of pests, parasites, and pathogens (PPP), pollution, harmful algal blooms, and climate change [21]. To control infections and maintain production yields, antibiotics are often used in aquaculture, not only to treat disease but also as growth promoters [24,25]. This widespread use of antibiotics in aquaculture has consequences that reach far beyond the industry itself, as these compounds can persist in water systems and promote the development of AMR in both aquatic and human pathogens [26]. Resistant bacteria and resistance genes can spread through waterways, potentially reaching human populations and contributing to the global AMR crisis [26,27]. As such, the reliance on antibiotics in aquaculture intensifies the AMR issue in humans, making it imperative to find alternative antimicrobial agents for sustainable aquaculture practices [28]. Among alternative strategies, aromatic and medicinal plants have shown promise in combatting fish pathogens while reducing reliance on traditional antibiotics [29,30]. Essential oils, for instance, contain bioactive volatile compounds that are effective against aquaculture pathogens. Eugenol-rich oil from Syzygium aromaticum and limonene-rich oil from Citrus limon, as well as thymol-rich oil from Thymus vulgaris, have demonstrated strong antibacterial activity against various fish pathogens in vitro [31,32]. These findings underscore the potential of herbal compounds not only in aquaculture but also in medical applications as eco-friendly alternatives to synthetic antibiotics. Therefore, this work aimed to investigate for the first time the chemical composition and antimicrobial potential of volatile compounds from P. furfuracea, harvested from two different locations in Morocco. Antimicrobial activity was screened against a wide range of microorganisms that included reference bacterial strains, multidrug-resistant clinical human isolates, fish pathogenic bacteria, and a pathogenic yeast.

2. Materials and Methods

2.1. Sample Collection, Volatile Compound Extraction, and Chemical Characterization

2.1.1. Sample Collection

Lichens (Figure S1) were collected in June 2021 from two different locations in Morocco, one on Quercus rotundifolia L. near Marrakech in the High Atlas region (31°28′19.5″ N 7°25′27.6″ W, 1644 m), and the other on Cedrus atlantica (Manetti ex Endl.) Carriere near Azrou in the Middle Atlas region (33°25′2″ N 5°11′10″ W, 1776 m). Biomass was harvested manually from the low branches of the different substrates. Lichen identification was conducted by one of the authors (Y.E.) using determination keys [33] and online resources [34]. The results were as follows: Medulla C−, K+, and cortex C−, K− for both samples. The lichens were identified as Pseudevernia furfuracea var. furfuracea. Voucher specimens were deposited in the personal herbarium of one of the authors (Y.E.) under the reference numbers 9 and 15.

2.1.2. Volatile Compound Extraction

Volatile compound extraction was performed according to the protocol of Sanad et al. [35]. Briefly, the collected biomass was stored under shaded conditions at room temperature (±25 °C) on a laboratory bench for one week to eliminate excess moisture. Manual sorting was then executed to eliminate contaminants such as branches, bark, debris, and other lichens. Following the drying and sorting processes, the biomass was ground using an electrical grinder. Subsequently, 100 g of the powdered lichens was mixed in a hydrodistillation apparatus with 4 L of deionized water. Distillation was conducted for a period of 4 h. The obtained distillate was then mixed thoroughly with dichloromethane in a separatory funnel. Following complete phase separation (with two clear phases visible in the funnel), the dichloromethane phase was collected and evaporated in a rotary evaporator at 30 °C. The obtained volatile compounds were therefore stored in amber glass vials at 4 °C. Pseudevernia furfuracea volatile compounds (PFVCs) from the High Atlas region were attributed the code 1, and PFVCs from the Middle Atlas region the code 2.

2.1.3. Gas Chromatography–Mass Spectrometry Protocol and Molecular Characterization

The chromatographic separation and subsequent identification of compounds were carried out using a gas chromatograph coupled to a quadrupole-Orbitrap hybrid mass spectrometer (GC-MS) QExactive (ThermoFisher Scientific, Waltham, MA, USA) equipped with an electron impact ionization source operating in positive mode. Xcalibur 4.3 software (ThermoFisher Scientific, Waltham, MA, USA) was employed for instrument control and data acquisition, while Trace Finder 5.1 software (ThermoFisher Scientific, Waltham, MA, USA) was used for result processing. GC analysis was conducted using an Agilent Technologies chromatographic column (30 m × 0.25 mm, 0.25 µm particle size). Helium was utilized as the carrier gas. The temperature ramp protocol consisted of an initial isothermal hold at 60 °C for 1 min, followed by a gradient increase of 5 °C/min up to 210 °C, then of 10 °C/min up to 280 °C, and finally, the temperature was maintained for 15 min. The injection volume was 1 µL, and the analysis was performed in Split mode with a Split ratio of 1:50. The injector temperature was set at 250 °C, and the carrier gas flow was held constant at 1 mL/min. Electron impact ionization was set to 70 eV, employing a Full Scan analysis method covering the m/z range of 35–650 in profile mode with a resolution of 60,000.
To determine the composition of both extracts, Compound Discoverer™ 3.2 software (Thermo Fisher Scientific, Waltham, MA, USA) was used. The GC-EI Deconvolution node was employed to differentiate and identify overlapping compounds by selecting the appropriate library (National Institute of Standards and Technology, NIST) for molecular matching. For compounds lacking chromatographic peaks, the workflow imputes area values, yielding a comprehensive dataset. Retention Index information was also included to improve identification accuracy. After processing the GC data, a differential analysis was conducted to compare variations in compound abundance between the different extracts, facilitating a clearer understanding of compositional differences. Finally, a Molecular Network visualization was generated to illustrate potential structural relationships among compounds, offering insights into chemical families or pathways within the extracts. This combined approach provides an in-depth analysis of extract composition, enabling both the detection and structural interpretation of its constituents.

2.2. Antimicrobial Activity

2.2.1. Reference Strains

The antimicrobial activity of PFVC1 and 2 was assessed against a group of five reference microorganisms: two Gram-positive strains (Staphylococcus aureus (ATCC 29213) and Bacillus subtilis (ATCC 6633)), two Gram-negative strains (Salmonella typhimurium (ATCC 25241) and Escherichia coli (ATCC 25922)), and one yeast (Candida albicans (ATCC 10231)). S. aureus, B. subtilis, S. typhimurium, and E. coli were grown on Mueller–Hinton agar (MH) (Biokar, Beauvais, France). C. albicans was grown on Sabouraud-Dextrose agar (SDA) (Biokar, Beauvais, France). Additional information on the culture conditions can be found in the Supplementary Materials (Table S1).

2.2.2. Clinical Strains

Four additional isolates from the Intensive Care Unit of the University Hospital Mohammed VI of Marrakesh (Morocco) were also used [36]. Those isolates originated from pus, blood, tracheal aspirate, and catheters and were identified as E. coli (E1), Klebsiella pneumoniae (K1), Acinetobacter baumannii (A1), and S. aureus (S1), respectively. Identification involved a comprehensive assessment of morphological, biochemical, and cultural characteristics, supported by the API library (Biomerieux, Marcy-l'Etoile, France). Antibiotic susceptibility testing was executed according to the guidelines recommended by the Antibiogram Committee of the French Microbiology Society [37] and the European Committee on Antimicrobial Susceptibility Testing EUCAST [38]. The resistance profiles of the individual strains are provided in the Supplementary Materials (Table S2). These strains were considered multidrug-resistant as per the criteria established by Magiorakos et al. [39] and were grown on Mueller–Hinton agar (MH) (Biokar, Beauvais, France) at 37 °C for 24 h.

2.2.3. Fish Pathogenic Strains

In addition, a group of 5 fish pathogenic strains were used: Aeromonas hydrophila (DSM 30187), Pseudomonas anguiliseptica (DSM 12111), Edwardsiella tarda (DSM 30052), Listonella anguillarum (ATCC 19264), and Yersinia ruckeri (ATCC 29473). These strains were grown on Tryptic Soy agar (TSA) (Biokar, Beauvais, France). The specific culture conditions are available in the Supplementary Materials (Table S1).

2.2.4. Disc Diffusion Assay

The disc diffusion assay was conducted according to Girão et al. [40]. Briefly, microorganism suspensions in the appropriate liquid medium were prepared and adjusted to 0.5 McFarland. Sterile cotton swabs were used to inoculate the suspensions on agar plates (MH, TSA, or SDA, depending on the microorganism). Blank 6 mm Whatman paper discs were placed on the surface of the inoculated agar plates, and 10 µL of PFVCs (24 mg/mL) were loaded onto the discs. Positive control discs were loaded with Enrofloxacin (1 mg/mL) for the reference bacterial strains and with Nystatin (1 mg/mL) for the yeast. Negative control discs were loaded with Dimethylsulfoxide (DMSO). The experiment was repeated three independent times. Antimicrobial activity was determined by measuring the inhibition zone diameter (IZD) formed around the discs after the appropriate incubation time and at the appropriate temperature for each microorganism (Table S1).

2.2.5. Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) was determined using the broth microdilution assay according to the CLSI guidelines M07-A10 for bacteria [41] and M27-A3 for yeasts [42]. Briefly, a PFVC stock solution was prepared in the appropriate liquid medium at a concentration of 2000 µg/mL with 2% DMSO. Two-fold dilutions were subsequently prepared from this stock, and 100 µL of each dilution was added to microwells previously inoculated with 100 µL of the microbial inoculum (prepared by diluting the 0.5 McFarland microorganism suspension fifty-fold). The positive growth control consisted of 100 µL of microbial inoculum and 100 µL of medium broth, and the negative growth control consisted of 200 µL of medium broth. The MIC was defined as the lowest concentration of PFVC that inhibited the macroscopic growth of the tested strains. For each extract, the MIC was determined in triplicate, and two independent experiments were performed.

3. Results

3.1. Chemical Composition of the Volatile Compounds

A total of 15 volatile compounds were detected in PFVC1 and PFVC2. Of these, five compounds were found in both samples, and five compounds were unique to each sample (Table 1). Most of the compounds detected are phenolic cyclic compounds. The corresponding chromatograms are presented in Figure 1 and Figure 2.
The primary compound in both samples was identified as atraric acid, comprising 73.53% and 56.95% of PFVC1 and PFVC2, respectively. Chloroatranol followed as the second most abundant compound, accounting for 19.80% and 24.38% of PFVC1 and PFVC2, respectively. Notably, atraric acid has been previously reported in acetone fractions of methanol extracts of lichens from the Parmeliaceae family, with varying relative abundances [43]. However, our extracts exhibited relatively higher proportions of atraric acid than in the existing literature.
Chloroatranol is derived from the degradation of chloroatranorin, commonly found in oakmoss and treemoss absolutes [12,44]. This compound is a well-known allergen and sensitizer in cosmetic products [45,46,47].
The remaining compounds detected in both extracts showed relative abundances of less than 3.77%. In PFVC1, δ-cadinene, naphthalene, and n-hexadecanoic acid were among the most abundant minor compounds, while in PFVC2, acetisoeugenol, ρ-cymene-8-ol, 2-furancarboxaldehyde, 5-(2-furanylmethyl)-, n-hexadecanoic acid, trans-verbenol, naphthalene, and abietatriene predominated.
Among the minor compounds detected, methyl haematommate has also been identified in volatile fractions of lichens of the Parmeliaceae family [43], whereas n-hexadecanoic acid and naphthalene have previously been reported in volatile compounds from Lichina pygmaea [35]. Moreover, abietatriene was reported in essential oils of Evernia prunastri and Evernia divaricata from Turkey [48], thus further corroborating the presence of these compounds in our samples.
In contrast, a total of eight compounds are reported here for the first time in lichens. Those compounds are as follows: trans-verbenol; ρ-cymene-8-ol; quinoline; 2-furancarboxaldehyde, 5-(2-furanylmethyl)-; δ-cadinene; himachalol; acetisoeugenol; and guaiol acetate. The novelty of these compounds may be attributed to the fact that this is the first report of volatile compounds from the lichen P. furfuracea L. (Zopf.) var. furfuracea from two different biotopes in Morocco using this novel extraction procedure.
This analysis sheds light on the chemical composition of PFVC1 and PFVC2, highlighting the relatively high abundance of atraric acid and chloroatranol, as well as the presence of novel minor compounds that have never been reported previously in lichens.

3.2. Antimicrobial Activity

Pseudevernia furfuracea volatile compounds were evaluated for their antimicrobial potential both qualitatively, using the disc diffusion method, and quantitatively, using the well microdilution assay.
Based on the observed diameters of inhibition zones (Table 2), PFVC1 and PFVC2 exhibited antimicrobial activity against all tested microorganisms, with inhibition zone diameters (IZDs) ranging from 6.63 to 40 mm. Notably, the largest IZDs were observed against P. anguiliseptica, with values of 36 and 40 mm for PFVC1 and PFVC2, respectively. Similarly, the second most susceptible strain in the solid medium to PFVC1 and PFVC2 was C. albicans, with IZD values of 23 and 32 mm, respectively. Conversely, the lowest activity in the solid medium was observed for PFVC1 against S. typhimurium and E. coli ATCC 25922, with IZDs of 6.63 ± 0.15 mm and 6.73 ± 0.15 mm, respectively.
Quantitatively, the MIC of the volatile compounds of P. furfuracea was determined, revealing high antimicrobial activity, with MIC values equal to or below 1000 µg/mL (Table 2). Notably, the most promising activity was observed against C. albicans, with MIC values of 62.5 and 31.25 µg/mL for PFVC1 and PFVC2, respectively. Similarly, promising activity was observed against E. tarda, with an MIC value of 125 µg/mL for both PFVC1 and PFVC2. In contrast, the highest MIC was observed for K. pneumoniae, with a value of 1000 µg/mL for PFVC2. Furthermore, no difference in MIC was observed between the two strains of E. coli and S. aureus tested in this study.
PFVC1 and PFVC2 exhibited the same MIC values against E. coli E1, S. aureus S1, S. aureus ATCC 29213, S. typhimurium, E. coli ATCC 29213, P. anguiliseptica, E. tarda, and Y. ruckeri. PFVC1 had a lower MIC value than PFVC2 against A. baumannii, K. pneumoniae, and L. anguilarum, whereas PFVC2 exhibited a lower MIC value than PFVC1 against B. subtilis, C. albicans, and A. hydrophila. Given that the major compounds of both PFVC1 and PFVC2 are the same, the difference in the observed MIC values is likely due to variations in the composition of minor compounds, or due to variations in the abundance of the major compounds.

4. Discussion

This work evaluates for the first time the composition and antimicrobial potential of the volatile compounds of the lichen P. furfuracea (L.) Zopf. var. furfuracea from two different biotopes in Morocco. To our knowledge, very few reports exist about volatile compounds from lichens, due to various reasons, such as the low volatility of lichenic compounds and the low yields of those compounds [49].
Our findings indicate that the major compound in PFVC1 and PFVC2 is atraric acid (73.53 and 56.95%, respectively), followed by chloroatranol (19.80 and 24.38%, respectively). Moreover, eight compounds are reported here for the first time in lichens. Despite the similarities in major compounds between PFVC1 and PFVC2, specifically atraric acid and chloroatranol, which account for 93.33 and 81.33% of their compositions respectively, there are differences in the proportions of these major compounds and the composition of minor compounds. These differences may be due to variations in environmental conditions, the substrate, and altitude between the lichens. Indeed, variation in environmental conditions can be responsible for variation in gene expression [50]. Moreover, variation in the substrate (tree species) can have an impact on the mineral content of lichens [51]. PFVC1 is derived from a lichen growing in a sub-humid region on Quercus rotundifolia bark at an altitude of 1644 m, while PFVC2 comes from a lichen developing in a humid region on Cedrus atlantica bark at an altitude of 1776 m [52]. Aoussar et al. [15,16] investigated the composition and antimicrobial potential of methanol and acetone extracts of P. furfuracea (L.) Zopf. from Khenifra, Morocco. These extracts contained physodalic acid, atranorin, and chloroatranorin. This highlights the unique composition of PFVC1 and PFVC2, which contain atraric acid, a compound from Moroccan P. furfuracea described here for the first time.
PFVC1 and PFVC2 exhibited strong antimicrobial activity against human and fish pathogens, with MIC values as low as 32.5 µg/mL against C. albicans and no higher than 1000 µg/mL against the multidrug-resistant clinical isolate K. pneumoniae. A very interesting result was the high antimicrobial activity against the fish pathogenic bacteria A. hydrophila, E. tarda, Y. ruckeri, and P. anguiliseptica. These findings are in opposition with the results of Tas et al. [53], which showed that methanol, acetone, and water extracts from Turkish P. furfuracea did not exhibit any antimicrobial potential in a solid medium against A. hydrophila, Aeromonas salmonicida, Streptococcus agalactiae, Y. ruckeri, Enterococcus faecalis, and Lactococcus garvieae. Therefore, this is the first report of the antimicrobial potential of compounds from P. furfuracea against fish pathogenic bacteria, highlighting at the same time the potential of volatile compounds from P. furfuracea as a source of antimicrobial compounds with a large spectrum of action, covering Gram-positive and Gram-negative bacteria as well as yeast. In addition, PFVC1 and PFVC2 exhibited similar MIC values for antibiotic-resistant and reference strains of E. coli and S. aureus, suggesting a mechanism of action independent of bacterial resistance pathways.
The antimicrobial potential of PFVC1 and PFVC2 can be partly attributed to their composition of shared and unique bioactive compounds. Atraric acid, the major compound shared between PFVC1 and PFVC2, has shown mixed results regarding its antimicrobial efficacy. While it exhibited no activity against Porphyromonas gingivalis and Streptococcus mutans at concentrations up to 80 µM when isolated from Stereocolon paschale [54], atraric acid from oakmoss absolute displayed strong antimicrobial effects against several Legionella strains, with MIC values ranging from 12 to 26.7 µg/mL across strains including L. pneumophila, L. bozemanii, and L. longbeachae [55].
Both PFVC1 and PFVC2 also contained chloroatranol and n-hexadecanoic acid, which contribute to their overall antimicrobial profile. Chloroatranol, isolated from Turkish P. furfuracea, demonstrated activity against a range of pathogens, including A. hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, S. aureus, Yersinia enterocolitica, C. albicans, and Candida glabrata, with MIC values between 7.7 and 30.6 mM [56]. Additionally, n-hexadecanoic acid showed antibacterial activity against S. aureus, B. subtilis, E. coli, and K. pneumoniae, with MIC values ranging from 2.65 to 6.87 µg/mL [57], and was active against Mycobacterium smegmatis, a tuberculosis model, with an MIC of 128 µg/mL [58].
Among the compounds unique to PFVC1, δ-cadinene and himachalol exhibited distinct antimicrobial potential. δ-Cadinene, a volatile compound also found in green tea, showed activity against B. subtilis, Bacillus ammoniagenes, Streptococcus mutans, and Propionibacterium acnes, with MIC values between 3.13 and 800 µg/mL, though it was less effective against pathogens like S. aureus and Pseudomonas aeruginosa [59]. Himachalol has shown antifungal activity with an MIC of 250 µg/mL against Aspergillus fumigatus and has demonstrated a 60% survival rate in treated mice when administered at 200 mg/kg [60]. Methyl haematommate, another compound specific to PFVC1, has also shown limited antimicrobial potential against S. mutans and P. gingivalis [54].
Finally, PFVC2 contains 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-, which contributes further to its antimicrobial potential. 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- has shown activity against Gram-positive bacteria such as Bacillus cereus and S. aureus, with MIC values of 20 and 50 µg/mL, respectively, though it was inactive against Gram-negative bacteria at concentrations up to 1000 µg/mL [61]. This compound also enhanced the efficacy of rifampicin against M. smegmatis, reducing its MIC by a factor of four, suggesting a potential synergistic effect [58].
Nevertheless, it is important to note that antimicrobial potential was evaluated for a mixture of compounds and not for isolated pure compounds. Therefore, the observed antimicrobial activities cannot be definitively attributed to any single compound, even to those present in high abundance. Consequently, we cannot exclude the possibility that the antimicrobial activity observed is the result of a synergistic interaction between the various constituents of the volatile compounds of P. furfuracea. Thus, a fractionation of the various components of PFVC1 and PFVC2 to obtain pure compounds and the evaluation of the antimicrobial potential of those pure compounds is needed to assess if there could be new candidates for antibiotic applications for human or animal use.

5. Conclusions

This study represents the first report on the chemical composition and antimicrobial potential of volatile compounds from P. furfuracea var. furfuracea from two different biotopes in Morocco. Major constituents identified in both volatile compounds were consistent, predominantly atraric acid and chloroatranol, while differences in minor compounds were observed. Atraric acid was reported for the first time in P. furfuracea from Morocco, and eight compounds were detected for the first time in lichens. The antimicrobial activity of volatile compounds was assessed using the disc diffusion and broth microdilution assays, against clinical antibiotic-resistant strains, reference strains, and fish pathogenic strains. This work constitutes the first report of the antimicrobial potential of P. furfuracea against fish pathogenic strains. Notably, the most promising activity against fish pathogens was observed in a liquid medium against A. hydrophila and E. tarda. In addition, potent activity was observed against C. albicans in a liquid medium. No significant difference was noted between the antimicrobial potential of PFVCs against antibiotic-resistant and reference strains of E. coli and S. aureus. However, further investigations are warranted to identify the specific compounds responsible for this antimicrobial potential. Such insights could facilitate the development of novel antimicrobial agents with broad-spectrum activity and potential clinical applications.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/microorganisms12112336/s1, Figure S1: Pictures of Pseudevernia furfuracea (L.) Zopf on Quercus rotundifolia L. Table S1: Culture conditions of the strains tested in the antimicrobial assays; Table S2: Resistance profile of the clinical isolates tested in this study following the guidelines recommended by the Antibiogram Committee of the French Microbiology Society [33] and the European Committee on Antimicrobial Susceptibility Testing [34]. R, indicates resistance to the tested antibiotic; S, indicates susceptibility to the tested antibiotic; ‘-’, indicates that the antibiotic was not tested against the strain. Table S3: Inhibition zone diameters obtained with the controls of the disc diffusion assay. ‘-’ not tested.

Author Contributions

Conceptualization, Y.E., A.H., A.C.-L., E.M.D., R.M. (Rosário Martins), A.R., A.J., A.M.C., V.V., A.C., F.E.K., B.O., M.B., and M.d.F.C.; methodology, Y.E., A.H., A.C.-L., M.G., E.M.D., R.M. (Rosário Martins), A.R., S.E.A.Z., Z.T., E.M.R., A.J., A.M.C., V.V., A.C., F.E.K., B.O., M.B., and M.d.F.C.; investigation, Y.E., A.H., A.C.-L., M.G., and E.M.D.; resources, R.M. (Rosário Martins), A.R., A.J., A.M.C., V.V., A.C., F.E.K., B.O., M.B., and M.d.F.C.; data curation, Y.E., A.H., and A.C.-L.; writing—original draft preparation, Y.E., A.H., and A.C.-L.; writing—review and editing, Y.E., A.H., A.C.-L., M.G., E.M.D., R.M. (Richard Mugani), A.R., S.E.A.Z., Z.T., E.M.R., A.J., A.M.C., V.V., A.C., F.E.K., B.O., M.B., and M.d.F.C.; visualization, Y.E. and A.C-L; supervision, R.M. (Rosário Martins), A.R., A.J., A.M.C., V.V., A.C., F.E.K., B.O., M.B., and M.d.F.C.; project administration, R.M. (Rosário Martins), A.R., A.J., A.M.C., V.V., A.C., F.E.K., B.O., M.B., and M.d.F.C.; funding acquisition, A.M.C., V.V., B.O., A.C., and M.d.F.C. All authors have read and agreed to the published version of the manuscript.

Funding

The authors would like to acknowledge the TOXICROP project (H2020-823860). Antonio Cascajosa-Lira would like to acknowledge the Spanish Ministerio de Universidades (FPU 2019-01247). MFC wishes to acknowledge the CEEC program supported by FCT (CEECIND/02968/2017), Fundo Social Europeu and Programa Operacional Potencial Humano. The authors also acknowledge Strategic Funding UIDB/04423/2020, through national funds provided by FCT and the European Regional Development Fund (ERDF) and Fundo Social Europeu, and the Project PID2023-147444OB-I00 (funded by the MICIU/AEI).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to express our sincere gratitude to Eva Barreno from the University of Valencia and Michel Bertrand, along with the Association Française de Lichénologie (AFL), for their invaluable assistance in confirming the identification of the lichen species. Our appreciation also goes to Ahmed Ouhammou for his significant contributions to this research. We are grateful to Hamza Zine and Sara Elgadi for their continuous support throughout the course of this study. Additionally, we would like to thank the Direction Provinciale des Eaux et Forêts et de la Lutte Contre la Désertification of Azrou for granting the necessary permissions for lichen specimen collection.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Honegger, R. Lichens and Their Allies Past and Present. In Plant Relationships: Fungal-Plant Interactions; Springer: Berlin/Heidelberg, Germany, 2022; pp. 133–183. [Google Scholar]
  2. Hawksworth, D.L.; Grube, M. Lichens redefined as complex ecosystems. New Phytol. 2020, 227, 1281–1283. [Google Scholar] [CrossRef] [PubMed]
  3. Honegger, R. 15 The Symbiotic Phenotype of Lichen-Forming Ascomycetes and Their Endo- and Epibionts; Springer: Berlini/Heidelberg, Germany, 2012; pp. 287–339. [Google Scholar]
  4. Lücking, R.; Hodkinson, B.P.; Leavitt, S.D. The 2016 classification of lichenized fungi in the Ascomycota and Basidiomycota–Approaching one thousand genera. Bryol. 2017, 119, 361–416. [Google Scholar] [CrossRef]
  5. Ureña-Vacas, I.; González-Burgos, E.; Divakar, P.K.; Gómez-Serranillos, M.P. Lichen Depsides and Tridepsides: Progress in Pharmacological Approaches. J. Fungi 2023, 9, 116. [Google Scholar] [CrossRef] [PubMed]
  6. Ren, M.; Jiang, S.; Wang, Y.; Pan, X.; Pan, F.; Wei, X. Discovery and excavation of lichen bioactive natural products. Front. Microbiol. 2023, 14, 1177123. [Google Scholar] [CrossRef] [PubMed]
  7. Maciąg-Dorszyńska, M.; Węgrzyn, G.; Guzow-Krzemińska, B. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis. FEMS Microbiol. Lett. 2014, 353, 57–62. [Google Scholar] [CrossRef]
  8. Ajaj, A.; Touhami, A.O.; Benkirane, R.; Douira, A. Contribution to the update catalogue of lichenized and lichenicolous fungi in Morocco. J. Anim. Plant Sci. JAPS 2013, 19, 2961–3025. [Google Scholar]
  9. Seaward, M.R.; Amrani, S. Checklist of lichens and lichenicolous fungi of Morocco. Herzogia 2022, 35, 564–612. [Google Scholar] [CrossRef]
  10. Egea, J.M. Catalogue of lichenized and lichenicolous fungi of Morocco. Bocconea 1996, 6, 19–114. [Google Scholar]
  11. Hale, M.E., Jr. A synopsis of the lichen genus Pseudevernia. Bryologist 1968, 71, 1–11. [Google Scholar] [CrossRef]
  12. Joulain, D.; Tabacchi, R. Lichen extracts as raw materials in perfumery. Part 2: Treemoss. Flavour Fragr. J. 2009, 24, 105–116. [Google Scholar] [CrossRef]
  13. Bellakhdar, J. Contribution à L’éTude de la Pharmacopée Traditionnelle AU Maroc: La Situation Actuelle, Les Produits, Les Sources du Savoir (Enquête Ethnopharmacologique de Terrain Réalisée de 1969 à 1992). Ph.D. Thesis, Université Paul Verlaine, Metz, France, 1997. [Google Scholar]
  14. Mitrovic, T.; Stamenkovic, S.; Cvetkovic, V.; Radulovic, N.; Mladenovic, M.; Stankovic, M.; Topuzovic, M.; Radojevic, I.; Stefanovic, O.; Vasic, S.; et al. Platismatia glauca and Pseudevernia furfuracea lichens as sources of antioxidant, antimicrobial and antibiofilm agents. Excli J. 2014, 13, 938–953. [Google Scholar] [PubMed]
  15. Aoussar, N.; Laasri, F.E.; Bourhia, M.; Manoljovic, N.; Mhand, R.A.; Rhallabi, N.; Ullah, R.; Shahat, A.A.; Noman, O.M.; Nasr, F.A.; et al. Phytochemical Analysis, Cytotoxic, Antioxidant, and Antibacterial Activities of Lichens. Evid.-Based Complement. Altern. Med. 2020, 2020, 8104538. [Google Scholar] [CrossRef] [PubMed]
  16. Aoussar, N.; Achmit, M.; Es-Sadeqy, Y.; Vasiljević, P.; Rhallabi, N.; Ait Mhand, R.; Zerouali, K.; Manojlović, N.; Mellouki, F. Phytochemical constituents, antioxidant and antistaphylococcal activities of Evernia prunastri (L.) Ach., Pseudevernia furfuracea (L.) Zopf. and Ramalina farinacea (L.) Ach. from Morocco. Arch. Microbiol. 2021, 203, 2887–2894. [Google Scholar] [CrossRef] [PubMed]
  17. World Health Organization. Antimicrobial Resistance. Available online: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (accessed on 6 November 2024).
  18. Tang, K.W.K.; Millar, B.C.; Moore, J.E. Antimicrobial Resistance (AMR). Br. J. Biomed. Sci. 2023, 80, 11387. [Google Scholar] [CrossRef] [PubMed]
  19. Salam, M.A.; Al-Amin, M.Y.; Salam, M.T.; Pawar, J.S.; Akhter, N.; Rabaan, A.A.; Alqumber, M.A.A. Antimicrobial Resistance: A Growing Serious Threat for Global Public Health. Healthcare 2023, 11, 1946. [Google Scholar] [CrossRef] [PubMed]
  20. Ikuta, K.S.; Swetschinski, L.R.; Robles Aguilar, G.; Sharara, F.; Mestrovic, T.; Gray, A.P.; Davis Weaver, N.; Wool, E.E.; Han, C.; Gershberg Hayoon, A.; et al. Global mortality associated with 33 bacterial pathogens in 2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2022, 400, 2221–2248. [Google Scholar] [CrossRef]
  21. Naylor, R.L.; Hardy, R.W.; Buschmann, A.H.; Bush, S.R.; Cao, L.; Klinger, D.H.; Little, D.C.; Lubchenco, J.; Shumway, S.E.; Troell, M. A 20-year retrospective review of global aquaculture. Nature 2021, 591, 551–563. [Google Scholar] [CrossRef]
  22. Naylor, R.L.; Goldburg, R.J.; Primavera, J.H.; Kautsky, N.; Beveridge, M.C.M.; Clay, J.; Folke, C.; Lubchenco, J.; Mooney, H.; Troell, M. Effect of aquaculture on world fish supplies. Nature 2000, 405, 1017–1024. [Google Scholar] [CrossRef]
  23. Reverter, M.; Sarter, S.; Caruso, D.; Avarre, J.-C.; Combe, M.; Pepey, E.; Pouyaud, L.; Vega-Heredía, S.; De Verdal, H.; Gozlan, R.E. Aquaculture at the crossroads of global warming and antimicrobial resistance. Nat. Commun. 2020, 11, 1870. [Google Scholar] [CrossRef]
  24. Dawood, M.A.O.; El Basuini, M.F.; Zaineldin, A.I.; Yilmaz, S.; Hasan, M.T.; Ahmadifar, E.; El Asely, A.M.; Abdel-Latif, H.M.R.; Alagawany, M.; Abu-Elala, N.M.; et al. Antiparasitic and Antibacterial Functionality of Essential Oils: An Alternative Approach for Sustainable Aquaculture. Pathogens 2021, 10, 185. [Google Scholar] [CrossRef]
  25. Irshath, A.A.; Rajan, A.P.; Vimal, S.; Prabhakaran, V.-S.; Ganesan, R. Bacterial Pathogenesis in Various Fish Diseases: Recent Advances and Specific Challenges in Vaccine Development. Vaccines 2023, 11, 470. [Google Scholar] [CrossRef] [PubMed]
  26. Yuan, X.; Lv, Z.; Zhang, Z.; Han, Y.; Liu, Z.; Zhang, H. A Review of Antibiotics, Antibiotic Resistant Bacteria, and Resistance Genes in Aquaculture: Occurrence, Contamination, and Transmission. Toxics 2023, 11, 420. [Google Scholar] [CrossRef]
  27. Xu, M.; Huang, X.H.; Shen, X.X.; Chen, H.Q.; Li, C.; Jin, G.Q.; Cao, J.S.; Xue, Z.X. Metagenomic insights into the spatiotemporal responses of antibiotic resistance genes and microbial communities in aquaculture sediments. Chemosphere 2022, 307, 135596. [Google Scholar] [CrossRef]
  28. Chen, J.M.; Sun, R.X.; Pan, C.G.; Sun, Y.; Mai, B.X.; Li, Q.X. Antibiotics and Food Safety in Aquaculture. J. Agric. Food Chem. 2020, 68, 11908–11919. [Google Scholar] [CrossRef]
  29. Kuebutornye, F.K.A.; Abarike, E.D. The contribution of medicinal plants to tilapia aquaculture: A review. Aquac. Int. 2020, 28, 965–983. [Google Scholar] [CrossRef]
  30. Tadese, D.A.; Song, C.; Sun, C.; Liu, B.; Liu, B.; Zhou, Q.; Xu, P.; Ge, X.; Liu, M.; Xu, X.; et al. The role of currently used medicinal plants in aquaculture and their action mechanisms: A review. Rev. Aquac. 2022, 14, 816–847. [Google Scholar] [CrossRef]
  31. Öntas, C.; Baba, E.; Kaplaner, E.; Küçükaydin, S.; Öztürk, M.; Ercan, M.D. Antibacterial Activity of Citrus limon Peel Essential Oil and Argania spinosa Oil Against Fish Pathogenic Bacteria. Kafkas Univ. Vet. Fak. Derg. 2016, 22, 741–749. [Google Scholar] [CrossRef]
  32. Navarrete, P.; Toledo, I.; Mardones, P.; Opazo, R.; Espejo, R.; Romero, J. Effect of Thymus vulgaris essential oil on intestinal bacterial microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum) and bacterial isolates. Aquac. Res. 2010, 41, e667–e678. [Google Scholar] [CrossRef]
  33. Clauzade, G.; Roux, C.; Houmeau, J.-M.; Roux, C. Likenoj de Okcidenta Eŭropo: Ilustrita Determinlibro; Société Botanique du Centre-Ouest: Nercillac, France, 1985. [Google Scholar]
  34. Cannon, P.; Divakar, P.; Yahr, R.; Aptroot, A.; Clerc, P.; Coppins, B.; Fryday, A.; Sanderson, N.; Simkin, J. Lecanorales: Parmeliaceae including the genera Alectoria, Allantoparmelia, Arctoparmelia, Brodoa, Bryoria, Cetraria, Cetrariella, Cetrelia, Cornicularia, Evernia, Flavocetraria, Flavoparmelia, Hypogymnia, Hypotrachyna, Imshaugia, Melanelia, Melanelixia, Melanohalea, Menegazzia, Montanelia, Nesolechia, Parmelia, Parmelina, Parmeliopsis, Parmotrema, Platismatia, Pleurosticta, Protoparmelia, Pseudephebe, Pseudevernia, Punctelia, Raesaenenia, Tuckermannopsis, Usnea, Vulpicida and Xanthoparmelia. In Revisions of British and Irish Lichens; British Lichen Society: London, UK, 2023; Volume 33. [Google Scholar]
  35. Sanad, H.; Belattmania, Z.; Nafis, A.; Hassouani, M.; Mazoir, N.; Reani, A.; Hassani, L.; Vasconcelos, V.; Sabour, B. Chemical Composition and In Vitro Antioxidant and Antimicrobial Activities of the Marine Cyanolichen Lichina pygmaea Volatile Compounds. Mar. Drugs 2022, 20, 169. [Google Scholar] [CrossRef]
  36. El mekes, A.; Zahlane, K.; Ait Said, L.; Tadlaoui Ouafi, A.; Barakate, M. The clinical and epidemiological risk factors of infections due to multi-drug resistant bacteria in an adult intensive care unit of University Hospital Center in Marrakesh-Morocco. J. Infect. Public Health 2020, 13, 637–643. [Google Scholar] [CrossRef]
  37. Bonnet, R.; Caron, F.; Cavallo, J.; Chardon, H.; Chidiac, C.; Courvalin, P.; Drugeon, H.; Dubreuil, L.; Jarlier, V.; Jehl, F. Comité de l’Antibiogramme de la Société Française de Microbiologie. Recommandations 2013, 19, 133–142. [Google Scholar]
  38. EUCAST. Breakpoint Tables for Interpretation of MICs and Zone Diameters. Version 5.0; EUCAST: Basel, Switzerland, 2015. [Google Scholar]
  39. Magiorakos, A.-P.; Srinivasan, A.; Carey, R.B.; Carmeli, Y.; Falagas, M.; Giske, C.; Harbarth, S.; Hindler, J.; Kahlmeter, G.; Olsson-Liljequist, B. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 2012, 18, 268–281. [Google Scholar] [CrossRef] [PubMed]
  40. Girão, M.; Ribeiro, I.; Ribeiro, T.; Azevedo, I.C.; Pereira, F.; Urbatzka, R.; Leão, P.N.; Carvalho, M.F. Actinobacteria Isolated From Laminaria ochroleuca: A Source of New Bioactive Compounds. Front. Microbiol. 2019, 10, 683. [Google Scholar] [CrossRef] [PubMed]
  41. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically-Tenth Edition; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2015; Volume 35. [Google Scholar]
  42. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Third Edition; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2008. [Google Scholar]
  43. Stojanovic, G.S. Volatile constituents of selected Parmeliaceae lichens. J. Serbian Chem. Soc. 2011, 76, 987–994, Erratum in J. Serbian Chem. Soc. 2017, 82, 1333. [Google Scholar] [CrossRef]
  44. Joulain, D.; Tabacchi, R. Lichen extracts as raw materials in perfumery. Part 1: Oakmoss. Flavour Fragr. J. 2009, 24, 49–61. [Google Scholar] [CrossRef]
  45. Andersen, F.; Andersen, K.H.; Bernois, A.; Brault, C.; Bruze, M.; Eudes, H.; Gadras, C.; Signoret, A.C.J.; Mose, K.F.; Muller, B.P.; et al. Reduced content of chloroatranol and atranol in oak moss absolute significantly reduces the elicitation potential of this fragrance material. Contact Dermat. 2015, 72, 75–83. [Google Scholar] [CrossRef]
  46. Avonto, C.; Chittiboyina, A.G.; Khan, S.I.; Dale, O.R.; Parcher, J.F.; Wang, M.; Khan, I.A. Are atranols the only skin sensitizers in oakmoss? A systematic investigation using non-animal methods. Toxicol. In Vitro 2021, 70, 105053. [Google Scholar] [CrossRef]
  47. Lopez-Nogueroles, M.; Chisvert, A.; Salvador, A. Determination of atranol and chloroatranol in perfumes using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry. Anal. Chim. Acta 2014, 826, 28–34. [Google Scholar] [CrossRef]
  48. Kahriman, N.; Yazici, K.; Arslan, T.; Aslan, A.; Karaoglu, S.A.; Yayli, N. Chemical Composition and Antimicrobial Activity of the Essential Oils from Evernia prunastri (L.) Ach. and Evernia divaricata (L.) Ach. Asian J. Chem. 2011, 23, 1937–1939. [Google Scholar]
  49. Le Pogam, P.; Herbette, G.; Boustie, J. Analysis of Lichen Metabolites, a Variety of Approaches. In Recent Advances in Lichenology: Modern Methods and Approaches in Biomonitoring and Bioprospection, Volume 1; Upreti, D.K., Divakar, P.K., Shukla, V., Bajpai, R., Eds.; Springer: New Delhi, India, 2015; pp. 229–261. [Google Scholar]
  50. Steinhäuser, S.S.; Andrésson, Ó.S.; Pálsson, A.; Werth, S. Fungal and cyanobacterial gene expression in a lichen symbiosis: Effect of temperature and location. Fungal Biol. 2016, 120, 1194–1208. [Google Scholar] [CrossRef]
  51. Asplund, J.; Ohlson, M.; Gauslaa, Y. Tree species shape the elemental composition in the lichen Hypogymnia physodes transplanted to pairs of spruce and beech trunks. Fungal Ecol. 2015, 16, 1–5. [Google Scholar] [CrossRef]
  52. Mokhtari, N.; Mrabet, R.; Lebailly, P.; Laurent, B. Spatialisation des bioclimats, de l’aridité et des étages de végétation du Maroc. Rev. Marocaine Des Sci. Agron. Et Vétérinaires 2014, 2, 50–66. [Google Scholar]
  53. Tas, I.; Yildirim, A.B.; Ozkan, E.; Ozyigitoglu, G.C.; Yavuz, M.Z.; Turker, A.U. Biological evaluation and phytochemical profiling of some lichen species. Acta Aliment. 2019, 48, 457–465. [Google Scholar] [CrossRef]
  54. Carpentier, C.; Queiroz, E.F.; Marcourt, L.; Wolfender, J.-L.; Azelmat, J.; Grenier, D.; Boudreau, S.; Voyer, N. Dibenzofurans and Pseudodepsidones from the Lichen Stereocaulon paschale Collected in Northern Quebec. J. Nat. Prod. 2017, 80, 210–214. [Google Scholar] [CrossRef] [PubMed]
  55. Nomura, H.; Isshiki, Y.; Sakuda, K.; Sakuma, K.; Kondo, S. The Antibacterial Activity of Compounds Isolated from Oakmoss against Legionella pneumophila and Other Legionella spp. Biol. Pharm. Bull. 2012, 35, 1560–1567. [Google Scholar] [CrossRef]
  56. Türk, H.; Yılmaz, M.; Tay, T.; Türk, A.Ö.; Kıvanç, M. Antimicrobial Activity of Extracts of Chemical Races of the Lichen Pseudevernia furfuracea and their Physodic Acid, Chloroatranorin, Atranorin, and Olivetoric Acid Constituents. Z. Für Naturforschung C 2006, 61, 499–507. [Google Scholar] [CrossRef]
  57. Ganesan, T.; Subban, M.; Christopher Leslee, D.B.; Kuppannan, S.B.; Seedevi, P. Structural characterization of n-hexadecanoic acid from the leaves of Ipomoea eriocarpa and its antioxidant and antibacterial activities. Biomass Convers. Biorefinery 2024, 14, 14547–14558. [Google Scholar] [CrossRef]
  58. Gröblacher, B.; Kunert, O.; Bucar, F. Compounds of Alpinia katsumadai as potential efflux inhibitors in Mycobacterium smegmatis. Bioorganic Med. Chem. 2012, 20, 2701–2706. [Google Scholar] [CrossRef]
  59. Kubo, I.; Muroi, H.; Himejima, M. Antimicrobial activity of green tea flavor components and their combination effects. J. Agric. Food Chem. 1992, 40, 245–248. [Google Scholar] [CrossRef]
  60. Chowdhry, L.; Khan, Z.K.; Kulshrestha, D.K. Evaluation of himachalol in murine invasive aspergillosis*. Mycoses 1996, 39, 449–452. [Google Scholar] [CrossRef]
  61. Lee, J.-Y.; Kim, Y.-S.; Shin, D.-H. Antimicrobial Synergistic Effect of Linolenic Acid and Monoglyceride against Bacillus cereus and Staphylococcus aureus. J. Agric. Food Chem. 2002, 50, 2193–2199. [Google Scholar] [CrossRef] [PubMed]
Microorganisms 12 02336 g001
Figure 1. Chromatograms of the main compounds present in PFVC1 obtained by GC-MS.
Figure 1. Chromatograms of the main compounds present in PFVC1 obtained by GC-MS.
Microorganisms 12 02336 g001
Microorganisms 12 02336 g002
Figure 2. Chromatograms of the main compounds present in PFVC2 obtained by GC-MS.
Figure 2. Chromatograms of the main compounds present in PFVC2 obtained by GC-MS.
Microorganisms 12 02336 g002
Table 1. Chemical composition of the volatile compounds from Pseudevernia furfuracea (L.) Zopf from two different locations in Morocco obtained by GC-MS. PFVC1, Pseudevernia furfuracea volatile compounds from the High Atlas region. PFVC2, Pseudevernia furfuracea volatile compounds from the Middle Atlas region.
Table 1. Chemical composition of the volatile compounds from Pseudevernia furfuracea (L.) Zopf from two different locations in Morocco obtained by GC-MS. PFVC1, Pseudevernia furfuracea volatile compounds from the High Atlas region. PFVC2, Pseudevernia furfuracea volatile compounds from the Middle Atlas region.
Relative Abundance (%)
NameFormulam/zRetention Time (min)Characterized m/z FragmentationPFVC1PFVC2
Trans-VerbenolC10H16O150.1043410.819137.09605; 109.0679; 67.05420-2.25
NaphthaleneC10H8128.0624811.906128.06195; 127.054221.232.2
ρ-Cymene-8-olC10H14O150.1043411.929135.08034; 132.09329-3.04
QuinolineC9H7N129.0577114.070129.05716; 128.049410.25-
2-Furancarboxaldehyde, 5-(2-furanylmethyl)-C10H8O3176.0471219.878110.05623; 53.046850.892.88
ChloroatranolC8H7ClO3186.0076720.039184.99997; 140.0022819.8024.38
δ-CadineneC15H24204.1876423.527161.13234; 133.101151.41-
HimachalolC15H26O204.1876723.825204.18712; 150.03104; 119.085520.27-
Methyl haematommateC10H10O5210.0527224.176210.05218; 178.025878; 150.031050.87-
Atraric acidC10H12O4196.073324.978196.07274; 177.03619; 164.0517373.5356.95
AcetisoeugenolC12H14O3238.0839725.993164.04664-3.77
Guaiol acetateC17H28O2204.1876426.634161.13238; 105.069870.82-
n-Hexadecanoic acidC16H32O2256.2401730.20073.028410.942.67
AbietatrieneC20H30270.2347431.946255.21075; 173.13241-1.62
9,12,15-Octadecatrienoic acid, (Z,Z,Z)-C18H30O2278.2242133.13079.05423; 55.05425-0.18
Total100100
Table 2. Antimicrobial activity of volatile compounds from Pseudevernia furfuracea (L.) Zopf. from two different locations in Morocco. IZD, inhibition zone diameter. MIC, minimum inhibitory concentration. PFVC1, Pseudevernia furfuracea volatile compounds from the High Atlas region. PFVC2, Pseudevernia furfuracea volatile compounds from the Middle Atlas region.
Table 2. Antimicrobial activity of volatile compounds from Pseudevernia furfuracea (L.) Zopf. from two different locations in Morocco. IZD, inhibition zone diameter. MIC, minimum inhibitory concentration. PFVC1, Pseudevernia furfuracea volatile compounds from the High Atlas region. PFVC2, Pseudevernia furfuracea volatile compounds from the Middle Atlas region.
MicroorganismIZDMIC (µg/mL)
PFVC1PFVC2PFVC1PFVC2
Acinetobacter baumannii (A1) 125250
Escherichia coli (E1) 500500
Klebsiella pneumoniae (K1) 5001000
Staphylococcus aureus (S1) 250250
Staphylococcus aureus (ATCC 29213) 250250
Bacillus subtilis (ATCC 6633) 500250
Salmonella typhimurium (ATCC 25241) 500500
Escherichia coli (ATCC 25922) 500500
Candida albicans (ATCC 10231) 62.531.25
Aeromonas hydrophila (DSM 30187) 250125
Pseudomonas anguiliseptica (DSM 12111) 250250
Edwardsiella tarda (DSM 30052) 125125
Listonella anguillarum (ATCC 19264) 250500
Yersinia ruckeri (ATCC 29473) 250250
Microorganisms 12 02336 i001: No halo; Microorganisms 12 02336 i002: <10 mm; Microorganisms 12 02336 i003: 10–20 mm; Microorganisms 12 02336 i004: >20 mm.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Essadki, Y.; Hilmi, A.; Cascajosa-Lira, A.; Girão, M.; Darrag, E.M.; Martins, R.; Romane, A.; El Amrani Zerrifi, S.; Mugani, R.; Tazart, Z.; et al. In Vitro Antimicrobial Activity of Volatile Compounds from the Lichen Pseudevernia furfuracea (L.) Zopf. Against Multidrug-Resistant Bacteria and Fish Pathogens. Microorganisms 2024, 12, 2336. https://doi.org/10.3390/microorganisms12112336

AMA Style

Essadki Y, Hilmi A, Cascajosa-Lira A, Girão M, Darrag EM, Martins R, Romane A, El Amrani Zerrifi S, Mugani R, Tazart Z, et al. In Vitro Antimicrobial Activity of Volatile Compounds from the Lichen Pseudevernia furfuracea (L.) Zopf. Against Multidrug-Resistant Bacteria and Fish Pathogens. Microorganisms. 2024; 12(11):2336. https://doi.org/10.3390/microorganisms12112336

Chicago/Turabian Style

Essadki, Yasser, Adel Hilmi, Antonio Cascajosa-Lira, Mariana Girão, El Mehdi Darrag, Rosário Martins, Abderrahmane Romane, Soukaina El Amrani Zerrifi, Richard Mugani, Zakaria Tazart, and et al. 2024. "In Vitro Antimicrobial Activity of Volatile Compounds from the Lichen Pseudevernia furfuracea (L.) Zopf. Against Multidrug-Resistant Bacteria and Fish Pathogens" Microorganisms 12, no. 11: 2336. https://doi.org/10.3390/microorganisms12112336

APA Style

Essadki, Y., Hilmi, A., Cascajosa-Lira, A., Girão, M., Darrag, E. M., Martins, R., Romane, A., El Amrani Zerrifi, S., Mugani, R., Tazart, Z., Redouane, E. M., Jos, A., Cameán, A. M., Vasconcelos, V., Campos, A., El Khalloufi, F., Oudra, B., Barakate, M., & Carvalho, M. d. F. (2024). In Vitro Antimicrobial Activity of Volatile Compounds from the Lichen Pseudevernia furfuracea (L.) Zopf. Against Multidrug-Resistant Bacteria and Fish Pathogens. Microorganisms, 12(11), 2336. https://doi.org/10.3390/microorganisms12112336

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop