Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm Staphylococcus aureus-Candida albicans.

The demand for antibiofilm molecules has increased over several years due to their potential to fight biofilm-associated infections, such as those including the interkingdom Staphylococcus aureus-Candida albicans occurring in clinical settings worldwide. Recently, we identified a pentacyclic triterpenoid compound, betulinic acid, from invasive macrophytes, with interesting antibiofilm properties. The aim of the present study was to provide insights into the mechanism of action of betulinic acid against the clinically relevant bi-species S. aureus-C. albicans biofilms. Microscopy examinations, flow cytometry and crystal violet assays confirmed that betulinic acid was effective at damaging mature S. aureus-C. albicans biofilms or inhibiting their formation, reducing biofilm biomass by 70% on average and without microbicidal activity. The results suggested an action of betulinic acid on cell membranes, inducing changes in properties such as composition, hydrophobicity and fluidity as observed in C. albicans, which may hinder the early adhesion step, biofilm growth and the physical interactions of both microbial species. Further results of real-time polymerase chain reaction argued in favour of a reduction in S. aureus-C. albicans physical interaction due to betulinic acid by the modulation of biofilm-related gene expression, as observed in early stages of biofilm formation. This study revealed the potential of betulinic acid as a candidate agent for the prevention and treatment of S. aureus-C. albicans biofilm-related infections.

Screening antibiofilm activity of invasive plants growing at the Slope Merapi Mountain, Central Java, against Candida albicans.

BACKGROUND: Candida albicans causes high-mortality candidiasis. Antifungal drug resistance demands the development of virulence factor-targeting drugs, particularly antibiofilm. This study screened the effects of five invasive plants growing in Indonesia (Mimosa pudica, Lantana camara, Acacia mangium, Ageratina riparia, and Mikania micrantha) against C. albicans biofilms. Antifungal activity, antiphospholipase activity, biofilm morphology of C. albicans, and cytotoxic capacity were also evaluated. METHODS: Maceration was used to extract the plants, and the most active extract inhibiting the biofilms was fractionated using liquid-liquid fractionation. Antibiofilm activity was determined by a colorimetric assay, MTT. Antifungal activity was tested using the broth microdilution method. A phospholipase assay was performed using the egg-yolk agar method. Influence on the C. albicans morphology was assessed using scanning electron microscopy (SEM). The cytotoxic effect was carried out against Vero and HeLa cell lines. RESULTS: M. pudica extracts showed the most potent antifungal efficacy with minimum inhibitory concentration (MIC) of 15.62 µg/mL and 7.81 µg/mL for aerial parts and roots, respectively. At high concentrations (500 µg/mL and 250 µg/mL), ethanol extract of M. pudica aerial parts strongly inhibited the phospholipase activity. Ethyl-acetate fraction of M. pudica aerial parts demonstrated the most potent antibiofilm activity against 24 h old biofilm of C. albicans with an inhibitory concentration (53.89%) of 62.5 µg/mL showed no cytotoxicity in both Vero and HeLa cells. This fraction affected the morphology of C. albicans and contained promising compounds for inhibiting the 24 h old biofilm of C. albicans. CONCLUSIONS: Invasive M. pudica plant inhibited the growth of planktonic C. albicans cells and its ethyl acetate fraction decreased the metabolic activity of C. albicans biofilms. This result demonstrates the potential of invasive M. pudica plant to reduce biofilm-associated candida infection.

Antibiofilm Activity of Invasive Plants against Candida albicans: Focus on Baccharis halimifolia Essential Oil and Its Compounds.

The extracts of five invasive plants were investigated for antifungal and antibiofilm activities against Candida albicans, C. glabrata, C. krusei, and C. parapsilosis. The antifungal activity was evaluated using the microdilution assay and the antibiofilm effect by measurement of the metabolic activity. Ethanol and ethanol-water extracts of Reynoutria japonica leaves inhibited 50 % of planktonic cells at 250 µg mL-1 and 15.6 µg mL-1 , respectively. Ethanol and ethanol-water extracts of Baccharis halimifolia inhibited >75 % of the mature biofilm of C. albicans at 500 µg mL-1 . The essential oil (EO) of B. halimifolia leaves was the most active (50 % inhibition (IC50 ) at 4 and 74 µg mL-1 against the maturation phase and 24 h old-biofilms of C. albicans, respectively). Oxygenated sesquiterpenes were the primary contents in this EO (62.02 %), with ß-caryophyllene oxide as the major component (37 %). Aromadendrene oxide-(2), ß-caryophyllene oxide, and (±)-ß-pinene displayed significant activities against the maturation phase (IC50 =9-310 µ mol l-1 ) and preformed 24 h-biofilm (IC50 =38-630 µ mol l-1 ) of C. albicans with very low cytotoxicity for the first two compounds. C. albicans remained the most susceptible species to this EO and its components. This study highlighted for the first time the antibiofilm potential of B. halimifolia, its EO and some of its components.

Valorization of Invasive Plant Extracts against the Bispecies Biofilm Staphylococcus aureus-Candida albicans by a Bioguided Molecular Networking Screening.

Invasive plants efficiently colonize non-native territories, suggesting a great production of bioactive metabolites which could be effective antibiofilm weapons. Our study aimed to look for original molecules able to inhibit bispecies biofilm formed by S. aureus and C. albicans. Extracts from five invasive macrophytes (Ludwigia peploides, Ludwigia grandiflora, Myriophyllum aquaticum, Lagarosiphon major and Egeria densa) were prepared and tested in vitro against 24 h old bispecies biofilms using a crystal violet staining (CVS) assay. The activities of the extracts reducing the biofilm total biomass by 50% or more were comparatively analyzed against each microbial species forming the biofilm by flow cytometry (FCM) and scanning electron microscopy. Extracts active against both species were fractionated. Obtained fractions were analyzed by UHPLC-MS/MS and evaluated by the CVS assay. Chemical and biological data were combined into a bioactivity-based molecular networking (BBMN) to identify active compounds. The aerial stem extract of L. grandiflora showed the highest antibiofilm activity (>50% inhibition at 50 µg∙mL−1). The biological, chemical and BBMN investigations of its fractions highlighted nine ions correlated with the antibiofilm activity. The most correlated compound, identified as betulinic acid (BA), inhibited bispecies biofilms regardless of the three tested couples of strains (ATCC strains: >40% inhibition, clinical isolates: ≈27% inhibition), confirming its antibiofilm interest.

Access to Anti-Biofilm Compounds from Endolichenic Fungi Using a Bioguided Networking Screening.

Endolichenic microorganisms represent a new source of bioactive natural compounds. Lichens, resulting from a symbiotic association between algae or cyanobacteria and fungi, constitute an original ecological niche for these microorganisms. Endolichenic fungi inhabiting inside the lichen thallus have been isolated and characterized. By cultivation on three different culture media, endolichenic fungi gave rise to a wide diversity of bioactive metabolites. A total of 38 extracts were screened for their anti-maturation effect on Candida albicans biofilms. The 10 most active ones, inducing at least 50% inhibition, were tested against 24 h preformed biofilms of C. albicans, using a reference strain and clinical isolates. The global molecular network was associated to bioactivity data in order to identify and priorize active natural product families. The MS-targeted isolation led to the identification of new oxygenated fatty acid in Preussia persica endowed with an interesting anti-biofilm activity against C. albicans yeasts.

Lichen Polyphenolic Compounds for the Eradication of Candida albicans Biofilms.

Lichens, due to their symbiotic nature (association between fungi and algae), constitute a chemical factory of original compounds. Polyphenolic compounds (depsides and depsidones) are the main constituents of lichens and are exclusively biosynthesized by these organisms. A panel of 11 polyphenols was evaluated for their anti-biofilm activity against Candida albicans biofilms on the maturation phase (anti-maturation) (MMIC50) as well as on preformed 24-h-old biofilm (anti-biofilm) (MBIC50) using the XTT assay. Minimum inhibitory concentrations of compounds (MICs) against C. albicans planktonic yeast were also determined using a broth microdilution method. While none of the tested compounds were active against planktonic cells (IC50 > 100 µg/ml), three depsides slowed the biofilm maturation (MMIC50 ≤12.5 µg/ml after 48 h of contact with Candida cells). Evernic acid was able to both slow the maturation and reduce the already formed biofilms with MBIC50 ≤12.5 µg/ml after 48 h of contact with the biofilm. This compound shows a weak toxicity against HeLa cells (22%) at the minimal active concentration and no hemolytic activity at 100 µg/ml. Microscopic observations of evernic acid and optimization of its solubility were performed to further study this compound. This work confirmed the anti-biofilm potential of depsides, especially evernic acid, and allows to establish the structure-activity relationships to better explain the anti-biofilm potential of these compounds.

Can Leaves and Stems of Rubus idaeus L. Handle Candida albicans Biofilms?

Candida albicans is an opportunistic pathogen involved in many infections, especially linked to implanted medical devices. Its ability to form biofilms complicates the treatment of these infections as few molecules are active against sessile C. albicans. The aim of this study was to evaluate the potential of leaves, three-month-old and one-year-old stems of Rubus idaeus L. against C. albicans biofilm growth. Extractions with a polarity gradient were carried out on hydroacetonic extracts and followed by fractionation steps. The obtained extracts and fractions were tested for their anti-biofilm growth activity against C. albicans using XTT method. Compounds of active subfractions were identified by LC-MS. The hexane extracts from leaves and stems were the most active against the fungus with IC50 at 500 and 250 µg/mL. Their bioguided fractionation led to 4 subfractions with IC50 between 62.5 and 125 µg/mL. Most of the components identified in active subfractions were fatty acids and terpenoïds.

Nanovectorized Microalgal Extracts to Fight Candida albicans and Cutibacterium acnes Biofilms: Impact of Dual-Species Conditions.

Biofilm-related infections are a matter of concern especially because of the poor susceptibility of microorganisms to conventional antimicrobial agents. Innovative approaches are needed. The antibiofilm activity of extracts of cyanobacteria Arthrospira platensis, rich in free fatty acids, as well as of extract-loaded copper alginate-based nanocarriers, were studied on single- and dual-species biofilms of Candida albicans and Cutibacterium acnes. Their ability to inhibit the biofilm formation and to eradicate 24 h old biofilms was investigated. Concentrations of each species were evaluated using flow cytometry. Extracts prevented the growth of C. acnes single-species biofilms (inhibition > 75% at 0.2 mg/mL) but failed to inhibit preformed biofilms. Nanovectorised extracts reduced the growth of single-species C. albicans biofilms (inhibition > 43% at 0.2 mg/mL) while free extracts were weakly or not active. Nanovectorised extracts also inhibited preformed C. albicans biofilms by 55% to 77%, whereas the corresponding free extracts were not active. In conclusion, even if the studied nanocarrier systems displayed promising activity, especially against C. albicans, their efficacy against dual-species biofilms was limited. This study highlighted that working in such polymicrobial conditions can give a more objective view of the relevance of antibiofilm strategies by taking into account interspecies interactions that can offer additional protection to microbes.

Anti-biofilm activity of a semi-synthetic molecule obtained from resveratrol against Candida albicans biofilm.

Candida albicans can form biofilm on tissues and medical devices, becoming, in that case, less susceptible to antifungal agents. Treatment of candidiasis associated with the formation of C. albicans biofilms is restricted to echinocandins and lipid forms of amphotericin B. This study investigated the activity of micafungin and resveratrol modified molecule (EB487) against C. albicans biofilms. The anti-biofilm growth (Bgrowth) and anti-preformed biofilm (Bpreformed) activities of micafungin (0 to 3.94 µM) and EB487 (0 to 20.32 mM) were comparatively studied separately and combined, using XTT, flow cytometry and cell counts approaches. Concentrations causing 50% inhibition of the studied steps (IC50) were evaluated. When tested separately, IC50 Bgrowth was obtained for 4.8 mM and 0.13 µM of EB487 and micafungin respectively, and IC50 Bpreformed for 3.6 mM and 0.06 µM of EB487 and micafungin respectively. Micafungin used alone was not able to totally eradicate fungi. Micafungin combined with EB487 displayed synergistic activity (both anti-growth- and anti-preformed biofilm-activities). Optimal combination concentrations were EB487 (≤9.12 mM -strain ATCC 28367™ or ≤8.12 mM -strain CAI4-p), micafungin (≤0.05 µM for both) and caused a total eradication of fungi. Dose reduction indexes obtained using these concentrations were at least 9 (micafungin) and 3.2 (EB487) for both anti-biofilm growth- and anti-preformed biofilm-activities. Combinations indexes were consistently below one, demonstrating a synergistic relationship between micafungin and EB487 in these conditions. This study demonstrated the strong anti-biofilm activity of EB487 and highlighted its synergistic potential when combined with micafungin. EB487 is a promising semi-synthetic molecule with prophylactic and curative interests in fighting C. albicans biofilms.

Candida albicans enhances initial biofilm growth of Cutibacterium acnes under aerobic conditions.

Candida albicans and Cutibacterium acnes are opportunistic pathogens that co-colonize the human body. They are involved in biofilm-related infections of implanted medical devices. The objective of this study was to evaluate the ability of these species to interact and form polymicrobial biofilms. SEM imaging and adhesion assays showed that C. acnes adhesion to C. albicans did not have a preference for a specific morphological state of C. albicans; bacteria adhered to both hyphal and yeast forms of C. albicans. C. albicans did not influence growth of C. acnes under anaerobic growth conditions, however under aerobic growth condition, C. albicans enhanced early C. acnes biofilm formation. This favorable impact of C. albicans was not mediated by secreted compounds accumulating in the medium, but required the presence of metabolically active C. albicans. The ability of these microorganisms to interact together could modulate the physiopathology of infections.

Collected mass spectrometry data on monoterpene indole alkaloids from natural product chemistry research.

This Data Descriptor announces the submission to public repositories of the monoterpene indole alkaloid database (MIADB), a cumulative collection of 172 tandem mass spectrometry (MS/MS) spectra from multiple research projects conducted in eight natural product chemistry laboratories since the 1960s. All data have been annotated and organized to promote reuse by the community. Being a unique collection of these complex natural products, these data can be used to guide the dereplication and targeting of new related monoterpene indole alkaloids within complex mixtures when applying computer-based approaches, such as molecular networking. Each spectrum has its own accession number from CCMSLIB00004679916 to CCMSLIB00004680087 on the GNPS. The MIADB is available for download from MetaboLights under the identifier: MTBLS142 ( https://www.ebi.ac.uk/metabolights/MTBLS142 ).

Do raspberry extracts and fractions have antifungal or anti-adherent potential against Candida spp.?

Candida spp., especially Candida albicans, is one of the main colonisers of the oral cavity. Due to its ability to form biofilms, it can be implicated in dental caries, periodontal disease and denture stomatitis. Microbial cells in biofilms are minimally impacted by conventional drugs. The aim of this study was to find new substances able to inhibit the adhesion of Candida spp. in order to prevent biofilm formation in the oral cavity. This study focused on the red raspberry (Rubus idaeus) fruit, known for its richness in potentially antimicrobial tannins. Extraction with a polarity gradient was performed on acetone extracts from frozen ripe and unripe fruits, resulting in eight extracts. The antifungal and anti-adhesion effects of the extracts were determined using broth microdilution and XTT methods, respectively, against C. albicans, Candida glabrata and Candida parapsilosis strains. Interestingly, four extracts (hexane and ethyl acetate) displayed anti-adhesion activity against C. albicans at low concentrations [50% inhibitory concentration (IC50) 15.6-62.5 µg/mL]. Bioassay-guided fractionation by chromatographic methods of the most active extract obtained from ripe fruit (ethyl acetate extract) led to two subfractions enriched in anti-adhesion compounds, identified by mass spectrometry analysis as hydrolysable and condensed tannins. Their activities were dose-dependent with maximum inhibition at 80% (IC50 = 25 µg/mL and 12.5 µg/mL). Regarding antifungal activity, no extract was active against planktonic cells of the tested strains. This work highlights for the first time the potential of raspberries to prevent oral C. albicans biofilms.

Cutibacterium acnes protects Candida albicans from the effect of micafungin in biofilms.

The aim of this study was to investigate the ability of Candida albicans and Cutibacterium acnes to grow together as a polymicrobial biofilm in vitro and to examine the influence of C. acnes on C. albicans susceptibility to micafungin. Mature 72-h-old single-species biofilms of C. albicans and polymicrobial biofilms involving both C. albicans and C. acnes were formed in brain-heart infusion and were observed by scanning electronic microscopy. Moreover, 24-h-old single-species and polymicrobial biofilms were treated for 24 h with micafungin (concentrations ranging from 0.75 mg/L to 12 mg/L) and the antibiofilm activity of micafungin was evaluated on fungal cells by flow cytometry following addition of propidium iodide. The results showed that C. albicans and C. acnes formed a polymicrobial biofilm in the tested conditions and that bacterial presence did not modify fungal viability. Micafungin induced a fungal mortality rate ranging from 70-95% in C. albicans single-species biofilms and from 35-40% in C. acnes-C. albicans polymicrobial biofilms. Mortality induced by micafungin was significantly reduced (P < 0.05 for micafungin at 6 mg/L and P < 0.001 for other micafungin concentrations) in polymicrobial conditions compared with single-species biofilms. In conclusion, this study showed that C. albicans and C. acnes are able to form polymicrobial biofilms together in a synergistic way and that this organisation increases yeast resistance to micafungin.

Fungal communities associated with Evernia prunastri, Ramalina fastigiata and Pleurosticta acetabulum: Three epiphytic lichens potentially active against Candida biofilms.

Fungal communities associated to three epiphytic lichens active against Candida, were investigated using culture-based methods We hypothetized that associated fungi would contribute to lichens activities. The ability of specific fungi to grow inside or outside lichens was investigated. To detect biogenesis pathways involved in the production of secondary metabolites, genes coding for nonribosomal peptide synthetase (NRPS) and polyketide synthase I (PKS I) were screened by PCR from fungal DNA extracts. Both endo and epilichenic communities were isolated from two fructicose (Evernia prunastri and Ramalina fastigiata) and one foliose (Pleurosticta acetabulum) lichens. A total of 86 endolichenic and 114 epilichenic isolates were obtained, corresponding to 18 and 24 phylogenetic groups respectively suggesting a wide diversity of fungi. The communities and the species richness were distinct between the three lichens which hosted potentially new fungal species. Additionally, the endo- and epilichenic communities differed in their composition: Sordariomycetes were particularly abundant among endolichenic fungi and Dothideomycetes among epilichenic fungi. Only a few fungi colonized both habitats, such as S. fimicola, Cladosporium sp1 and Botrytis cinerea. Interestingly, Nemania serpens (with several genotypes) was the most abundant endolichenic fungus (53% of isolates) and was shared by the three lichens. Finally, 12 out of 36 phylogenetic groups revealed the presence of genes coding for nonribosomal peptide synthetase (NRPs) and polyketide synthase I (PKS I). This study shows that common lichens are reservoirs of diverse fungal communities, which could potentially contribute to global activity of the lichen and, therefore, deserve to be isolated for further chemical studies.

Antifungal and Anti-Biofilm Activities of Acetone Lichen Extracts against Candida albicans.

Candida albicans is a commensal coloniser of the human gastrointestinal tract and an opportunistic pathogen, especially thanks to its capacity to form biofilms. This lifestyle is frequently involved in infections and increases the yeast resistance to antimicrobials and immune defenses. In this context, 38 lichen acetone extracts have been prepared and evaluated for their activity against C. albicans planktonic and sessile cells. Minimum inhibitory concentrations of extracts (MICs) were determined using the broth microdilution method. Anti-biofilm activity was evaluated using tetrazolium salt (XTT) assay as the ability to inhibit the maturation phase (anti-maturation) or to eradicate a preformed 24 h old biofilm (anti-biofilm). While none of the extracts were active against planktonic cells, biofilm maturation was limited by 11 of the tested extracts. Seven extracts displayed both anti-maturation and anti-biofilm activities (half maximal inhibitory concentrations IC50_mat and IC50_biof ≤ 100 µg/mL); Evernia prunastri and Ramalina fastigiata were the most promising lichens (IC50_mat < 4 µg/mL and IC50_biof < 10 µg/mL). Chemical profiles of the active extracts performed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) have been analyzed. Depsides, which were present in large amounts in the most active extracts, could be involved in anti-biofilm activities. This work confirmed that lichens represent a reservoir of compounds with anti-biofilm potential.

Natural Sources as Innovative Solutions Against Fungal Biofilms.

Fungal cells are capable of adhering to biotic and abiotic surfaces and form biofilms containing one or more microbial species that are microbial reservoirs. These biofilms may cause chronic and acute infections. Fungal biofilms related to medical devices are particularly responsible for serious infections such as candidemia. Nowadays, only a few therapeutic agents have demonstrated activities against fungal biofilms in vitro and/or in vivo. So the discovery of new anti-biofilm molecules is definitely needed. In this context, biodiversity is a large source of original active compounds including some that have already proven effective in therapies such as antimicrobial compounds (antibacterial or antifungal agents). Bioactive metabolites from natural sources, useful for developing new anti-biofilm drugs, are of interest. In this chapter, the role of molecules isolated from plants, lichens, algae, microorganisms, or from animal or human origin in inhibition and/or dispersion of fungal biofilms (especially Candida and Aspergillus biofilms) is discussed. Some essential oils, phenolic compounds, saponins, peptides and proteins and alkaloids could be of particular interest in fighting fungal biofilms.

Efficacy of dental unit waterlines disinfectants on a polymicrobial biofilm.

Due to their high surface-volume ratio, their laminar flow and frequent stagnation periods, dental unit waterlines (DUWL) foster the attachment of microorganisms and the development of biofilm, resulting in the continuous contamination of the outlet water from dental units; this contamination may be responsible for a potential risk of infection due to the exposure of patients and medical staff to droplet inhalation or splashed water. In this study, the anti-biofilm activity of three disinfectants recommended by dental unit manufacturers -Calbenium(©), Oxygenal 6(©) and Sterispray(©) - was evaluated. A dynamic model simulating DUWL conditions was developed and polymicrobial biofilms containing bacteria (Pseudomonas aeruginosa), fungi (Candida albicans) and Free Living Amoeba (FLA: Vermamoeba vermiformis) were allowed to form. The ability of disinfectants to reduce biofilm formation or to eradicate an already formed biofilm was evaluated. Results showed the various effects of the tested disinfectants according to their composition, concentration and the targeted species. V. vermiformis was resistant to disinfectants, regardless of the tested concentrations and the concentrations recommended by manufacturers were not the most appropriate. Results also showed that Calbenium(©) was the most effective disinfectant to reduce already formed biofilms; its maximum efficiency was observed from 0.5% on both P. aeruginosa and C. albicans compared to 2 and 3% respectively for Sterispray(©). The maximum efficiency of Oxygenal(©) was observed from 3% on P. aeruginosa but Oxygenal(©) was unable to totally eliminate C. albicans in the tested conditions, contrary to other disinfectants. Calbenium(©) was able to prevent biofilm formation efficiently even if it displayed no prophylactic activity against V. vermiformis. Overall, the FLA survival may contribute to maintaining other species. Finally the tested disinfectants were partially active against sessile microorganisms and more suitable concentrations could be used to increase their efficacy. Their use in a prophylactic rather than curative way should be recommended.

Novel strategies against Candida biofilms: interest of synthetic compounds.

A biofilm is a consortium of microbial cells that are attached to a substratum or an interface. It should be considered a reservoir that may induce serious infections. Indeed, Candidaspp. biofilms may be involved in the persistence or worsening of some chronic inflammatory diseases as well as in systemic infections, which may lead to high morbidity and mortality rates. New strategies are currently being explored, utilizing several synthetic compounds to prevent or fight these Candida biofilms. This article focuses on active synthetic compounds classified with regards to their modes of action: inhibition of early adherence phase, inhibition or control of biofilm maturation and finally elimination of already formed biofilms. Some of them show promise in fighting biofilm.

Comparative evaluation of the antimicrobial activity of 19 essential oils.

In our research on natural compounds efficient against human pathogen or opportunist microorganisms contracted by food or water, the antimicrobial activity of 19 essential oils (EOs) was investigated against 11 bacterial species (6 Gram positive, 5 Gram negative) and 7 fungal species (2 dermatophytes, 1 mould, 4 yeasts) using microdilution assays. Five essential oils were obtained from Tunisian plants (EOtun): Artemisia herba-alba Asso, Juniperus phoenicea L., Rosmarinus officinalis L., Ruta graveolens L. and Thymus vulgaris L., whereas others were commercial products (EOcom). Overall, T. vulgaris EOtun was the most efficient EO against both bacteria (Gram negative: MIC ≤ 0.34 mg/mL; Gram positive: MIC ≤ 0.70 mg/mL) and fungi (yeasts: MIC ≤ 0.55 mg/mL; mould: MIC = 0.30 mg/mL; dermatophytes: MIC ≤ 0.07 mg/mL). Two EOcom displayed both acceptable antibacterial and antifungal potency, although weaker than T. vulgaris EOtun activity: Origanum vulgare EOcom (bacteria: MIC ≤ 1.13 mg/mL, fungi: MIC ≤ 1.80 mg/mL), and Cymbopogon martinii var. motia EOcom (bacteria: MIC ≤ 1.00 mg/mL, fungi: MIC ≤ 0.80 mg/mL). Bacillus megaterium, Legionella pneumophila, Listeria monocytogenes and Trichophyton spp. were the most sensitive species to both EOcom and EOtun. This study demonstrated the noteworthy antimicrobial activity of two commercial EOs and points out the remarkable efficiency of T. vulgaris EOtun on all tested bacterial and fungal species, certainly associated with its high content in carvacrol (85 %). These three oils could thus represent promising candidates for applications in water and food protections.

Activity of Six Essential Oils Extracted from Tunisian Plants against Legionella pneumophila.

The aim of this study was to investigate the composition of six essential oils extracted from Tunisian plants, i.e., Artemisia herba-alba Asso, Citrus sinensis (L.) Osbeck, Juniperus phoenicea L., Rosmarinus officinalis L., Ruta graveolens L., and Thymus vulgaris L., and to evaluate their activity against Legionella pneumophila (microdilution assays). Eight Legionella pneumophila strains were studied, including the two well-known serogroup 1 Lens and Paris strains as controls and six environmental strains isolated from Tunisian spas belonging to serogroups 1, 4, 5, 6, and 8. The essential oils were generally active against L. pneumophila. The activities of the A. herba-alba, C. sinensis, and R. officinalis essential oils were strain-dependent, whereas those of the J. phoenicea and T. vulgaris oils, showing the highest anti-Legionella activities, with minimum inhibitory concentrations (MICs) lower than 0.03 and lower than or equal to 0.07 mg/ml, respectively, were independent of the strains' serogroup. Moreover, the microorganisms treated with T. vulgaris essential oil were shorter, swollen, and less electron-dense compared to the untreated controls. Isoborneol (20.91%), (1S)-α-pinene (18.30%) ß-phellandrene (8.08%), α-campholenal (7.91%), and α-phellandrene (7.58%) were the major components isolated from the J. phoenicea oil, while carvacrol (88.50%) was the main compound of the T. vulgaris oil, followed by p-cymene (7.86%). This study highlighted the potential interest of some essential oils extracted from Tunisian plants as biocides to prevent the Legionella risk.