Antimicrobial and Antibiofilm Activities of the Essential Oil from the Inflorescence of Etlingera elatior (Zingiberaceae) and Its Main Constituents (Dodecanal and 1-Dodecanol)Abstract
The spread of antibiotic-resistant pathogens had motivated the search for plant-derived antimicrobial agents, such as essential oils. This work investigated the antibacterial, antifungal and antibiofilm properties of the essential oil from the inflorescence of Etlingera elatior and its major compounds. Dodecanal (42.46%) and 1-dodecanol (25.84%) were the major compounds. The oil, dodecanal and 1-dodecanol were active against Staphylococcus aureus, with minimum bactericidal concentrations of 124, 31 and 62 µg mL-1, respectively. For Pseudomonas aeruginosa, the essential oil had an inhibitory effect against a multidrug resistant isolate (minimum inhibitory concentration (MIC): 4,000 µg mL-1). Dodecanal had an MIC of 60,000 µg mL-1 for all Candida species, but fungicidal activity was only detected for Candida albicans (minimum fungicidal concentration: 60,000 µg mL-1). 1-Dodecanol (60,000 µg mL-1) exhibited fungistatic activity for C. albicans. Biofilm inhibition linked to an anti-adhesive effect of the oil and dodecanal was only detected for the standard S. aureus isolate and C. albicans, respectively. In conclusion, the essential oil from the inflorescence of E. elatior and its major components were more active against nonresistant strains S. aureus. Essential oil, dodecanol and dodecanal showed excellent activity against S. aureus, however, these same materials showed low activity against P. aeruginosa.
Keywords:
Etlingera elatior; essential oil; antibacterial activity; antifungal activity; antibiofilm
Introduction
Yeasts and bacteria are responsible for various diseases and annual economic losses. Combating these organisms generally involves the use of chemical compounds. However, the growing and recurrent emergence of resistant strains makes elimination difficult and increases costs. With regards to pathogens in humans, essential oils (EOs) have potential antimicrobial and antifungal properties and can be used as natural alternatives, as EOs are capable of altering the permeability of fungal cells, inhibiting functions, impairing protein synthesis and disturbing the balance of the cell membrane.1-4
Diverse constituents of EOs enable the simultaneous occurrence of multiple mechanisms of antimicrobial and antifungal action, thus ensuring less likelihood of the development of resistance as well as less toxicity compared to the use of a single active compound.5-7 Therefore, EOs have potential action against the emergence of resistant strains resulting from the indiscriminate use of antibiotics, which constitutes a global public health problem.
This situation is aggravated by the reduction in the number of new antibiotics developed and approved for clinical use, thus motivating numerous studies focused on the identification of new compounds with antimicrobial activity.8 EOs constitute a promising source of chemical compounds that are candidates for antimicrobial and antifungal agents, such as aldehydes and alcohols.9 Thus, EOs have been increasingly employed in different fields, such as agriculture, medicine and industry, expanding the importance of these substances, which are easily acquired at low cost, renewable, biodegradable and less toxic to the population and environment.10,11
Pseudomonas aeruginosa and Staphylococcus aureus are bacteria of considerable medical importance responsible for infections, such as pneumonia, meningitis, endocarditis, toxic shock syndrome and septicemia.12-14 S. aureus is the most prevalent pathogen in childhood, adolescence and early adulthood, while P. aeruginosa affects more humans in adulthood.15 These bacteria form biofilms that can adhere to hospital materials, causing severe infections that are difficult to control due to the increase in resistance to antibiotics.16,17 Candida albicans, Candida tropicalis and Candida krusei are pathogens of considerable relevance to human health and cause a common fungal infection denominated candidiasis, which can affect oral and vaginal mucosae or internal organs. Systemic infections are denominated candidemia and usually pose a high risk.18
Essential oils and extracts obtained from the botanical family Zingiberaceae exhibit antimicrobial properties against bacteria and fungi.19 Essential oil from the rhizomes of Aframomum sceptrum exhibited bacteriostatic activity specifically against Gram-positive bacteria.20 Yusoff et al.21 reported the antibacterial effects of essential oils from the rhizomes of Alpinia ligulata and Alpinia nieuwenhuizii against Proteus vulgaris, Escherichia coli and S. aureus. The essential oil from the leaves of Etlingera fimbriobracteata exhibited antimicrobial activity against Bacillus subtilis, Bacillus spizizenii, S. aureus and E. coli.15
Etlingera elatior (Jack) R.M. Smith (Red Torch Ginger), which is also known as the “emperor’s staff”, is an aromatic, ornamental, tropical plant, with reports of the extraction of EOs from different parts of the plant.22,23 This plant is from the family Zingiberaceae, whose origin is in the Indo-Pacific region. The family comprises 53 genera and around 1300 species. Although not a native Brazilian plant, E. elatior is widely used ornamentally due to its large inflorescences, which are typically reddish or pinkish in color. In 2016, Bezerra Silva et al.24 extracted essential oil from the inflorescence of E. elatior (red, pink and porcelain) by hydrodistillation, which exhibited oviposition-deterrent activity against Aedes aegypti. Wijekoon et al.25 studied the antibacterial activity of the essential oil (17.5 ± 1.35% odecanal and 25.17 ± 3.4% dodecanol) and extracts from the inflorescence of E. elatior and both samples exhibited activity against Gram positive bacteria (S. aureus, Bacillus cereus, Listeria monocytogenes and Bacillus subtilis). Ferreira et al.26 reported moderate antibacterial activity of the EO of this plant and dodecanal (58.31%) as well as promising activity of dodecanol (20.33%) against strains of Staphylococcus aureus. Therefore, the aim of the present study was to investigate the antimicrobial, antifungal and antibiofilm activities of the EO from the inflorescence of E. elatior and its two major components against species of the genus Candida as well as resistant and non-resistant strains of S. aureus and P. aeruginosa.
Experimental
Plant material
In September 2016, our research group purchased fresh inflorescences of Etlingera elatior from Florix Flora Tropical in the city of Recife, northeastern Brazil. The plantation is in the rural area of Monjope, Igarassú, Pernambuco (7°50’2.76’’ S, 34°54’21.816’’ W”). A voucher was deposited in the herbarium of Recife Botanical Gardens (voucher number 1654). The SisGen number is A29F849.
Microorganisms
Pseudomonas aeruginosa (standard (ATCC-27853) and multiple drug-resistant (UFPEDA-262) isolates) and Staphylococcus aureus (standard (ATCC-6538) and oxacillinresistant (UFPEDA-672) isolates) were obtained from the culture collection (WDCM 114) of the Department of Antibiotics of Universidad Federal de Pernambuco (UFPE) in the city of Recife, Brazil. Candida albicans (URM 5901), Candida glabrata (URM 4246), Candida tropicalis (URM 6551) and Candida parapsilosis (URM 6345) isolates were obtained from the culture collection the UFPE Department of Mycology. Stock cultures were maintained at -80 °C in sterile nonfat milk powder with 10% (v/v) glycerol. For the assays, the bacteria were cultured overnight in Mueller-Hinton agar (MHA) at 37 °C and the yeasts were cultured in Sabouraud dextrose agar (SDA) at 28 °C. The colonies were then transferred to sterile 0.9% NaCl to obtain a suspension equivalent to 106 or 108 colony forming units (CFUs) per mL with an optical density at 600 ηm (OD600).
Essential oil and compounds
Fresh inflorescences (approximately 625 g) were powdered in a blender and submitted to hydrodistillation for 3 h in a Clevenger-type apparatus. The essential oil was separated, dried over anhydrous sodium sulfate (99.0% purity, Vetec, Rio de Janeiro, Brazil) and stored in an amber-glass vial at -5 °C. The yield was calculated from the ratio between the mass of oil obtained and the quantity of inflorescence used. Hydrodistillation was carried out as described in the European Pharmacopoeia.27 The antimicrobial and cytotoxic activities of the oil were investigated using stock solutions prepared in 5% dimethyl sulfoxide (DMSO). The compounds dodecanal (92% purity) and 1-dodecanol (≥ 98.0% purity) were purchased from Sigma-Aldrich (Gillingham, United Kingdom) and stock solutions in 5% DMSO (≥ 99.7% purity, Sigma Aldrich, Sao Paulo, Brazil) were also prepared for the bioassays. The entire procedure was repeated three times.
GC (quantitative) and GC-MS (qualitative) analysis of essential oil composition
Gas chromatography-mass spectrometry (GC-MS) and gas chromatography coupled to a flame ionization detector (GC-FID) was used for the analysis of the chemical composition of the oil, as described by Bezerra-Silva et al.24 GC-MS analysis was performed in a 5975C Series GC/MSD quadrupole instrument (Agilent Technologies, Palo Alto, CA, USA) equipped with capillary column (Agilent J&W CS columns; non-polar DB-5, 30 m × 0.25 mm × 0.25 μm). 1 μL of sample was injected in split mode (50:1) with an injector temperature of 250 °C. Oven temperature was initially held at 40 °C (2 min) then increased to 230 °C at 4 °C min-1 and held at 230 °C (5 min). Helium was the carrier gas, with a flow rate of 1 mL min 1. MS operating at 70 eV recorded in the range of 35 350 m/z at 1.0 scans s-1. The identification of compounds was performed by comparisons to the Van den Dool and Kratz retention indices (calculated by co-injection of a standard sample of C9-C30 (alkane standards, Sigma-Aldrich, Milwaukee, Wisconsin, USA)). The mass spectrum of each compound was compared to spectra reported in the literature (MS library: MassFinder 4, NIST08 and Wiley Registry™ 9th edition and Adams28). GC-FID analysis was performed in a Thermo Trace GC Ultra (Milan, Italy) gas chromatograph equipped with a VB-5 column (60 m × 0.25 mm; film thickness: 0.25 μm) under the same conditions as those used for GC-MS analysis.
Antimicrobial assays
Determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC)
The broth microdilution assays were performed in 96 well microplates. 80 µL of sterile distilled water was added to the second to the twelfth well of a row. Next, 80 µL of the sample (essential oil, dodecanal or 1-dodecanol in 5% DMSO) at a concentration of 16,000 µg mL-1 (for bacteria) or 60,000 µg mL-1 (for yeasts) were added to the third well and twofold serial dilutions were performed until the end of the row. In the second well, which corresponded to the 100% growth control, a 5% DMSO solution was diluted at a 1:1 proportion in the previously added distilled water.
The medium (40 µL) (Mueller-Hinton broth (MHB) for bacteria or Sabouraud dextrose broth (SDB) for yeasts) was added to all wells of the row except the first, which was filled with 200 µL of culture medium and served as the sterility control. Lastly, the microbial suspension (80 µL; 106 CFU mL-1) was added to second to twelfth wells. The microplates were incubated for 24 h at 37 °C (bacteria) or 28 °C (yeasts) and the experiments were performed in triplicate. Optical density of a sample measured at a wavelength of 600 nm (OD600) was measured at time zero and after 24 h using a microplate reader. The MIC was defined as the lowest concentration of the sample able to promote a ≥ 50% reduction in optical density in comparison to the 100% growth control. For determination of the MBC and MFC, aliquots (20 µL) from the wells corresponding to concentrations ≥ MIC were inoculated in Petri dishes containing either MHA or SDA. The dishes were then incubated for 24 h at either 37 or 28°C, respectively. The MBC and MFC corresponded to the lowest sample concentration able to reduce the number of CFUs by 99.9% compared to the initial inoculum. Each assay was conducted in triplicate and three independent experiments were performed.
Antibiofilm assay
The effects on biofilm formation were determined as described by Trentin et al.29 in 96-well polystyrene microplates. 40 µL of MHB or SDB, 80 µL of the microbial suspension (108 CFUs mL-1) and 80 µL of the essential oil, dodecanal or 1-dodecanol (30 4000 µg mL-1 for bacteria and 470-60000 µg mL-1 for yeast) were added to each well. The negative control was 80 µL of 5% DMSO. Ampicillin and tetracycline (80 µL at 8 µg mL-1) were used as positive controls for bacteria and fluconazole (80 µL at 64 µg mL-1) was used as the positive control for species of Candida. OD600 was determined at the beginning of the incubation time (24 h at 37 or 28 °C) as well as after this period. Next, the liquid content was removed from each well, which was then washed three times with 0.9% NaCl. The remaining attached cells (biofilm) were fixed with methanol for 30 min, followed by a heat fixation step at 50 °C for 60 min. The adherent biofilm layer was then stained with 0.4% (m/v) crystal violet for 25 min at 25 ºC. After washing with water, the stain bound to the biofilm was solubilized with ethanol (incubation: 15 min) and absorbance was measured at 570 nm. Three independent experiments were performed in triplicate.
Statistical analysis
Data were expressed as mean or mean percent ± standard deviation (SD). Statistically significant differences in antibiofilm assays were determined using Tukey’s test. In all cases, a p-value < 0.05 was considered indicative of statistical significance.
Results and Discussion
The essential oil from the inflorescence of E. elatior was colorless at room temperature and the extraction yield was 0.060% (m/m). The yield was higher than that reported by Jaafar et al.22 which was 0.0334%, but lower than that reported by Bezerra-Silva et al.24 which was 0.094%. In all these studies, the oils were obtained by hydrodistillation as the extraction method. Sixteen components were identified, corresponding to 98.38% of the total oil (Table 1). The major constituents were dodecanal (42.46 ± 0.39%) and 1-dodecanol (25.84 ± 0.73%).
Dodecanal and 1-dodecanol have previously been reported as the major components of essential oils from E. elatior inflorescences. Abdelmageed and co-workers23 identified 20 compounds in the essential oil, the major constituents of which were 1-dodecanol (54.48%) and dodecanal (16.53%). The major components reported by Zoghbi and Andrade30 were also dodecanol (42.5%) and dodecanal (14.5%). Bezerra-Silva et al.24 identified 27 compounds, also with these compounds as the major constituents, but dodecanal (49.37%) was found at higher concentration than 1-dodecanol (31.53%), as occurred in the present study. Conversely to these works, the essential oil from E. elatior flowers described by Jaafar et al.22 contained cyclododecane (47.28%) and 1,1-dodecanediol diacetate (24.38%) as the major components.
Essential oils from seeds, flowers and fruits have various biotechnological and pharmacological applications, including as antimicrobial agents.31-33 In the present work, we tested the hypothesis that the essential oil from the inflorescence of E. elatior would have antimicrobial activity against standard and resistant strains of P. aeruginosa and S. aureus as well as against species of Candida. The activity of the major constituents (dodecanal and 1-dodecanol) was also investigated.
The essential oil, dodecanal and 1-dodecanol were all active against the standard S. aureus isolate, with MBCs of 124, 31 and 62 µg mL-1, respectively. This result suggests that the activity of the oil against this bacterium was due to the presence of these two major compounds. No effect on the oxacillin-resistant isolate was found. For P. aeruginosa, the essential oil exhibited an inhibitory effect on the multi-resistant isolate, with an MIC of 4000 µg mL-1 but had no bactericidal action. The oil was not active against the standard isolate and the major compounds had no effect on either of the P. aeruginosa isolates, indicating that the activity of the oil may be related to other constituents.
Compared to data available in the literature, the present results for S. aureus were better than those described by Wijekoon et al.,25 who studied the bacteriostatic effects of the EO from the inflorescence of E. elatior on strains of B. subtilis, B. cereus, S. aureus, Listeria monocytogenes and Klebsiella pneumoniae, reporting MIC values ranging from 780 for B. subtilis to 1560 μg mL-1 for the other bacteria studied. This difference is related to the chemical composition of the oils; although the concentration of 1-dodecanol (25.17%) was similar to that found in the present investigation (25.84%), the major compounds of the oil tested in the study by Wijekoon et al.25 were not the same, with 1-dodecanal (17.5%) and dodecanoic acid (20.40%) as the other two major compounds. In contrast, dodecanal accounted for 42.46% and dodecanoic acid corresponded to only 2.03% of the EO in the present study. Several differences were also found with regards to the minor compounds in these oils. In the present study, dodecanal had a lower MIC in comparison to the oil and 1-dodecanol, lending support to the hypothesis that this compound was responsible for the lower MIC values. Moreover, differences in susceptibility among the isolates indicate differences in the bacteriostatic power of the essential oils.
The EOs from different parts of the congener Etlingera fimbriobracteata (K. Schum.) R.M.Sm. are reported to have antimicrobial activity against the bacteria Bacillus subtilis, B. spizizenii, S. aureus and E. coli as well as the yeast C. albicans, with respective MIC ranges of 19.5-78, 19.5 156, 39-156, 156-625 and 39-156 μg mL-1 for the oils from the leaves, stems and rhizomes.15 A comparison of the results indicates that the EO from E. elatior and its two major constituents were more efficient than the EOs from the leaves, stems and rhizomes of E. fimbriobracteata. According to Swamy et al.34 the effectiveness of essential oils at inhibiting or killing microorganisms depends on the ability of the constituents to penetrate the cell membrane and other cell structures. When penetrating the cellular barrier of microorganisms, the constituents of an oil can cause the loss of chemiosmotic control.
As an example of harm to the cell membrane, a study of the antibacterial activity against S. aureus conducted by Togashi et al.35 found greater growth inhibition for alcohols with 12 and 13 carbon atoms, whereas those with 11 carbon atoms exhibited better bactericidal activity. 1-Dodecanol was previously found to be an antibacterial agent against S. aureus, with MIC and MBC values of 8 and 16 µg mL 1, respectively, but its activity was not associated with membrane damage.
The literature also offers reports of the antibacterial activity of 1-dodecanol against Streptococcus mutans, with MIC and MBC values of 6.25 and 12.5 µg mL-1, respectively. According to the authors cited,35,36 aliphatic alcohols can affect the permeability of lipid bilayers, affecting the structure of the membrane and its function, where the efficiency of this antimicrobial activity will depend on the size of the carbon chain.
The EO exhibited no fungistatic activity. However, dodecanal had an inhibitory effect on all species of Candida tested (C. albicans, C. tropicalis, C. glabrata and C. parapsilosis), but with a high MIC (60000 µg mL 1), whereas fungicidal activity was only detected for C. albicans (MFC: 60000 µg mL-1). At a concentration of 60000 µg mL-1, 1-dodecanol also exhibited fungistatic activity only against C. albicans, whereas the EO exhibited no fungistatic or fungicidal activity. The difficulty in inhibiting or eliminating yeasts is due to the fact that these organisms are more complex than bacteria, which are prokaryotic. In contrast, fungi are eukaryotic and have a varied cellular morphology, impeding the entrance of external compounds into the interior of the cell and thus, avoiding the emptying of the cell contents.37
No studies were found in the literature that investigated antifungal activity against species of Candida in which dodecanal was the major constituent of the EO. However, one study38 was found on the EO from Coriandrum sativum L., but dodecanal accounted for only 2.36% of the oil, placing it in tenth position among the major constituents of the oil, which exhibited antifungal activity against four species of Candida (C. albicans, C. tropicalis and C. krusei), with respective MFC ranges of 31.25 to 250 µg mL-1, 31.25 to 250 µg mL-1 and 31.25 to 125 µg mL 1. We also found no fungicidal or fungistatic studies on dodecanal alone. However, Saracino et al.39 reported that α-pinene was the third major constituent of the EO from the inflorescence of E. elatior (15.89%) and the authors found fungicidal and fungistatic activity of five isolates (one aldehyde (cinnamaldehyde) and four monoterpenes (α-pinene, limonene, eucalyptol and eugenol)) against strains of C. albicans, C. glabrata and C. lusitaniae. The authors reported 100% fungistatic activity for cinnamaldehyde and eugenol, 88.9% for α-pinene and 33.3% for limonene, whereas eucalyptol had no activity. MIC and MFC ranges for cinnamaldehyde, eugenol and α-pinene were respectively 50050 to 109260 µg mL-1, 455420 to 690090 µg mL-1 and 195410 to 251270 µg mL-1.39 Thus, the concentration ranges tested experimentally in the present study (31 to 4000 µg mL-1 for bacteria and 470 to 60000 µg mL-1 for yeasts) are far lower than the ranges reported in the literature for fungicidal or fungistatic action of the oil and its three major constituents.
In addition, although the results obtained against Candida species and resistant bacteria showed a high concentration ranging from 4000 to 60000 µg mL-1 for the E. elatior EO and its major compounds (1-dodecanol and dodecanal), which were thus considered to have low activity, they can be modified using nanotechnology in order to improve the solubility and bioavailability of these bioactive compounds, allowing them to be more efficient at lower concentrations, thus resulting in a sustainable alternative for the use of natural compounds. E. elatior is therefore a sustainable source for essential oil extraction, especially in a context of growing resistance of fungi and bacteria to synthetic antifungals and antibiotics, and whose extraction of EO raises the importance of the biodiversity of plants that are adapted to the Brazilian tropical climate and are little studied. Such awareness promotes the conservation of local flora and strengthens the economy of local communities, causing positive social and environmental impacts.
The antibiofilm assay was performed with microorganisms/samples for which the MIC was determined. Thus, the EO (MIC: 124 and 4000 µg mL-1, respectively) was tested for S. aureus (non-resistant) and P. aeruginosa (resistant), whereas the isolated compounds dodecanol and dodecanal (MIC: 62 and 31 µg mL-1, respectively) were only tested for S. aureus (non-resistant). The values obtained for the control corresponded to 100% biofilm formation or bacterial/fungal growth. The results indicated that the EO was capable of impairing growth and biofilm formation by the multi-resistant isolate of P. aeruginosa (Figure 1a) and by the standard isolate of S. aureus (Figure 1b). For S. aureus, the oil was more effective on the reduction in the number of adherent cells than the inhibition of bacterial growth at all concentrations tested. Bacteriostatic action was found for P. aeruginosa, with up to 30% inhibition at all concentrations tested (30-4000 µg mL-1) in comparison to 59% for the antibiotic (tetracycline) at a concentration of 8 μg mL-1. The oil exhibited greater efficiency in the reduction of biofilm development (around 40% at 4000 µg mL-1), whereas tetracycline did not inhibit the growth of biofilm (Figure 1a). For S. aureus, the oil significantly reduced bacterial growth and the formation of biofilm. After treatment with the EO in a concentration range of 30 to 4000 µg mL-1, a 19 to 53% reduction was found in bacterial growth as well as a more than 50% reduction in biofilm formation at all concentrations tested in comparison to the control (untreated cells) (Figure 1b). This result indicates that the imparity of biofilm formation, especially at lower concentrations (30-120 µg mL-1) was related to the interference in the adhesion process and not merely the reduction in the number of cells. Moreover, the reduction in the formation of biofilm by P. aeruginosa was accompanied by a reduction in growth (Figure 1a). Exposure to the oil did not affect adhesion, but rather cell replication.
Effect of essential oil from inflorescence of E. elatior on bacterial growth and biofilm formation of resistant strains of P. aeruginosa (a) and standard strains of S. aureus (b). Control for Staphylococcus aureus: ampicillin; control for Pseudomonas aeruginosa: tetracycline.
Figure 2 shows the antibiofilm activity of the major constituent (dodecanal), which corresponded to 42.46% of the oil, for the standard strain of S. aureus (Figure 2a) and all species of Candida used in this study (C. albicans, C. tropicalis, C. glabrata and C. parapsilosis) (Figures 2b to 2e). Dodecanal reduced the bacterial growth of the non-resistant S. aureus (Figure 2a), C. albicans (Figure 2b) and C. tropicalis (Figure 2c) at the highest concentration (4000 µg mL-1) of the compound for the bacterial strain and at 60000 µg mL-1 for the fungal strains. In this assay, growth inhibition of 86, 92 and 50% was found for the respective species. No inhibitory effect was found for C. albicans at the lowest concentrations (930 and 470 µg mL 1). Fluconazole at a concentration of 64 μg mL-1 exhibited a similar capacity to inhibit the growth of C. tropicalis (by approximately 50%), which demonstrates a similar inhibitory effect between dodecanal and the commercial medication most widely used for the treatment of candidiasis (fluconazole).
Effect of dodecanal on bacterial growth of non-resistant strain of S. aureus (a) and biofilm formation and fungal growth of C. albicans (b), C. tropicalis (c), C. glabrata (d) and C. parapsilosis (e). Control for Candida: fluconazole; control for Staphylococcus aureus: ampicillin.
The inhibition of the growth of C. glabrata (Figure 2d) and C. parapsilosis (Figure 2e) was minimal-not exceeding 20% after treatment with dodecanal. Fluconazole at a concentration of 64 μg mL-1 also exhibited little effectiveness against C. parapsilosis but was effective against C. glabrata, with 11 and 44% growth inhibition, respectively. With regards to biofilm formation, dodecanal was tested with S. aureus (Figure 2a) and C. albicans (Figure 2b) at concentrations ranging from 30 to 4000 µg mL-1 and 470 to 60000 µg mL-1, respectively. For these species, the antibiofilm effect ranged from 30 to 53%, with better results achieved at a concentration of 4000 µg mL-1 for the bacterium (43%) and 15000 µg mL-1 for the yeast (53%). Biofilm inhibition by dodecanal (15000 µg mL-1) was similar to that found for fluconazole (64 μg mL-1), which inhibited no more than 70% of the biofilm formed by the species studied.
With regards to C. tropicalis (Figure 2c), dodecanal at concentrations ranging from 47 to 750 µg mL-1 stimulated biofilm growth, whereas no changes were found at concentrations ranging from 1500 to 6000 µg mL-1. For C. glabrata (Figure 2d), a concentration of 6000 µg mL-1 also led to an increase in biofilm formation, as occurred at concentrations of 47 to 375 µg mL-1. For C. glabrata, concentrations of 30150 and 7500 µg mL-1 caused a reduction in biofilm. For C. parapsilosis (Figure 2e), a significant reduction in biofilm formation was found in all concentration ranges tested. The results were generally significant for microbial cells treated with dodecanal compared to those treated with fluconazole (64 μg mL 1), which stimulated biofilm formation. There are no studies on the antibiofilm activity of the essential oil from Etilingera elatior, dodecanal, or essential oils in which dodecanal is a major constituent, only studies on the antibiofilm activity of dodecanol, which makes the present investigation a pioneering study in this regard.
The antibiofilm action of the compound 1-dodecanol was also tested against the microorganisms S. aureus (Figure 3a) and C. albicans (Figure 3b). Growth inhibition was found for S. aureus after treatment with the compound, especially at a concentration of 4000 µg mL-1, with 70% inhibition compared to the untreated cells (control).
Effect of 1-dodecanol on biofilm formation and microbial growth of S. aureus (a) and C. albicans (b) strains. Control for Staphylococcus aureus: ampicillin: Control for Candida: fluconazole.
These results are compatible with findings described by Kubo et al.,36 due to the high availability and relatively simple structure of this compound. After treatment with 1-dodecanol, the growth of C. albicans was also affected, but to a lesser degree, with 30% inhibition when using a concentration of 30000 µg mL-1 (Figure 2b). Fluconazole achieved 80% inhibition of this yeast after a 24-h incubation period.
The formation of biofilm by S. aureus was inhibited after the use of 1-dodecanol at all concentrations tested, reaching approximately 70% inhibition when using a concentration range of 120 to 4000 µg mL-1. This effect was lower than that found with the use of ampicillin (8 μg mL 1). However, 1-dodecanol (30000-60000 µg mL-1) did not have a substantial effect on the formation of biofilm by C. albicans compared to fluconazole, which achieved 70% inhibition.
Conclusions
The essential oil from the inflorescence of Etlingera elatior exhibited antibacterial activity against isolates of Staphylococcus aureus (124 µg mL-1) and low activity for Pseudomonas aeruginosa (4000 µg mL-1). The main constituents-dodecanal and 1-dodecanol-are the active components against Staphylococcus aureus (31 and 62 µg mL-1, respectively). Only the major compounds isolated from the oil showed activity against the Candida species tested at a concentration of 60,000 µg mL-1, although this was still low. The oil is also a promising antibiofilm agent against Staphylococcus aureus and dodecanal was able to inhibit the adhesion of Candida albicans to a surface at the MIC, MBC, MFC concentrations studied. Despite the high MIC, MBC, MFC values, the oil and its main isolated major components have potential antimicrobial, fungicidal and antibiofilm activity which can be further explored with the use of nanotechnology to improve the efficiency of these studied activities.
Acknowledgments
The authors wish to thank the Brazilian fostering agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE); APQ-0443-1.06/15; APQ-0108-2.08/14; APQ 0661-2.08/15) and the Center for Technological Platforms (NPT-1) of the Aggeu Magalhães Institute (IAM) for financial support.
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