1. Introduction
Antibiotics are one of the main defense weapons in our fight against pathogenic microorganisms; however, the abuse of these drugs in both medical and industrial fields [
1,
2] have led to the emergence of multidrug resistant pathogenic bacteria which is one of the most serious public health problems of this century [
3,
4]. Among the bacteria that pose the greatest threat to world public health are both methicillin resistant
Staphylococcus aureus (MRSA) and Shiga toxin-producing
Escherichia coli (STEC), with serotype O157:H7, being the most prevalent with 1.2 annual cases per 100,000 inhabitants since 2010 [
5,
6].
Not only
E. coli O157:H7 but MRSA are also responsible of the majority of nosocomial and community-associated infections [
7,
8], occupying the first and second place among the most common pathogens with 15 and 12% of total cases, respectively [
9]. They are also commonly associated with outbreaks of foodborne diseases, which can cause a wide range of illnesses, such as endocarditis, osteomyelitis, pneumonia, septicemia, skin infections, food poisoning, and hemolytic uremic syndrome, among others [
10,
11,
12].
E. coli O157:H7 and MRSA have also ability to adhere on living or inert surfaces [
13,
14,
15], forming amorphous mono or poly microbial structures called biofilm, which is mostly composed of an extracellular polymeric substance of proteins, polysaccharides, nucleic acids, and water [
13]. Biofilm constitutes a resistance strategy that prevents the permeability of the drug and increases drug tolerance hundreds of times [
16,
17,
18]. Recent investigations point out that at least 65% of all bacterial infectious diseases and 70% of chronic infections in humans could involve biofilms [
19,
20].
For this reason, it is necessary to join efforts in the search and development of new antimicrobial agents in order to inhibit or eradicate the formation of bacterial biofilm. In this aspect, the essential oils (EOs) from aromatic plants constitute a viable alternative due to their different modes of action that affect several bacterial cell targets at the same time, decreasing the possibility of the emergence of resistant microorganisms [
21]. In particular, EOs are composed primarily of aldehydes, phenols and terpene alcohols that are associated with high antimicrobial activity [
22], therefore, they constitute a source of molecules with bactericidal, fungicidal, antiparasitic, and insecticidal activity [
23,
24]. Food and Drug Administration (FDA) generally considers that many individual components of the EO are safe, which has allowed its use in multiple applications in medical, pharmaceutical, food, cosmetic, and health industry [
25,
26].
Thymol and carvacrol are monoterpenes present mainly in the EO of the
Origanum genus and some
Lippia origanoides chemotypes. These metabolites exhibit antibacterial, antifungal, anti-inflammatory, and analgesic activities and therefore, during the last decades, they have been of great interest as candidate molecules for developing pharmaceutical products [
27].
Moreover, EOs from plants that are produced in the Chicamocha river canyon in Colombia have shown a significant variation on their main constituents (chemotypes) [
28], causing variations in its biological activity and potency [
29]. This is because of external factors, such as soil quality and climatic conditions [
30]. Due to the vital importance of identifying relevant and promising alternatives for the treatment of microbial infections, in this work, we evaluated 15 EOs corresponding to 12 species of Colombian and foreign aromatic plants against the pathogenic strains MRSA and
E. coli O157:H7 in both planktonic and sessile states (biofilm).
3. Discussion
In this work it was found that Gram-positive bacteria were more susceptible to EOs than Gram-negative bacteria, this is a characteristic that has been documented in other studies [
37], and this may be due to Gram-negative cell wall does not allow for the entrance of hydrophobic molecules as readily as Gram-positive bacteria. Nazzaro et al. described in their study the effect of EO on pathogenic bacteria, a phenomena that occur at cellular level; thus, EOs are less able to affect the cell growth of the Gram-negative bacteria. Due to the wide variety of molecules present in the natural extracts, the antimicrobial activity of the EOs cannot be attributed to a single mechanism. The effects of EOs usually lead to the destabilization of the phospholipid bilayer, the destruction of the plasma membrane function and composition, the loss of vital intracellular components and the inactivation of enzymatic mechanisms. In some cases, EOs also alter membrane permeability by destroying the electron transport system, and a number of components of the EOs, such as carvone, thymol, and carvacrol, lead to an increase in the intracellular concentration of ATP, an event that is linked to the destruction of the microbial membrane. When antimicrobial compounds are present in the environment surrounding microorganisms, the bacteria may react by altering the synthesis of fatty acids and membrane proteins to modify the fluidity of the membrane. The hydrophobicity of the EOs and their components allow them to diffuse through the double lipid layer of the membrane. The EOs can alter both the permeability and function of membrane proteins. The alteration of membrane permeability and the defects in the transport of molecules and ions result in a “disbalance” within the microbial cell. This subsequently leads to cytoplasm coagulation, the denaturation of several enzymes and of cellular proteins and the loss of metabolites and ions [
21].
Only two EOs obtained from LOT and LOC had a high antibacterial activity against both bacteria (
Table 2), one of the common characteristics of these EOs was their high content of thymol and carvacrol (
Table 1), which have been studied extensively because of their antifungal and antibacterial properties [
38]. It has been determined that the antibacterial activity of thymol is greater against
E. coli,
S. aureus,
Pseudomonas aeruginosa, and
Salmonella enterica in comparison to those of carvacrol,
trans-cinnamic acid, and eugenol [
39,
40]. These results obtained in our studies are consistent with those published by other authors, because EOs with highest antibacterial activity (
Table 2) are oils richer in thymol content (LOC: 32.7% and LOT: 22.1%).
Nevertheless, it was also observed that EO from TV, despite having a high content of thymol (23%), did not have the same antibacterial activity against both bacteria, being ineffective against MRSA (MIC
50 > 3). In this regard, other authors suggest that the effect of a single compound may not be as effective in inhibiting the growth and formation of biofilm, as may be two or more compounds, posing a potential synergistic effect between the components of the same oil [
41], as it is the case of carvacrol and thymol present in LOC and LOT which can enhance its activity against MRSA [
24].
EOs from
Cymbopogon spp showed antimicrobial activity against
E. coli (CM, MIC
50 = 1.4 mg/mL) and MRSA (CF, MIC
50 = 2.4 mg/mL), which may be related to the high content of geranial (33%) in CF and geraniol (38.7%) in CM; these compounds have been studied by other authors due to their many biological activities useful as antibacterial, antifungal, pesticides, insecticides and anticancer [
42,
43,
44,
45,
46,
47]. It has also been shown that geranial is the compound with the highest antimicrobial activity in this plant genus, while other components such as geraniol and geranyl acetate play a secondary role [
48]. However, in this work we observed a low antibacterial activity of the CF (geranial, 33%) against
E. coli, while the activity of CM (geraniol 38.7%) was higher, which suggests that these compounds may have different action mechanisms that may be related to the bacterial cell wall conformation. Finally, EO from RO presented antibacterial activity against MRSA (MIC
50 = 2.5 mg/mL), which has been previously documented by other authors, and it has even been shown that EO from RO has a specific inhibitory activity against
S. aureus [
49]. This may be due to the presence of α-Pinene (12.7%) which has shown to present inhibitory activity against MRSA in previous experiments [
50].
In this work it was also possible to determine the great antibiofilm potential activity of the EOs from LOT, LOC and TV, which can be attributed to high content of aromatic monoterpenes (thymol and carvacrol) present in these EOs. These oils showed a significant bactericidal activity when analyzed separately [
40,
51,
52]. Due to the hydrophobic nature of carvacrol and thymol; these compounds can interact with the lipid bilayer of the cytoplasmic membranes, causing the loss of integrity and the leakage of cellular material such as ions, ATP and DNA [
53,
54].
Another attribute of carvacrol and thymol is their ability to diffuse through the polysaccharide matrix of the biofilm and destabilize it thanks to its strong intrinsic antimicrobial properties [
55]. Knowles et al. (2005) suggests that the continuous exposure of
S. aureus to non-biocidal concentrations of carvacrol interrupts the normal development of the biofilm, preventing the accumulation of protein mass and stopping micro-colony stage [
56]. Alternatively, these compounds could interact with the protein surfaces, leading to an alteration of the bacterial cell surface and compromising the initial binding phase to the surfaces [
57].
Other Eos, such as EO from CM, showed an interesting antibiofilm activity, attributable to the presence of eugenol, which can interfere with bacterial mobility, adhesion, and biofilm formation in
E. coli [
58,
59]. Finally, the EO from CO showed a good antibiofilm activity, despite this EO did not show an apparent antibacterial activity (MIC
50 > 3 mg/mL and MBC > 3 mg/mL). These results are similar to those obtained by Lee et al., which indicates that EO from CO negatively regulates the expression of the HLA gene of α-hemolysin in
S. aureus [
60], which is necessary for the formation of
S. aureus biofilm [
61].
Despite the high antimicrobial and antibiofilm activities, the EOs at high concentrations presented cytotoxicity. Cytotoxicity of EOs from LOT, LOC, and TV may be due to their high content of thymol. It is necessary to highlight that thymol has a limited use in drugs due to its moderate cytotoxicity, which it has been demonstrated both in vitro in human and animal cells, and in vivo in animal studies [
38,
62,
63,
64]. On the contrary, EO from CF presented a cytotoxic activity very similar to that other essential oils, which may be because of its high content on citral (neral and geranial) which has been demonstrated in in vitro cytotoxic tests with Vero and other cell lines, such as HeLa and MCF-7 [
65,
66]. Similar results have also been reported in other species of the
Cymbopogon genus, such as
C. citratus and
C. nardus, which is attributed to the high content of geranial and neral [
66].
Finally, the biofilm of SARM treated with the EO showed a significant decrease in the formation of biofilm, this effect may be due to the activity of thymol on the lipid bilayer of the bacterial cell membrane, which can cause disturbances and permeabilization of bacterial cell membrane, with the consequent loss of cellular content, irregular morphology and decrease in size of the bacteria [
40,
67,
68,
69]. On the other hand, it is also worth noting a low formation of biofilm in
E. coli on the glass coupon, which is consistent with the results obtained by Adetunji et al. (2012), where it is concluded that
E. coli O157:H7 is not a bacterium with great capacity to form biofilm in comparison with other bacteria, such as
Salmonella sp. [
70]. It should be mentioned that in biofilm formation, hydrophobicity of the bacteria and the surface are important aspects [
71]. In our study, a significantly lower biofilm formation was observed in glass, which may be due to its high hydrophobicity, which makes difficult the adhesion and subsequent formation of the biofilm [
70].
4. Materials and Methods
4.1. Materials
Bacterial culture media: Brain Heart Infusion Agar (BHI), Tripticase Soy Broth (TSB) and D-glucose ≥ 99.5% were purchased from Sigma-Aldrich (Milwaukee, WI, USA). For the cell viability assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole (MTT) bromide was used in Hank’s Balanced Salt Solution (HBSS).
4.2. Plant Material and Extraction
Aromatic plants used in this work (
Table 1) were obtained in different areas of the State of Santander (Colombia) and cultivated in experimental plots located in the Pilot Agroindustrial Complex (CENIVAM, N 07° 08,442, WO 73° 06,960 977 above mean sea level (amsl)) at the Universidad Industrial de Santander (UIS, Bucaramanga, Santander, Colombia). Taxonomic characterization of the plants was carried out in the Institute of Natural Sciences of the Universidad Nacional de Colombia (UNAL, Bogotá, Colombia).
Extraction of the essential oils was carried out by hydro-distillation in a Clevenger type equipment adapted to a microwave heating system (Samsung, MS-1242zk), with an output power of 1600 W and a radiation frequency of 2.4 GHz. The plant material (200 g of each plant) was suspended in water (300 mL) in a 2 L balloon, which was connected to a Clevenger-type glass device, with a Dean-Stark distillation reservoir. The plant sample was heated by microwave irradiation in three consecutive series of 15 min (45 min total). Extracted essential oil was dried with anhydrous sodium sulfate, weighed, and stored in an amber colored flask at 4 °C. All extractions were done by triplicate (
n = 3) [
28].
4.3. EOs Analysis by Gas Chromatography-Mass Spectrometry (GC/MS)
The EOs were analyzed by gas chromatography (Agilent Technologies 6890N Series Network System, Palo Alto, CA, USA), coupled to mass spectrometry (Agilent Technologies MSD 5975 Inert XL). A fused-silica capillary column DB-5MS (60 m × 0.25 mm id × 0.25 μm df), with a stationary phase of 5% -phenyl-poly (dimethylsiloxane), and a polar capillary column DB-WAX (60 mm × 0.25 mm id × 0.25 μm df), with stationary phase of polyethylene glycol were used. GC oven temperatures were programmed differently for each column as follows: 45 (5 min) to 150 °C (2 min) at 4 °C/min, then at 250 °C (5 min) at 51 °C/min, and finally, at 275 °C (15 min) at 10 °C/min, for the DB-5MS column and 45 (5 min) up to 150 °C (3 min) at 3 °C/min, then at 220 °C (5 min) at 4 °C/min for the DB-WAX column. The temperatures of the transfer line, the ionization chamber, and the injection port were set at 285 °C, 230 °C, and 250 °C, respectively. Helium was used as a carrier of gas (99.995% AP gas, Linde, Bucaramanga, Colombia), with a constant volumetric flow of 1 mL/min.
Mass spectra were obtained by electron ionization (EI) with electron energy of 70 eV. Mass spectra, total ionic currents (TIC) and extracted ion (EIC) were obtained with a quadrupole analyzer, by means of automatic radiofrequency scanning (full scan) in the mass range of
m/z 40–350 (5, 5 spectra/s). Components of the essential oils were identified by comparison of their mass spectra, obtained by GC/MS, and linear retention indexes (LRI) in the two columns; polar and apolar ones, calculated using the homologous series of
n-alkanes C10-C25 (Sigma-Aldrich, Milwaukee, WI, USA) and compared to the mass spectra from the MS-ChemStation G1701-DA data system, which include the WILEY, NIST 2014, and QUADLIB 2007 spectral libraries. In order to assess identification of components in essential oils and, the following standard substances were used: geranyl acetate (98%), aromadendrene (97%), benzyl benzoate, β-caryophyllene (98.5%), 1,8-cineol (99%),
p-cymene (99%), α-copaene (90%),
trans-farnesol (96%), geraniol (98%), hexanal (98%), α-humulene (96%), linalool (97%), (R)-(+)-limonene (97%), menthol (99%), (+)-menthone (98.5%),
trans-nerolidol (85%), caryophyllene oxide (95%), α-pinene (98%). All chemical standards were acquired from Sigma-Aldrich (Milwaukee, WI, USA) [
28].
4.4. Bacterial Strains
E. coli O157:H7 ATCC and MRSA strains were acquired from Strain Collection of Pontificia Universidad Javeriana, Colombia and School of Bacteriology and Clinical Laboratory of the Universidad Industrial de Santander (UIS), respectively. Cell culture maintenance of these strains was carried out in BHI medium at 37 °C.
4.5. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
Evaluation of MIC and MBC of the pathogenic bacteria
E. coli O157:H7 and MRSA, was carried out following the broth microdilution protocol standardized in the GIBIM [
34], which is based on the protocol of the Clinical and Laboratory Standards Institute (CLSI) document M100 S20 [
72].
Values of MIC
50 and MBC were defined as the minimum concentration of EO being able to inhibit 50% and totally inhibits bacterial growth, respectively [
73]. Additionally, it was determined if the activity of EOs was bacteriostatic; it was considered bacteriostatic if the value of the MBC was four times higher than the value of the MIC (MBC/MIC > 4) [
74].
For MIC determinations, a pre-inoculum was prepared from fresh culture of tested strains using TSB and TSB supplemented with 0.25% (
w/
v) glucose for
E. coli O157:H7 and MRSA, respectively. The pre-inoculums were incubated during 12 h at 37 °C and 200 rpm, until reaching a bacterial concentration equivalent to 4.4–5 × 10
9 CFU/mL, using the Mcfarland scale as a reference [
75]. Inoculum was prepared from the pre-inoculum, taking it to a final volume of 10 mL with sterile medium, until obtaining an absorbance between 0.07 and 0.1 (equivalent to ~5 × 10
5 CFU) [
35].
Subsequently, bacterial growth kinetics was monitored in 96-well ELISA microplates (Bio-Rad, Imarck), where 100 μL of the bacterial inoculum was plated along with 100 μL of each EO in serial concentrations from 0.18 to 3 mg/mL for both strains. These microplates were incubated at 37 °C with constant stirring (200 rpm), and microbial growth was evaluated measuring the absorbance every hour up to complete 24 h using an ELISA microplate reader at 595 nm. Wells containing bacterial cultures without EO were used as negative controls.
MBC was determined once cell growth kinetics of the pathogenic strains were evaluated. Aliquots of 100 μL from each well containing different EO concentrations were taken and plated in 2 mL Eppendorf tubes containing 900 μL of BHI; subsequently, these tubes were incubated at 37 °C for 24 h. To corroborate the bactericidal effect, an aliquot of 10 μL was taken from each tube and was transferred to BHI agar plates at 37 °C for 24 h.
4.6. Inhibition of Biofilm Formation
Potential biofilm inhibition of EOs was evaluated in a 96-well U-bottom microplate until reaching a final volume of 200 μL. This was performed by adding 100 μL of each EO at different concentrations and 100 μL of bacterial culture (~5 × 10
5 CFU/mL) in every well. Wells containing bacterial cultures without EO were used as negative controls, and the microplates were incubated at 37 °C for 48 h without agitation, allowing the adherence of bacteria to the surface [
57].
After incubation, liquid content from the wells was removed, and the microplate was rinsed three times with sterile saline solution 0.9% in order to eliminate planktonic bacteria; then, microplates were dried in an oven at 60 °C for 45 min. Subsequently, each well was stained with 200 μL of crystal violet 0.4% and incubated at a room temperature for 15 min. Thereafter, microplates were rinsed three times with sterile saline solution 0.9% to remove excess of crystal violet and 200 μL of acetic acid 30% in ultra-pure water [
76]. The liquid content of each well was transferred to a new flat bottom microplate and the absorbance was measured at 595 nm using an ELISA microplate reader (Bio-Rad, lmarck version 1.02.01, Hercules, CA, USA). Each assay was performed by triplicate (
n = 3). Minimum biofilm inhibition concentration (MBIC
50) was defined as the minimum concentration of EO that can inhibit biofilm formation by 50%. Finally, inhibition percentages of each EO were calculated using the following formula [
77,
78]:
4.7. EO Cytotoxicity Assay in VERO Cell Line
EOs with the high antibacterial and antibiofilm activities were tested to determine their cytotoxicity in non-tumor Vero cell line (African green monkey kidney, ATCC No. CCL-81) following the MTT methodology as was described by Mosman [
79]. Vero cell line was maintained in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum at 37 °C in 5% CO
2. Then, 1 × 10
4 cells/mL were seeded in a polystyrene microplate 96-well flat bottom and incubated for 24 h. These cells were treated with serial dilutions of EOs. After 48 h of incubation, the supernatant was discarded, and 200 μL of MTT (500 μg/mL in HBSS) was added to each well and incubated for 3 h, and then, the supernatant was discarded and 200 μL of di-methyl-sulfoxide (DMSO) was added to solubilize the formazan crystals inside the cells. Absorbance at 550 nm of each well was measured in a microplate reader (Multiskan™ GO microplate spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). The wells with DMSO without cells were used as blank. Cytotoxic Concentration 50% (CC
50) was defined as the concentration of the compound that reduces the viability of the cell line by 50% [
80,
81]. Finally, the Selectivity Index (SI) was calculated as the division of IC
50 over MIC
50 [
66]. All experiments were performed by triplicate (
n = 3). The results are presented as the mean ± standard deviation. Cell viability of Vero cells cultured without EOs was considered as 100%.
4.8. Visualization of the Morphological Alterations by Scanning Electron Microscopy (SEM)
Observations of the possible morphological changes of both bacteria was done by SEM, following the protocol of Singh et al. with some modifications [
82]. Frosted glass coupons (10 × 15 × 2 mm) were used in order to facilitate the biofilm formation, which were deposited in a flat-bottomed 12 well microplate. Subsequently, 1500 μL of the bacterial inoculum in 1/100 dilution was added and incubated at 37 °C for 48 h, in presence and absence of the EOs [
83]. After 48 h, these glass coupons were rinsed three times with sterile saline solution 0.9% to eliminate the planktonic cell. Then, they were fixed with 2.5% glutaraldehyde for 60 min and dehydrated with an isopropanol gradient (10–95%) for 10 min [
84]. Coupons were coated with gold and observed by SEM using a Quanta 650 FEG scanning electron microscope (SEM, Thermo Fisher Scientific, Waltham, MA, USA), which was equipped with an Everhart Thornley ETD detector. SEM images were taken under the following conditions: High vacuum, acceleration voltage 15 kV, and magnification between 600× and 25,000×.
4.9. Data Analysis
All the experiments were performed in triplicate and one-way analysis of variance (ANOVA) was used to analyze the differences among the treatments. In all cases, the level of significance was 0.05. Assumption of normality and equally of variances of data was previously tested using Shapiro–Wilk and Levene’s test, respectively.
