Investigating the Antimicrobial Activity of Essential Oils Against Pathogens Isolated from Sewage Sludge of Southern Lebanese Villages

Abstract

Introduction: Due to the high load of pathogens in sewage, seeking for effective treatments became a priority. In this regard, testing the sensitivity of microorganisms isolated from sewage against essential oils (EOs) is suggested. In Lebanon, little evidence supports bacteria isolated from sewage reveals a sensitivity to EOs. Due to this fact, the present investigation aims at determining the sensitivity of microbes isolated from sewage sludge to three EOs: lettuce, coconut, and almond. Methods: Bacterial isolates were identified by VITEK screening. Yeast was identified by germ tube assay. The chemical components of the oils were identified by gas chromatography—mass spectrometry (GC-MS). Susceptibility of the microbial isolates was assessed by the agar well diffusion assay. Bacteriostatic and bactericidal effects of EOs were detected by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) broth microdilution assay. The activity of EOs on biofilms was detected by antibiofilm screening. Results: The identified microorganisms include Gram-negative isolates (Escherichia coli, Citrobacter freundii, Citrobacter braakii, Leclercia adecarboxylata, and Stenotrophomonas maltophilia), Gram-positive isolates (Enterococcus faecium, Streptococcus intermedius, Staphylococcus aureus, Staphylococcus capitis, and Staphylococcus haemolyticus), and Candida albicans. Thirty oils’ chemical components were identified. Among the antibiotics, doxycycline exhibited the best inhibitory effect. The three EOs were effective against bacterial isolates and yeast at concentrations ranging between 3.125% and 50%. They exhibited a bacteriostatic activity. Lettuce and coconut oils were effective against biofilm formation and the three oils were effective on pre-formed biofilms. Conclusions: The results reflected the significant antimicrobial and antibiofilm activities of the oils, thus suggesting their potential antimicrobial applications.

Introduction

Sewage production is increasing worldwide and the need for alternative disposal approaches emerges [1]. This method is applied in the agricultural field in many countries and used as fertilizer and soil fixative, after recycling the valuable components. The latter are considered essential for soil fertility by enhancing soil microbial and enzymatic activities [1].

The content of sewage sludge is variable [1]. This variability depends on the origin of wastewater, the methods of assessment and treatment, and the biological treatment medium [1]. In general, sewage contains water (99.9%), and traces of organic and inorganic matter (0.1%). However, sewage carries excessive quantities of toxic chemicals, heavy metals and various microorganisms. The latter can be transmitted to the environment and raise serious potential health problems [1,2].

The two main constraints in sludge reuse are pathogenic microorganisms and heavy metals. Aiming to safely use sludge on land, the concentrations of heavy metals and pathogenic microorganisms should not exceed the allowable limits [1,3]. In the present investigation, the main concern is the presence of pathogenic microorganisms in wastewater, particularly bacteria.

Sludge microorganisms include viruses, bacteria, parasites, and fungi [3]. Among these, bacteria and more particularly antibiotic-resistant ones are the most frequently detected [2]. This is due to their ability to re-multiply in treated sludge, replicate, and maintain their virulence [1,2]. The most common bacteria include P. aeruginosa, E. coli, C. jejuni, S. typhi, S. aureus, fecal coliforms, S. dysenteriae, S. flexneri, Y. enterocolitica, V. cholerae, C. perfringens, and L. monocytogenes. Recently, contamination of sewage in Lebanon has been due to high load of fecal coliforms, Salmonella, Shigella, Klebsiella, Pseudomonas, Staphylococcus, and Enterococcus [1,3].

Microorganisms present in sewage exhibit increased resistance to synthetic drugs and antibiotics [4,5]. Thus novel antimicrobial agents from natural sources are needed. Essential oils (Eos), among many other natural compounds, are evaluated for their antimicrobial activity against microorganisms in sewage. EOs are concentrated complex natural products present in all plant organs, and have wide biological activities.

The chemical composition of EOs is affected by the geographical location of the plant, the environment, the stage of maturity, and the extraction techniques [4,5]. The effectivity of EOs depends on the type of pathogens and the methods of extraction [4].

EOs are also characterized by a prominent antibacterial activity. Their hydrophobic nature helps cross the bacterial cell membrane leading to bacterial death [4]. Gram-positive bacteria are more resistant to EOs because of the abundance of peptidoglycans linked to teichoic acid [4,6]. In addition, the activity of EOs is based on different biomedical reactions in the microbial cells [4]. Many mechanisms are identified in this context including cell wall degradation, cytoplasmic membrane damage, and many others [4,5,6].

In this regard, the present study suggests the use of EOs as potential antimicrobial agents against pathogenic microorganisms present in the sewage in Lebanon.

Methods

Collection of wastewater samples

Wastewater samples were collected in Falcon tubes from a station related to South Lebanon Water Establishment (SLWE). The collection process was performed during the winter season in December 2020.

Identification of microorganisms

Isolation of bacteria and yeast

Bacteria were isolated from wastewater by spreading 100 µL of the samples on different selective media. Plates were incubated at 37 °C for 24 hours. After isolation, bacteria were identified by VITEK assay. Yeast was isolated on Sabouraud dextrose agar (SDA), then identified by germ tube assay.

VITEK assay: A bacterial suspension was prepared in sodium chloride (NaCl), adjusted to 0.5 McFarland, and inserted into VITEK cards for biochemical tests. The levels of identification were classified as excellent (96%-99%), very good (93%-95%), good (89%-92%), and acceptable (85%-88%).

Germ tube assay: Yeast was identified by germ tube assay. Human serum was added to a test tube and a colony of yeast was emulsified in it. The tube was then incubated for 2-4 hours at 37 °C. A drop of the serum was then placed on a slide and observed under the microscope.

Antibiogram assay

Antibiotic susceptibility testing was performed by agar well diffusion assay performed with four antibiotics: levofloxacin, amoxicillin, tetracycline, and doxycycline, against the ten isolates. A final concentration of 250 µg/mL of each antibiotic was applied. A total of 100 µL of 0.5 McFarland bacterial suspension was spread on tryptone soy agar (TSA) over the entire plate by a loop and kept 10 min to dry. Plates were then punched with a cork-borer (6 mm) and 100 µL of each antibiotic was added into the wells. The plates were then incubated at 37 °C for 24 hours. The antimicrobial effect was determined by measuring the diameters of inhibition [7]. The data obtained by the measurement of zone diameters were converted to susceptibility categories (sensitive, intermediate, and resistant). The diameters were compared to the standards of the Clinical and Laboratory Standards Institute (CLSI) [8]. The assays were repeated three times.

Essential oils collection and sample preparation

Three EOs, lettuce (Lactuca sativa), coconut (Cocos nucifera), and almond (Prunus dulcis), were purchased from the “Soap and Natural Oils House” shop. They were imported from Pakistan. The oils were half-fold diluted (v/v) with 5% methanol at room temperature to obtain concentrations ranging between 3.125% and 50% [9].

Gas chromatography—mass spectrometry (GC-MS): GC-MS analysis of EOs was performed using an Agilent 7890A GC coupled with a Headspace Sampler Agilent 7697A and an HP5-MS column (30 m x 250 μm x 0.25 μm, Agilent 19091S-433). Helium gas was used as the carrier gas at a constant flow rate of 3 mL/min and an injection volume of 1 μL was employed (split ratio of 10:1) using automatic liquid sampler (ALS) syringe. The column was initially held at 90 °C for 1min, then increased to 205 °C at an increase rate of 8 °C/min for 1 min, then increased to 240 °C at 5 °C/min for 1 min, and finally increased to 300 °C at8 °C/min and maintained for 30 min. The total running time of the GC-MS system was 61.875 min. Peak identification of EOs were performed by comparison with retention times of standards and the mass spectra obtained were compared with those available in Adams Library of GC-MS [10].

Agar well diffusion assay

Agar well diffusion assay was performed in triplicates for the three EOs on ten bacterial isolates using TSA and one yeast using SDA. A standard inoculum of each microbial isolate was prepared as stated in the antibiogram assay. The plates were inoculated with 100 µL of each microbial suspension and spread evenly on the agar plate. Plates were then punched with a 6 mm cork-borer. A total of 100μL of each EO at different concentrations ranging between 3.125% and 50% were pipetted into the wells and the plates were incubated at 37 °C for 24 hours. For each well, the diameter of the zone of inhibition was measured. An obtained zone of inhibition higher than 7 mm was considered of a good activity [7].

MIC and MBC broth microdilution assay

The EOs’ MICs were detected against all microorganisms using the microwell dilution method. The test was done in sterile 96-well microplates by introducing into each well 90 µL of tryptone soy broth (TSB) and 10 µL of microbial suspensions adjusted to 0.5 McFarland. Then 100 µL of each EO of concentrations ranging between 3.125% and 50% were added to the wells. The plates were incubated at 37 °C for 24 hours, and the optical density (OD) was measured at 595 nm, using an ELISA microtiter plate reader. MIC was defined as the lowest concentration of the EOs that inhibited visible growth of the tested microorganisms. A total of 10 µL of the clear wells of bacteria and yeast were transferred to TSA and SDA plates respectively, and incubated at 37 °C for 24 hours to detect the MBCs/MFCs [11]. All experiments were performed three times.

Antibiofilm screening

Inhibition of biofilm formation: The ability of the EOs to prevent biofilm formation was investigated through the biofilm inhibition assay. A total of 100 µL standard concentration of cultures of the different microbial isolates were added into 96-well microtiter plates and incubated at 37 °C for 4 hours. Then 100 µL of EOs of different concentrations ranging between 3.125% and 50% were added into the wells. Culture medium without any inoculum was used as negative control. The plates were then incubated at 37 °C for 24 hours. Crystal violet (CV) staining was used to quantify the biomass. After incubation, the plates were washed five times with sterile distilled water, air-dried, then oven-dried at 60 °C for 45 min. The wells were then stained with 100 µL of 1% CV and incubated at room temperature for 15 min. Then, the plates were washed with sterile distilled water five times to remove the stain that was unabsorbed. Biofilms were observed as purple rings at the sides of the wells. A total of 100 µL of 95% ethanol was then added to de-stain the wells. The absorbance was measured at 595 nm using an ELISA microplate reader [12]. All experiments were repeated three times.

The percentage of inhibition of biofilm formation was determined using the following equation:

$$\% \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}={\displaystyle \frac{OD negativecontrol-OD experimental}{oD negative control}}\times 100$$

Percentage of inhibition above 10% reflected a good prevention of biofilm formation. Negative values reflected the enhancement of the biofilms’ formation.

Destruction of pre-formed biofilms: The ability of the EOs to destroy pre-formed biofilms was also investigated. A total of 100 µL of the standard concentrations of cultures of the different microbial isolates were added into 96-well microtiter plates and incubated at 37 °C for 30 hours to form biofilms. Following the incubation period, 100 µL of EOs of different concentrations ranging between 3.125% and 50% were added into the wells of 96-well microtiter plates and further incubated at 37 °C for 24 hours. Culture medium without any inoculum was used as negative control. CV staining assay was used to assess the biofilm biomass and the percentage of inhibition was determined as mentioned in the inhibition of biofilm formation [12]. All experiments were repeated three times.

Statistical analysis

Statistical significance was determined by t-test using Excel software (2016) from Microsoft. Differences with p˂0.05 were considered statistically significant.

Results

Identification of bacterial isolates and yeast

Ten bacterial isolates and one yeast were obtained. VITEK analysis data was obtained for the ten bacterial isolates. Gram-positive bacteria included Streptococcus intermedius (94%), Enterococcus faecium (86%), Staphylococcus aureus (99%), Staphylococcus capitis (99%), and Staphylococcus haemolyticus (99%). Gram-negative bacteria included Escherichia coli (99%), Citrobacter freundii (97%), Citrobacter braakii (95%), Stenotrophomonas maltophilia (99%), and Leclercia adecarboxylata (88%). Germ tube analysis showed that the yeast was Candida albicans (95%).

Antibiotic susceptibility of the bacterial isolates

The results revealed that S. intermedius, E. faecium, S. haemolyticus, E. coli, C. braakii, S. maltophilia, and L. adecarboxylata were sensitive to levofloxacin, while S. aureus, S. capitis, and C. freundii showed an intermediate susceptibility. None of the isolates was resistant to levofloxacin. In addition, E. coli and L. adecarboxylata were sensitive to amoxicillin, and all of the other isolates were resistant. Furthermore, E. faecium, S. capitis, E. coli, S. maltophilia and L. adecarboxylata were sensitive to tetracycline, S. intermedius, S. haemolyticus and C. braakii showed an intermediate susceptibility, and S. aureus and C. freundii were resistant. On the other hand, E. faecium, S. aureus, S. capitis, S. haemolyticus, E. coli, C. freundii, C. braakii, S. intermedius, and L. adecarboxylata were sensitive to doxycycline and S. intermedius showed an intermediate susceptibility. None of the isolates was resistant to doxycycline. The diameters of the inhibition zones are shown in

Table 1. Antibiotic susceptibility of the bacterial isolates. 

Bacterial isolates	Levofloxacin	Amoxicillin	Tetracycline	Doxycycline
Gram-positive bacteria
S. intermedius	25.6 ± 0.3 (S)	0 (R)	20.3 ± 0.3 (I)	21.6 ± 0.3 (I)
E. faecium	32.3 ± 0.3 (S)	0 (R)	20.6 ± 0.6 (S)	29.6 ± 0.6 (S)
S. aureus	16.6 ± 0.3 (I)	0 (R)	0 (R)	20 (S)
S. capitis	15.3 ± 0.3 (I)	0 (R)	25.6 ± 0.3 (S)	27.6 ± 0.3 (S)
S. haemolyticus	27.6 ± 0.3 (S)	0 (R)	14.3 ± 0.3 (I)	21 ± 0.5 (S)
Gram-negative bacteria
E. coli	37.6 ± 0.3 (S)	19.6 ± 0.6 (S)	26.6 ± 0.3 (S)	20.3 ± 0.3 (S)
C. freundii	17.3 ± 0.3 (I)	0 (R)	0 (R)	20.3 ± 0.3 (S)
C. braakii	34.6 ± 0.3 (S)	0 (R)	14 (I)	24.3 ± 0.3 (S)
S. maltophilia	32.6 ± 0.3 (S)	0 (R)	31.6 ± 0.6 (S)	41.3 ± 0.6 (S)
L. adecarboxylata	30.3 ± 0.3 (S)	20.3 ± 0.3 (S)	23.3 ± 0.6 (S)	21 (S)
Total S	7	2	5	9
% Sensitive	70	20	50	90
Total I	3	0	3	1
% Intermediate	30	0	30	10
Total R	0	8	2	0
% Resistant	0	80	20	0

Zone of inhibition ± standard error of the mean. S—sensitive; I—intermediate; R—resistant.

and Figure 1.

Oil susceptibility of the bacterial and fungal isolates

Among the ten bacterial isolates, S. aureus, S. capitis, E. coli, C. freundii, C. braakii, S. maltophilia, and L. adecarboxylata showed sensitivity to lettuce oil. S. aureus, E. coli, and L. adecarboxylata were the most sensitive and S. capitis was the most resistant (Figure 2). As for coconut oil, S. intermedius, S. aureus, S. capitis, S. haemolyticus, E. coli, S. maltophilia and L. adecarboxylata, showed sensitivity. Among these, S. intermedius and S. aureus were the most sensitive and S. capitis showed the least sensitivity (Figure 3). Furthermore, S. intermedius, S. aureus, S. capitis, S. haemolyticus, E. coli, S. maltophilia, and L. adecarboxylata, showed sensitivity to almond oil. S. intermedius and S. aureus were the most susceptible and S. capitis was the least sensitive (Figure 4). C. albicans showed sensitivity to lettuce oil at a concentration of 50%, to coconut oil at a concentration of 6.25%, and to almond oil at a concentration of 25% (Figure 5). The agar well diffusion results are shown in

Table 2. Agar well diffusion of different concentrations of EOs against the bacterial isolates. 

Bacterial/fungal isolates	Concentrations of oils (%) and antimicrobial diameters (mm) of inhibition
	3.125%	6.25%	12.5%	25%	50%
Lettuce oil
S. aureus	8.1±0.48 (p=0.050)	8.2±0.24 (p=0.050)	8.2±0.24 (p=0.050)	8.3±0.24 (p=0.003)	8.3±0.2 (p=0.002)
S. capitis	0±0.0 (p=0.002)	0±0.0 (p=0.002)	6.6±0.3 (p=0.002)	7±0.0 (p=0.03)	8.6±0.3 (p=0.002)
E. coli	8±0.0 (p=0.006)	8±0.0 (p=0.037)	8±0.0 (p=0.03)	8.2±0.3 (p=0.002)	8±0.0 (p=0.050)
C. freundii	0±0.0 (p=0.015)	6.75±0.25 (p˂0.001)	7.8±0.24 (p˂0.001)	7.8±0.24 (p=0.015)	8±0.0 (p=0.015)
C. braakii	0±0.0 (p=0.015)	6.85±0.3 (p˂0.001)	6.75±0.25 (p˂0.001)	8±0.0 (p˂0.001)	8±0.0 (p=0.015)
S. maltophilia	0±0.0 (p=0.002)	6.26±0.25 (p=0.009)	9.5±0.5 (p=0.013)	9.5±0.5 (p=0.005)	9.75±0.25 (p=0.002)
L. adecarboxylata	7.6±0.24 (p=0.005)	7.4±0.24 (p=0.005)	7.6±0.24 (p=0.004)	8±0.0 (p=0.005)	8±0.0 (p=0.001)
C. albicans	0±0.0 (p˂0.001)	0±0.0 (p˂0.001)	6.75±0.25 (p˂0.001)	6.7±0.25 (p˂0.001)	8±0.0 (p=0.015)
Coconut oil
S. intermedius	8±0.0 (p=0.002)	7.75±0.25 (p=0.003)	8±0.0 (p=0.002)	8.2±0.25 (p=0.002)	8±0.0 (p=0.050)
S. aureus	8±0.0 (p=0.002)	8±0.0 (p=0.002)	8±0.0 (p=0.002)	8±0.0 (p=0.003)	8±0.0 (p=0.050)
S. capitis	0±0.0 (p=0.002)	6±0.0 (p=0.037)	6±0.0 (p=0.037)	7.2±0.25 (p=0.057)	7.3±0.3 (p=0.002)
S. haemolyticus	6±0.0 (p=0.015)	6.6±0.3 (p=0.002)	11±0.0 (p˂0.001)	10±0.0 (p=0.001)	11±0.0 (p˂0.001)
E. coli	0±0.0 (p=0.050)	0±0.0 (p=0.050)	8±0.3 (p=0.006)	8.3±0.3 (p=0.006)	8±0.0 (p=0.002)
S. maltophilia	0±0.0 (p=0.050)	6.75±0.25 (p=0.003)	7.5±0.25 (p=0.002)	7.5±0.25 (p=0.003)	7.8±0.28 (p=0.050)
L. adecarboxylata	6.6±0.24 (p=0.003)	9.2±0.48 (p=0.015)	10.4±0.97 (p=0.016)	10.5±0.95 (p=0.030)	10±0.0 (p=0.002)
C. albicans	7±0.0 (p˂0.001)	7.75±0.25 (p=0.015)	7.45±0.35 (p˂0.001)	7.25±0.25 (p=0.050)	7.3±0.3 (p˂0.001)
Almond oil
S. intermedius	8±0.0 (p=0.002)	8±0.0 (p=0.002)	8±0.0 (p=0.003)	8±0.0 (p=0.015)	7.3±0.3 (p˂0.001)
S. aureus	8±0.0 (p=0.003)	8±0.0 (p=0.001)	8±0.0 (p=0.002)	7.4±0.24 (p=0.001)	7.5±0.25 (p=0.050)
S. capitis	0±0.0 (p=0.001)	6±0.0 (p=0.001)	6±0.0 (p=0.001)	7.5±0.3 (p=0.050)	7.5±0.3 (p=0.050)
S. haemolyticus	6±0.0 (p=0.002)	6.6±0.3 (p=0.002)	11±0.0 (p=0.015)	10±0.0 (p=0.019)	11.6±0.3 (p=0.002)
E. coli	0±0.0 (p=0.050)	0±0.0 (p=0.050)	8±0.3 (p=0.050)	8±0.0 (p=0.013)	8±0.0 (p=0.050)
S. maltophilia	0±0.0 (p=0.003)	6.75±0.25 (p=0.003)	7.75±0.25 (p=0.003)	7.75±0.25 (p=0.003)	8±0.0 (p=0.003)
L. adecarboxylata	6.6±0.24 (p=0.002)	9.2±0.48 (p=0.002)	10±0.0 (p=0.003)	10.4±0.97 (p=0.001)	9.5±0.75 (p=0.002)
C. albicans	0±0.0 (p˂0.001)	0±0.0 (p˂0.001)	7±0.0 (p=0.015)	7.5±0.25 (p=0.050)	7.5±0.25 (p˂0.001)

Zone of inhibition ± standard error of the mean.

. All results were significant with p˂0.05.

Determination of the MIC and MBC/MFC of the tested EOs against bacterial and fungal isolates

The results revealed that lettuce oil did not affect S. intermedius, E. faecium, and S. haemolyticus. However, it showed a bacteriostatic activity on S. aureus (MIC=12.5%), S. capitis (MIC=50%), E. coli (MIC=25%), C. freundii (MIC=12.5%), C. braakii (MIC=25%), and S. maltophilia (MIC=12.5%)—Figure 6. Lettuce oil also exhibited a bactericidal activity against L. adecarboxylata (MBC=50%)—Figure 7. Coconut oil showed no effect on E. faecium, C. freundii, and C. braakii, and exerted a bacteriostatic effect on S. intermedius (MIC=25%), S. aureus (MIC=12.5%), S. capitis (MIC=25%), S. haemolyticus (MIC=12.5%), E. coli (MIC=25%), S. maltophilia (MIC=12.5%), and L. adecarboxylata (MIC=12.5%). Coconut oil had no bactericidal effect. As for almond oil, it had no effect on E. coli and S. maltophilia, while it showed a bacteriostatic activity against E. faecium (MIC=3.125%), S. aureus (MIC=25%), S. haemolyticus (12.5%), and L. adecarboxylata (MIC=25%). This oil also showed a bactericidal effect on S. intermedius (MBC=12.5%), S. capitis (MBC=12.5%), C. freundii (MBC=12.5%), and C. braakii (MBC=25%). On the other hand, the EOs exerted a fungistatic activity rather than a fungicidal activity on C. albicans with MIC=50% for lettuce oil, MIC=6.25% for coconut oil, and MIC=12.5% for almond oil. The MIC and MBC/MFC results are shown in

Table 3. MIC and MBC results of the tested EOs against the bacterial isolates. 

			MICs and MBCs (%)
Bacterial/fungal isolates	Lettuce oil		Coconut oil		Almond oil
	MIC	MBC	MIC	MBC	MIC	MBC
Gram-positive bacteria
S. intermedius	-	-	25%	-	6.25%	12.5%
E. faecium	-	-	-	-	3.125%	-
S. aureus	12.5%	-	12.5%	-	25%	-
S. capitis	50%	-	25%	-	6.25%	12.5%
S. haemolyticus	-	-	12.5%	-	12.5%	-
Gram-negative bacteria
E. coli	25%	-	25%	-	-	-
C. freundii	12.5%	-	-	-	3.125%	12.5%
C. braakii	25%	-	-	-	6.25%	25%
S. maltophilia	12.5%	-	12.5%	-	-	-
L. adecarboxylata	25%	50%	12.5%	-	25%	-
Yeast
C. albicans	50%	-	6.25%	-	25%	-

MIC—minimum inhibitory concentration; MBC—minimum bactericidal concentration.

. All results were significant with p˂0.05.

Antibiofilm screening results

Inhibition of the formation of bacterial and fungal biofilms: Lettuce oil showed antibiofilm effect against S. maltophilia. Coconut oil showed effectivity against S. haemolyticus, E. coli, and S. maltophilia. C. albicans biofilm was sensitive to coconut oil only. However, almond oil did not affect any of the bacterial and fungal biofilms. The antibiofilm formation results are shown in

Table 4. Effect of the EOs on the inhibition of biofilm formation and the destruction of pre-formed biofilms. 

	Concentrations of oils (%) and % of inhibition of biofilms
Bacterial/fungal biofilms	Inhibition of formation of the biofilms		Destruction of pre-formed biofilms
	Inhibitory concentration of oil (%)	% of formation inhibition (%)	Destruction concentration of oil (%)	% of destruction (%)
		Lettuce oil
E. faecium	-	-	50	65±0.03 (p=0.001)
S. haemolyticus	-	-	3.125	54±0.01 (p=0.003)
C. freundii	-	-	12.5	56±0.003 (p=0.041)
C. braakii	-	-	50	58±0.001 (p=0.004)
S. maltophilia	3.125	31±0.02 (p=0.001)	25	27±0.03 (p=0.001)
C. albicans	-	-	50	65±0.03 (p=0.050)
		Coconut oil
S. intermedius	-	-	12.5	51±0.01 (p=0.050)
E. faecium	-	-	12.5	56±0.02 (p=0.038)
S. aureus	-	-	25	21±0.02 (p=0.009)
S. capitis	-	-	12.5	51±0.01 (p=0.001)
S. haemolyticus	50	31±0.03 (p=0.032)	6.25	64±0.02 (p˂0.001)
E. coli	6.25	33±0.0006 (p=0.004)	-	-
C. freundii	-	-	50	42±0.001 (p=0.011)
C. braakii	-	-	3.125	58±0.02 (p=0.008)
S. maltophilia	6.25	67±0.01 (p˂0.001)	25	53±0.003 (p=0.003)
L. adecarboxylata	-	-	3.125	42±0.03 (p=0.015)
C. albicans	3.125	59±0.02 (p˂0.001)	12.5	51±0.01 (p=0.013)

and Figure 8. All results were significant with p˂0.05.

Inhibition of pre-formed bacterial and fungal biofilms: Lettuce oil was effective against E. faecium, S. haemolyticus, C. freundii, C. braakii, and S. maltophilia. Coconut oil showed a significant activity against S. intermedius, E. faecium, S. aureus, S. capitis, S. haemolyticus, C. freundii, C. braakii, S. maltophilia and L. adecarboxylata. On the other hand, almond oil did not exhibit any inhibitory effect. As for C. albicans, pre-formed biofilm was sensitive to lettuce and coconut oils only. The results of inhibition of pre-formed biofilms are shown in Table 4 and Figure 9. All results were significant with p˂0.05.

GC-MS analysis showing the different major components of the tested EOs

The chemical analysis of lettuce oil revealed the presence of nine major chemical compounds. Among these, (E)-9-Tetradecen-1-ol, Hexadecanoic acid, 1,2-Benzenedicarboxylic acid, dibutyl ester, Hexadecanoic acid, Oleic acid, methyl ester, and Cholest-5-en-3-ol are of significant effect. As for coconut oil, fourteen major chemical compounds were detected, in which Myristic acid; Hexadecanoic acid, Ricinoleic acid, Hexadecanoic acid, butyl ester, Oleic acid, methyl ester, Palmitic acid, butyl ester, Hydrocortisone 21-acetate, and Myristic acid, tetradecyl ester are abundant. In addition, almond oil showed the presence of six major chemical compounds: hexadecenoic acid; Oleic acid, methyl ester; Stearic acid; Palmitin-1-mono-; and Digitoxin in which all are significant. The GC-MS results are shown in

Table 5. List of the chemical components identified in the tested EOs by GC-MS. 

Compound name	Molecular formula	Molecular weight	Relative abundance (%)	Retention time (min)
Lettuce oil [index value: HG-30/390]
(E)-9-Tetradecen-1-ol	C12H28O	212	32.99	7.10
1,2-Benzenedicarboxylic acid, dibutyl ester	C16H22O4	278	33.06	13.28
Hexadecanoic acid	C16H32O2	256	78.85	14.19
Oleic acid, methyl ester	C19H36O2	296	78.12	15.93
Rescinnamine	C35H42N12O9	634	19.04	24.35
Palmitic acid, butyl ester	C20H40O2	312	7.14	24.71
Cholest-5-en-3-ol	C27H46O	386	38.14	25.91
Coconut oil [index value: HG-30/306]
Myristic acid	C14H28O	228	86.22	12.5
Hexadecanoic acid	C16H32O2	256	79.91	14.18
Ricinoleic acid	C18H34O3	298	20.74	15
Hexadecanoic acid, butyl ester	C20H40O2	312	57.24	15.41
Oleic acid, methyl ester	C19H36O2	296	86.17	15.87
Palmitic acid, butyl ester	C18H36O2	284	86.59	20.94
Rescinnamine	C35H42N2O9	634	8.94	25.71
Hydrocortisone 21-acetate	C23H32O6	404	44.20	26.14
Myristic acid, tetradecyl ester	C28H56O2	424	25.01	28.56
Almond oil [index value: HG-30/308]
Hexadecanoic acid	C16H32O2	256	72.13	14.27
Oleic acid, methyl ester	C19H36O2	296	95.36	18.55
Stearic acid	C18H36O2	284	86.38	16.24
Palmitin, 1-mono-	C19H38O4	330	82.78	17
Digitoxin	C41H64O13	764	82.53	20.21

.

Discussion

Sewage sludge is widely used in agriculture in many countries. However, the high content of pathogenic microorganisms in the Lebanese sewage limits its reuse [13]. The resistance of pathogens to many antibiotics raises the need to find alternatives for pathogens’ treatment. Therefore, this study aimed at investigating the antimicrobial effect of EOs against pathogens.

Previous studies on the Lebanese wastewater have shown that all the identified bacterial pathogens, except S. maltophilia and L. adecarboxylata, are frequently present in the environment, especially in feces [3]. L. adecarboxylata is sensitive to several antibiotics, with some resistant strains reported [14]. S. maltophilia infection is difficult to treat due to its ability to colonize tissues [15]. Previous studies have also revealed the presence of the identified C. albicans in the Lebanese wastewater. It has shown great resistance to many antifungal agents [16].

From the antibiotic-resistance profiles, antibiotics with a broad-spectrum activity are used in this study. Levofloxacin, a member of the quinolone family, acts by inhibiting bacterial topoisomerase IV and DNA gyrase enzymes, thus inhibiting DNA replication, transcription, and repair [17]. Amoxicillin belongs to the beta-lactam family and acts by inhibiting transpeptidation, thus destroying the cell wall of bacteria [18]. In addition, tetracycline and doxycycline, members of the tetracyclines family, act by inhibiting the 30S ribosomal subunit and inhibiting protein synthesis, respectively, thus leading to the inability of bacterial cells to function [19]. Among the tested antibiotics, doxycycline had the broadest activity spectrum because all tested bacterial isolates were susceptible to it. This result is similar to a previous study that tested the susceptibility of bacteria to doxycycline [19]. Many previous studies revealed the high antibacterial activity of the mentioned antibiotics against the tested bacterial isolates, except amoxicillin which has shown decreased activity against many bacteria [1]. The resistance raises a public health problem and, in turn, the need for novel antimicrobial agents with a unique mode of action. In this regard, plant-derived EOs have been used as effective antimicrobial agents [11,20]. Many EOs tested previously showed inhibitory effects against bacteria and yeast. Most importantly, EOs are environmentally safe and non-toxic [5]. This study revealed that the EOs exhibited antibacterial and antifungal activities. Almond oil possessed bactericidal effect due to the presence of aminoglycosides, which are used in many countries to treat microbial pathogens. Lettuce and coconut oils’ activity was assessed by the presence of flavones, saponins, and sterols [21]. Upon agar well diffusion, lettuce and coconut oils displayed a better antibacterial activity on Gram-negative bacteria. This result is consistent with previous studies that showed that Gram-positive bacteria are more resistant to EOs because of their thick peptidoglycan layer [5]. On the other hand, almond oil exhibited better antibacterial activity on Gram-positive bacteria. Such resistance could be due to the lipopolysaccharides of the cell wall of Gram-negative bacteria [5]. Regarding bacterial susceptibility, L. adecarboxylata and S. aureus were the most sensitive. On the other hand, S. haemolyticus and E. coli were the most resistant. This is due to the composition of the bacterial cell wall, in which many previous studies reported that the efficiency of EOs differs based on the type of bacterial isolate [20]. C. albicans showed susceptibility to the three EOs. The best susceptibility was noticed for almond oil. In contrast, susceptibility to lettuce and coconut oils was detected at higher concentrations. These susceptibility differences are due to the chemical composition of oils, as well as the eukaryotic characteristics of C. albicans [11].

Among the tested EOs, lettuce and coconut oils exhibited a bacteriostatic effect. However, almond oil had a bactericidal effect. The bacteriostatic and bactericidal effects of an oil depend on the composition of the bacteria. This means that the major components of the tested oils reacted with the cell wall of bacterial cells through different mechanisms [7,20]. They work by penetrating the cell membrane and inducing structural changes on the surface of bacterial cells, thus causing leakage of electrolytes and leading to bacterial death [22].

The effect of EOs on the inhibition of biofilm formation wasn’t relatively significant. All tested EOs mostly enhanced the biofilm formation. S. maltophilia biofilm was the most sensitive. This result is attributed to the fact that the composition of the biofilm differs between bacterial isolates [12]. A higher effect was observed for lettuce and coconut oils, but not for almond oil. Interestingly, almond oil exhibited a better effect on bacteria rather than on the biofilm. This might be due to the fact that biofilms produce proteins and exopolysaccharides, which increases their resistance [11,12]. C. albicans biofilm formation was inhibited by coconut oil only. The anti-biofilm activity of EOs depends on their compounds which act by different mechanisms, involving a series of chemical reactions in the microbial cells. Previous studies reported that when EOs are applied before the formation of the biofilm, they interact with microbial proteins and prevent the attachment [11,12].

Many recent studies have shown that EOs are better at preventing biofilm formation than at destroying pre-formed biofilms [11,12]. This study showed that the tested EOs can inhibit pre-formed biofilms. Lettuce and coconut oils exerted inhibitory effects on almost all biofilms. It is assumed that the major components of the EOs interact with the exopolysaccharides secreted by biofilms to inhibit their attachment [12]. Briefly, biofilms form great resistance to antimicrobial agents and remain a global threat to health. They over-produce polysaccharides which protect the biofilm cells. The inhibitory concentrations detected were MIC independent [22]. The oils were able to inhibit biofilms at different concentrations. These results are consistent with previous studies stating that the anti-biofilm effects are not concentration-dependent [22]. This variation might be due to the absorbance and cell enumeration [22]. In addition, our study showed that the anti-biofilm effect is oil-dependent. The significant effect might be due to the fact that the components of the EOs penetrate the cells of the biofilms, leading to their destruction [22].

All the previous antimicrobial effects of EOs are related to the presence of specific compounds. These compounds might be responsible for the different actions of the oils against the tested microbial isolates. Previous studies reported that fatty acids and fatty acyl esters cause leakage of fatty acids and potassium ions in the membrane [23]. In addition, steroids are known to alter membrane fluidity [24]. Glycosides inhibit the plasma membrane sodium and calcium ATPases. This increases the levels of sodium and calcium in the membrane, leading to cell death [25]. Further studies are needed to elaborate precisely on the compounds responsible for the antimicrobial activity of these EOs.

This study could be limited by the following issues. The oils are imported from outside Lebanon, which might make them expensive when needed in large amounts. In addition, the mode of action of EOs in the bacterial morphological setup is still not very clear. So, the significant results obtained in this study open new doors to perform more studies in the purpose of investigating the antimicrobial effect of the mentioned oils against other microbial isolates, which could be helpful in treating both environmental as well as health problems.

Conclusions

This study showed that wastewater in South Lebanon contains various pathogens, including rare bacteria. A significant antimicrobial activity was revealed for three EOs derived from lettuce, coconut, and almond, which have not been previously investigated for their potential antimicrobial activity. EOs exhibited a significant antimicrobial activity against different bacteria, most importantly against Gram-negative bacteria. This study sheds the light on the use of EOs against microorganisms present in the Lebanese southern wastewater to prevent bacterial and fungal growth, and biofilms formation.