2.1. Antibacterial Effect of Essential Oils
Because of the problems of microbial resistance to synthetic antibiotics, several studies have focused on the antimicrobial power of EOs. In the current study, the antibacterial activity of eight essential oils (
E. globulus,
E. camaldulensis,
A. absinthium,
M. communis,
M. pulegium,
T. ammi,
C. citratus, and
T. capitatus) against extended-spectrum β-lactamase (ESBL)-producing
E. coli, isolated from foods, was investigated. This antimicrobial potential was evaluated by determination of diameters of inhibition zones (IZ) (
Table 1) and MIC and MCB values (
Table 2).
As illustrated in
Table 1, the obtained results show that the diameters of IZ generated by EOs varied significantly, according to the tested EO and evaluated
E. coli strains. Generally, EO is considered active when the diameters of IZ engendered in its presence were greater than or equal to 15 mm [
11]. Based on this condition, we can conclude that EOs of
T. capitaus,
E.globulus,
E. camaldulensis,
T. ammi, and
M. pulegium exert an important antimicrobial effect against tested bacterial strains. In contrast, with inhibition diameters less than 15 mm, EOs of
A. absinthium,
C. citratus and
M. communis show an intermediate activity.
Indeed, the analysis of obtained data demonstrated that EO of
T. capitatus had the most important inhibitory effect against
E. coli strains, since it represented the highest inhibition zone (27 mm) and the lowest MIC value (0.0975%) (
Table 2). This remark is consistent with previous work carried out by Aouadhi et al. [
6], which showed that
T. capitatus is very effective against different bacterial species (
E. coli,
S. typhimurium,
S. aureus,
P. aeruginosa,
A. hydrophila,
L. monocytogenes, and
B. cereus), with a maximum inhibition zone and MIC value varied between 17–40 mm and 0.025–0.4% (
v/
v), respectively.
Major components of
T. capitatus, such as thymol, carvacrol, ocimene, and terpiene may be responsible for important antimicrobial activities. In fact, all of these compounds are well known for their antimicrobial properties. Indeed, several authors showed that EOs rich in phenolic derivatives, such as carvacrol and thymol, have strong antimicrobial activities [
12,
13]. In this same context, Dorman et al. [
14] demonstrated that thymol had a broader spectrum of antibacterial activity against 25 different bacteria species. In addition, studies carried out by the World Health Organization highlighted that this constituent had strong antifungal and antibacterial activities against many species, including
Aspergillus spp.,
S. aureus and
E. coli. In fact, Lambert et al. [
15] and Juven et al. [
16] explained this phenomenon by the fact that thymol binds to membrane proteins and increases the permeability of the bacterial cell membrane. Other work suggested that this volatile compound is responsible for the inactivation of enzymes, including those involved in energy production and the synthesis of structural components [
17].
Furthermore, the results in
Table 1 and
Table 2 signaled that EO of
T. ammi had the lowest antimicrobial power compared to
T. capitaus, with the IZ and MIC values in the range of 20 to 22 mm and 0.39 to 0.78% (
v/
v), respectively.
Concerning EO of
E. camaldulensis, the obtained data showed that it is endowed with remarkable antimicrobial power against tested strains, with the IZ and MIC values varied between 16–18 mm and 0.0975–0.78% (
v/
v) respectively. Several studies have already demonstrated the antimicrobial activity of
Eucalyptus compounds in different fields of life sciences [
18,
19]. These results are in agreement with those found by Farah et al. [
20], who demonstrated that at a concentration of 0.2% (
v/
v),
E. coli,
S. aureus and
Aspergillus niger are sensitive to EO of
E. camaldulensis. According to Bouanoun et al. [
21], the antimicrobial activity of
Eucalyptus EO is attributed to eucalyptol or 1.8-cineole, which is a monoterpene belonging to the class of ethers. It had antioxidant and antibacterial properties and, therefore, explains the origin of its antimicrobial activity [
22].
By comparing IZ diameters generated by
E. globulus EO to that of
E. camaldulensis, variability in their antibacterial effects against
E. coli strains was observed. Indeed, IZ and MIC values of
E. globulus were in the range of 15–18 mm and 0.195–0.78% (
v/
v), respectively. A study carried out in Morocco reported that IZ and MIC of
E. globulus in the presence of
E. coli and
S. aureus were 48.15 and 13.50 mm and MICs of 0.15 and 0.75 mg/mL, respectively [
18]. These results do not corroborate those obtained during this study, and this can be attributed to the chemical composition of our oil and to its origin.
The aromatogram produced with the
M. pulegium EO demonstrates that
E. coli is noticeably sensitive. Indeed, the potential inhibitors of the oil were in the range of 0.195–0.78% (
v/
v) and the IZ were in the order of 20–21 mm. These results are in agreement with those of Ghazghazi et al. [
7], who studied the antimicrobial activity of
M. pulegium against 10 microorganisms (
E. coli,
S. typhimurium,
S. aureus,
P. aeruginosa,
A. hydrophila,
L. monocytogenes,
B. cereus,
Aspergillus niger,
Aspergillus flavus, and
C. albicans). This study showed that the IZ and MIC values for the tested microorganisms were in the range of 15–30 mm and 0.05–0.8% (
v/
v), respectively. In addition, previous studies with the same plant, collected in Tunisia, Iran, Portugal and Morocco, demonstrated that these EO possess very important antimicrobial power, but there is great variation in the intensity of these activities against Gram-negative and Gram-positive bacteria. Such differences may be due to the chemical composition of EO. For example, Mahboubi and Haghi [
23] showed that the volatile oil of Iranian
M. pulegium had potent antimicrobial activity against Gram-positive bacteria, particularly against
L. monocytogenes, where the inhibition zones were in the range of 8–21 mm, while the least susceptible bacteria were
E. coli and
S. typhimurium [
7]. Our results confirm this previous work, which demonstrated significant antibacterial activities in the essential oil of mint in Algeria and, in particular, against
E. coli [
24].
As shown in
Table 1 and
Table 2, the results of the antimicrobial activity of EO of
A. absinthium, showed an intermediate sensitivity of
E. coli. Indeed, IZ were in the range of 11.33–13.67 mm. The antimicrobial power of
A. absinthium has been previously reported by Riahi et al. [
25], who showed that the EO of
A. absinthium, observed in the Gafsa region, had a maximum inhibition zone of 11 mm and MIC value of around 25%, but for the Kasserine region, El Kef and Ghar Dimaou, the maximum inhibition zones and MIC values were around 14 mm and 12.5–25% (
v/
v) respectively.
Concerning the
M. communis EO, it can be signaled that this oil can inhibit the microbial growth of
E. coli, with IZ and MIC values in the range of 13.33 mm and 0.39–1.56% (
v/
v), respectively. This result is in agreement with the previous work of Chebaibi et al. [
26], who revealed that the EO of
M. communis had an antimicrobial activity against diverse bacterial species (
E. coli,
B. subtilis,
M. luteus and
S. aureus) when their growth was totally inhibited in the presence of 0.5%
v/
v of the EO. This antimicrobial activity is mainly due to the richness of the evaluated EO in a mixture of terpenols (α-terpineol and myrtenol) and 1,8-cineole. This fraction, which constitutes about 20% of the total oil, can be used in the food industry, as a means of prevention against pathogens that contaminate foodstuffs or in other pharmacological and medical fields [
27].
The results obtained during the study of the antimicrobial activity of
C. citratus EO were more or less average compared to other tested EOs. The maximum inhibition zones and MIC values were in the ranges of 12.33 mm and 0.78% (
v/
v), respectively. These results are in agreement with those obtained by Zulfa et al. [
28], who showed that
C. citratus EO can be used as an antimicrobial agent against
E. coli, with a diameter inhibition zone and MIC values of 7.5 mm and 0.63% (
v/
v), respectively. Previous studies reported that
C. citratus oil was more effective against Gram-positive bacteria, such as
S. aureus and
B. cereus. Based on the study of Bin et al. [
29], it can be concluded that all of the Gram-negative bacteria have lipopolysaccharides on the outer layer and they have a negative charge all over the cell, which results in less affinity with the negative phenolic compounds. On the contrary Gram-positive bacteria have different structures and, for this, are perhaps more sensitive. The resistance of Gram-negative bacteria is attributed to their hydrophilic outer membrane, which can block the penetration of hydrophobic compounds into the target cell membrane [
30,
31].
Based on the obtained data, it can be signaled that the effect of different EOs against strains of
E. coli used is low compared to that found in other studies in the presence of reference bacteria. These can be explained by two hypotheses. First,
E. coli is a Gram-negative bacterium, so it is more resistant to the effects of antimicrobial agents than the Gram-positive ones. In this context, Dorman et al. [
14] showed that Gram-positive bacteria exhibit IZ greater than those observed in Gram-negative bacteria. This can be explained by the fact that the cell wall structure of Gram-negative bacteria makes them less sensitive to the action of essential oils. The presence of an outer membrane, highly hydrophilic, constitutes a barrier to hydrophobic and amphiphilic macromolecules, which enter into the composition of these oils. Associated with the plasma membrane, which limits the passage of hydrophilic molecules, the intracellular penetration of all these compounds is considerably restricted in these types of bacteria [
32]. This specific structure gives Gram-negative bacteria intrinsic resistance to most antibacterial molecules, including antibiotics, and these may explain the resistance of
E. coli, which is a Gram-negative bacterium, observed during this study with certain EOs.
Second, the used isolates of E. coli are multi-antibiotic-resistant strains, capable of producing broad spectrum β-lactamase (ESBL)-type enzymes, which can destroy antibiotics prior to their penetration inside the cell or can modify the target of the antibiotic. The presence of this enzyme may explain the significant resistance of these strains to different Eos, in comparison to strains of the same species or different species.