Essential Oils and Their Components as Modulators of Antibiotic Activity against Gram-Negative Bacteria

Gram-negative bacteria cause infections that are difficult to treat due to the emergence of multidrug resistance. This review summarizes the current status of the studies investigating the capacity of essential oils and their components to modulate antibiotic activity against Gram-negative bacteria. Synergistic interactions are particularly discussed with reference to possible mechanisms by which essential oil constituents interact with antibiotics. Special emphasis is given to essential oils and volatile compounds that inhibit efflux pumps, thus reversing drug resistance in Gram-negative bacteria. In addition, indifference and antagonism between essential oils/volatile compounds and conventional antibiotics have also been reported. Overall, this literature review reveals that essential oils and their purified components enhance the efficacy of antibiotics against Gram-negative bacteria, being promising candidates for the development of new effective formulations against Gram-negative bacteria.


Introduction
Infectious diseases are the second leading cause of death worldwide, being responsible for approximately 15 million deaths each year [1]. This high mortality rate is mainly due to antibiotic resistance. Frequent and improper use of antibiotics favored the emergence and spread of single-and multidrug-resistant microbial strains [2]. In 2009, several European countries reported that 25%-50% of Staphylococcus aureus isolates from patients with nosocomial infections were resistant to methicillin (oxacillin). In 2012, some of these countries (Portugal and Romania) reported percentages higher than 50% for methicillin-resistant S. aureus isolates. Resistance to carbapenems, broad-spectrum β-lactam antibiotics, has alarmingly increased. From 2009-2012, the percentages of carbapenem-resistant Klebsiella pneumoniae isolates increased dramatically from very low values, such as below 1% in Romania and 1%-5% in Italy, to values of 10%-25% and 25%-50%, respectively [3].
Combination therapy, combining conventional antibiotics and natural products, represents a promising strategy in overcoming antibiotic resistance [4][5][6]. Such antimicrobial combinations may exhibit synergistic, additive, indifferent or antagonistic effects. Synergy, addition and antagonism occur when the overall effect is greater than, equal to and less than the sum of the individual effects, infections [21,22]. Immortelle (Helichrysum italicum (Roth) G. Don., Asteraceae) EO reduced the resistance to chloramphenicol of A. baumannii ATCC 19606 and the AB1 strain isolated from central venous catheters of critical care patients [37,38]. In the case of both strains, immortelle EO reduced by eight-fold the MIC value of chloramphenicol in combination, whereas phenylalanine-arginine β-naphthylamide (PaβN), a potent efflux pumps inhibitor, caused only a two-fold reduction. Geraniol (Figure 1), a component of immortelle EO, is an inhibitor of bacterial efflux pumps, which are responsible for both antibiotic resistance and resistance to host innate immune defense [37,39]. Cinnamon (Cinnamomum zeylanicum Nees., Lauraceae) and lemon (Citrus limon L. Burm. F., Rutaceae) EOs showed strong synergistic effects in combination with amikacin against a multidrug-resistant A. baumannii clinical isolate (A-06 strain) [40]. The study provides no information on the chemical composition of both cinnamon and lemon EOs. However, a permeabilization of the bacterial membrane by certain EO constituents followed by an increased penetration of amikacin might explain, at least in part, these synergistic effects.
Medicines 2016, 3, 19 4 of 33 Geraniol (Figure 1), a component of immortelle EO, is an inhibitor of bacterial efflux pumps, which are responsible for both antibiotic resistance and resistance to host innate immune defense [37,39]. Cinnamon (Cinnamomum zeylanicum Nees., Lauraceae) and lemon (Citrus limon L. Burm. F., Rutaceae) EOs showed strong synergistic effects in combination with amikacin against a multidrug-resistant A. baumannii clinical isolate (A-06 strain) [40]. The study provides no information on the chemical composition of both cinnamon and lemon EOs. However, a permeabilization of the bacterial membrane by certain EO constituents followed by an increased penetration of amikacin might explain, at least in part, these synergistic effects.

Essential Oils and Volatile Compounds Modulating Antibiotic Activity against Escherichia coli
Escherichia coli is a facultative anaerobic, non-spore-forming Gram-negative bacterium, commonly abundant in the normal intestinal tract flora. Pathogenic strains are spread mainly by contaminated food and water being responsible for a wide range of infections. Identification of O (lipopolysaccharide) and H (flagellar) antigens allowed the classification of enteric E. coli into seven pathovars responsible for certain diseases: diarrhea in children (enteropathogenic, diffusely adherent and enteroaggregative E. coli), hemolytic-uremic syndrome (enterohemorrhagic E. coli), traveler's diarrhea (enterotoxigenic E. coli), dysentery (enteroinvasive E. coli) and Crohn's disease (adherent invasive E. coli) [41,42]. There are also two pathovars of the extraintestinal type, which are involved in urinary tract infections (uropathogenic E. coli) and meningitis in newborns (neonatal meningitis E. coli) [43]. Cell adhesion through pili or fimbriae is characteristic for all pathovars, except for enteroinvasive E. coli Besides, specific virulence factors have been identified for different pathovars, such as hemolysin (uropathogenic and enteroinvasive E. coli) [44,45], Shiga toxins (enterohemorrhagic and enteroinvasive E. coli) [45] and heat-labile and heat-stable toxins (enterotoxigenic E. coli) [46]. E. coli is intrinsically resistant to penicillin G due to the outer membrane barrier, but extended spectrum β-lactamase production is responsible for the resistance to a broad spectrum of β-lactams, including penicillins, cephalosporins (third-and fourth-generation cephalosporins) and monobactams [47]. Carbapenem resistance of E. coli is also a therapeutic challenge because many nosocomial isolates have been reported to synthesize carbapenemases [48]. The alteration of quinolone targets (DNA gyrase, topoisomerase IV), the decrease in membrane permeability and the overexpression of efflux pumps explain the resistance of E. coli to quinolones [49]. The production of acetyltransferases, nucleotidyltransferases, phosphotransferases and methylation of 16S rRNA are responsible for the resistance to aminoglycosides [42]. Treatment options of infections caused by extended spectrum β-lactamase-producing E. coli include imipenem, meropenem and third-generation cephalosporins in combination with an enzyme inhibitor (clavulanic acid) [50].
A feasible approach to reduce the antibiotic resistance of E. coli is the combination of conventional antibiotics with EOs or volatile compounds (Table 2). A strong synergy was reported for two Moroccan thyme (Thymus maroccanus L. and Thymus broussonetii L., Lamiaceae) EOs in combination with chloramphenicol against E. coli strains: AG100 (wild-type E. coli K-12) and AG102 (AG100 derivative overexpressing AcrAB pump responsible for resistance to tetracycline, chloramphenicol, ampicillin, nalidixic acid and rifampicin) [51,52]. Both EOs decreased the MIC values of chloramphenicol against E. coli AG100 and AG102 strains by four-and 32-fold, respectively. The synergy could be attributed to the alteration of efflux pump activity by certain EO constituents, but also to the ability of carvacrol (76.35% in T. maroccanus EO, 39.77% in T. broussonetii EO) to cause the permeabilization and depolarization of the E. coli cytoplasmic membrane, thus facilitating the passage of the antibiotic to the bacterial cell [52,53]. Carvacrol ( Figure 1) is a well-known antibacterial agent [54]; in the case of Gram-negative bacteria, carvacrol disintegrates the polysaccharidic capsule, followed by an increase in the fluidity and permeability of the cytoplasmic membrane [55]. In addition, it acts as a proton exchanger, thus affecting the cytoplasmic pH and proton motive force with a subsequent reduction of ATP and DNA synthesis and inhibition of cytoplasmic enzymes, leading to bacterial cell death [56,57]. Similar results were obtained for another Moroccan thyme species (T. riatarum L., Lamiaceae) EO in combination with chloramphenicol against E. coli AG100 and AG100A (AG100 derivative with AcrAB pump deleted, but resistant to kanamycin). T. riatarum EO decreased the MIC values of chloramphenicol against AG100 and AG100A by four-and two-fold, respectively; the synergy could be attributed to efflux pump blocking activity of T. riatarum EO, thus increasing the activity of chloramphenicol [58]. The lemon thyme (Thymus pulegioides L., Lamiaceae) EO showed synergistic effects with tetracycline and chloramphenicol against E. coli ATCC 25922. Surprisingly, the combination between lemon thyme EO and streptomycin displayed an antagonistic effect related to a possible competition for the same target: the bacterial 30S ribosomal subunit; binding of this subunit leads to inhibition of protein synthesis. Lemon thyme EO contained geraniol (66.59%) as a major constituent. Geraniol has also been reported in immortelle (Helichrysum italicum (Roth) G. Don, Asteraceae) EO [59]. Immortelle EO restored the chloramphenicol susceptibility of E. coli AG100A Tet r overexpressing AcrEF efflux pumps, responsible for tetracycline resistance. The MIC value of chloramphenicol was decreased by 256-fold in combination with immortelle EO, suggesting the ability of the latter to inhibit AcrEF efflux pumps [37]. Other oxygenated monoterpene-rich EOs, such as savory (Satureja kitaibelii Wierzb. ex Heuff., Lamiaceae), rosewood (Aniba rosaeodora Ducke, Lauraceae), geranium (Pelargonium graveolens L., Geraniaceae) and tea tree (Melaleuca alternifolia (Maiden and Beach) Cheel., Myrtaceae) EOs exhibited synergistic effects in combination with different antibiotics (chloramphenicol, tetracycline, gentamicin) against E. coli ATCC 25922 [34,60]. Synergistic effects might be correlated with the damage of the microbial membrane lipids by terpene alcohols [34] followed by an increased penetration of antibiotics into the bacterial cell. Modulatory effects of rosemary (Rosmarinus officinalis L., Lamiaceae) EO on the E. coli EC27 clinical isolate resistant to aminoglycosides have been investigated. The antimicrobial activities of amikacin, neomycin and gentamycin were increased by rosemary EO [61]. 1,8-Cineol (eucalyptol) (Figure 1), the main component of rosemary EO, has been reported to disintegrate the outer membrane and reduce the nucleoplasm [39]. On the other hand, all three aminoglycosides bind to the 30S ribosomal subunit, blocking protein synthesis [10]. Modulation of antibiotic activity by EOs through gaseous contact is a new approach that should be taken into consideration especially, in the case of respiratory infections. Cidreira-do-mato [62], also known as cidreira-brava [63] (Hyptis martiusii Benth., Lamiaceae), EO increased the susceptibility to tobramycin of E. coli ATCC 25922 and the EC27 multidrug-resistant surgical wound isolate by 76.5% and 80%, respectively; in addition, it considerably reduced the resistance to gentamicin and amikacin of the EC27 isolate (by 40% and 82.4%, respectively). The main constituents of cidreira-brava EO were bicyclogermacrene (10.6%), trans-caryophyllene (9.2%), caryophyllene oxide (7.4%), 1,8-cineole (7%) and δ-2-carene (6.8%) [64]. Neither for cidreira-brava EO, nor for its main constituents studies were performed in order to assess the mechanisms of synergism with aminoglycosides, but the permeabilization of the bacterial membrane by EO components might be partially responsible for the increase in antibiotic activity.
Several studies have reported interactions between EO constituents and conventional antibiotics against E. coli strains. Gallucci et al. [65] detected a potent synergism between eugenol/thymol and penicillin against E. coli resistant to β-lactam antibiotics. Thymol ( Figure 1) also acted synergistically with penicillin against multidrug-resistant E. coli N00 666 [66]. These synergisms might be attributed to the ability of terpene phenols to increase the antibiotic permeation into the bacterial cell. Both thymol and carvacrol showed more synergistic interactions with antibiotics against E. coli strains in comparison with eugenol [66]. This difference in synergistic potential is mainly due to different electron delocalization. In the case of thymol and carvacrol, the delocalized electrons enable the release of the proton belonging to the phenolic hydroxyl group. The released proton acts as a proton exchanger and reduces the pH gradient across the cytoplasmic membrane, thus decreasing the bacterial membrane potential and causing ATP depletion [56]. Eugenol ( Figure 1) has been reported to target both the bacterial membrane, leading to a non-specific increase in antibiotic penetration, and different metabolic pathways [18]. However, in the case of eugenol, the presence of the methoxyl group impairs the release of the phenolic hydroxyl proton. This structural pattern might explain not only the weaker antimicrobial activity of eugenol in comparison with thymol and carvacrol [67], but also the reduced number of synergisms between eugenol and conventional antibiotics. Cinnamaldehyde (Figure 1), a volatile phenylpropanoid, increased the antibiotic susceptibility of E. coli N00 666 [66]. The effect could be attributed, at least in part, to the ability of cinnamaldehyde to inhibit ATP-ase activity with a consequent alteration in bacterial membrane permeability [10,68]. An antagonistic interaction against E. coli has been reported for myrcene-penicillin combination [65]. Myrcene is a monoterpene present in different percentages in EOs isolated from hemp (Cannabis sativa L., Cannabaceae) (21%-35%) [69], hop (Humulus lupulus L., Cannabaceae) (6%-10%) [70], chameleon plant/Chinese lizard tail/fishwort/heartleaf (Houttuynia cordata Thunb., Saururaceae) (up to 30.8%) [71,72] and lemon grass (Cymbopogon citratus (DC.) Stapf., Poaceae) (3%-8%) [73]. The antagonism might be attributed to a competition between myrcene and penicillin for the same targets, namely penicillin-binding proteins [10].

Essential Oils and Volatile Compounds Modulating Antibiotic Activity against Klebsiella pneumoniae
Klebsiella pneumoniae is a rod-shaped, encapsulated, non-motile, facultative anaerobic and lactose-fermenting bacillus, an opportunistic pathogen, being asymptomatically present in the gastrointestinal tract, mouth, nasopharynx and skin of healthy persons [77,78]. This Gram-negative bacterium is associated with both community-acquired and nosocomial infections, such as severe forms of pneumonia, occurring mainly in alcoholic and diabetic patients [78], pyogenic liver abscess with severe extrahepatic metastasis, including septic endophthalmitis and meningitis [79], urinary tract infections [80], nosocomial pneumonia, wound and surgical site infections, peritonitis and endocarditis, followed by subsequent bacteremia [78,81]. K. pneumoniae virulence factors include the capsule polysaccharides, fimbriae, outer membrane lipopolysaccharide and proteins and siderophores [78]. The capsule polysaccharides express antiphagocytic functions and resistance to the lytic activity of the serum complement, being essential for bacterial pathogenicity [82]. The severity of K. pneumoniae infections is also due to the biofilm formation on both biotic and abiotic surfaces [78,82,83]. Over the last two decades, numerous reports have described K. pneumoniae carbapenemase-producing strains. The production of these extended-spectrum β-lactamases and modification of the outer membrane permeability (porin defects, upregulation of efflux pumps) are responsible for the emergence of multidrug-resistant K. pneumoniae phenotypes [84,85]. As carbapenemases are able to hydrolyze almost all β-lactam antibiotics and even β-lactamase inhibitors, carbapenem-resistant K. pneumoniae remains susceptible only to a few second-line antibiotics, such as colistin, tigecycline, fosfomycin, polymyxin B and gentamicin, administered alone or in combination [83,86]. Recently, colistin-resistant K. pneumoniae has been reported in Italian hospitals [87]. In the case of infections caused by carbapenem-resistant K. pneumoniae, the combination therapy including a carbapenem and a second-line antibiotic significantly improved the patient survival [85,88]. Selection of second-line anti-K. pneumoniae agents should also consider particular toxicity risks for patients (e.g., colistin is known for its nephrotoxic and neurotoxic effects, and gentamicin possesses the risk of nephrotoxicity) [85].

Essential Oils and Volatile Compounds Modulating Antibiotic Activity against Pseudomonas aeruginosa
Pseudomonas aeruginosa is an aerobic, rod-shaped, non-spore forming, oxidase positive and non-lactose fermenting Gram-negative bacterium [93]. It is a common agent for nosocomial infections being responsible for urinary tract, corneal, surgical and wound infections, but also for pneumonia and endocarditis. This pathogen is involved in chronic respiratory infections in cystic fibrosis patients. In addition, it is the second pathogen responsible for ventilator-associated pneumonia [94]. Bacteremia caused by P. aeruginosa leads to a mortality rate of 30%-50% in cancer patients who develop neutropenia after chemotherapy [95,96]. The pili, proteases, exoenzyme S, siderophores and alginate pseudocapsule are the main virulence factors of P. aeruginosa [97]. Amoxicillin, first and second generation cephalosporins are naturally inactive against P. aeruginosa. The most problematic aspect is the resistance of P. aeruginosa strains to many antibiotics. Piperacillin and ceftazidime resistance is explained by β-lactamase production, but strains resistant to quinolones and aminoglycosides have also been reported [95]. Besides the alteration of antibiotic target, another mechanism responsible for antibiotic resistance is the efflux pump overexpression [98].
Numerous studies have been conducted in order to assess if combinations between EOs and conventional antibiotics are able to increase the antibiotic susceptibility of P. aeruginosa strains ( Table 4). Strong synergistic effects have been reported for the combinations between basil (Ocimum basilicum L., Lamiaceae) EO and imipenem/ciprofloxacin against P. aeruginosa ATCC 25853 and the 1662339 clinical isolate. Both linalool, the main ingredient of basil EO (55.2%), and imipenem act on the same target, the bacterial cell membrane, whereas ciprofloxacin inhibits DNA gyrase and topoisomerase IV [99,100]. Thyme (Thymus vulgaris L., Lamiaceae) and marjoram (Origanum majorana L., Lamiaceae) EOs restored piperacillin susceptibility of P. aeruginosa ATCC 9027 (resistant to piperacillin, but susceptible to cefepime, meropenem, gentamicin and norfloxacin). Both EOs acted synergistically in combination with cefepime, meropenem and gentamicin. Thyme and marjoram EOs had high contents of oxygenated terpenes, thymol (33.6%) and 4-terpineol (21.3%), respectively. Weaker synergistic effects have been detected for sage (Salvia officinalis L., Lamiaceae) EO in combination with piperacillin, meropenem and gentamicin. 1,8-Cineole (29%) (Figure 1) was the major oxygenated terpene in sage EO [101]. Alecrim pimento (Lippia sidoides Cham., Verbenaceae) EO, rich in thymol (84.9%), reduced the MIC values of gentamicin and neomycin by four-and two-fold, respectively [102]. Phenolic terpenoids, such as thymol, alter the bacterial membrane, facilitating the antibiotic uptake. Moreover, thymol is distributed in the bilayer lipid membrane causing loss of K + and ATP [103]. Additional mechanisms involve the alteration of periplasmic proteins and the citrate metabolic pathway [104]. Monoterpene alcohols, such as 4-terpineol, also have antipseudomonal activity, as they inhibit the microbial oxygen uptake and oxidative phosphorylation. Terpenoid ethers (1,8-cineole) have a lower capacity to sensitize the pathogen to the antibiotic activity as they lack free hydroxyl groups, a structural feature present in both phenolic and alcoholic terpenoids [101,105]. Three Thymus saturejoides Coss. (Lamiaceae) EOs significantly increased the susceptibility of AmpC β-lactamase producing P. aeruginosa ATCC 27853 to cefixime. All EOs were characterized by high contents of carvacrol (25.3%, 26.5% and 45.3%) and borneol (19.7%, 20.1% and 7.5%) [76]. Other carvacrol-rich EOs, Thymus maroccanus L. and Thymus broussonetii L. (Lamiaceae) EOs, acted synergistically with ciprofloxacin and gentamicin against a P. aeruginosa clinical isolate. These synergistic effects are mainly due to carvacrol, a cytoplasmic membrane permeabilizer and an ATP, DNA and enzyme inhibitor [55][56][57]. Lippia gracilis Schauer. (Verbenaceae) EO, containing thymol (44.4%) and carvacrol (22.2%) as major constituents, significantly increased the susceptibility of P. aeruginosa ATCC 15442 to amikacin, tobramycin and gentamicin if the combinations were tested by direct contact. A marked difference was detected when tested in gaseous contact: high dilutions of Lippia gracilis EO (25%, 12% and 6%) increased the activity of gentamycin, but did not increase the activity of tobramycin. Tobramycin and gentamicin belong to the same class of antibiotics, but tobramycin is more polar than gentamicin, and it can hardly cross the lipid membrane. Moreover, in contrast with direct contact, in the case of gaseous contact, the interaction between bacteria and EO vapors is significantly influenced by the concentration of the latter [106]. It is possible that the high dilutions of Lippia gracilis EO used in the study were not enough to facilitate the entrance of tobramycin within the cell [107]. The susceptibility of P. aeruginosa ATCC 15442 to tobramycin, amikacin and gentamicin was increased by gaseous contact with Hyptis martiusii Benth. (Verbenaceae) EO. The synergistic effect with amikacin in gaseous contact was converted into an antagonistic one in the case of direct contact. A possible explanation would be related to the fact that the gaseous contact involves a series of qualitative and quantitative changes in EO composition. Unstable volatile constituents undergo degradation, generating new substances with different biological effects; for example, bicyclogermacrene converts into spathulenol, which increases the antibacterial activity of Hyptis martiusii EO [64]. Synergistic effects against the same β-lactamase producing strain (P. aeruginosa ATCC 27853) have been reported for geraniol, savory (Satureja kitaibelii Wierzb. ex Heuff., Lamiaceae) and lemon thyme (Thymus pulegioides L., Lamiaceae) EOs in combination with chloramphenicol or tetracycline. Geraniol is a major constituent in savory and lemon thyme EOs (50.4% and 66.59%, respectively) [60,74]. Immortelle (Helichrysum italicum (Roth) G. Don., Asteraceae) EO restored the susceptibility to chloramphenicol of two highly chloramphenicol-resistant P. aeruginosa strains (PAO1 and PA124). Immortelle EO decreased the MICs of chloramphenicol against PAO1 and PA124 by 16and eight-fold, respectively. These effects are undoubtedly due to the ability of geraniol, a constituent of EO, to inhibit efflux pumps responsible for drug resistance. As a result, geraniol increased the intra-bacterial concentration of antibiotics [37]. Nevertheless, lemon thyme EO acted antagonistically with streptomycin. Antagonism is often due to a competition for the same target. It is possible that some constituents in lemon thyme EO have the same target as streptomycin, namely the 30S ribosomal subunit [74]. P. aeruginosa ATCC 15442 is usually used to assess the effectiveness of disinfectants and antimicrobial preservatives. Gentamicin activity against this bacterial strain was significantly enhanced by gaseous contact with oxygenated sesquiterpene-rich EOs (Zanthoxylum articulatum, Vanillosmopsis arborea, Croton zehntneri EOs) [106,108,109]. Oxygenated sesquiterpenes (viridiflorol, α-bisabolol, trans-anethole) (Figure 1) are lipophilic compounds that accumulate into the phospholipid bilayers of bacterial membrane, causing its disruption, with a subsequent increase in the penetration of hydrophilic substances, such as gentamicin [110,111]. Viridiflorol exerts antibacterial activity through an additional mechanism, including interference with the respiratory chain reaction and energy production [106]. Antagonistic effects have been detected for Vanillosmopsis arborea EO in combination with tetracycline or tobramycin (gaseous contact). These effects could be attributed to chemical interactions between EO constituents and tetracycline/tobramycin, which led to antibiotic inactivation [108]. Similar results have been reported for Lippia microphylla EO, rich in 1,8-cineole (18.1%) and β-ocimene (15.2%) [112]. A strong synergistic effect against P. aeruginosa ATCC 15442 was recorded for amikacin in association with Lantana montevidensis Briq. (Verbenaceae) EO containing 31.5% β-caryophyllene (Figure 1). This non-polar compound might also affect the bacterial membrane and interfere with the mitochondrial respiratory chain reaction [113,114]. It is worthy to note eugenol (Figure 1), a volatile phenylpropanoid, which enhanced the activity of different penicillins, fluoroquinolones and macrolides against P. aeruginosa NCIM 5029. Bacterial cells treated with eugenol at a concentration of 1 mM had a 50% loss of membrane integrity. Therefore, eugenol synergized with penicillins, antibiotics targeting the bacterial wall. Acting as a membrane permeabilizer, eugenol facilitated the uptake of antibiotics that target ribosome (erythromycin, tetracycline, chloramphenicol, rifampicin) or DNA synthesis (norfloxacin). The synergistic effect could also be attributed to the intrinsic antimicrobial activity of eugenol. The hydroxyl group of eugenol is responsible for the inhibition of some enzymes, such as ATP-ase, histidine decarboxylase or proteases, thus impairing the bacterial metabolism [104,115].

Essential Oils and Volatile Compounds Modulating Antibiotic Activity against Other Gram-Negative Bacteria
Enterobacter aerogenes and E. cloacae are facultative anaerobic, rod-shaped and non-spore-forming Gram-negative bacteria with clinical significance, being responsible for hospital-acquired infections [116,117]. These infections are usually associated with a high fatality rate due to antibiotic resistance. E. aerogenes and E. cloacae are naturally resistant to aminopenicillins, and numerous clinical isolates exhibited resistance to other β-lactam antibiotics due to the production of extended-spectrum β-lactamases. Analysis of several E. aerogenes clinical isolates revealed resistance to quinolones, tetracycline and chloramphenicol due to the overexpression of efflux mechanisms [118]. Several EOs have been evaluated in order to assess their potential as antibiotic resistance modifying agents for E. aerogenes and E. cloacae strains ( Table 5). Thymus maroccanus L. and Thymus broussonetii L. (Lamiaceae) EOs decreased the MIC value of chloramphenicol against E. aerogenes ATCC 13048 and the EA27 clinical isolate (overexpressing AcrAB pump responsible for multidrug-resistance) by four-and eight-fold, respectively. The results were comparable to those obtained with PaβN. It seems that both EOs inhibited the efflux pumps [52]. For T. maroccanus EO, there has also been reported a membranotropic effect on Gram-negative bacteria involving the permeabilization of both inner and outer bacterial membranes, whereas polymyxin B permeabilizes only the outer membrane; no degradation of cellular constituents was detected [119]. Both thyme EOs acted synergistically with ciprofloxacin, gentamicin and pristinamycin against an E. cloacae clinical isolate [57]. Carvacrol, the main component of both EOs (76.35% and 39.77%, respectively), is known to increase the bacterial membrane permeability and inhibit ATP-ase activity [10], thus facilitating the activity of conventional antibiotics that act on different targets. Immortelle (Helichrysum italicum (Roth) G. Don., Asteraceae) EO increased the susceptibility to chloramphenicol of several E. aerogenes strains: E. aerogenes ATCC 13048 (susceptible to many antibiotics, but resistant to amoxicillin), E. aerogenes EAEP289 (overexpressing the AcrAB pump, but susceptible to kanamycin), E. aerogenes EAEP294 (having the AcrAB pump deleted, but expressing other efflux pumps involved in antibiotic resistance) and E. aerogenes CM-64 (overexpressing the AcrAB pump). It is worthy to note a significant reduction in the MIC of chloramphenicol against the E. aerogenes EAEP294 strain (128-fold reduction). Geraniol, a component of immortelle EO, has been reported to increase the susceptibility of E. aerogenes EAEP294 to other antibiotics (ampicillin, penicillin, norfloxacin) [37]. Obviously, the efflux pump inhibitory activity of geraniol plays an important role in the synergy between immortelle EO and antibiotics against E. aerogenes strains. Synergistic effects against E. aerogenes NCIM 5139 have been reported for eugenol in combination with antibiotics acting on different targets [115,120].
Proteus vulgaris is an aerobic and facultatively anaerobic member of the Enterobacteriaceae family. Urinary tract, burn, bloodstream, wound and respiratory tract infections are the most common infections caused by P. vulgaris. Adherence through pili or fimbriae and cytotoxic hemolysins are the most important pathogenicity factors of P. vulgaris [121,122]. P. vulgaris is naturally resistant to benzylpenicillin, oxacillin, macrolides and tetracycline. Acquired resistance has been reported for ampicillin, third-generation cephalosporins (ceftazidime, cefotaxime, ceftriaxone), but also for aztreonam, monobactam due to extended-spectrum β-lactamase production. P. vulgaris strains are usually susceptible to aminoglycosides and quinolones [123]. Eugenol has been reported to act synergistically in combination with several antibiotics against P. vulgaris NCIM 2813 (Table 5).     Salmonella spp. are motile Gram-negative bacteria. Ninety-nine-point-five percent of the pathogenic Salmonella strains belong to S. enterica subsp. enterica. Infections caused by Salmonella are treated with ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole, but multidrug-resistant strains have also been isolated. Fluoroquinolones, extended-spectrum cephalosporins [125] and azithromycin [126] have been used in order to treat multidrug-resistant S. enterica subsp. enterica serovar typhi (S. typhi). Synergistic effects between EOs or volatile compounds and antibiotics against Salmonella strains have been reported ( Table 5). The combinations between Thymus maroccanus EO and ciprofloxacin or cefixime, Thymus broussonetii EO and pristinamycin or cefixime exhibited total synergism against Salmonella sp. CCMM B 17 (laboratory collection) [57]. As carvacrol, the main component of both EOs, and nalidixic acid (at low concentrations) target the bacterial membrane; their combination significantly increased the susceptibility of nalidixic acid-resistant Salmonella strains (including S. typhimurium, S. derby, S. enteritidis, S. minnesota) [124]. Both carvacrol and thymol acted synergistically with different antibiotics (ampicillin, penicillin, tetracycline, erythromycin, bacitracin, novobiocin) against tetracycline-resistant S. typhimurium SGI1, whereas eugenol acted synergistically only with tetracycline and novobiocin [66]. In a study conducted by Hemaiswarya et al. [115], eugenol was found to increase the antibiotic susceptibility of S. typhimurium NCIM 2501. Allyl isothiocyanate (Figure 1), a constituent of EOs isolated from Brassicaceae species [127], acted synergistically in combination with ampicillin, erythromycin and bacitracin against S typhimurium SGI1 [66]. Allyl isothiocyanate targets the same bacterial element as ampicillin and bacitracin: the cell wall [128]. Besides, the central highly electrophile carbon atom of allyl isothiocyanate is a structural pattern that induces the damage of cytoplasmic proteins [104]. Proteic damage caused by allyl isothiocyanate could explain the synergism with erythromycin (inhibitor of protein synthesis through 50S ribosomal subunit binding) [10].

Conclusions
In conclusion, EOs and their components can enhance the antibiotic activity against Gram-negative bacteria. Most volatile compounds disrupt the bacterial membrane, thus facilitating antibiotic penetration. Enzyme inhibition, reduction of ATP and DNA synthesis and alteration of metabolic pathways are other mechanisms by which EOs and pure volatile compounds act in synergy with conventional antibiotics against Gram-negative bacteria. By inhibiting efflux pumps, volatile compounds, such as geraniol, restore the antibiotic susceptibility of Gram-negative-resistant strains. The mechanisms of synergy of EOs with conventional antibiotics are very complex and still not completely understood as they involve multiple interactions between individual EO constituents, on the one hand, and individual EO constituents and antibiotics, on the other hand. EOs usually possess different antibacterial effects than their major components, which suggests synergistic, additive or antagonistic interactions between their constituents and also an undoubted contribution of minor components to the antibacterial activity. For example, eucalyptus and lavender EOs showed higher antibacterial effects against Staphylococcus aureus, Bacillus subtilis and Escherichia coli compared to their major components, 1,8-cineole and linalyl acetate, respectively. On the contrary, thyme EO was less active against the same bacterial strains than its main constituent, thymol [129]. Both thymol and carvacrol have been detected in EOs isolated from certain Lippia, Thymus and Origanum species [57,76,107,[130][131][132][133]. Surprisingly, these two monoterpene phenols might modulate the antibiotic activity in a different manner. For instance, thymol showed excellent synergistic effects with penicillin against Escherichia coli (resistant to some β-lactam antibiotics), whereas the combination of carvacrol with penicillin resulted in an indifferent effect [65]. The complexity of interactions generated when combining EOs and antibiotics is evident. Overall, the results of the in vitro assays are very promising for the development of new effective formulations against Gram-negative bacteria. Obviously, further in vivo studies are required to evaluate the bioavailability, efficacy and toxicity of EO/volatile compound-antibiotic combinations.