Antibiofilm Potential of Natural Essential Oils

Abstract

Commonly available essential oils (EOs) are pure aromatic substances derived from natural plant sources (fresh or dried raw materials), with broad biological activity, including antimicrobial activity. The activity of EOs is of great interest due to the serious problem of diseases caused by microorganisms. Pathogenic microorganisms (PAMs) show increased resistance to physical and chemical factors due to their association with a form of specific biological membrane called biofilm (BF), which is resistant to extreme conditions and significantly hinders effective therapy. The decreasing effectiveness of antibiotics, combined with the increasing resistance of microorganisms, has prompted the search for alternative antimicrobial (AM) therapies. EOs and some of their components are currently considered as potential agents useful in the prevention, treatment, and control of infections transmitted by microbial BF. In combination with antibiotics, EOs can prevent the transfer of resistance to AM agents due to the synergistic antibiofilm (ABF) effect. BF inhibition by EO is not based on killing bacterial cells but on the inhibition of the quorum sensing (QS) pathway. EOs also affect growth regulation, nutritional balance, and energy conversion in bacteria. It can be assumed that this group of substances will be of significant importance in the treatment of infectious diseases in the near future. This article reviews the results of the latest research on essential oils and their main components as potential factors limiting/inhibiting the development of PAMs.

1. Introduction

The human body is a preferred host for multiple pathogens and poses the risk of developing infectious diseases. Despite the enormous progress in diagnostics, treatment, and prevention, infectious diseases still remain the leading cause of death worldwide, especially in developing countries [1]. The major causes of these diseases include biological factors (bacteria, viruses, fungi, and protozoa) and substances they produce (bacterial exotoxins and endotoxins) [2]. Some bacterial infections are mild and resolve spontaneously without the need for specific AM treatment. In many cases, however, the course of infections of this etiology is rapid and very dynamic [3]. The tendency of increasing bacterial resistance, which is becoming a large-scale health issue worldwide, entailing an increase in morbidity and mortality, is currently a problem of utmost clinical significance. The WHO predicts that in the near future, bacteria such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, as well as Salmonella spp., will exhibit a high degree of resistance with little or no treatment options [4]. This article presents a review of the latest research on the current development of infectious diseases caused by PAMs, including antibiotic-resistant strains, the role of EOs, and their main components as potential agents limiting or inhibiting PAM development. In the review, special attention was paid to BF, a specific structure produced by many PAMs, which is considered one of the probable causes of antibiotic resistance. Furthermore, not only was the role of essential oils as potential AM and ABF agents highlighted, but their main components may also prove helpful in this regard. Scientific information was obtained by searching available databases to review and consolidate current research on infectious diseases, their causative agents, the therapeutic role of natural remedies, including EOs, and possible mechanisms of their action.

2. Biofilm: The Role in the Pathogenesis of the Disease

Microorganisms (bacteria, fungi, protozoa) become increasingly resistant to physical and chemical factors due to their association in the form of a specific biological membrane called a biofilm (BF). BFs are ubiquitous in the natural environment. They are formed by bacteria in natural and industrial systems as part of their survival strategy, which protects them against AM chemical substances, environmental bacteriophages, and phagocytes. BFs are resistant to extreme conditions and are capable of protecting microorganisms against UV radiation, high pressure, or nutrient deficiency [5]. They represent a complex, cross-linked structure, the components of which affect many of their properties, like hydrophobicity and reduced gas permeability [6]. BF formation is regulated by genetic and environmental factors, and the main stages of this process include the attachment of microorganisms to a solid surface, enhanced production of the extracellular polymeric substance (EPS), and BF detachment (Figure 1 and Figure S1). The balance between BF attachment, growth, and detachment is important in the formation and maintenance of a functional community [7].

In a BF, microbial cells are connected to each other by EPS containing polysaccharides (the largest fraction), proteins, nucleic acids, surfactants, lipids, and water [8]. BFs are of great importance to public health as their development is one of the key mechanisms of bacteria resistance to AM agents [6]. According to data from the National Institutes of Health, USA, BFs are responsible for almost 80 types of infections induced by microorganisms and 65 types of hospital infections [9]. The infections caused by BF-producing bacteria include such conditions as, e.g., cystic fibrosis, otitis media, infectious endocarditis, and chronic inflammatory diseases [10]. Bacterial BFs are found on wound surfaces, are implicated in wound healing failure, and contribute to the chronic inflammatory condition. Pseudomonas aeruginosa can be a major contributor to wound inflammation due to the production of rhamnolipids [11]. Recent meta-analyses have shown the likely involvement of BFs in the pathogenesis of atherosclerosis. The presence of BF has also been speculated to contribute to an increased risk of atherosclerotic plaque rupture [12]. Bacteria and fungi have the ability to estimate their own density in a given environment due to the quorum sensing phenomenon (QS), which is a process of chemical communication between bacterial cells, affected by the bacterial population density and mediated by small signaling molecules called autoinducers. Microorganisms synthesize and secrete signaling substances into the environment. One of the tasks of these substances is to coordinate the control of the expression of genes involved in BF formation. The traits of bacteria regulated by QS include, among other things, virulence, BF formation, sporulation, genetic competence, and bioluminescence [13]. QS allows the bacteria to monitor the environment in terms of the presence of other bacteria and to adjust their behavior at the entire population scale in response to changes in the number and/or species composition of a bacterial community [14]. The QS phenomenon may occur in both Gram-positive and Gram-negative bacteria as well as in fungi (e.g., Candida albicans) [6,15].

Biological Profile of Biofilm

BF is formed by a multicellular community, often heterogenous in terms of species and featuring a spatial character. It may include from one to several, or even a dozen different species. This structure is formed not only within representatives of the same kingdoms, as interspecific and intergeneric BFs are also likely, e.g., S. mutant and C. albicans [16]. Poyrazoğlu Çoban and Onur [17] have isolated 67 bacteria from food products sold at the market, of which seven were strong and two were moderate BF producers (Enterococcus faecalis, Bacillus cereus, C. freundii, S. epidermidis, Pantoea conspicua, E. gallinarum, and E. faecium, respectively). BFs may serve positive functions (as an example, S. epidermidis, a commensal organism preventing the colonization of pathogenic bacteria); however, most of them are implicated in triggering infections and diseases [18]. Coagulase-negative staphylococci, E. coli, S. aureus, P. aeruginosa, and Enterobacteriaceae are mentioned as common pathogens involved in BF-borne infections, including in particular S. aureus [10]. The emergence of S. aureus bacteria resistant to antibiotics, including especially methicillin (MRSA), has urged the need for identifying new compounds potent to inhibit this clinically important pathogen [19]. Another example of a microorganism capable of BF formation is P. aeruginosa, an opportunistic pathogen often involved in respiratory tract infections (pneumonia, chronic obstructive pulmonary disease, cystic fibrosis) [20]. It is an important causative agent of hospital infections associated with BF, often difficult to treat due to its innate resistance and ability to undergo mutations and adaptations [21]. The ability to produce BF affords important advantages, including resistance to host defense mechanisms, antibiotics, and disinfectants [20]. Due to the ability to grow in the form of BF, bacterial uropathogens are able to effectively and persistently colonize the tissues of a macroorganism, as well as adhere to many polymers that are components of urological catheters. The analyses of BF derived from urological catheters [22] have demonstrated frequent prevalence of Edwardsiella, Moraxella, Yersinia, Burkholderia, Corynebacterium, Achromobacter, Citrobacter, or Stenotrophomonas. All of the above-mentioned groups of microorganisms are capable of growing in the form of BF, which is one of the main virulence factors responsible for dangerous disease states and severely limiting effective urological therapy [22].

3. New Natural AM Strategies

The decreasing efficacy of antibiotics in the face of increased microbial resistance has led to the search for alternative AM therapies, including antiviral drugs, i.e., molecules that can affect virulence factors and not cell growth. BF inhibition would boost the efficacy of antibiotics and would make pathogens more sensitive to the immune response of the host. Novel therapeutic approaches are focused on preventing the synthesis or breaking down constituents of the BF matrix, thereby making the bacteria more susceptible to antibiotic therapy [23,24]. Various natural products, like lantibiotics (nisin, subtilin, epidermin), AM peptides (LL-37, Burford-II, PR-39), plant-derived active substances (tannins, flavonoids, flavones, flavonols), bacteriophages and enzymes (DNases, depolymerases, lactonases, and bacteriophage-based endolysins), have been extensively investigated for their BF-forming capability [18]. Natural products derived from plants, bacteria, fungi, and marine organisms have been shown to be effective as anti-BF (ABF) agents by inhibiting the formation of a polymeric matrix, cell adhesion and attachment, and the production of virulence factors, acting as QS inhibitors. More than 90 ABF compounds from the groups of alkaloids, polyphenols, terpenes, and EOs have been identified in different morphological parts of plants [25,26,27]. Some studies have reported, among others, the capability of eugenol (EUG), garlic, and phenolic extract from Rubus rosifolius Smith for attenuating BF formation by P. aeruginosa and Serratia marcescens [15]. Sulfated polysaccharides (fuoidan), carotenoids (zeaxanthin and lutein), lipids, fatty acids (γ-linolenic acid and linoleic acid), and phlorotannins can inhibit cell attachment, reduce cell growth, impede QS by blocking associated enzymes, and disrupt extracellular polymeric substances [27]. Vikram et al. [28] have indicated the potential modulation of bacterial cell-to-cell communication, BF formation by E. coli O157:H7, and virulence of Vibrio harveyi by flavonoids, including in particular naringenin, quercetin, sinensetin, and apigenin. Among the flavonoids tested, naringenin turned out to be a strong and, probably, non-specific inhibitor of autoinducer-mediated cell-to-cell signaling. Maastor et al. [19] demonstrated that salicylaldehyde, vanillin, α-methyl-trans-cinnamic aldehyde, and trans-4-nitrocinnamic acid were potent (15–92%) inhibitors/reducers of BF of drug-resistant S. aureus isolates at a concentration of 1–10 mg/mL. They also emphasized the significant suppression of gene expression by phenylpropenes and phenolic aldehyde, which can inactivate bacterial adhesion and reduce BF-formation by S. aureus. These molecules may attenuate virulence factors and serve as potential AM and ABF drugs [19].

4. ABF Activity of EOs and Their Components

4.1. EOs as Antimicrobial Agents (Mechanisms of Action)

EOs with strong and multi-faceted biological activity are considered as potential alternatives in clinical settings for the prevention, treatment, and control of infections transmitted by microbial BF. Most EOs modify the bacterial cell wall or membrane, causing the release of lipopolysaccharides, which alters the ATP balance and influences pH fluctuations, protein synthesis, and internal cytoplasmic changes, such as coagulation of cytoplasmic material, DNA disruption, and QS inhibition. Khan et al. [29] showed that Tagetes minuta EO (TMEO) significantly inhibited virulence factors associated with QS sensing, such as pyocyanin production, protease production, and swarming motility. TMEO proved to be relatively effective in inhibiting BF even at a very low concentration (20 μg/mL), confirming that BF inhibition by EO is not based on killing bacterial cells but on the inhibition of the QS pathway. EOs also affect growth regulation, nutritional balance, and energy conversion in bacteria [23]. It should be noted that BF remains in a continuous balance between accumulation and degradation and is exposed to the action of various intra- and extracellular factors. The same EO can act synergistically or anti-synergistically, stimulating or inhibiting BF development [20]. Moreover, attention should be paid to the variability of the chemical composition and biological activity of EOs caused by various factors [30].

Microorganisms are destroyed by various compounds administered in different concentrations. The minimum inhibitory concentration (MIC) is the lowest concentration (in μg/mL) of an agent that inhibits the growth of a given strain of bacteria. The sub-minimum inhibitory concentration (sub-MIC) is defined as concentrations lower than the MIC values. Many studies have shown that sub-MICs of antibiotics can act as signal molecules and may alter their physicochemical characteristics and expression of bacterial virulence. Some scientific evidence confirms the high antibacterial and ABF activity of EOs (Table 1). The most commonly studied in this respect are clove, thyme, tea tree, oregano, and citrus fruit essential oils. They are highly effective against some viruses, whereas EO mixtures are effective against influenza and herpes simplex viruses [23].

Thymus vulgaris EO (TEO) exhibits strong AM and ABF activity against Haemophilus influenzae, H. parainfluenzae, and P. aeruginosa [30], which has also been confirmed by Myszka et al. [31], who demonstrated the potential of TEO and its components against QS and BF using a model strain P. fluorescens KM121. When applied in concentrations slightly below the sub MIC of the tested TEO (20.0 μL/mL), sub-MIC of carvacrol (2.0 μL/mL), and sub-MIC of thymol (4.0 μL/mL) caused significant reductions in the production of QS autoinducers (90%, 80%, and 78%, respectively), significantly inhibited bacterial motility, and reduced mRNA level of flagella gene (−0.672, −0.776, and −0.576 for TEO, carvacrol, and thymol, respectively). The authors posited TEO as a potential biocide against food-spoiling BF produced by P. fluorescens. THM-rich chemotype of TEO (TH > 50%) can be considered an effective AM and ABF agent in the in vitro experiments. It is worth emphasizing that this is the first report linking the anti-QS properties of TEO, thymol, and carvacrol with the inhibition of both the flagella gene expression and the BF formation capacity of the P. fluorescens KM121 strain. In addition, Proškovcová et al. [32] considered TEO and Origanum vulgare L. EO to be effective antifungal agents in the case of planktonic cells at MIC 0.4 mg/mL and potential therapeutic or preventive agents due to their capability to reduce pathogen resistance.

Table 1. Antimicrobial and antibifilm activity of essential oils (selected examples).

Essential Oil Source	Pathogenic Microorganism	MIC	Reference
Croton conduplicatus Kunth (Euphorbiaceae)	S. aureus ATCC 25923 (MSSA)	256 μg/mL	[33]
	S. aureus ATCC 33591 (MRSA)	512 μg/mL	[33]
Foeniculum vulgare Mill. (Apiaceae)	S. aureus ATCC 29213 (MSSA)	71.3 ±1.90 mg/mL	[34]
	S. aureus ATCC 43300 (MRSA)	60.30 ± 0.00 mg/mL	[34]
	S. aureus ATCC 6538 (MSSA)	100.4 ± 34.80 mg/mL	[34]
Hyssopus officinalis L. (Lamiaceae)	C. albicans	0.9 ± 0.3 mg/mL	[32]
Origanum vulgare L. (Lamiaceae)	C. albicans *	0.4 mg/mL	[32]
	S. aureus *	5 μL/mL	[35]
	S. aureus **	10 μL/mL	[35]
Rosmarinus officinalis L. (Lamiaceae)	S. aureus *	0.04% (v/v)	[36]
	S. aureus ATCC 9144	1.25–2.5 μL/mL	[37]
	S. epidermidis S61	0.312–0.625 μL/mL	[37]
Tagetes minuta L. (Asteraceae)	P. aeruginosa PAO1	312.5 μg/mL	[29]
Tetraclinis articulata (Vahl) Masters (Cupressaceae)	S. aureus ATCC 25923	0.38 nL/mL	[38]
Thymbra capitata L. (Lamiaceae)	P. aeruginosa (BL)	1.11%	[39]
	P. aeruginosa 10145	1.11%
Thymus vulgaris L. (Lamiaceae)	P. fluorescens KM121	20.0 μL/mL	[31]
	S. aureus *	0.02% (v/v)	[36]
	S. aureus **	0.63% (v/v)	[36]
	Candida albicans *	0.4 mg/mL	[32]
	Haemophilus influenzae	0.156 mg/mL	[30]
	H. parainfluenzae	0.156 mg/mL	[30]
	P. aeruginosa	1.50 mg/mL	[30]
Zataria multiflora Boiss. (Lamiaceae)	P. aeruginosa **	4 μL/mL	[40]
Zingiber officinale Roscoe (Zingiberaceae)	S. mutans	21.25 μL/mL	[41]

MIC—Minimum Inhibitory Concentration; MSSA—methicillin-sensitive S. aureus; MRSA—methicyllin-resistant S. aureus; BL—beta lactamase producing. * planktonic cells; ** biofilm cells.

Table 1. Antimicrobial and antibifilm activity of essential oils (selected examples).

Essential Oil Source	Pathogenic Microorganism	MIC	Reference
Croton conduplicatus Kunth (Euphorbiaceae)	S. aureus ATCC 25923 (MSSA)	256 μg/mL	[33]
	S. aureus ATCC 33591 (MRSA)	512 μg/mL	[33]
Foeniculum vulgare Mill. (Apiaceae)	S. aureus ATCC 29213 (MSSA)	71.3 ±1.90 mg/mL	[34]
	S. aureus ATCC 43300 (MRSA)	60.30 ± 0.00 mg/mL	[34]
	S. aureus ATCC 6538 (MSSA)	100.4 ± 34.80 mg/mL	[34]
Hyssopus officinalis L. (Lamiaceae)	C. albicans	0.9 ± 0.3 mg/mL	[32]
Origanum vulgare L. (Lamiaceae)	C. albicans *	0.4 mg/mL	[32]
	S. aureus *	5 μL/mL	[35]
	S. aureus **	10 μL/mL	[35]
Rosmarinus officinalis L. (Lamiaceae)	S. aureus *	0.04% (v/v)	[36]
	S. aureus ATCC 9144	1.25–2.5 μL/mL	[37]
	S. epidermidis S61	0.312–0.625 μL/mL	[37]
Tagetes minuta L. (Asteraceae)	P. aeruginosa PAO1	312.5 μg/mL	[29]
Tetraclinis articulata (Vahl) Masters (Cupressaceae)	S. aureus ATCC 25923	0.38 nL/mL	[38]
Thymbra capitata L. (Lamiaceae)	P. aeruginosa (BL)	1.11%	[39]
	P. aeruginosa 10145	1.11%
Thymus vulgaris L. (Lamiaceae)	P. fluorescens KM121	20.0 μL/mL	[31]
	S. aureus *	0.02% (v/v)	[36]
	S. aureus **	0.63% (v/v)	[36]
	Candida albicans *	0.4 mg/mL	[32]
	Haemophilus influenzae	0.156 mg/mL	[30]
	H. parainfluenzae	0.156 mg/mL	[30]
	P. aeruginosa	1.50 mg/mL	[30]
Zataria multiflora Boiss. (Lamiaceae)	P. aeruginosa **	4 μL/mL	[40]
Zingiber officinale Roscoe (Zingiberaceae)	S. mutans	21.25 μL/mL	[41]

MIC—Minimum Inhibitory Concentration; MSSA—methicillin-sensitive S. aureus; MRSA—methicyllin-resistant S. aureus; BL—beta lactamase producing. * planktonic cells; ** biofilm cells.

The efficacy of EOs in combating BF-related infections in chronic wounds is often assessed using standard microbiological media and conditions that do not reflect the actual chronic wound environment. To address this problem, Brożyna et al. [36] cultured S. aureus BF in the In Vitro Wound Milieu (IVWM) medium, which closely resembles the chronic wound environment. The BF cultured in a standard Tryptic Soy Broth (TSB) medium served as a control. The cultured bacterial strains were exposed to TEO and Rosmarinus officinalis EO (REO), and a larval model (Galieria mellonella L.) was deployed to assess TEO cytotoxicity and AM activity in vivo. The conducted analyses showed that the key characteristics of BF-forming cells (biomass, metabolic activity, cell number, and live/dead cell ratio) were poorer in BF from the IV medium than in that from the TS medium. Conversely, BF thickness was greater (up to 25%) when the BF was cultured in the IV medium. These differences resulted in various BF responses to EO exposure. The use of TEO led to a greater (up to 2-fold) reduction of 67% of the BF-forming strains in the IV medium compared to the TS medium, while biofilm exposure to REO caused a greater reduction (up to 2.6-fold) of 83% of the BF-forming strains in TS than in the IV medium. TEO was not only non-toxic to G. mellonella larvae, but also increased the survival rate of larvae infected with staphylococci (from 48 to 85%). These results suggest that EOs are promising agents for the treatment of BF-related wound infections. Abdullah et al. [42] demonstrated the AM and ABF effects of the Elletaria cardamomum L. EO (ECEO) containing α-terpinyl acetate (34.95%) and eugenol (25.30%). The EO used in the 1% concentration inhibited the growth of E. coli O157:H7 and P. aeruginosa ATCC 14213. The evaluation of the ABF activity showed that the EO concentration of 0.015, 0.031, 0.062, and 0.125% caused 64.29, 65.98, 70.41, and 85.59% inhibition of BF formation by E. coli and 6.13, 45.50, 49.45, and 100 inhibition of BF formation by S. Typhimurium, respectively. The authors pointed out that ECEO could be used as a safe natural AM agent inhibiting microbial BF and counteracting multidrug-resistant microorganisms, as well as a safe alternative to chemical agents in the food industry. In turn, Achmit et al. [38] showed the AM and ABF effects of an EO from Tetraclinis articulata (Vahl) Masters sawdust (containing 34.37% carvacrol and 11.07% cedrol). Nine strains were tested, including eight clinical isolates from catheter-related infections and one ATCC 25923. The analyzed EO showed strong AM activity against all strains, including one multidrug-resistant (MDR) S. aureus strain and two methicillin-resistant S. aureus (MRSA) strains, as well as BF-inhibiting activity.

4.2. EOs Compounds as Antimicrobial Agents

4.2.1. Phenolic Compounds

Phenolic compounds (PCs) are widespread secondary metabolites with strong biological activity [43]. The phenolic components of EOs (carvacrol, eugenol, and thymol) (Figure 2) affect the cytoplasmic membrane, electron flow, proton forces, active transport, and coagulation of cell contents. EOs are complex mixtures of different active compounds that exert additive, antagonistic, or synergistic effects on each other. The identification of the mechanism of EO action depends on its chemical composition [23]. ABF activity has also been demonstrated for eugenol, which was found, among others, in clove oil. It can inactivate enzymes, react with the cell membrane, and disrupt the functionality of genetic material, energy production, and synthesis of structural components. Also, it inhibits BF formation in the intermediate and maturation phases, and eliminates BF formed by a group of microorganisms (S. aureus, E. coli, P. aeruginosa, and C. albicans). In addition, eugenol shows a concentration-dependent ABF activity in single and mixed BFs formed by drug-resistant strains (C. albicans and S. mutans) [44], and has been shown to be active against planktonic cells and BF of Vibrio parahaemolyticus, a widespread foodborne pathogen that can cause severe gastroenteritis [45]. Studies by Kim et al. [46] have indicated that eugenol at a concentration of 0.005% (v/v) significantly inhibits BF formation by E. coli O157:H7 (EHEC) without suppressing the growth of planktonic cells. EHEC forms BFs tolerant to AM agents and is a causative agent of serious health problems worldwide. Similar effects were also observed for eugenol derivatives: isoeugenol, 2-methoxy-4-propylphenol, and 4-ethylguaiacol. These findings indicate that the C-1 hydroxyl unit and the C-2 methoxy unit, as well as the C-4 alkyl or alkane chain in the benzene ring of eugenol, play an important role in the ABF activity. Transcription analysis has shown that eugenol downregulated 17 of the 28 genes analyzed, including those required for BF formation (csgABDFG, fimCDH, espD, escJ, escR, and tir). In the nematode Caenorhabditis elegans model, clove oil and eugenol attenuated the virulence of EHEC [46]. The complexity of the structure of individual EOs components may be one of the main reasons for their effective AM and ABF activity and, ultimately, the effective therapeutic effect of EOs.

4.2.2. Terpene Compounds

Terpenes have been found to exert antimicrobial effects on antibiotic-sensitive and resistant bacteria, mainly through their ability to promote cell rupture and inhibit protein and DNA synthesis [47]. Linalool and eucalyptol (Figure 2) have been reported to reduce BF-formation in both reference and clinical isolates, S. aureus and P. aeruginosa [48,49]. Merghni et al. [50] demonstrated the anti-staphylococcal activity and antibacterial efficacy of eucalyptol, in which the minimum inhibitory concentrations were equal to 7.23 mg/mL. This compound also enhanced membrane permeability, with a 5.36-fold increase in nucleic acid and protein leakage compared to untreated strains, and caused an increase in ROS generation along with a decrease in the activity of oxidative stress enzymes. The authors suggest that eucalyptol has the ability to damage membrane integrity and induce ROS-mediated oxidative stress in MRSA cells, which may be helpful in reversing antibiotic resistance. Other studies [15] indicate that limonene at a concentration of 0.1% mg/mL caused 41% inhibition of BF formation by P. aeruginosa and 33% inhibition of AHL signaling. Artini et al. [20] evaluated the modulation of BF growth exerted by 61 commercial EOs and found that limonene and other hydrophobic components (α-pinene and p-cymene) could act as enhancers (either positive or negative) for other EO components. Linalool was found to be the component most likely involved in the strong inhibition of BF formation, followed by eucalyptol, linalyl anthranilate, geranyl acetate, bornyl acetate, cis-geraniol, sabinene, and cis-3-pinanone. LIN and geranyl acetate were the major compounds of the palmarosa EO, which showed a strong BF-reducing potential (Figure 2). Furthermore, when combined mainly with eucalyptol and geranyl acetate, linalool present in other EOs with a stronger BF-reducing capability probably exhibited synergistic action [20]. β-caryophyllene, α-pinene, limonene, and p-cymene have been found important in diminishing BF formation, but could also negatively affect its inhibition. In turn, β-pinene and carvacrol have been demonstrated to only negatively modulate BF inhibition [20]. Cheruvanachari et al. [51] evaluated the AM and ABF activities of terpinen-4-ol against S. aureus and Klebsiella pneumoniae, and their reference strains MTCC-740 and MTCC-109. The minimum inhibitory concentrations (MICs) of terpinen-4-ol against S. aureus and K. pneumoniae were determined at 50 and 25 mM, respectively. At the MIC level, terpinen-4-ol showed AM activity against both reference strains, whereas at the sub-MIC concentration (i.e., ½ MIC), it caused a significant reduction (at the level of 67.51 ± 1.29%) in exopolysaccharide (EPS) synthesis and a significant reduction in BF thickness. Based on these findings, the authors considered terpinen-4-ol as a potential drug candidate in combating chronic infections associated with BF and drug resistance.

5. Synergistic Effect of EO and Antibiotics

Bacteria in biofilm undergo phenotypic changes that make them several-fold more resistant to antimicrobials. Owing to the heterogeneous nature of the BF, it is likely that there are multiple resistance mechanisms at work within a single community [52]. Combining existing antimicrobials with any ingredient (synergistic interaction), which can be a compound, a mixture of compounds (EOs), an antibiotic, antimicrobial peptides, and even any other antimicrobial agent, can be an effective form of neutralization of the resistance mechanism. There are many synergistic combinations used to define MIC and to prevent drug resistance, as well as to reduce toxicity. An example is flavon baikalin, which reduced MIC of ampicillins, methylicins, and cefotaxis in S. aureus (MRSA) resistant to many drugs, as well as in penicillin-resistant S. aureus [53]. Shareef et al. [54] found various interactions (synergistic and indifferent) between plant extracts and antibiotics. The best synergistic ability was demonstrated between erythromycin and sesame EO. MIC values of each antibiotic were reduced by half when used in combination with the tested plant extracts. Therefore, comprehensive actions seem to be particularly interesting, especially when using multi-component products such as EOs. EOs in combination with antibiotics can prevent the transfer of resistance to AM agents due to the synergistic ABF effect [23]. In turn, certain phytonutrients can influence the formation and development of bacterial BF by acting as factors controlling bacterial adhesion and QS [55]. Due to their amphipathic nature, menthol, thymol, and eucalyptol can pervade the exopolysaccharide matrix of BF and enhance the antibiotic’s activity by disrupting membrane permeability. Dominant synergy was observed for mixtures of mupirocin and menthol or thymol, whereas the mupirocin-eucalyptol mixture exerted a neutral or additive effect on the inhibition of BF formation by S. aureus. Monoterpenes probably inhibited Staphylococci adherence, allowing mupirocin diffusion into bacteria. Considering the anti-inflammatory properties of monoterpenes, mupirocinmonoterpene combinations could have a dual benefit of reducing both BF growth and inflammation. Monoterpenes applied in sub-inhibitory concentrations prior to BF formation could interact with staphylococcal surface proteins, compromising the initial attachment phase as well as interfering with the QS system [48]. Asadi et al. [56] demonstrated the potential AM and ABF activities of carvacrol as well as its synergistic interaction with an antibiotic (cefixime) against E. coli. Carvacrol and cefixime significantly inhibited BF formation at MIC/2 (125 and 62.5 µg/mL), MIC/4 (62.5 and 31.25 µg/mL), and MIC/8 (31.25 and 15.625 µg/mL), respectively. Coelhoi and Pereira [21] evaluated the ABF activity of three EOs: cinnamon EO, tea tree EO (TTO), and palmarosa EO, and the synergistic effect of TTO and its major component terpinen-4-ol in combination with ciprofloxacin (CIP) against 24 h BF formed by P. aeruginosa. They showed that P. aeruginosa BF treatment with the EOs, T4ol, and CIP individually promoted a significant reduction in BF biomass and cell count in BF (mainly in the case of the higher concentrations tested). The AM synergism of TTO or T4ol with CIP proved to be more effective, causing a significant disruption of BF at lower concentrations. The enhancement of CIP anti-BF activity with TTO could be explained by a multi-target effect (cytoplasmic membrane, DNA synthesis inhibitors) or the reversal of antibiotic resistance, inhibiting active efflux pumps. In the authors’ opinion, the combination of terpinen-4-ol and CIP may offer a useful therapeutic option in the treatment of infections associated with P. aeruginosa BF.

The synergistic inhibitory effects between EOs and drugs would aim to reduce the side effects of antibiotic therapy, such as decreased immunity, inflammation, diarrhea, mycosis, and thrush. Rosato et al. [57] studied the ABF activity of Cinnammonum zeylanicum, Mentha × piperita, O. vulgare, and T. vulgaris EOs on S. aureus ATCC 29213, E. faecalis ATCC 29212, S. epidermidis IG4, S. aureus IG22 BF, and synergistic effect in combination with antibiotic drugs (norfloxacin, oxacillin, and gentamicin). The association of EOs-antibiotics showed a strong destruction of the BF growth of the four bacterial species considered, wherein the interaction of norfloxacin with EOs was the most effective. The authors demonstrated that the amount of antibiotic can be significantly reduced by EOs, thus adding to the achievement of the desired therapeutic effects. Furthermore, they noted the large reduction in the quantities of EOs employed to attain the association with respect to the quantity of the EOs used alone to inhibit the strain. Salvagno et al. [58] report that there is a potential synergistic effect between M. × piperita EO (MPEO) and three widely used antibiotics (gentamicin, oxacillin, norfloxacin) on S. aureus ATCC 29213, E. faecalis ATCC 29212, S. epidermidis IG4, S. aureus IG22 BFs formation and growth. All the combinations of MPEO with antibiotics are able to produce a substantial inhibition of bacterial BF growth. Kwiatkowski et al. [34] found reduced MIC values for Foeniculum vulgare Mill. EO (FEO) in combination with H₂O₂ compared to FEO alone in BF formation by reference S. aureus strains. The coupled use of FEO and H₂O₂ exerted synergistic effects on all reference strains (MSSA and MRSA). Sub-inhibitory concentrations of FEO (0.5 MIC and 0.25 MIC) alone and in combination with 0.5 MIC H₂O₂ significantly inhibited BF formation by S. aureus and suppressed the metabolic activity of the attached cells. It seems that the combination of FEO containing 80% trans-anethole and H₂O₂ affords potential possibilities for trial use in wound treatment. EOs-drug mixtures can be a good option for topical applications. Topical antiseptics seem to be more beneficial compared to systemic antibiotics because they do not cause multidrug resistance, have a broader spectrum of AM activity, and are less likely to cause allergic reactions [50]. In turn, Oliveira et al. [33] investigated the possible synergism of EOs and antibiotics against S. aureus, using EO from Croton conduplicatus Kunth (EOCC), oxacillin (OXA), and ampicillin (AMP). MIC values of EOCC were 256 and 512 μg/mL for methicillin-sensitive and resistant S. aureus strains (MSSA and MRSA), respectively. The combinations of EOCC with OXA and AMP were synergistic (OXA/EOCC and AMP/EOCC combined decreased the OXA MIC and AMP MIC to 0.5 and 0.25 for MSSA, respectively, and OXA/EOCC and AMP/EOCC combined decreased the OXA MIC and the AMP MIC to 1 and 0.5 for MRSA, respectively) and could modify the resistance profile of MSSA and MRSA strains. The combinations of EOCC with OXA and AMP were able to reverse resistance to these antibiotics, indicating that at concentrations lower than MIC, OXA and AMP became active against the strains when combined with EOCC. The results indicated that EOCC was also able to partially inhibit biofilm formation by damaging the cell membrane with extravasation of intracellular contents, whereas beta-lactam antibiotics act by inhibiting penicillin-binding proteins. The synergistic effect of EOs and antibiotics can be associated with the creation of complex chemical products that can be very effective in inhibiting many species of microorganisms by preventing the synthesis of the cell wall or can lead to lysis and ultimately death. Mixing EOs with antibiotics strengthens their antibacterial effects, raising the possibility of reducing the dose of the drug, which reduces side effects. They also increase antibiotic spectrum, preventing the development of resistance and reducing toxicity, often showing antibacterial effects better than estimated for each antibiotic separately [54].

6. Toxic Activity of EOs

EOs in the form of natural preservatives are generally recognized as safe according to the recommendations of the Food and Drug Administration (FDA) and are permitted for use. However, some of the EOs components are considered toxic compounds (including carvacrol, pulegone, safrole, psoralen, bergapten, camphor). Very few positive genotoxic results have been reported in the literature. Studies on the toxicological evaluation of EOs are limited to animal models, and in vivo evidence is very scarce [59]. Judzentiene and Garjonyte [60] determined the toxicity of mugwort essential oils using a test on brine shrimp (Artemia sp.). LC50 values (10.3–23.1 μg/mL) obtained for the oils after 24 h of exposure showed that oils containing significant amounts of D germacrene, 1,8-cineole, camphor, and davanone were particularly toxic. EOs derived from Salvia lavandifula, Mentha pulegium, Satureja hortensis, Chenopodium, and Thuja spp. have significant toxic effects with LD50 values ranging from 0.1 to 1 g/kg in an animal model (rats) and require prescribed precautions before their use [59]. In addition, the phototoxic risk of EOs should be taken into account; an example is citrus oils containing phototoxic compounds, mainly psoralens or furanocoumarins, which are present in some EOs, followed by a skin reaction intensified by heat, moisture, and exposure to sun/UV radiation [61,62].

7. Conclusions

Foodborne pathogens, recurrent bacterial infections, and antibiotic resistance have prompted researchers to analyze natural compounds as safe alternative AM agents. EOs, which are mixtures of different active compounds, are particularly promising in this respect. Their efficacy is due not only to their strong AM activity, but also to their potential to damage the structure of BF. These effects are related to the activity of individual EOs’ components and/or their synergistic effects. The action mechanisms of EOs include disruption of the expression of genes involved in QS, motility, adhesion, and virulence. EOs disrupt the communication system (e.g., by regulating gene expression), thus influencing the formation of BF. Furthermore, EOs modulate genes involved in bacterial adhesion and motility, as well as genes responsible for the synthesis of intercellular polysaccharide adhesin. The mechanism of action of EOs on a wide group of PAM results, among others, from their complex composition and the structure of individual components. Combinations of EOs and/or their components with antibiotics or other AM agents may also prove effective in the therapies for infectious diseases. The synergistic effects of such combinations result from different mechanisms and areas of action, resulting in an effective AM and ABF strategy.

Research on the possibility of using EOs in the prevention and treatment of infectious diseases should extend into the following avenues: analysis of the chemical composition of EOs, their extraction techniques, as well as their pharmacodynamics and pharmacokinetics, with particular emphasis on potential cytotoxic effects and safety of use. As a result of the assessment of available data, it can be said that EOs can be helpful in overcoming antibiotic resistance, which is one of the main health problems in the world. There are many types of EOs differing in the content of main compounds and biological activity. We cannot always associate a specific type of activity (e.g., AM and/or ABF) with the dominant compounds of the EOs. It is necessary to continue research on the chemical composition and biological activity of EOs and factors of variability and also to determine correlations between the main compounds of EOs and AM and ABF in relation to specific microorganisms. Research in this area should still be conducted to solve the problem of infectious diseases and antibiotic resistance. Current research in this area is very promising and indicates a clear direction for the search for alternative therapies using natural ABF agents.