Harnessing Monoterpenes and Monoterpenoids as Weapons against Antimicrobial Resistance

Sim et al. (2019) reported the antimicrobial efficacy of oregano oil, carvacrol, thyme oil, and thymol against bacterial isolates from dogs with otitis externa, showing MIC₉₀ values ranging from 200 to 2,292 μg/ml for various MDR strains, including methicillin-resistant Staphylococcus pseudintermedius and P. aeruginosa. Carvacrol was notably effective, with an MIC₉₀ of 146 μg/ml against S. pseudintermedius. Similarly, Sharma et al. (2023) found that thymol and eugenol had MICs of 125–250 μg/ml and 500–1,000 μg/ml against S. aureus, respectively.

Muniz et al. (2021) highlighted the potential of synthetic and natural eugenol derivatives to inhibit the NorA efflux pump in S. aureus, with their studies suggesting mechanisms involving hydrogen bonds and hydrophobic interactions. The findings suggest that eugenol holds promise for development in antibacterial drug formulations; mainly targeting strains carrying the NorA efflux pump. Additionally, Buru et al. (2022) demonstrated that eugenol exposure leads to downregulated luxS expression in MRSA, potentially enhancing biofilm production and secondary metabolite biosynthesis through the upregulation of the argC gene, indicating potential targets for antibacterial development against drug-resistant strains.

Kwiatkowski et al. (2020) evaluated the antimicrobial efficacy of 1,8-cineole and linalyl acetate against S. aureus, with MIC values ranging from 28,800– 57,600 μg/ml and 28,200–112,600 μg/ml, respectively. Caballero Gómez et al. (2022) studied various compounds including cinnamaldehyde, thymol, carvacrol, limonene, and geraniol against Enterococcus, Pseudomonas, and Staphylococcus strains, finding cinnamaldehyde the most effective with MICs of 10 to 50 μg/ml. Carvacrol and thymol demonstrated increased MIC₉₀ values of 292–400 μg/ml against β-hemolytic Streptococcus compared to staphylococcal isolates (146–200 μg/ml) (Sim et al. 2019). Thymol showed the highest MIC₉₀ (800 μg/ml) against P. aeruginosa, and displayed comparable activity against Proteus mirabilis at MIC₉₀ 200–292 μg/ml. Additionally, de Souza et al. (2021) reported that carvacrol showed inhibitory effects against carbapenem and polymyxin-resistant Klebsiella pneumoniae, with MICs of 130–260 μg/ml. In murine models, carvacrol treatment significantly improved survival and reduced bacterial counts in infections, indicating its potential for modulating host immune responses and reducing infection severity compared to polymyxin B treatment.

Owen et al. (2019) highlighted carvacrol’s potent antimicrobial properties, exhibiting the lowest MICs (0.99–15.81 mM) across various microorganisms and bactericidal effects. Although ineffective against carbapenem-resistant E. coli and P. aeruginosa, carvacrol showed no significant cross-resistance with antibiotics, positioning it as a promising candidate against AMR. Linalool exhibited higher activity against antibioticsensitive P. aeruginosa (MIC 228.20 mM) than resistant strains (MIC 912.90 mM), while cuminaldehyde showed higher activity against antibiotic-sensitive E. coli (MIC 2.10 mM) compared to resistant strains (MIC 8.40 mM). Moreover, cuminaldehyde displayed better efficacy against vancomycin-susceptible enterococci (VSE) (MIC 134.41 mM) than resistant strains (MIC 537.65 mM).

Šimunović et al. (2020) further demonstrated that monoterpenes such as carvacrol, thymol, and thymoquinone possess significant antimicrobial activity, each with MICs of 31.25 μg/ml, while p-cymene and γ-terpinene showed reduced efficacy with MICs of 1,000 μg/ml. Notably, p-cymene also inhibited efflux pumps in C. jejuni while carvacrol displayed a weaker effect. Rossi et al. (2021) reported that thymol, carvacrol, eugenol, geraniol, and cinnamaldehyde effectively inhibited two Vibrio species, Vibrio anguillarum, and Vibrio harveyi, with MICs ranging from 0.94–7.5 mM, whereas eucalyptol, linalool, menthol, α-pinene, and limonene showed no activity at the tested concentrations.

Giovagnoni et al. (2020) investigated thymol and carvacrol’s protective effects against Salmonella Typhimurium, revealing a dual mechanism of action with MICs of 1.87 mM. These compounds enhanced epithelial barrier integrity while directly inhibiting Salmonella growth and modulating virulent genes. Noumi et al. (2023) explored limonene’s antimicrobial properties, demonstrating inhibition of bacterial growth and biofilm formation at a MIC of 48 μg/ml, surpassing the efficacy of the whole Anethum graveolens EO. Limonene also showed anti-adhesion activity and inhibited violacein production, highlighting its potential for targeted interventions.

Stringaro et al. (2022) explored the antimicrobial effects of Oregano vulgare EO (OVEO) against Candida species, finding variable sensitivity across strains, with C. albicans being less sensitive than Candida glabrata, Candida tropicalis, and Candida krusei. Carvacrol showed superior antimicrobial activity compared to OVEO and thymol, with MIC values of 97.5–195 μg/ml and 195–390 μg/ml for thymol, respectively. Larvae viability studies using Galleria mellonella revealed that OVEO slightly reduced survival rates by about 30% in C. albicans. Post-infection treatment with OVEO, carvacrol, or thymol generally improved survival, notably with carvacrol treatment increasing survival by 100% in C. albicans, while thymol and OVEO showed lesser improvement.

Sousa Silveira et al. (2020) demonstrated the antibacterial efficacy of thymol and carvacrol against S. aureus, with MIC values of 72 and 256 μg/ml, respectively. Shaban et al. (2020) reported that carvacrol was more effective against Candida auris than thymol, with MICs of 125 μg/ml and 312 μg/ml, respectively. The study also noted that carvacrol inhibited C. auris adherence to host cells and reduced proteinase production in C. auris and C. albicans, even at sub-inhibitory concentrations.

Touil et al. (2020) explored carvacrol and cuminaldehyde against amphotericin B-resistant C. albicans, individually and in combination, in single- or mixedinfections. They found variable inhibitory effects on C. albicans yeast formation compared to hyphae development, with carvacrol exhibiting MIC values ranging from 250–1,000 μg/ml and cuminaldehyde from 2000–4000 μg/ml), varying among Candida isolates. Carvacrol showed greater efficacy than cuminaldehyde, particularly against C. albicans isolates. Both compounds exhibited inhibitory effects against bacteria co-isolated with C. albicans, with MIC values of 1,000 μg/ml for carvacrol and 1,000–4,000 μg/ml for cuminaldehyde. These results highlight the potential of carvacrol and cuminaldehyde, alone or in combination, in combating AMR in C. albicans infections.

In assays against C. albicans, different cell forms, such as hyphal or yeast are influenced by environmental conditions such as pH, temperature, and nutrient availability. The studies reviewed here primarily focused on the hyphal form of C. albicans, which is the infectious form, with the exception of Sharifzadeh et al. (2019), Iraji et al. (2020), and Stringaro et al. (2022). Table II summarizes research on monoterpenes, detailing their minimum inhibitory concentration (MIC) against Gram-positive, Gram-negative, and fungal pathogens.

Panagiotopoulos et al. (2021) found that p-cymene binds to the nuclear localization signal of SARS-CoV-2, impairing its nuclear translocation and viral replication, achieving up to 90% inhibition in Vero cells at non-toxic concentrations (0.0125 to 200 μg/ml). Similar effects were observed against influenza H1N1, where p-cymene at 20 μg/ml reduced virus protein expression and impaired nuclear translocation. These findings propose p-cymene as a potential antiviral agent, either as a standalone treatment or as an adjuvant in treating COVID-19 and other RNA virus infections.

Recently, Wang et al. (2024) identified 100 μM eugenol as optimal concentration for inhibiting Singapore grouper iridovirus (SGIV) infection by reducing mRNA expression and protein synthesis. Eugenol’s mechanism involves inhibiting the MAPK signaling pathway, reducing inflammatory factor expression (IL-1β, IL-6, TNF-α), and upregulating interferon-related genes while reducing oxidative stress by suppressing intracellular reactive oxygen species.

Wang et al. (2020a) demonstrated that HSV-2 infection reduced intracellular protein ubiquitination, a process reversed by carvacrol, suggesting its role in modulating the ubiquitin-proteasome system. Carvacrol inhibited HSV-2 replication, lowering virus titers, and reducing the virus release rate to 33.67% at 1 mmol/l. It downregulated the expression of key HSV-2 replication proteins (ICP4, ICP27, VP16, gB, UL30), and induced increase in TNF-α and reduced RIP3 and MLKL protein expressions via the RIP3-mediated necrosis pathway. These findings suggest that carvacrol exerts its antiviral activity by interfering with the replication process of HSV-2, making it a potential therapeutic agent against HSV-2 infections.

Similarly, Mediouni et al. (2020) found that carvacrol and thymol disrupted the cholesterol content of the viral envelope membrane, blocking HIV-1 entry into target cells without affecting other stages of the viral life cycle. Carvacrol exhibited significant antiviral potency with an IC50 of 16 μM, while thymol displayed a slightly higher IC50 of 25.2 μM. Pretreatment of HIV with carvacrol reduced viral infectivity by 60.6%, underscoring its effectiveness in altering viral particles. Testing against viruses using the CCR5 coreceptor further demonstrated that carvacrol and thymol inhibited HIV replication without affecting cell viability or receptor endocytosis.

Kumar et al. (2021) demonstrated that thymoquinone (TQ) inhibited Chikungunya virus (CHIKV) replication with an EC50 value of 4.478 μM. A plaque reduction assay revealed that TQ significantly reduced CHIKV titer in a dose-dependent manner, with over 90% reduction observed at 20 μM concentration. Additionally, immunofluorescence assays showed reduced expression of CHIKV glycoprotein in cells treated with 10 μM TQ, indicating lower viral load. Time-of-addition and time-of-elimination studies confirmed TQ’s inhibitory action in the late stages of the CHIKV life cycle (8–12 hours post-infection). These findings underscore TQ’s significant antiviral activity by disrupting CHIKV replication at both molecular and cellular levels.

Šimunović et al. (2020) demonstrated promising synergistic effects in various monoterpenes such as carvacrol, thymol, and thymoquinone combinations. Combinations like carvacrol + thymoquinone (fractional inhibitory concentration indices (FICI) 0.5), carvacrol + thymol (FICI 0.2), and thymol + thymoquinone (FICI 0.3) showed robust synergistic activities. All other tested combinations exhibited additive effects without any antagonistic effects observed. Similarly, Touil et al. (2020) observed promising synergistic effects of carvacrol and cuminaldehyde against C. albicans and co-isolated bacteria. The combination significantly reduced MIC values to 60–250 μg/ml for carvacrol and 500–2,000 μg/ml for cuminaldehyde, showing synergistic interactions (FICI values ranged 0.36–0.5) for 12 strains of C. albicans and indifferent interactions (FICI values between 0.62–1.0) for four Candida strains.

Owen et al. (2020) investigated the synergy between vancomycin and monoterpenes, particularly carvacrol and cuminaldehyde, against VSE and VRE. Significant synergy was found in binary combinations of vancomycin with carvacrol or linalool against VSE, with a substantial four to eight-fold reduction in vancomycin’s MIC. The combination of carvacrol and cuminaldehyde with vancomycin showed bactericidal activity against VSE, resulting in a 5.87 log₁₀ reduction, indicating strong synergy, while ternary combinations of two monoterpenes with vancomycin demonstrated significant reductions in MIC (1,024-fold) against VRE.

Kwiatkowski et al. (2019) identified carvacrol as highly potent against MRSA strains, with 1,8-cineole exhibiting synergy when combined with mupirocin against mupirocin-susceptible (FICI 0.44) and mupirocin low-level resistant strains (FICI 0.28), while (–)-menthone showing synergistic activity against mupirocin-susceptible MRSA strains (FICI 0.38). Additionally, Kwiatkowski et al. (2020) explored the synergistic potential of 1,8-cineole and linalyl acetate with conventional antibiotics against MRSA strains, revealing synergistic effects of 1,8-cineole with penicillin G (FICI 0.1) and additive activities of linalyl acetate in combination with methicillin and penicillin G at FICI 0.4 and FICI 0.6, respectively against all MRSA isolates, highlighting the versatility of these combinations.

AMR, especially carbapenem resistance in bacteria like K. pneumoniae, presents a formidable treatment challenge (Köse 2022). Köse (2022) investigated combining carvacrol with meropenem against carbapenem-resistant Klebsiella pneumoniae (CRKP) strains. Their findings revealed synergy between carvacrol and meropenem against 8 of 25 CRKP strains, confirmed by checkerboard assays (FICI = 0.5) and time-kill assays. This combination induced significant membrane damage to CRKP cells, as shown by live-dead tests and spectrophotometric measurements. Sousa Silveira et al. (2020) demonstrated a notable decrease in tetracycline MIC against S. aureus, from 114–101 μg/ml, when combined with thymol. Although thymol and carvacrol exhibited antibiotic activity, they did not act as efflux pump inhibitors (EPIs), suggesting alternative mechanisms for overcoming resistance apart from the TetK efflux pump.

Dhara and Tripathi (2020) investigated the effects of eugenol on extended-spectrum-β-lactamase producing quinolone resistant (ESBL-QR) strains of E. coli and K. pneumoniae, revealing distinct responses in cellular morphology. ESBL-QR K. pneumoniae exhibited cellular stress and autolysis upon eugenol treatment, accentuated when combined with cefotaxime/ciprofloxacin, indicating synergistic interactions. The combination therapy also suppressed the expression of the acrB and beta-lactamase genes (bla_TEM and bla_CTX-M) in K. pneumoniae, suggesting a multifaceted approach to combating AMR.

Aleksic Sabo et al. (2021) demonstrated the potent antimicrobial activity of carvacrol, thymol, and eugenol, against Acinetobacter baumannii, comparable to antibiotics, particularly when combined with ciprofloxacin (FICI range 0.25–0.32). Binary combinations of gentamicin with carvacrol or thymol showed additive effects, while combinations with ciprofloxacin displayed synergy against both reference and MDR strains.

The study by Shaban et al. (2020) underscores carvacrol’s potent antifungal properties with a median MIC of 125 μg/ml, revealing synergistic and additive effects when combined with established antifungal agents like fluconazole, amphotericin B, nystatin, and caspofungin. Notably, carvacrol not only reduced the MIC values of these drugs but also inhibited critical virulence factors of C. auris by reducing its adherence and proteinase production. Sharifzadeh et al. (2019) demonstrated synergistic potential between carvacrol and voriconazole against Candida species, minimizing the risk of side effects from high drug concentrations. The reported FICI values against all tested C. albicans isolates were at FICI 0.370–0.853 and C. glabrata at FICI 0.412–0.625. Sharifzadeh and Shokri (2021) further confirmed synergy between eugenol and voriconazole against C. tropicalis and C. krusei isolates at FICI 0.25–0.88 and 0.19–0.63, respectively. Schlemmer et al. (2019) explored synergistic interactions of monoterpenes with antifungal agents, revealing primary synergies in combinations like carvacrol + nystatin, thymol + nystatin, and carvacrol + miconazole at a rate of 80%. Some combinations showed indifference such as thymol + terbinafine and cinnamaldehyde + terbinafine, while antagonistic effects were observed in carvacrol + ketoconazole, thymol + ketoconazole, and cinnamaldehyde + ketoconazole, emphasizing the need for careful combination selection for effective antifungal strategies.

Biernasiuk et al. (2022) revealed significant synergistic effects when cinnamaldehyde and eugenol were combined with cetylpyridinium chloride against Candida spp. strains, resulting in a notable reduction in MICs by 4–8 fold and 2–4 fold, respectively, across various strains. Similar synergistic outcomes were observed with chlorhexidine at FICI 0.375–0.5 for all strains except Candida parapsilosis (addition at FICI 0.562). These compounds act by binding to ergosterol in the fungal membrane, increasing ion permeability and leading to cell death, offering potential for topical antifungal preparations.