Antibacterial Activity and Antibiofilm of Essential Oils of Clove and Thyme Against Clinical Isolates of Multidrug-Resistant Acinetobacter Baumannii Associated with Nosocomial Infections

Nihal EZZARIGA^{*, 1}, Oumaima ZOUHARI², Amal RHARS¹, Asmaa WAKRIM³, Mohamed AGHROUCH⁴, Zohra LEMKHENTE¹

¹Faculty of Medicine and Pharmacy of Agadir, University Ibn Zohr, Agadir 80060, Morocco.

²Municipal Office of Cleaning of Agadir, Morocco.

³Private University of Health and Sciences of Agadir, Morocco.

⁴Hassan II Regional Hospital Center of Agadir, Morocco.

^*Corresponding author: Nihal EZZARIGA, Faculty of Medicine and Pharmacy of Agadir, University Ibn Zohr, Agadir 80060, Morocco.

Received: 28 February 2025; Accepted: 03 March 2025; Published: 11 March 2025

Introduction

Acinetobacter baumannii is a Gram-negative opportunistic bacterium with a remarkable ability to colonize humans. It is now a major nosocomial pathogen due to its resistance to conventional antibiotic treatments and its persistence in hospital environments [1]. Classified as a priority pathogen by the World Health Organization (WHO) since 2017, A. baumannii represents a global health threat, particularly due to its resistance to carbapenems, which are often considered the last therapeutic option against multidrug-resistant infections [2]. Infections caused by multidrug-resistant A. baumannii strains are particularly difficult to treat and are associated with higher mortality rates than those caused by other resistant pathogens [3]. Its ability to form biofilms on hospital surfaces and persist in healthcare settings promotes its spread, especially in intensive care units [4]. These biofilms, composed of complex microbial structures surrounded by an extracellular matrix rich in polysaccharides, provide A. baumannii with increased protection against antimicrobial agents, making the treatment of biofilm-associated infections particularly challenging [5,6]. Thus, the persistence of this bacterium and its ability to cause severe nosocomial infections refractory to treatment make it a major public health concern.

In response to this issue, essential oils (EOs), such as those of Syzygium aromaticum and Thymus vulgaris, are gaining increasing interest due to their antimicrobial properties. They contain bioactive compounds, such as eugenol and thymol, capable of disrupting bacterial cell membranes, altering enzymatic processes, and inducing cell lysis. Moreover, these compounds can penetrate the protective barrier of biofilms, disperse them, and directly target bacterial cells. Thanks to these properties, EOs are increasingly studied as potential alternatives to conventional antibiotics, with a reduced risk of resistance development. They are particularly used for wound disinfection, treatment of skin and respiratory infections, and the management of nosocomial infections caused by multidrug-resistant strains such as A. baumannii [7,8]. Thus, the present study aims to evaluate the antibacterial and antibiofilm activity of Syzygium aromaticum and Thymus vulgaris essential oils against clinical isolates of multidrug-resistant A. baumannii, collected from the hospital environment of the intensive care units of the Regional Hospital Center of Agadir, Morocco.

Materials and Methods

Essential oils

The essential oils of Syzygium aromaticum (clove) and Thymus vulgaris (thyme) were evaluated for their antibacterial and antibiofilm activities. These oils were extracted through hydrodistillation and were subsequently purchased from a commercial supplier. The oils were solubilized in a 10% dimethyl sulfoxide (DMSO) solution to prepare working stock solutions.

Bacterial strains

The antibacterial and antibiofilm activities of the essential oils were tested against Acinetobacter baumannii strains, including one reference strain (ATCC 19606) and six clinical isolates associated with nosocomial infections. The clinical isolates were obtained from the following sources: one strain from protected distal sampling (PDS), three strains from cytobacteriological examinations of urine (CBU), and two strains from catheters. All isolates were sourced from patients in intensive care units and were confirmed to be A. baumannii through standard microbiological identification techniques.

Antibacterial activity

Antibacterial activity was assessed using the disk diffusion method [9]. Sterile filter paper discs (6 mm diameter) were impregnated with 10 µL of each essential oil and placed on Mueller-Hinton agar plates inoculated with bacterial suspensions standardized to 10⁶ CFU/mL. The plates were incubated at 37°C for 24 hours, and the inhibition zone diameters (IZDs) were measured in millimeters. Bacterial sensitivity was classified as follows [10]:

Non-sensitive: IZD ≤ 8 mm Sensitive: 9 mm ≤ IZD ≤ 14 mm

Very sensitive: 15 mm ≤ IZD ≤ 19 mm Extremely sensitive: IZD ≥ 20 mm

Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC)

Minimum Inhibitory Concentrations (MIC, µL/mL) and Minimum Bactericidal Concentrations (MBC) were determined using the broth microdilution method in 96-well microplates [11]. Serial dilutions of the essential oils were prepared in nutrient broth, and bacterial suspensions standardized to 10⁶ CFU/mL were added. After 24 hours of incubation at 37°C, the MIC was recorded as the lowest concentration that inhibited visible bacterial growth. The MBC was identified as the lowest concentration at which no bacterial growth occurred upon subculturing onto agar plates. The MBC/MIC ratio was calculated to evaluate the bactericidal effect of HEs if MBC/MIC = 1-2 or its bacteriostatic effect if MBC/MIC = 4-16 [12]. All experiments were performed in triplicate, and results were expressed as mean values.

Antibiofilm activity

The biofilm formation was evaluated by the TCP (Tissue Culture Plate) method. The bacterial suspensions were inoculated into 96-well microplates, and after incubation for 24 hours at 37°C, the planktonic phase was gently removed, the wells were washed and dried, and then stained with purple crystal. The optical density of the biofilms formed was measured at 550 nm. Biofilm production was classified as negative, weak, moderate and strong based on the optical density threshold value, calculated according to the following formula [13].

Threshold value DO = DO of the negative control (3 × standard deviation of the DOs of the negative control) The criteria used were: DOm ≤ Threshold value DO Non-biofilm-former

Threshold value DOm ≤ 2 × Threshold value DO Low biofilm

2 × Threshold value DOm ≤ 4 × Threshold value DO Moderate biofilm-former

DOm 4 × Threshold value DO High biofilm formation

With: DOm: the average of the optical density of the three wells.

Finally, the inhibition of the initial attachment of bacterial cells was studied using a method adapted from Bazargani and Rohloff [14]. Semi-logarithmic dilutions of the essential oils corresponding to the MIC, MIC/2 and MIC/4 values were applied in 96-well microplates, followed by the addition of bacterial suspensions. After incubation at 37°C for 8 hours, the wells were rinsed, dried and then stained with purple crystal. Absorbance was measured at 590 nm to calculate the percentage inhibition of bacterial attachment compared to positive controls.

%Inhibition = (Mean OD control - Mean OD sample) ×100 / The Mean OD control

Statistical analysis

The values were obtained as mean ± standard deviation (SD) from three repetitions. Optical density values obtained for the different bacterial strains were organized in an Excel table, and the statistical analysis was carried out using an analysis of variance. The results were analyzed using Excel, where a p-value of less than 0.05 was considered significant.

Results

Antibacterial activity of essential oils

Diameter of inhibition zones

The essential oils of Syzygium aromaticum and Thymus vulgaris showed significant antibacterial activity against A.baumannii strains, measured by the diameters of the inhibition zones (± SD) (Table 1). Thymus vulgaris exhibits varying antibacterial properties against different strains of Acinetobacter baumannii. Syzygium aromaticum shows inhibition zones (IZD) ranging from 14 ± 1.2 mm for strain N6 (sensitive) to 31 ± 1.5 mm for strain N2 (extremely sensitive), with notable effectiveness against strains ATCC 19606, N2, and N4. Thymus vulgaris demonstrates more pronounced activity, with IZDs ranging from 16 ± 1.5 mm to 39 ± 2.1 mm, and "extremely sensitive" effectiveness against strains ATCC 19606, N1, N2, N3, and N4. In comparison, T. vulgaris appears more effective, but S. aromaticum still performs well, particularly against strains ATCC 19606, N2, and N4. These results suggest that both essential oils have significant antibacterial potential against A. baumannii, although their mechanisms of action or intrinsic effectiveness may differ.

Table 1: Antibacterial activity of EHs Against seven strains of A.baumannii ± SD

Abbreviations: diameter of the inhibition zone (DIZ) ± SD and n=3.

Determination of minimum inhibitory and bactericidal concentrations

Minimum inhibitory (MIC) and bactericidal (BMC) concentrations of Syzygium aromaticum (Table 2) and Thymus vulgaris (Table 3) essential oils were determined to evaluate their efficacy against A.baumannii strains. The determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Syzygium aromaticum (Table 2) and Thymus vulgaris (Table 3) essential oils reveals their efficacy against Acinetobacter baumannii. For Syzygium aromaticum, MIC values range from 0.78 µL/mL (N5, N6) to 12.5 µL/mL (N3, N4), and MBC values range from 0.78 µL/mL to 25 µL/mL. Strains N5 and N6 exhibit a bactericidal effect (MIC = MBC = 0.78 µL/mL), whereas others, such as ATCC 19606 and N3, show bacteriostatic activity (high MBC/MIC ratio). For Thymus vulgaris, MIC values range from 0.19 µL/mL (N1) to 12.5 µL/mL (N4, N6), and MBC values range from 0.39 µL/mL to 50 µL/mL. Strains N1, N2, and N5 demonstrate a bactericidal effect, while N3, N4, and N6 show bacteriostatic effects. In conclusion, both essential oils exhibit significant antibacterial activity against A. baumannii. Syzygium aromaticum is particularly effective against N5 and N6, while Thymus vulgaris acts more strongly on N1 and N2. The MBC/MIC ratios highlight differences in their mechanisms of action.

Table 2: Minimum inhibitory and bactericidal concentrations of Syzygium aromaticum essential oil against A.baumannii strains.

Abbreviations: MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration

Table 3: Minimum inhibitory and bactericidal concentrations of Thymus vulgaris essential oil against A.baumannii strains.

Abbreviations: MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration

Antibiofilm activity of essential oils

Biofilm formation

The potential for biofilm formation and antibiotic resistance of six strains of A. baumannii isolated from different clinical sites were evaluated (Table 4) to better understand their resistance and behaviour in the presence of the essential oils studied. The table highlights the biofilm-forming potential and antibiotic resistance profiles of six A. baumannii strains from different clinical sites, revealing a strong correlation between biofilm formation and antimicrobial resistance. Five strains (N°2 to N°6) exhibit strong biofilm-forming ability and multidrug resistance, while the weak biofilm-forming strain (N°1) shows comparatively lower resistance, particularly to imipenem, norfloxacin, and gentamicin. Resistance to carbapenems (ertapenem and meropenem) is universal, with imipenem effective only against N°1. Fluoroquinolones (ciprofloxacin and levofloxacin) exhibit widespread ineffectiveness, with norfloxacin effective against strains N°1 and N°5. Among aminoglycosides, amikacin and gentamicin show limited efficacy, and tobramycin resistance is universal. The strong biofilm-forming capacity of most strains underscores their role in enhancing resistance, complicating treatment options and emphasizing the need for tailored strategies, including the exploration of antibiofilm agents and combination therapies, to combat these resilient strains.

Table 4: Biofilm formation in A. baumannii strains and their antibiotic resistance profiles

Abbreviations: CBU: cytobacteriological examination of urine. PDS: Protected distal sampling. ETP: ertapenem. MEM: meropenem. IMP: imipenem. Nor: norfloxacin. CIP: ciprofloxacin. LEV: levofloxacin. AK: amikacin. TOB: tobramycin. GEN: gentamicin. R: Resistant. S: Sensitive.

Determination of biofilm inhibition

Inhibition of biofilm formation, was determined to evaluate the efficacy of Syzygium aromaticum (Figure 1) and Thymus vulgaris (Figure 2) essential oils against A.baumannii strains. The percentages of inhibition obtained illustrate the effectiveness of these oils in preventing bacterial fixation. Both figures demonstrate the antibiofilm effects of Syzygium aromaticum and Thymus vulgaris essential oils against Acinetobacter baumannii, showing a concentration-dependent inhibition (MIC, MIC/2, MIC/4). In both cases, the highest inhibition occurs at MIC, with the ATCC reference strain exhibiting the strongest response, followed by clinical isolates No. 1 and No. 4, which also show significant inhibition. Other isolates (No. 2, No. 3, No. 5, and No. 6) display moderate to low sensitivity, with reduced inhibition at lower concentrations. The variations in response suggest strain-specific differences in biofilm formation capabilities. These findings highlight the potential of both essential oils as promising antibiofilm agents, though their effectiveness varies among bacterial isolates.

Figure 1: Percentage inhibition of A. baumannii fixation by sublethal concentrations of Syzygium aromaticum essential oil.

Figure 2: Percentage inhibition of A.baumannii fixation by sublethal concentrations of Thymus vulgaris essential oil.

Discussion

The risk of antibiotic-resistant hospital-acquired infections is particularly severe in intensive care units and other hospital settings, posing a significant threat to patient health [15]. Multidrug-resistant bacteria such as Acinetobacter baumannii and Pseudomonas aeruginosa are major contributors to these infections [16]. All the isolates analyzed exhibited resistance to one, two, or all three classes of antibiotics (carbapenems, fluoroquinolones, and aminoglycosides), classifying them as multidrug-resistant and associated with nosocomial infections. These findings align with previously reported studies. Plants have long been a valuable source of bioactive compounds, offering promising alternatives for biomedical applications. Essential oils contain highly concentrated bioactive molecules capable of exerting antimicrobial effects at low concentrations against multidrug-resistant bacteria [15]. Our results demonstrated that both essential oils exhibited strong activity against all A. baumannii strains, with inhibition zone diameters (IZD) ranging from 14 to 31 mm for Syzygium aromaticum (Myrtaceae) and from 17 to 39 mm for Thymus vulgaris (Lamiaceae), classifying them as highly to extremely sensitive. These findings are consistent with previous research, such as the study by Al Janabi and Asaad [17], which reported a 30 mm IZD for S. aromaticum essential oil against Acinetobacter strains. Similarly, Zeshan [18] observed an IZD of 15.4 mm, while Haddouchi and Benmansour [19] reported an IZD of 34 mm for T. vulgaris essential oil against A. baumannii. Additionally, Boukhatem et al. [20] recorded a 16 mm IZD against A. baumannii strains.

These findings corroborate those of Nzeako et al. [21], who highlighted the antimicrobial properties of clove and thyme essential oils, attributing their activity to the presence of bioactive compounds such as thymol, eugenol, flavones, phenolic monoterpenoid glycosides, and aliphatic alcohols. Determination of minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) confirmed the potent antibacterial activity of S. aromaticum and T. vulgaris essential oils against clinical multidrug-resistant A. baumannii strains. The MIC and MBC values exhibited variability, demonstrating both bacteriostatic and bactericidal effects depending on the strain and the essential oil used. For S. aromaticum, MIC values ranged from 0.78 to 12.5 µl/ml, while MBC values varied between 0.78 and 25 µl/ml. The oil exhibited bacteriostatic activity against A. baumannii ATCC 19606, N2, N3, and N4 strains, whereas it showed bactericidal activity against strains N1, N5, and N6. These results align with the findings of Zeshan [18], who reported MIC values between 2 and 6.25 µl/ml for S. aromaticum essential oil against A. baumannii. Similarly, Faujdar et al. [22] reported a MIC of 0.78 mg/ml and an MBC of 0.39 mg/ml for all A. baumannii isolates. For T. vulgaris, MIC values ranged from 0.19 to 12.5 µl/ml, while MBC values varied between 0.39 and 50 µl/ml. The oil exhibited bacteriostatic activity against A. baumannii ATCC 19606, N3, N4, N5, and N6 strains, while it showed bactericidal activity against strains N1 and N2. These results are consistent with previous research by Saghi et al. [23], which demonstrated a MIC of 0.44 µg/ml for Thymus syriacus against A. baumannii, as well as the findings of Safi et al. [24], who observed a MIC of 0.04 µl/ml for T. vulgaris against Gram-negative bacteria including A. baumannii.

Prolonged use of medical devices significantly increases the risk of infectious complications, leading to extended hospital stays and higher healthcare costs. Consequently, most patients requiring medical devices experience negative outcomes due to treatment failures [13]. A. baumannii accounts for approximately 20% of intensive care unit infections [25]. Our study confirms that all tested A. baumannii strains demonstrated biofilm-forming capacity. Notably, isolates from catheters in intensive care units exhibited strong biofilm formation, whereas strain N1, isolated from a different biological sample (CBU), formed a weak biofilm. Our findings reveal that all studied A. baumannii isolates exhibited resistance to one, two, or all three antibiotic classes (carbapenems, fluoroquinolones, and aminoglycosides) and formed biofilms, with five strains (83.3%) demonstrating strong biofilm formation, while only one strain (N1) displayed weak biofilm formation (16.7%). A study in India also established a correlation between antibiotic resistance and biofilm formation, showing that multidrug-resistant A. baumannii strains formed more biofilm than antibiotic-sensitive strains [26]. Carbapenems, a subclass of beta-lactam antibiotics, have been considered an effective treatment option, particularly imipenem. However, increasing resistance to imipenem and ciprofloxacin has been widely reported [27]. Interestingly, strain N1, which exhibited weak biofilm formation, was more susceptible to imipenem, suggesting a potential inverse relationship between biofilm formation and antibiotic susceptibility. This observation aligns with findings by Gedefie et al. [28], who noted a statistically significant correlation between A. baumannii biofilm formation and imipenem resistance.

Krystova and colleagues [34] studied the effect of Thymus vulgaris essential oil on various typical bacterial strains. The antibiofilm activity test showed that the bacterial biofilm was reduced by 53% after exposure to the lowest concentration of thyme EO, and use at higher concentrations resulted in a reduction in structure by 76%. There was a case where the anti-adhesion activity did not depend on the concentration used, as for the Ab6 strain where MIC/2 had the same percentage inhibition to eliminate the adherent cells as the MIC itself. This finding suggests that the antibacterial effect is not solely responsible for the inhibition of adhesion. These results are consistent with those found by Kerekes et al. [35], when biofilms were significantly inhibited by EOs at MIC/2 concentration. The antibiofilm activity of essential oils of the Myrtaceae family against multidrug-resistant A. baumannii has been reported for several species. For example, Cinnamomum zeylanicum essential oil (cinnamon) showed significant inhibition of A. baumannii biofilms (80%) [37]. Similarly, essential oils of Pimenta dioica and Pimenta racemosa demonstrated notable antibiofilm activity against strains of A. baumannii, with inhibition rates of 85% [38].

The Lamiaceae family has also demonstrated antibiofilm activity against antibiotic-resistant A. baumannii in several species. Mentha pulegium L. [8], Ziziphora tenuior L. [7], and Salvia glutinosa L. [39] have all shown promising results. M. pulegium was observed to damage biofilms formed by A. baumannii strains, with inhibition rates ranging from 26% to 91% [8]. Similarly, the minimum biofilm inhibitory concentration of Z. tenuior essential oil affected A. baumannii biofilms at rates between 51% and 84% [7]. Additionally, S. glutinosa essential oil exhibited antibacterial efficacy with MICs between 1.25 and 2.5 µl/ml and MBCs between 5 and 10 µl/ml, along with notable antibiofilm activity against A. baumannii, with minimum biofilm inhibitory concentrations ranging from 0.3 to 2.5 µl/ml [39]. The antibacterial therapeutic potential of thyme and clove is based on their major active compounds, thymol and eugenol [21,32,34]. In summary, future studies should focus on deepening the understanding of the major active components of thyme and clove essential oils, their specific mechanisms of action against biofilms through molecular biology approaches, and the optimization of their clinical application to effectively combat nosocomial infections caused by A.baumannii.

Conclusion

In conclusion, this study highlights the importance of antibiotic resistance and biofilm formation in Acinetobacter baumannii, as well as their role in the persistence of nosocomial infections. It also emphasizes the therapeutic potential of clove and thyme essential oils, which exhibit antimicrobial and antibiofilm properties. These findings pave the way for new therapeutic approaches aimed at complementing conventional antibiotic treatments, particularly in the face of the growing threat of multidrug-resistant bacteria.

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