Skip to main content
NCBI home page
Veterinary Medicine and Science logoLink to Veterinary Medicine and Science
. 2025 May 23;11(3):e70407. doi: 10.1002/vms3.70407

Evaluating the Antimicrobial and Antibiofilm Efficacy of Lavender Essential Oil and Linalool on Dual Candida albicans Biofilms With Staphylococcus aureus and Staphylococcus epidermidis From Canine External Otitis

Navid Neisari 1, Aghil Sharifzadeh 1,✉, Bahar Nayeri Fasaei 1,✉, Sepideh Asadi 2, Alireza khosravi 1, Abolfazl Rafati Zomorodi 3,4, Javad Malakootikhah 1
PMCID: PMC12099306  PMID: 40406870

ABSTRACT

Background

Biofilm formation significantly contributes to the rise of antimicrobial resistance, treatment failures and recurrent infections. Essential oils (EOs), particularly lavender EO (LEO), have gained attention for their antimicrobial and antibiofilm activities. This study investigates the effects of LEO and linalool on Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), and Candida albicans (C. albicans) isolates.

Materials and Methods

The chemical composition of LEO was analysed using gas chromatography‐mass spectrometry (GC‐MS). Eight clinical and reference microorganisms were tested, including four C. albicans, three S. aureus, and three S. epidermidis isolates, to assess their biofilm‐producing potential with the tissue microtiter plate method. Antimicrobial and antibiofilm activities of LEO and linalool were evaluated in planktonic, single‐biofilm, and dual‐biofilm phases through microbroth dilution and scanning electron microscopy (SEM).

Results

The minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) of LEO in the planktonic phase were 1250 µg/mL and 2500 µg/mL against Staphylococcus isolates, respectively, while the corresponding value for C. albicans isolates was 5000 µg/mL. 90% biofilm inhibition was achieved at concentrations of 5000 µg/mL and 40,000 µg/mL for Staphylococcus and C. albicans, respectively. LEO completely inhibited dual biofilms formed by C. albicans/S. aureus and C. albicans/S. epidermidis at 20,000 µg/mL, whereas linalool attained 100% inhibition at 40,000 µg/mL.

Conclusion

LEO demonstrates significant antimicrobial and antibiofilm activity against Staphylococcus and C. albicans isolates, effective in both planktonic and biofilm phases.

Keywords: antibiofilm, antimicrobial, essential oil, lavender, linalool

The minimum inhibitory concentration of antimicrobial and anti‐biofilm activity of lavender essential oil (LEO) was determined. LEO exhibited robust antimicrobial and anti‐biofilm activity against clinically relevant Staphylococcus spp. and Candida albicans (C. albicans), including both planktonic and biofilm forms.

graphic file with name VMS3-11-e70407-g005.jpg

1. Introduction

Otitis externa (OE) is considered one of the most prevalent infections affecting dogs globally, with an estimated frequency ranging from 5% to 20% (Ponn et al. 2024). The clinical signs in OE cases include head shaking, purulent discharge, malodour, pain and swelling resulting from inflammation, as well as erythema of the pinnae, external meatus and lining of the external canal. In recurrent or chronic OE cases, clinical signs may progress to stenosis of the external ear canal and occlusion (Vercelli et al. 2021). Although bacteria and yeasts are not the primary causes of OE, they are responsible for persistent inflammation that contributes to the chronicity of the pathology (Secker et al. 2023).

Staphylococcus spp., Pseudomonas spp., Corynebacterium spp., Proteus spp., and Escherichia coli, along with Malassezia pachydermatis and Candida albicans (C. albicans), naturally inhabit the ear epithelium as commensal microorganisms (Rosales et al. 2024). However, the ability of some of these microorganisms, particularly Staphylococcus spp., Pseudomonas spp. and C. albicans, to form biofilms contributes to increased antimicrobial resistance and impaired immune responses, leading to treatment failures and chronic and recurrent infections (Luciani et al. 2023). The Minimum Inhibitory Concentration (MIC) of currently available antibiotics is measured in the planktonic phase of microorganisms; however, in the biofilm phase, these MIC values can increase up to 1000‐fold (Verderosa et al. 2019).

The treatment strategy for OE typically involves the empirical use of antibacterial, antifungal, and anti‐inflammatory drugs (Chan et al. 2019). However, the suboptimal use of antimicrobial agents in medicine, veterinary care and agriculture has led to an increase in multidrug‐resistant microorganisms (MDROs), including extensively and pan‐drug‐resistant strains (Meshkat et al. 2021, Rafati Zomorodi et al. 2020). In recent years, the assessment of natural products, including essential oils (EOs), has gained significant attention as a novel strategy to address the declining antimicrobial pipeline (Aljaafari et al. 2021). Numerous studies report promising antimicrobial activities of EOs, such as cinnamon against Porphyromonas gingivalis (Wang et al. 2018), peppermint EO against the planktonic and biofilm phases of Staphylococcus aureus (S. aureus) (Kang et al. 2019), lemongrass EO against the planktonic and biofilm phases of Candida tropicalis (Sahal et al. 2020), and an oregano EO against Vibrio vulnificus (Luo et al. 2022).

Linalool (C10H18O), a monoterpene alcohol, has been identified as one of the major volatile compounds extracted from over 200 plant species globally (Herman et al. 2016). The antibacterial and antifungal activities of linalool have been established; moreover, this compound also exhibits antioxidant, anti‐inflammatory, and anti‐cancer properties (Gao et al. 2019, Guo et al. 2021, Guo et al. 2021). Additionally, linalool has been recognised as a vital component in domestic products, cosmetic preservatives, food preservatives and the synthesis of vitamins A and E, with an estimated annual usage of 1000 metric tonnes worldwide. However, the application of linalool and essential oils remains challenging due to their high volatility, low utilisation rate and solubility issues (Herman et al. 2016, Zhong et al. 2021).

This investigation aimed to assess the antimicrobial and antibiofilm activity of lavender EO (LEO) against S. aureus, Staphylococcus epidermidis (S. epidermidis), and C. albicans isolated from dogs with clinical OE. Additionally, the study compared the antimicrobial and antibiofilm efficacy of LEO with that of linalool.

2. Material and Methods

2.1. Preparation of LEO and Linalool

The aerial parts of cultivated Lavandula angustifolia (batch number 1092) were sourced from Pakan Bazar Company in Isfahan, Iran. Subsequently, the plant was taxonomically identified and catalogued with the herbarium number PMP‐2327 by botanists at the Herbarium of the Faculty of Pharmacy, University of Tehran. To prepare the essential oil, 100 grams of the lavender's aerial parts were washed, dried at room temperature, and then ground into a powder using a grinder. Linalool was purchased from Sigma‐Aldrich (UK; 98%; CAS number STBJ6366). The preparation of LEO was conducted as follows: 50 g of dried lavender plant powder was sampled and suspended in 600 mL of distilled water in a sterile 2 L Erlenmeyer flask. Hydrodistillation was then performed using a Clevenger apparatus for 4 h. Finally, the extracted oil was dehydrated using sodium sulphate and stored in a capped container at 4°C.

2.2. Gas Chromatography‐mass Spectrometry (GC‐MS) Analysis

The chemical composition of the LEO was analysed using GC‐MS. An AGILENT 6890 gas chromatograph, coupled with an AGILENT 5973 series mass selective detector, was employed for this purpose. The column used had a length of 30 metres, an inner diameter of 250 mm, and a film thickness of 25 mm. The injector and mass detector temperatures were set at 250°C and 230°C, respectively. The oven temperature programme started at 40°C was held for 1 min, then increased to 250°C at a rate of 3°C per minute, and maintained at 250°C for 20 min. Helium served as the carrier gas at a flow rate of 1.0 mL/min. The oil's compounds were identified by comparing their retention indices (RIs) and mass spectra fragmentation patterns with those in the Wiley 7 n.1 Mass Computer Library.

2.3. Microorganisms and Culture Conditions

In this investigation, a total of five clinical microorganisms were included, comprising three clinical isolates of C. albicans, two isolates of S. aureus, and two isolates of S. epidermidis; all isolates were obtained from dogs with otitis. The C. albicans isolates were previously identified using the RapID Yeast Plus System (Remel, USA) and were stored as a collection at the Department of Mycology, Veterinary School of Tehran University. Additionally, the S. aureus and S. epidermidis isolates were obtained from a collection in the Department of Microbiology, Veterinary School of Tehran University. The C. albicans ATCC 10331, S. aureus ATCC 29213, and S. epidermidis ATCC 12228 strains were used as quality controls. All microorganisms were preserved in the Brain Heart Infusion broth (BHI) (Merck, Germany) medium supplemented with 30% glycerol (Merck, Germany) at ‐70°C. The deferrisation of C. albicans and Staphylococcus isolates was conducted by culturing them on Sabouraud dextrose agar (SDA) (Merck, Germany) supplemented with chloramphenicol (Sigma Aldrich, USA) (50 µg/L) and in Luria–Bertani broth (LB) (Merck, Germany), respectively; also, incubation was performed for 48 h at 30°C and for 24 h at 37°C, respectively.

2.4. Biochemical Confirmation of Microorganisms

After the initial incubation, C. albicans was confirmed by culturing growth colonies on Candida chrome agar, with plates incubated for 48 h at 35°C. The colonies that appeared green were identified as C. albicans for further experiments. Additionally, confirmation of the Staphylococcus strains was performed using standard biochemical tests, including Gram staining (showing Gram‐positive cocci), catalase/oxidase tests (both species were +ve /‐ve), coagulase tests (+ve for S. aureus and ‐ve for S. epidermidis), DNase tests (+ve for S. aureus and ‐ve for S. epidermidis), and susceptibility to novobiocin (both species were susceptible) (Zomorodi et al. 2024).

2.5. Determination of Minimum Inhibitory Concentration of LEO and Linalool Against Planktonic Phase of C. albicans and Staphylococcus Isolates

The antimicrobial activity of LEO and linalool was assessed against C. albicans and Staphylococcus isolates in the planktonic phase, as recommended by CLSI guidelines (M60‐Ed1 (CLSI 2020) and M100‐Ed31 (CLSI 2021), respectively). First, the LEO and linalool were suspended in RPMI 1640 medium (Merck, Germany) (pH 7) using 0.1% dimethyl sulfoxide (DMSO) (Merck, Germany) to prepare an initial concentration of 80 mg/mL. The investigated concentration range was adjusted from 0.156 to 80 mg/mL using serial dilution in a 96‐well microplate. The final inoculum size of C. albicans was adjusted to 0.5–2.5 × 103 CFU/mL in RPMI 1640 medium. For Staphylococcus isolates, the initial inoculum was prepared in trypticase soy broth (TSB; Merck, Germany) at 1 × 106 CFU/mL and then diluted 1:2 in Mueller–Hinton Broth (MHB) to achieve a final concentration of 5 × 105 CFU/mL for MIC testing. The test was conducted using MHB medium (Merck, Germany) for Staphylococcus isolates and RPMI 1640 for C. albicans isolates. Two wells in each row were designated as positive and negative controls. The positive control was filled with only the medium and the microorganism inoculum, while the negative control contained only the medium and either LEO or linalool suspensions. After overnight incubation at 37°C, the well containing the lowest concentration of LEO or linalool that exhibited no visible growth turbidity was interpreted as the MIC.

2.6. Determination of Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC)

In continuation of the previous section, 10 µL samples were taken from the MIC wells, as well as from wells containing higher concentrations. These samples were then utilised to perform spread plate cultures on Mueller–Hinton agar (Merck, Germany) for Staphylococcus isolates and SDA for C. albicans. The plates were incubated at 37°C for 24 h for Staphylococcus isolates and at 30°C for 48 h for C. albicans isolates to evaluate potential growth. The culture corresponding to the lowest concentration of LEO and linalool, which showed no growth of colonies, was identified as the MBC or MFC.

2.7. Assessment of Biofilm Production of C. albicans and Staphylococcus Isolates

The tissue microtiter plate assay was conducted to demonstrate biofilm production by the tested microorganisms, as previously described (Lohse et al. 2017, Stepanović et al. 2007). Two to three colonies of freshly cultured C. albicans and Staphylococcus isolates were inoculated into yeast peptone dextrose (YPD) and tryptic soy broth (TSB) (Merck, Germany), both of which were supplemented with 0.5% glucose. After overnight incubation at 37°C, the media were centrifuged at 3000 rpm for 10 minutes; the supernatants were discarded, and the pellets were washed gently twice with phosphate‐buffered saline (PBS, pH 7.2). The pellet cells were resuspended in RPMI 1640 for C. albicans and in TSB for Staphylococcus isolates, adjusted to 1 × 106 CFU/ml using a hemocytometer slide. Next, 100 µL of each microbial suspension was added to a 96‐well polystyrene microtiter plate, with the exception of column 12, which was designated as the negative control. Following another overnight incubation at 37°C, the medium was aspirated, and the wells were washed three times with PBS. The wells were fixed with 150 µL methanol for 20 min, then stained with 150 µL of 0.1% crystal violet for 10 min before washing. Finally, the dye was resolubilised with 150 µL of 33% glacial acetic acid. The optical density (OD) at 570 nm was measured using an ELISA reader, and biofilm production was reported based on the formula in Table 1.

TABLE 1.

Interpretation of biofilm potential production.

The formula Results
OD ≤ ODC * No biofilm producer
ODC < OD ≤ 2 × ODC Weak biofilm producer
2 × ODC < OD ≤ 4 × ODC Moderate biofilm producer
4 × ODC < OD Strong biofilm producer
Open in a new tab

Abbreviations: ODC, cut‐off OD; OD, optical density; SD, standard division.

*

ODC is defined as the average OD of the negative control plus three standard deviations of the negative control.

2.8. Anti‐biofilm Activity of LEO and Linalool Against C. albicans and Staphylococcus Isolates

In this section, the anti‐biofilm effects of different concentrations of LEO and linalool suspensions were separately investigated on C. albicans and Staphylococcus isolates. As described previously, varying concentrations of LEO and linalool were prepared to a final volume of 100 µL in each well. Subsequently, 100 µL of C. albicans and Staphylococcus isolate suspensions, each equal to 1 × 106 CFU/mL, were added to the wells, with the positive control containing only the microbial suspension. After incubation at 37°C for 24 h, the percentage of biofilm inhibition was calculated using the following formula: 100–((OD at 560 nm of treated wells) / (mean OD at 560 nm of control wells without antimicrobial agent) × 100) (Esfandiary et al. 2024).

2.9. Anti‐biofilm Activity of LEO and Linalool Against Dual Biofilm Producing of C. albicans and Staphylococcus Isolates

This method represents the anti‐biofilm activity of LEO and linalool separately when exposed to combinations of subjected microorganisms, which were C. albicans ATCC 10331 and one clinical S. aureus isolates and C. albicans ATCC 10331 with clinical S. epidermidis isolates. The percentage of biofilm inhibition was calculated as explained above.

2.10. Scanning Electronic Microscopic (SEM) Visualisation

The cell and biofilm structures of the tested microorganisms after treatment with LEO and linalool suspension (each applied separately), as well as in the absence of treatment, were demonstrated using SEM. Sample preparation began with placing 7 mm PVC slides into the wells of a 24‐well cell culture plate. Subsequently, 1 mL of LEO and linalool suspension, which was suspended in buffered RPMI‐1640 with 0.1% DMSO at a MIC/2 concentration, was added to each well containing 1 mL of the prepared microorganism suspension as previously described. Additionally, C. albicans and Staphylococcus isolate suspensions were separately mixed in the following wells with culture medium without LEO and linalool suspension. Furthermore, yeast and bacteria were mixed in a 1:1 ratio and added to wells containing culture medium without LEO and linalool suspension. After an incubation period of 48 h at 37°C, the medium was discarded from the wells, and the slides were gently washed with PBS. Fixation was accomplished by adding 2.5% glutaraldehyde for 2 h at 4°C. The samples were then dehydrated with alcohol in 30%, 70%, 80%, 90% and 95% concentrations. Finally, the samples were coated with a gold layer, and their three‐dimensional structures were captured using a JEOL JSM‐840 scanning electron microscope.

2.11. Statistical Analysis

All experiments were performed in triplicate, and the results were expressed as mean ± standard deviations. Statistical analysis was carried out using SPSS version 25.0 (SPSS, Chicago, IL, USA). Data were analysed using one‐way analysis of variance (ANOVA), followed by multiple comparisons between treatments using the Bonferroni test. A p‐value of less than 0.05 was considered statistically significant for all analyses.

3. Results

3.1. GC‐MS Analysis

A total of 37 different components were detected in LEO using GC‐MS analysis. The most predominant component was linalool (42.33%), followed by alpha‐pinene (12.47%), alpha‐terpinolene (8.01%) and limonene (7.89%) (Table 2).

TABLE 2.

GC‐MS analysis of lavender essential oil.

No RT (min) Area% Name Quality CAS Number
1 4.376 0.07 Cyclofenchene 96 000488‐97‐1
2 4.859 0.01 2‐Bornene 96 000464‐17‐5
3 4.963 0.07 Bornylene 95 000464‐17‐5
4 5.131 0.12 (4E)‐2,6‐Dimethyl‐4‐octene 94 062960‐76‐3
5 5.456 0.47 Tricyclene 96 000508‐32‐7
6 6.022 12.47 Alpha‐PINENE 89 000080‐56‐8
7 6.379 4.23 Camphene 98 000079‐92‐5
8 6.955 0.01 Delta‐2‐carene 95 000000‐00‐0
9 7.029 0.01 Linaloyl oxide 62 000000‐00‐0
10 7.207 0.65 Beta‐pinene 97 018172‐67‐3
11 7.464 0.24 Delta‐4(8)‐Menthene 96 001124‐27‐2
12 7.763 0.41 Beta‐Myrcene 96 000123‐35‐3
13 8.009 0.15 p‐Menth‐3‐ene 93 000500‐00‐5
14 8.229 0.44 l‐Phellandrene 97 000099‐83‐2
15 8.449 0.43 Delta‐3‐Carene 97 013466‐78‐9
16 8.869 7.57 Alpha‐Terpinene 98 000099‐86‐5
17 9.241 4.43 Cymene 94 000527‐84‐4
18 9.451 7.89 Dl‐Limonene 97 000138‐86‐3
19 10.053 0.09 Sabinene 93 003387‐41‐5
20 10.509 1.98 Gamma‐Terpinene 97 000099‐85‐4
21 10.955 0.07 (1R)‐(+)‐Trans‐Isolimonene 58 005113‐87‐1
22 11.909 8.02 Alpha‐Terpinolene 98 000586‐62‐9
23 12.963 42.33 Linalool L 97 000078‐70‐6
24 13.062 0.11 Endo‐Isofenchol 91 000534‐35‐0
25 13.157 0.15 D‐Fenchyl alcohol 98 001632‐73‐1
26 14.105 0.39 Dihydrolinalool 80 000000‐00‐0
27 14.383 4.53 Camphor 98 000076‐22‐2
28 14.818 0.93 Isoborneol 96 000124‐76‐5
29 15.159 0.39 BORNEOL L 95 000464‐45‐9
30 15.704 0.02 Beta‐Phellandrene 64 000555‐10‐2
31 16.354 0.61 Alpha‐Terpineol 90 010482‐56‐1
32 17.796 0.01 Carveol 56 000099‐48‐9
33 18.598 0.10 Trifluoroacetyl‐isopulegol 49 028587‐54‐4
34 18.949 0.02 2,5‐Dimethoxytoluene 64 024599‐58‐4
35 20.626 0.35 4,5,6,7‐Tetrahydrophthalide 72 066309‐76‐0
36 23.394 0.03 Linalyl isobutyrate 86 000078‐35‐3
37 24.637 0.01 Geranyl butyrate 80 000106‐29‐6
Open in a new tab

3.2. Determination of MIC and MBC/MFC of LEO and Linalool Against Planktonic Phase of C. albicans and Staphylococcus Isolates

In general, linalool was more effective against all tested microorganisms in the planktonic phase compared to LEO (Table 3). Interestingly, the MIC of both LEO and linalool for yeast was higher than those for bacteria. Although the MIC of LEO and linalool was similar among Staphylococcus isolates, the MIC/MBC ratio for LEO and linalool was 1:2 and 1:1, respectively. In contrast, the MIC/MFC ratio for LEO and linalool was 1:1 and 1:4, respectively.

TABLE 3.

The MIC and MBC of lavender ES and linalool against C. albicans and Staphylococcus isolates in planktonic phase.

Lavender ES Linalool
Microorganisms MIC (µg/ml) MBC/MFC (µg/ml) Ratio MIC (µg/ml) MBC/MFC (µg/ml) Ratio
S. aureus 1 1250 2500 2 1250 1250 1
S. aureus ATCC 29213 1250 2500 2 1250 1250 1
S. epidermidis 1 1250 2500 2 1250 1250 1
S. epidermidis ATCC 12228 1250 2500 2 1250 1250 1
C. albicans 1 5000 5000 1 2500 10,000 4
C. albicans 2 5000 5000 1 2500 10,000 4
C. albicans 3 5000 5000 1 2500 10,000 4
C. albicans ATCC 29213 5000 5000 1 2500 10,000 4
Open in a new tab

Abbreviations: ES, essential oil; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration.

3.3. Biofilm Production of Tested Microorganisms

All subjected microorganisms in the present study were biofilm producers. There were only two moderate biofilm producers, including S. aureus ATCC 29213 and C. albicans 1 (Tables 4 and 5)

TABLE 4.

The results of biofilm production.

Microorganisms OD (Mean) SD OD of Negative control Results
S. aureus 1 1.82 0.37 0.2 Strong
S. aureus ATCC 29213 0.94 0.17 0.25 Moderate
S. epidermidis 1 1.44 0.19 0.24 Strong
S. epidermidis ATCC 12228 1.13 0.45 0.25 Strong
C. albicans 1 0.76 0.07 0.23 Strong
C. albicans 2 1.44 0.19 0.27 Moderate
C. albicans 3 1.1 0.17 0.28 Strong
C. albicans ATCC 29213 0.87 0.10 0.21 Strong
Open in a new tab

Abbreviations: OD, optical density; SD, standard division.

TABLE 5.

The MIC and MBC/MFC of lavender ES and linalool against single‐ and dual‐biofilm formation of C. albicans and Staphylococcus isolates.

Lavender ES Linalool
Microorganisms

MIC90

(µg/ml)

MBC/MFC90

(µg/ml)

MIC90

(µg/ml)

MBC/MFC90

(µg/ml)
C. albicans ATCC 29213 + S. aureus 1 2500 10,000 5000 20,000
C. albicans + S. epidermidis 1 5000 10,000 10,000 20,000
C. albicans ATCC 29213 20,000 20,000 40,000 40,000
S. aureus 1 5000 20,000 5000 10,000
S. epidermidis 1 5000 20,000 5000 10,000
Open in a new tab

Abbreviations: ES, essential oil; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration.

3.4. The Effect of LEO and linalool on Biofilm

Table 4 presents the MIC and MBC/MFC of LEO and linalool against single and dual‐biofilm yeast and bacteria. A comparison of results revealed a consistent ratio of MIC to MBC/MFC (1:4) for both agents against dual‐biofilms of yeast and bacteria, indicating that the MBC/MFC was four‐fold higher than the MIC. Additionally, linalool demonstrated greater effectiveness at higher concentrations compared to LEO.

The dose‐dependent effects of LEO and linalool against single biofilms of S. aureus and S. epidermidis isolates were significant at a concentration of 1250 µg/ml, which inhibited 50% of biofilm formation (p ≤ 0.05). Furthermore, a 90% inhibition of biofilm formation was observed at a concentration of 5000 µg/ml. Complete inhibition was achieved at concentrations of 20,000 µg/ml for LEO and 10,000 µg/ml for linalool (Figures 1 and 2). Notably, these values were even higher in the context of biofilm formation by C. albicans isolates. Specifically, 50% and 90% inhibition of biofilm formation against C. albicans isolates was achieved at concentrations of 10,000 and 40,000 µg/ml for LEO and at 2500 and 40,000 µg/ml for linalool, respectively (Figures 3 and 4)

FIGURE 1.

FIGURE 1

Open in a new tab

Mean inhibitory effect of lavender essential oil on biofilm formation by S. aureus and S. epidermidis isolates. Different letters on each column indicate a significant difference from other columns.

FIGURE 2.

FIGURE 2

Open in a new tab

Mean inhibitory effect of linalool on biofilm formation by S. aureus and S. epidermidis isolates. Different letters on each column indicate a significant difference from other columns.

FIGURE 3.

FIGURE 3

Open in a new tab

Mean inhibitory effect of lavender essential oil on biofilm formation by C. albicans isolates. Different letters on each column indicate a significant difference from other columns.

FIGURE 4.

FIGURE 4

Open in a new tab

Mean inhibitory effect of linalool on biofilm formation by C. albicans isolates. Different letters on each column indicate a significant difference from other columns.

The LEO demonstrated a 50% inhibition of dual biofilms formed by C. albicans + S. aureus and C. albicans + S. epidermidis at lower concentrations compared to linalool. As illustrated in Figure 5, LEO at concentrations of 2500 and 5000 µg/ml inhibited the biofilm of C. albicans + S. aureus by 69.57% ± 1.8 and that of C. albicans + S. epidermidis by 51.07% ± 2.4, respectively. In contrast, linalool exhibited inhibition of the dual biofilm of C. albicans + S. aureus at a concentration of 5000 µg/ml, achieving an inhibition rate of 60.5% ± 1.8, and inhibited the C. albicans + S. epidermidis biofilm at a concentration of 10,000 µg/ml with an inhibition rate of 69.47% ± 1.8.

FIGURE 5.

FIGURE 5

Open in a new tab

Inhibitory effect of lavender essential oil and linalool on dual biofilms formed by C. albicans + S. aureus and C. albicans + S. epidermidis.

3.5. SEM Analysis

As demonstrated in captured photos from control samples (Figure 6), a high density of dual biofilm consisted of C. albicans hyphae (Figure 6: A and B; arrows g, b) and ballistoconidia (Figure 6: A and B; arrows c, e) that Staphylococcus isolates were attached to the hyphae (Figure 6: A and B; arrow d) or ballistoconidia (Figure 6: A and B; arrows a, f). Treatment with MIC/2 concentration of LEO generally reduced the density of biofilms, certainly hyphae structures. Also, the morphology of Staphylococcus isolates was deformed and cell accumulation was decreased. In comparison, treatment with linalool in MIC/2 concentration revealed higher anti‐biofilm activity against yeast and bacteria.

FIGURE 6.

FIGURE 6

Open in a new tab

SEM images showing various dual biofilms of C. albicans/S. aureus (A) and C. albicans/S. epidermidis (B) as control groups. (C, D) depict the dual biofilms of C. albicans/S. epidermidis and C. albicans/S. aureus, respectively, after treatment with lavender essential oil at MIC/2 concentration. Disruption of integrity and pore formation are evident among the hyphae (arrows e, g); ballistoconidia were observed with reduced cell accumulation, increased cell wrinkling, and membrane disruption (arrows a, c, h, f). Disturbances in bacterial cells are indicated (arrows b, d, i). (E, F) show dual biofilms of C. albicans/S. aureus and C. albicans/S. epidermidis, respectively, following treatment with linalool at MIC/2 concentration. Notable disruptions and cell membrane lysis of hyphae are observed (arrows a, h, e, f), along with pore formation on the cell membrane of C. albicans (arrows c, b, g). Bacterial cells appear disturbed and detached from the yeast structures (arrows b, d).

4. Discussion

OE is the most frequently diagnosed condition in pet animals, particularly in dogs. Factors contributing to the persistence of otitis once established, which necessitates treatment to prevent recurrence, include those associated with chronic diseases, such as chronic inflammation and the progressive pathological changes within the ear canal and associated otitis media (Tambella et al. 2020). However, biofilm formation by certain microorganisms, such as S. aureus, S. epidermidis and C. albicans, can lead to tolerance to antimicrobial agents and subsequent treatment failure (Nocera et al. 2023). Therefore, a new approach that includes the use of EOs with appropriate antimicrobial and antibiofilm activity has recently been highlighted. In the present study, the antimicrobial and antibiofilm activity of the LEO was assessed against S. aureus, S. epidermidis and C. albicans isolates.

The LEO consists of linalool in a range of 20% to 45%, as reported by ISO (Lis‐Balchin and Hart 1999). This finding supports our GC‐MS analysis of the LEO, which identified linalool as the most prevalent component, with a frequency of 42.33% among 37 different identified chemical compounds. Additionally, Valkova et al. found linalool to be the most common component in their study of 44 identified chemical compounds in LEO, with a frequency of 32.7% (Valková et al. 2021). However, these findings contradict those of Puvaca et al., who reported a linalool frequency of only 10.71% in LEO (Puvača et al. 2021). Another frequently identified component in the present study was α‐pinene (12.47%), along with α‐terpinolene (8.01%), limonene (7.89%), and camphor (4.53%), all of which were higher than the values published by ISO (α‐pinene, 0%; α‐terpinolene, 0–2%; limonene, 0–1%; camphor, 0–1.5%). However, the amounts of β‐phellandrene (0.02%) and α‐terpineol (0.61%) were in agreement with the ISO values (0‐1% and 0–2%, respectively) (Lis‐Balchin and Hart 1999).

In the present survey, LEO demonstrated a growth inhibitory effect against S. aureus and S. epidermidis at an MIC of 1250 µg/ml, while the MIC for C. albicans isolates was 5000 µg/ml. The MBC and MFC of LEO were determined to be 2500 µg/ml and 5000 µg/ml, respectively. Furthermore, the antimicrobial activity of linalool, the most prevalent compound in LEO, was evaluated. The MIC and MBC values for linalool against Staphylococcus isolates were found to be 1250 µg/ml and 2500 µg/ml, respectively. In contrast, the results for C. albicans differed, with MIC and MFC values of 2500 µg/ml and 10,000 µg/ml, respectively. According to the systematic literature review, the antimicrobial activity of EOs has been well established, with LEO being extensively investigated (Aprotosoaie et al. 2017, Malloggi et al. 2022). Cruz Sanchez et al. determined that increasing the concentration of LEO significantly enhanced its inhibitory effect against S. aureus, S. epidermidis and C. albicans strains. Additionally, they tested LEO at various concentrations on biosynthetic membranes and did not find significant differences (Cruz Sánchez et al. 2024). However, several studies have indicated that using LEO coated on different biosynthetic membranes increases its inhibitory effect due to the antimicrobial activity of the tested membranes (Danila et al. 2021, Liu et al. 2022, Sukhodub et al. 2023).

The lower determination of the MIC and MBC/MFC of LEO in some experiments compared to linalool may be attributed to the presence of other compounds. For instance, the antimicrobial activity of α‐pinene, which is the second most prevalent compound in the current study, has been observed in previous investigations (Leite‐Sampaio et al. 2022, Nóbrega et al. 2021). However, linalool is a major compound found in lavender, basil, and other essential oils known for their antimicrobial activity (Mahizan et al. 2019).

The mechanism of antimicrobial activity of linalool was previously demonstrated by Yang et al., who stated that linalool disrupts cell membrane permeability by inducing oxidative stress. This finding was recorded by measuring the zeta potential of the tested K. pneumoniae isolates; treatment with linalool increased the zeta potential to ‐7.83 compared to the non‐treated condition, which had a value of ‐12.1 (Yang et al. 2021). SEM images captured in the current study revealed significant disruption of the cell membrane in the tested isolates after exposure to linalool and LEO.

Interestingly, we observed the antimicrobial activity of linalool and LEO in both the single‐ and dual‐biofilm phases of Staphylococcus and C. albicans isolates. Generally, the MIC and MBC/MFC ratio of LEO and linalool against Staphylococcus isolates in the biofilm phase was 1:2 and 1:1, respectively, while these values against C. albicans isolates were 1:1 and 1:4, respectively. Our observations indicated that linalool exhibited higher antimicrobial activity compared to LEO against Staphylococcus isolates, whereas LEO was more effective than linalool against C. albicans isolates in the biofilm phase. These findings do not confirm the earlier investigation by Brożyna et al., which indicated that LEO emulsions is inactive against S. aureus isolates in the biofilm phase (Brożyna et al. 2021). However, a recently conducted systematic review indicated that emulsified LEO with Tween 20 did not exhibit antibiofilm activity, while non‐emulsified LEO was able to eradicate biofilms of S. aureus in the range of 40% to 70% (Truong and Mudgil 2023). This discrepancy may be explained by differences in sample size, the characteristics of the tested microorganisms, the methodology for extracting EOs, and variety of used lavender types.

Although LEO was effective against all microorganisms tested in the current study, C. albicans isolates exhibited greater resistance to LEO. In general, there are few mechanisms identified as exhibiting antibiofilm activity of EOs in fungi, which include fungicidal effects, prevention of adhesion, disruption of intracellular connections, and modulation of morphogenesis in dimorphic fungi (Budzyńska et al. 2017). The biofilm inhibition of LEO among C. albicans isolates at the MIC was estimated to be approximately 60%. This value is lower than the previous findings, which reported a 75% –80% inhibition of biofilm growth of C. albicans at the MIC of LEO (Karpiński et al. 2023, Santos et al. 2024).

Investigating the antibiofilm activity of LEO in dual biofilms formed by fungi and bacteria is one of the few studies conducted in this area. Our results demonstrated that LEO completely inhibited the dual biofilms formed by C. albicans/S. aureus and C. albicans/S. epidermidis at a concentration of 20,000 µg/mL. Furthermore, the synergistic effect of essential oils in the dual biofilm phase of C. albicans/S. aureus against various antibiotics and antifungal agents has been evaluated. Li et al. demonstrated the synergistic effect of clove oil when combined with fluconazole or mupirocin against the dual biofilm of C. albicans/S. aureus (Li et al. 2015).

Limitations highlight the challenges associated with collecting data on the expression of biofilm‐mediated genes following exposure to LEO. Also, there is a need for a comprehensive evaluation of the antimicrobial and antibiofilm activity of LEO against Gram‐negative bacteria. Furthermore, further investigations are warranted to assess the antimicrobial and antibiofilm efficacy of essential oils, particularly LEO, in combination with various antibiotics and antifungal agents. Additionally, the evaluation of certain antibacterial and antifungal agents as controls could be used to compare the antimicrobial and antibiofilm activities of LEO and other essential oils.

5. Conclusion

In summary, LEO has exhibited antimicrobial and antibiofilm activity against S. aureus, S. epidermidis, and C. albicans isolates in both planktonic and biofilm phases. Our findings are promising, suggesting the potential of LEO in novel therapeutic approaches to combat the threat of antimicrobial resistance. However, further in vivo investigations are necessary to evaluate the effective dosage of LEO and its potential toxic effects before it can be considered a viable therapeutic option for humans and/or animals.

Author Contributions

N.N.: conceptualization, data curation, funding acquisition, investigation and methodology. A.A. and B.N.F.: conceptualisation, data validation, formal analysis, project administration and supervision. S.A. and A.A.: investigation, review and editing of the manuscript and visualisation. A.R.Z.: conceptualisation, writing the draft of the manuscript and formal analysis. J.M.: conceptualization, data curation and visualisation.

Conflicts of Interest

The authors declare no conflicts of interest.

Ethics Statement

The experiment was conducted on archived isolates; therefore, no ethical approval was required.

Peer Review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/vms3.70407.

Supporting information

Supporting Information

VMS3-11-e70407-s001.docx (13.8KB, docx)

Funding: The authors received no specific funding for this work.

See Author's Additional Information in Supporting Information.

Contributor Information

Aghil Sharifzadeh, Email: asharifzadeh@ut.ac.ir.

Bahar Nayeri Fasaei, Email: nayerib@ut.ac.ir.

Data Availability Statement

The datasets generated and analysed during this research are available by contacting the corresponding author.

References

  1. Aljaafari, M. N. , AlAli A. O., Baqais L., et al. 2021. “An Overview of the Potential Therapeutic Applications of Essential Oils.” Molecules 26, no. 3: 628. 10.3390/molecules26030628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aprotosoaie, A. C. , Gille E., Trifan A., Luca V. S., and Miron A. 2017. “Essential Oils of Lavandula Genus: a Systematic Review of Their Chemistry.” Phytochemistry Reviews 16, no. 761–799. 10.1007/s11101-017-9517-1. [DOI] [Google Scholar]
  3. Brożyna, M. , Paleczny J., Kozłowska W., et al. 2021. “The Antimicrobial and Antibiofilm in Vitro Activity of Liquid and Vapour Phases of Selected Essential Oils Against Staphylococcus aureus.” Pathogens 10, no. 9: 1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Budzyńska, A. , Różalska S., Sadowska B., and Różalska B. 2017. “Candida albicans/Staphylococcu s aureus Dual‐Species Biofilm as a Target for the Combination of Essential Oils and Fluconazole or Mupirocin.” Mycopathologia 182, no. 989–995. 10.1007/s11046-017-0192-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chan, W. Y. , Hickey E. E., Page S. W., Trott D. J., and Hill P. B. 2019. “Biofilm Production by Pathogens Associated With Canine Otitis Externa, and the Antibiofilm Activity of Ionophores and Antimicrobial Adjuvants.” Journal of Veterinary Pharmacology and Therapeutics 42, no. 6: 682–692. 10.1111/jvp.12811. [DOI] [PubMed] [Google Scholar]
  6. CLSI . 2020. Performance Standards for Antifungal Susceptibility Testing of Fungi, M60. 1st ed., Clinical and Laboratory Standards Institute. [Google Scholar]
  7. CLSI . 2021. Performance Standards for Antimicrobial Susceptibility Testing, M100. 31st ed., Clinical and Laboratory Standards Institute. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cruz Sánchez, E. , García M. T., Gracia I., et al. 2024. “Antibacterial Activity Assessment of Chitosan/Alginate Lavender Essential Oil Membranes for Biomedical Applications.” Membranes 14, no. 1: 12. 10.3390/membranes14010012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Danila, A. , Muresan E. I., Ibanescu S.‐A., et al. 2021. “Preparation, Characterization, and Application of Polysaccharide‐based Emulsions Incorporated With lavender Essential Oil for Skin‐Friendly Cellulosic Support.” International Journal of Biological Macromolecules 191, no. 405–413. 10.1016/j.ijbiomac.2021.09.090. [DOI] [PubMed] [Google Scholar]
  10. Esfandiary, M. A. , Khosravi A. R., Asadi S., Nikaein D., Hassan J., and Sharifzadeh A. 2024. “Antimicrobial and Anti‐biofilm Properties of Oleuropein Against Escherichia coli and Fluconazole‐resistant Isolates of Candida albicans and Candida glabrata.” BMC Microbiology 24, no. 1: 1–12. 10.1186/s12866-024-03305-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gao, Z. , Van Nostrand J. D., Zhou J., Zhong W., Chen K., and Guo J. 2019. “Anti‐listeria Activities of Linalool and Its Mechanism Revealed by Comparative Transcriptome Analysis.” Frontiers in Microbiology 10, no. 2947. 10.3389/fmicb.2019.02947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guo, F. , Liang Q., Zhang M., et al. 2021. “Antibacterial Activity and Mechanism of Linalool Against Shewanella Putrefaciens.” Molecules 26, no. 1: 245. 10.3390/molecules26010245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guo, F. , Chen Q., Liang Q., et al. 2021. “Antimicrobial Activity and Proposed Action Mechanism of Linalool Against Pseudomonas Fluorescens.” Frontiers in Microbiology 12, no. 562094. 10.3389/fmicb.2021.562094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Herman, A. , Tambor K., and Herman A. 2016. “Linalool Affects the Antimicrobial Efficacy of Essential Oils.” Current Microbiology 72, no. 165–172. 10.1007/s00284-015-0933-4. [DOI] [PubMed] [Google Scholar]
  15. Kang, J. , Jin W., Wang J., Sun Y., Wu X., and Liu L. 2019. “Antibacterial and Anti‐biofilm Activities of Peppermint Essential Oil Against Staphylococcus aureus.” LWT 101, no. 639–645. 10.1016/j.lwt.2018.11.093. [DOI] [Google Scholar]
  16. Karpiński, T. M. , Ożarowski M., Seremak‐Mrozikiewicz A., and Wolski H. 2023. “Anti‐Candida and Antibiofilm Activity of Selected Lamiaceae Essential Oils.” Frontiers in Bioscience‐Landmark 28, no. 2: 28. 10.31083/j.fbl2802028. [DOI] [PubMed] [Google Scholar]
  17. Leite‐Sampaio, N. F. , Gondim C. N., Martins R. A., et al. 2022. “Potentiation of the Activity of Antibiotics Against ATCC and MDR Bacterial Strains With (+)‐α‐Pinene and (‐)‐Borneol.” BioMed Research International 2022, no. 1: 8217380. 10.1155/2022/8217380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li, H. , Zhang C., Liu P., Liu W., Gao Y., and Sun S. 2015. “In Vitro Interactions Between Fluconazole and Minocycline Against Mixed Cultures of Candida albicans and Staphylococcus aureus.” Journal of Microbiology, Immunology and Infection 48, no. 6: 655–661. 10.1016/j.jmii.2014.03.010. [DOI] [PubMed] [Google Scholar]
  19. Lis‐Balchin, M. , and Hart S. 1999. “Studies on the Mode of Action of the Essential Oil of LavenderLavandula angustifolia P. Miller.” Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives 13, no. 6: 540–542. . [DOI] [PubMed] [Google Scholar]
  20. Liu, J. , Liu Y., Shao S., Zheng X., and Tang K. 2022. “Soluble Soybean Polysaccharide/Carboxymethyl Chitosan Coatings Incorporated With lavender Essential Oil: Structure, Properties and Application in Fruit Preservation.” Progress in Organic Coatings 173, no. 107178. 10.1016/j.porgcoat.2022.107178. [DOI] [Google Scholar]
  21. Lohse, M. B. , Gulati M., Valle Arevalo A., Fishburn A., Johnson A. D., and Nobile C. J. 2017. “Assessment and Optimizations of Candida albicans in Vitro Biofilm Assays.” Antimicrobial Agents and Chemotherapy 61, no. 5: 10. 10.1128/aac.02749-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luciani, L. , Stefanetti V., Rampacci E., et al. 2023. “Comparison Between Clinical Evaluations and Laboratory Findings and the Impact of Biofilm on Antimicrobial Susceptibility in Vitro in Canine Otitis Externa.” Veterinary Dermatology 34, no. 6: 586–596. 10.1111/vde.13197. [DOI] [PubMed] [Google Scholar]
  23. Luo, K. , Zhao P., He Y., et al. 2022. “Antibacterial Effect of Oregano Essential Oil Against Vibrio Vulnificus and Its Mechanism.” Foods 11, no. 3: 403. 10.3390/foods11030403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mahizan, N. A. , Yang S.‐K., Moo C.‐L., et al. 2019. “Terpene Derivatives as a Potential Agent Against Antimicrobial Resistance (AMR) Pathogens.” Molecules 24, no. 14: 2631. 10.3390/molecules24142631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Malloggi, E. , Menicucci D., Cesari V., Frumento S., Gemignani A., and Bertoli A. 2022. “Lavender Aromatherapy: a Systematic Review From Essential Oil Quality and Administration Methods to Cognitive Enhancing Effects.” Applied Psychology: Health and Well‐Being 14, no. 2: 663–690. 10.1111/aphw.12310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meshkat, Z. , Salimizand H., Amini Y., et al. 2021. “Detection of Efflux Pump Genes in Multiresistant Acinetobacter baumannii ST2 in Iran.” Acta Microbiologica Et Immunologica Hungarica 68, no. 2: 113–120. 10.1556/030.2021.01314. [DOI] [PubMed] [Google Scholar]
  27. Nóbrega, J. R. , Silva D. F., Andrade Júnior F. P., et al. 2021. “Antifungal Action of α‐pinene Against Candida Spp. Isolated From Patients With Otomycosis and Effects of Its Association With Boric Acid.” Natural Product Research 35, no. 24: 6190–6193. 10.1080/14786419.2020.1837803. [DOI] [PubMed] [Google Scholar]
  28. Nocera, F. P. , Pizzano F., Masullo A., Cortese L., and De Martino L. 2023. “Antimicrobial Resistant staphylococcus Species Colonization in Dogs, Their Owners and Veterinary Staff of the Veterinary Teaching Hospital of Naples, Italy.” Pathogens 12, no. 8: 1016. 10.3390/pathogens12081016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ponn, P. C. , Tipold A., and Volk A. V. 2024. “Can We Minimize the Risk of Dogs Developing Canine Otitis Externa?—A Retrospective Study on 321 Dogs.” Animals 14, no. 17: 2537. 10.3390/ani14172537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Puvača, N. , Milenković J., Galonja Coghill T., et al. 2021. “Antimicrobial Activity of Selected Essential Oils Against Selected Pathogenic Bacteria: in Vitro Study.” Antibiotics 10, no. 5: 546. 10.3390/antibiotics10050546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rafati Zomorodi, A. , Rad M., Hashemitabar G. R., and Salimizand H. 2020. “Molecular Typing of Cephalosporin Resistant Serovars of Salmonella Enterica From Poultry and Farm Animals.” Bulgarian Journal of Veterinary Medicine 23, no. 2. 10.15547/bjvm.2196. [DOI] [Google Scholar]
  32. Rosales, R. S. , Ramírez A. S., Moya‐Gil E., de la Fuente S. N., Suárez‐Pérez A., and Poveda J. B. 2024. “Microbiological Survey and Evaluation of Antimicrobial Susceptibility Patterns of Microorganisms Obtained From Suspect Cases of Canine Otitis Externa in Gran Canaria, Spain.” Animals 14, no. 5: 742. 10.3390/ani14050742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sahal, G. , Woerdenbag H. J., Hinrichs W. L., et al. 2020. “Antifungal and Biofilm Inhibitory Effect of Cymbopogon Citratus (lemongrass) Essential Oil on Biofilm Forming by Candida tropicalis Isolates; an in Vitro Study.” Journal of Ethnopharmacology 246, no. 112188. 10.1016/j.jep.2019.112188. [DOI] [PubMed] [Google Scholar]
  34. Santos, A. , Oliveira‐Filho A., Teixeira B., et al. 2024. “Lavandula Dentata L. essential Oil: A Promising Antifungal and Antibiofilm Agent Against Oral Candida albicans.” Brazilian Journal of Biology 84, no. e283646. 10.1590/1519-6984.283646. [DOI] [PubMed] [Google Scholar]
  35. Secker, B. , Shaw S., and Atterbury R. J. 2023. “Pseudomonas Spp. in Canine Otitis Externa.” Microorganisms 11, no. 11: 2650. 10.3390/microorganisms11112650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stepanović, S. , Vuković D., Hola V., et al. 2007. “Quantification of Biofilm in Microtiter Plates: Overview of Testing Conditions and Practical Recommendations for Assessment of Biofilm Production by Staphylococci.” APMIS 115, no. 8: 891–899. 10.1111/j.1600-0463.2007.apm_630.x. [DOI] [PubMed] [Google Scholar]
  37. Sukhodub, L. , Kumeda M., Sukhodub L., Bielai V., and Lyndin M. 2023. “Metal Ions Doping Effect on the Physicochemical, Antimicrobial and Wound Healing Profiles of Alginate‐based Composite.” Carbohydrate Polymers 304, no. 120486. 10.1016/j.carbpol.2022.120486. [DOI] [PubMed] [Google Scholar]
  38. Tambella, A. M. , Attili A. R., Beribè F., et al. 2020. “Management of Otitis Externa With an Led‐illuminated Gel: a Randomized Controlled Clinical Trial in Dogs.” BMC Veterinary Research 16: 1–14. 10.1186/s12917-020-02311-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Truong, S. , and Mudgil P. 2023. “The Antibacterial Effectiveness of Lavender Essential Oil Against Methicillin‐resistant Staphylococcus aureus: A Systematic Review.” Frontiers in Pharmacology 14, no. 1306003. 10.3389/fphar.2023.1306003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Valková, V. , Ďúranová H., Galovičová L., Vukovic N. L., Vukic M., and Kačániová M. 2021. “In Vitro Antimicrobial Activity of Lavender, Mint, and Rosemary Essential Oils and the Effect of Their Vapours on Growth of Penicillium Spp. in a Bread Model System.” Molecules 26, no. 13: 3859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vercelli, C. , Pasquetti M., Giovannetti G., et al. 2021. “In Vitro and in Vivo Evaluation of a New Phytotherapic Blend to Treat Acute Externa Otitis in Dogs.” Journal of Veterinary Pharmacology and Therapeutics 44, no. 6: 910–918. 10.1111/jvp.13000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Verderosa, A. D. , Totsika M., and Fairfull‐Smith K. E. 2019. “Bacterial Biofilm Eradication Agents: A Current Review.” Frontiers in Chemistry 7, no. 495483. 10.3389/fchem.2019.00824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang, Y. , Zhang Y., Shi Y.‐Q., Pan X.‐H., Lu Y.‐H., and Cao P. 2018. “Antibacterial Effects of Cinnamon (Cinnamomum zeylanicum) Bark Essential Oil on Porphyromonas gingivalis.” Microbial Pathogenesis 116, no. 26–32. 10.1016/j.micpath.2018.01.009. [DOI] [PubMed] [Google Scholar]
  44. Yang, S.‐K. , Yusoff K., Ajat M., et al. 2021. “Combinatorial Antimicrobial Efficacy and Mechanism of linalool Against Clinically Relevant Klebsiella pneumoniae.” Frontiers in Microbiology 12: 635016. 10.3389/fmicb.2021.635016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhong, W. , Tang P., Liu T., Zhao T., Guo J., and Gao Z. 2021. “Linalool Nanoemulsion Preparation, Characterization and Antimicrobial Activity Against Aeromonas Hydrophila.” International Journal of Molecular Sciences 22, no. 20:11003. 10.3390/ijms222011003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zomorodi, A. R. , Motamedifar M., Rahmanian K., et al. 2024. “Investigation of Integron Classes 1, 2, and 3 Among Multi‐Drug Resistant Staphylococcus Aureus Isolates in Iran: A Multi‐Center Study.” BMC Infectious Diseases 24: 1430. 10.1186/s12879-024-10311-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

VMS3-11-e70407-s001.docx (13.8KB, docx)

Data Availability Statement

The datasets generated and analysed during this research are available by contacting the corresponding author.

Articles from Veterinary Medicine and Science are provided here courtesy of Wiley

RESOURCES