Anti-adhesion and antibiofilm activities of Lavandula mairei humbert essential oil against Acinetobacter baumannii isolated from hospital intensive care units

Abstract This study aimed to assess, for the first time, the anti-adhesion and antibiofilm effects of Lavandula mairei Humbert essential oil against multidrug resistant Acinetobacter baumannii. Scanning electron microscope was used for visualizing its antibiofilm activity and the effect of this oil on surface physicochemical parameters was examined as a possible anti-adhesive target. Chemical analysis of Lavandulaa mairei essential oil showed a high content of carvacrol composition (79.12%). The oil tested exhibited antibacterial efficacy with inhibition diameters of 33 to 37.33 mm and minimum inhibitory and bactericidal concentrations of 1.56 µl ml−1. The oil inhibited adhesion by 83.66%, detach 73.30% of adherent cells and eliminated 64.02% of the biofilm compared to the untreated control. Lavandula mairei essential oil has proven its possible application as a preventive strategy by intervening in the initial adhesion of Acinetobacter baumannii to polystyrene.


Introduction
Acinetobacter baumannii is a ubiquitous bacterium and an effective Gram-negative human colonizer. This microorganism has become a problematic nosocomial pathogen by its persistence in the hospital environment and its ability to survive conventional antibiotic treatments (Perez et al. 2007). It represents 20% of intensive care unit infections (Garnacho-Montero and Timsit 2019). Therefore, the World Health Organization has listed A. baumannii as one of the highest priority pathogens for research and as a target for the development of new drugs. This pathogen adopts ways to overcome antibiotic therapy including the production of b-lactamases, efflux pumps, and target mutations (Kyriakidis et al. 2021). A. baumanni threatens global healthcare since it is resistant to classes of antibiotics such as carbapenems, which are used as a last line of defense against multidrug resistant diseases (Codjoe and Donkor 2017). Indeed, multidrug resistant A. baumanni infections are one of the most difficult-to-treat resistance phenotype, and result in increased mortality compared to other resistant pathogens (Kadri et al. 2018;Babiker et al. 2021).
Making the situation more challenging is that A. baumanni has a sophisticated survival and resistance mechanism which involves adhering to surfaces and forming biofilms (Elkheloui et al. 2022). Biofilms are a community of bacteria adhering to biotic or abiotic surfaces in a protective matrix of biopolymers (Lee Ro et al. 2017). This phenomenon begins with the adhesion of the bacteria to a suitable surface. This step can involve several factors of which the surface physicochemical properties are a major one (Zineba et al. 2014). Adhesion is followed by cell aggregation and proliferation to form biofilm microcolonies and, in order to obtain a mature biofilm, the bacterial cells produce extracellular biopolymers matrix (exopolysaccharides, proteins and extracellular DNA) (Toole et al. 2000). This biofilm forming ability allows A. baumanni to adapt and survive on various surfaces and under critical environmental conditions such as antibiotic therapy, high temperature variations, different intensities of ultraviolet and ionizing radiation in hospitals (Rampelotto 2010). Furthermore, biofilm forming bacteria can cause chronic infections, as this bacterium is recognized to be a cause of nosocomial infections, such as ventilator-associated pneumonia, bacteremia, meningitis, urinary tract and wound infections .
Consequently, growing concern about the resistance and survival attributed by the biofilm as well as the development of antibiotic resistance of A. baumannii has induced an urgent need for alternative strategies. In this regard, plants and their derivatives, such as essential oils (EOs), have been proposed as a possible alternative approach for the control of bacterial pathogens. Plants have always provided an excellent source of bioactive substances, which represent a great opportunity for biomedical science. Essential oils contain highly concentrated bioactive substances that are able to act in very low concentrations against multidrug resistant pathogens and with a wide spectrum of antimicrobial activities. Several studies have proven the effectiveness of EOs to affect the biofilms of several bacteria such as A. baumannii (Celik et al. 2015;Tutar 2016;Tutar et al. 2016). Consequently, these EOs were probably able to cross the protective barrier of the biofilm to disperse it and reach the bacterial cells, which most antibiotics cannot achieve. The EO currently used to combat biofilms have not be given the attention that truly reflects the possible and effective application of these natural products. The genus Lavandula contains 9 species and subspecies including 5 endemic to Morocco (Fennane et al. 2014). Lavandula mairei Humbert is one of these 5 species and is considered to be rare (Fennane and Tattou 1998). It is an aromatic plant widely used in traditional medicine for the treatment of various diseases such as gastrointestinal diseases, microbial infections, cough, asthma and fever (Abouri et al. 2012). L. mairei EO has been shown to have biological activity such as antibacterial, antioxidant and antifungal activities (El Hamdaoui et al. 2018;Boubaker et al. 2019;Ngasotter et al. 2022).
The present work was therefore undertaken to test the antibacterial, anti-adhesion and antibiofilm effects of L. mairei EO against multidrug resistant A. baumanni isolated from the hospital environment of the intensive care units of the Regional Hospital Center of Agadir-Morocco. To the best of our knowledge, this is the first investigation of anti-adhesion and antibiofilm properties of L. mairei EO. Additionally, the effect of this oil on surface physicochemical properties of surface tension and hydrophobicity, was also investigated as a possible EO target due to their important application in bacterial adhesion (Ksontini et al. 2013;Zineba et al. 2014).

Plant material
Aerial parts of L. mairei were harvested in the May 2020 from Tafraoute region (Western Anti Atlas) and a voucher specimen was deposited in the laboratory of Biotechnology and Valorization of Natural Resources, Faculty of Sciences, Ibn Zohr University, Agadir, Morocco.

Extraction of essential oil
The L. mairei EO was obtained from dried aerial plant material by hydrodistillation using a Clevenger type apparatus. The EO recovered was stored in the dark at 4 C until used.

Gas chromatography/mass spectrometry (GC/ MS) analyses
The EO composition was determined by GC/MS analysis according to El Hamdaoui et al. (2018). Identification of compounds was based on the comparison of their mass spectra with those of Wiley and NIST libraries as well as by comparison of their retention indices with those of authentic samples. Oil chemical relative composition was determined based on the peak areas for compounds contributing more than 0.1% of the total composition.

Bacterial strains
Six multidrug resistant A. baumannii (Ab) strains, isolated from medical devices of the intensive care units of the Regional Hospital Center of Agadir-Morocco (Laktib et al. 2018), were studied (Tables 1  and 2).

Disk diffusion method
The antibacterial activity of the EO was determined using the disk diffusion agar medium method (Fadli et al. 2012). The bacterial suspension at a concentration of approximately 10 8 UFC ml À1 (% 0.5 McFarland) was prepared from a 18-24 h Trypticase soy agar (TSA) culture of the A. baumannii strains in sterile saline solution. Ten microliters of EO were applied to sterile filter paper disks (Whatman No 1; 6 mm in diameter) and placed on the surface of the petri dishes with Mueller Hinton agar medium inoculated with one of the A. baumannii strains. The plates were incubated at 37 C for 24 h. Antibacterial activity was determined by measuring the diameter of the inhibition zone formed around the disc. Discs of 30 mg of Amikacin was used as positive controls. All the tests were performed in triplicate.

Minimum inhibitory concentration and minimum bactericidal concentration
Minimum inhibitory concentration (MIC) analysis was performed in Mueller Hinton Broth (MHB) via broth micro-dilution technique with 96-well microtiter plate (Bazargani and Rohloff 2016). The bacterial suspensions were adjusted to a concentration approximately 10 6 CFU ml À1 in fresh sterile MHB. Stock solution of the EO at a concentration of 50 ml ml À1 was prepared in MHB with 0.5% Tween 80. To each well of sterile 96-well microplates, 100 ml of MHB was added. Then 200 ml of stock solution was placed in the first well of a 96-well microplate and two-fold serially diluted in sterile MHB to obtain a final concentration range of 25-0.048 ml ml À1 . 100 ml of bacterial suspension was inoculated to each well. Each plate was incubated at 37 C for 24 h. Each plate had a set of negative controls (bacteria without EO). The plates were prepared in three replicates. Following incubation, 20 ml CTT (2,3,5-chloride tetrazolium triphenyl) (20 mg ml À1 ) was added to each well and incubated at room temperature for a further 10-15 min. Bacterial growth was observed as a pink-red coloration of the wells. The well of lowest concentration of EO in which bacterial growth was prevented (no pink-red coloration) and the corresponding concentration was referred to as the MIC value (Bazargani and Rohloff 2016). 10 ml from each no visible growth wells were plated and incubated. MBC was defined as the lowest concentration which caused total cell inactivation.

Biofilm formation
200 ml of A. baumannii culture (10 8 CFU ml À1 ) prepared in Trypticase soy broth (TSB), was added to each well in 96-well microtiter plate and incubated for either 24, 48 or 72 h at 37 C to allow biofilm formation (the medium was replaced each 24 h). Negative controls contained 200 ml of sterile medium instead of bacteria. Following incubation, the contents of each well were removed. Wells were washed three times with sterile distilled water to remove nonattached cells. The wells were oven-dried at 60 C for 45-60 min and stained with 200 ml of 1% crystal violet for 15 min. The plates were again rinsed three times to remove unabsorbed stain. The wells were distained by adding 200 ml of ethanol and the absorbance was measured at OD 570nm using a microplate ELISA reader (Thermo Scientific Multiskan). Biofilm production was classified as negative, weak, moderate, and strong based on the cutoff value, calculated according to the following formula (Folliero et al. 2021): ODcutoff ¼ Average OD of the negative control þ ð3 Â standard deviation of the ODs of the three repetitions of the negative controlÞ:  The used criteria were as follows:

Contact angle measurements and hydrophobicity
Contact angle measurements using a goniometer and the sessile drop method were estimated for polystyrene surfaces either treated or untreated with L. mairei EO. Three to six contact angle measurements were made on each surface for all probe liquids including formamide (99%), diiodomethane (99%) and distilled water. The Lifshitz-Van der Waals (c LW ), electron donor (c À ) and electron acceptor (c þ ) components of the surface tension of the surfaces were estimated from the approach proposed by Van Oss et al. (1988). In this approach the contact angles (h) can be expressed as: h is measured by the contact angle. (S) and (L) denote surface and liquid respectively.
The surface hydrophobicity was evaluated through contact angle measurements and using the approach of Van Oss and co-workers Van Oss et al. (1988). In this approach, the degree of hydrophobicity of a given material (i) is expressed as the free energy of interaction between two entities of that material when immersed in water (w) DG iwi . If the interaction between the two entities is stronger than the interaction of each entity with water DG iwi < 0, the material is considered hydrophobic. Conversely, if DG iwi > 0, the material is hydrophilic. DG iwi can be calculated through the surface tension components of the interacting entities, according to:

Anti-adhesion activity
The EO was evaluated for its inhibition potential against A. baumannii cell attachment by treating the wells before (adhesion prevention) and after (cell detachment) adherence of the bacteria with different concentrations of EO: MIC, MIC/2 and MIC/4.

Adhesion prevention
200 ml of each EO concentration was added to the wells of a 96-well microplate for 1 h. The negative control contained 200 ml of Tween 80 (0.5%) instead of EO. After 1 h, the wells were then rinsed one time with sterile distilled water before adding 200 ml of bacterial suspension (10 8 CFU ml À1 ). The plates were incubated at 37 C for 3 h without shaking. Following incubation, the contents of each well were removed. Wells were washed three times to remove nonattached cells.
Cell detachment 200 ml of bacterial suspension (10 8 CFU ml À1 ) was deposited to each well of a 96-well microplate and the plates were incubated at 37 C for 3 h of adhesion without shaking. After 3 h of adhesion, the wells were then rinsed three times before adding 200 ml of each EO concentration for 1 h. The negative control was treated with 200 ml of Tween 80 (0.5%) instead of EO. Subsequently, the EO was removed and the wells were rinsed. For both adhesion prevention and cell detachment, the plates were oven-dried at 60 C for 45-60 min. The wells were then stained with 200 ml of crystal violet (1%) and incubated at room temperature for 15 min. The plates were rinsed three times again to remove unabsorbed stain. The wells were distained by adding 200 ml of ethanol and the distaining solution was then transferred to a new plate and the absorbance was measured at OD 570 nm using a microplate ELISA reader (Thermo Scientific Multiskan). The percentage inhibition was then calculated (Bazargani and Rohloff 2016):

Antibiofilm activity
Biofilm formation inhibitory measurements were carried out according to Bazargani and Rohloff (2016) method with same modifications. 200 ml of A. baumannii culture (10 8 CFU ml À1 ) was added to each well in 96-well microtiter plate and incubated for 72 h at 37 C to allow biofilm formation (the medium was replaced each 24 h). Following incubation, the contents of each well were removed. Wells were washed three times to remove non-attached cells. After that,

Scanning electron microscopy
Polystyrene surfaces, with preformed biofilm treated and untreated with oil, were prepared for observation by Scanning Electron Microscopy. Briefly, polystyrene coupons of 1cm 2 were exposed to a bacterial culture of 10 8 CFU ml À1 prepared in TSB for 3 days. Afterwards, they were treated with EO at different concentrations for 24 h, followed by a fixation with 2.5% of glutaraldehyde for 1 h, then a gradual ethanol dehydration (50, 70, 90 and 100%). The samples were then coated with gold and processed under SEM (JEOL JSM IT10).

Statistical analysis
The values were obtained as mean ± Standard Deviation (SD) as a result of three repetitions. The results were analyzed using Anova test. A p-value of less than 0.05 was considered as significant.

Disk diffusion method
According to disc diffusion method results (Figure 1, Table 4), it clearly appears that all tested strains of A.

Minimum inhibitory concentration and minimum bactericidal concentration
For minimum inhibitory concentration and minimum bactericidal concentration assay, the results are given in  (Table 5).

Biofilm formation
According to the results of Figure 2, all the strains are strong biofilm producers, whether at 24 h or after 72 h of incubation. It is clear that as time passes, the biofilm increases significantly in strength. Basically, it appears that the Ab6 strain is the most biofilm producing strain after 72 h of incubation, even if it was Note: a: compounds listed in order of elution, b: retention indices measured relative to n-alkanes (C-9 to C-24) on a non-polar DB5-MS column.
the weakest after 24 h. Meanwhile, the Ab3 and Ab4 strains were the highest biofilm producers after 24 and 48 h of incubation ( Figure 2).

Contact angle measurements and hydrophobicity
In agreement with the results given in Table 6, it is evident that the EO treatment have slightly affected some parameters of the studied surface. The electron donor character cincreased remarkably, especially for MIC and MIC/4. However, for electron acceptor c þ character, there is no important difference between the negative control and the treated surfaces. In general, the polystyrene surface has changed from a hydrophobic to a hydrophilic character following the oil treatment with MIC and MIC/4 (Table 6).

Anti-adhesion activity
The anti-adhesion activity was detected by comparing the absorbance of crystal violet after adhesion of A. baumannii strains during 3 h (negative control) with those detected by treating the surfaces before or after adhesion of bacterial cells with EO (MIC, MIC/2 and MIC/4).

Adhesion prevention
Anti-adhesion tests were carried out by crystal violet assay in order to evaluate EO inhibition potential against cell attachments at different concentrations basing on MIC value (MIC, MIC/2 and MIC/4). Results indicated that L. mairei EO could inhibit bacteria cell attachment of multidrug resistant A. baumannii with different percentages (Figure 3). The tested EO generally displayed percentage inhibition in a range of 12.33 (Ab5 with MIC/4) to 83.66% (Ab2 with MIC). In addition, MIC was the more effective concentration in all strains, except for the Ab1 strain for which the MIC/2 concentration was more active than the MIC. A high significant difference was detected between strains and also between EO concentrations (p ¼ 0.0001).

Cell detachment
In attempt to detach cells, A. baumannii adhered cells for 3 h were treated with MIC, MIC/2 and MIC/4 of L. mairei EO. All the concentrations were able to detach bacteria from the microplate surface (11.76-74.30%) (Figure 4). This cell detachment was very expressed for Ab3 whatever the EO used concentration. From the obtained results, the adherent cells removal following the application of the L. mairei EO was not concentration-dependent, especially in the case of Ab4, Ab5 and Ab6 where MIC/2 or MIC/4 were more active than MIC itself. Statistically, a high significant difference was detected (p ¼ 0.0001).

Antibiofilm activity
The results obtained for the antibiofilm activity are shown in the Figure 5. The data show that the investigated EO possess inhibitory effects on multidrug resistant A. baumannii biofilm formation (7.5 to 64.02%). Especially, the MIC was the most active concentration against A. baumannii biofilm. Basically, the antibiofilm activity was concentration-dependent since the ability of L. mairei EO to detach the formed biofilm was enhanced as the concentration used increased ( Figure 5). Furthermore, the Ab6 biofilm was the most sensitive to the treatment, while the biofilm formed by Ab3 remained undamaged.

Scanning electron microscopy
SEM analysis showed that the strains all successfully adhered and formed biofilms on the studied surface ( Figure 6, negative control). Obviously, depending on the strain, the forming biofilm ability differed as proven before (Figure 2). According to the images, there was also a difference in the dispersal pattern of the Table 6. Contact angles of water (h w ), formamide (h F ), diiodomethane (h D ), the surface tension of Lifshitz-Van der Waals (c LW ), electron donor (c -), electron acceptor (c þ ) and hydrophobicity of polystyrene surfaces treated and untreated with L. mairei EO.     cells on the surface, such as Ab1 and Ab6 whose cells were more aggregated than the other strains. To analyze the effects of L. mairei EO on the biofilm, the strains were treated with different concentrations of this oil (MIC, MIC/2 and MIC/4). This SEM visualization has demonstrated that A. baumannii biofilms were dispersed after the treatment with the L. mairei EO ( Figure 6) as already established by the quantitative analysis ( Figure 5). The oil treatment impacted on cell clustering in the biofilm especially for Ab1 and Ab6.

Discussion
The resistance of A. baumannii against antibiotic agents, owing to their intensive usage, and the biofilm formation ability of this bacterium are together a worrying problem. Indeed, the effectiveness of recently developed antimicrobial agents against this bacterium seems limited because of its resistance to almost all the antimicrobial agents used for treatment today. Therefore, there is a clear need to discover a new efficient agent for the treatment of this pathogen. Natural products have been used as alternative medicines to conventional therapy. Currently, researchers are focusing on the therapeutic activities of plant products. The Lavandula species are recognized as a potential source of antimicrobial activity. This genus has been used since antiquity to flavor and preserve foods and to cure diseases (Celep et al. 2018).
In this study, the antibacterial, anti-adhesion and antibiofilm activities of EO of one of the Moroccan rare endemic plants; L. mairei were researched. L. mairei EO chemical composition showed a high content of carvacrol composition (79.12%) which is in agreement with previous studies (El Hamdaoui et al. 2018;Boubaker et al. 2019). The oil had an effective antibacterial activity on the multidrug resistant A. baumannii strains tested, with an inhibition zone diameter of 33-37.33 mm. The MIC/MBC ratio was 1 (MIC ¼ MBC ¼ 1.56 ml ml À1 ). A study by El Hamdaoui et al. (2018), reported inhibition zones varying from 2.5 to 35.6 mm and MIC values from 0.60 to 1.20 mg ml À1 for L. mairei EO against Listeria innocua, Listeria monocytogenes, Staphylococus aureus, Bacillus subtilis, Proteus vulgaris and Pseudomonas aeruginosa (El Hamdaoui et al. 2018). Ghanimi et al. (2021) investigated the antibacterial activities of EOs of three Moroccan species of the genus Lavandula; L. dentata L., L. mairei Humbert and L. stoechas L. against several bacteria including A. baumannii and reported that L. mairei EO had a significant anti-A.
baumannii activity with a 10 mm of inhibition zone and a MIC of 0.624 mg ml À1 . The effectiveness of L. mairei EO against A. baumannii activity is postulated to be due to high carvacrol content (Table 3). Indeed, carvacrol was recognized to possess an important antimicrobial activity (El Hamdaoui et al. 2018;Boubaker et al. 2019;Ghanimi et al. 2021). Carvacrol is characterized by its hydrophobic nature and with a hydroxyl group in its chemical structure, it thus has a significant effect on the membrane of bacterial cells (Nazzaro et al. 2013). Carvacrol causes disintegration of external membrane of Gram-negative bacteria affecting a series of critical functions, especially energy for transformation processes, nutrient processing, synthesis of structural macromolecules and many key enzymes for growth, finally leading to the death of the bacterium (Faleiro 2011).
One of the characteristics, which leads to ineffective treatment of A. baumannii infections, is the ability of this bacterium to form biofilms. The initial attachment of the cells is influenced by several factors including bacterial determinants, environmental conditions and the surface physicochemical parameters. Once the cells are attached, they can proliferate to form microcolonies by producing the extracellular matrix of the biofilm. Consequently, the biofilm becomes increasingly mature and resistant to various environmental stressor. This framework provides enhanced survival and protection against the mechanisms of host defense, antibiotics and other environmental hazards. To combat a bacterial biofilm, two tactics can be implemented, namely to prevent bacterial adhesion, and consequently inhibit biofilm installation, or to eliminate a preformed biofilm. Hence, the present work assessed the capacity of the EO to prevent A. baumannii adhesion by treating the surface before bacterial intervention, as well as its ability to detach adherent cells. The ability of the L. mairei EO to eliminate A. baumannii preformed biofilm was also investigated.
The anti-adhesion activity showed quite significant efficiencies whether treating the surface before adhesion (inhibiting adhesion by 11.76-74.30%) or detaching 12.33-83.66% of adhering cells (Figures 3 and 4). Comparing the percentages of inhibition with those of cell detachment, the involvement of oil before adhesion showed its effectiveness for most strains (Ab1, Ab2, Ab4 and Ab6). Thus, it was concluded that once the cells were attached, they became more resistant. However, Ab3 and Ab4 strains were more sensitive to the post-adhesion treatment than the preadhesion treatment. The attachment of a bacterial cell to a surface is a complicated process that is still poorly understood and has yet to completely defined, but it is accepted that physicochemical properties (surface tension and hydrophobicity) related to the surface are key determinants of initial adhesion (Ksontini et al. 2013;Zineba et al. 2014). The modification of these parameters could be the cause of the increase or decrease of the adhesion potential on the surface. For that reason, the physicochemical properties of the surface were determined before and after L. mairei EO treatment.
Treatment of the surface before bacterial colonization modified the physicochemical properties of the surface (Table 6). Interestingly, the polystyrene surface changed from a hydrophobic to a hydrophilic character by applying L. mairei EO (MIC and MIC/ 4). These findings can provide an indication of the probable pathway whereby the oil acts on the tested surface. Bennouna et al. (2018), analyzed various wood species physicochemical properties before and after treatment with EOs of Mentha pulegium, Rosmarinus officinalis and Cananga odorata and demonstrated that the hydrophobicity decreased considerably using Mentha pulegium EO followed by Cananga odorata and Rosmarinus officinalis Eos (Bennouna et al. 2018).
Similarly, Barkai et al. (2015) have also demonstrated that treating cedar wood with EO components (carvacrol and carvone) changed its surface properties from a hydrophobic to a hydrophilic character. However, the surface remained hydrophobic after the carvone treatment but with a strong increase of the electron donor character (Barkai et al. 2015). In the same context, contact angle measurement of the cedar wood after Cedrusatlantica EO treatment showed that the surface maintained its hydrophobic character, with an increased electron donor character (Bennouna et al. 2020).
In the current work, there were cases where the anti-adhesion activity did not depend on the concentration used as for Ab4, Ab5 and Ab6 where MIC/2 or MIC/4 were better at removing the adherent cells than MIC itself. This finding suggests that the antibacterial effect is not the only one responsible for the inhibition of adhesion. These results are in agreement with those found by Kerekes et al. (2019) when biofilms were significantly inhibited by the EOs in MIC/ 2 concentration.
Once a biofilm has formed it is difficult to eradicate through conventional antibiotic therapy. Hence, the effect of L. mairei EO on biofilm formed by A. baumannii were also investigated. The biofilm formed by Ab6 showed the highest percentage of elimination, whereas the biofilms formed by Ab1 and Ab5 were less affected. Furthermore, it appeared that the Ab3 strain formed the most resistant biofilm even though its adherent cells were the easiest to detach. The detection of a mass decrease of the biofilms formed by the A. baumannii studied strains, is an indirect expression of the EO's ability to cross the barrier or the biofilm matrix that surrounds the bacterial cells. Although, further research to understand the mechanism of action of this EO against A. baumannii biofilm is required.
As mentioned above, there are no previous studies concerning L. mairei Humbert EO anti-adhesion and antibiofilm activities. However, the antibiofilm activity of other Lamiaceae family EOs against multidrug resistant A. baumannii has been reported for Mentha pulegium L. , Ziziphora tenuior L. (Celik et al. 2015) and Salvia glutinosa L. (Tutar 2016). It was observed that Mentha pulegium damaged biofilms formed by A. baumannii strains at MIC by 26-91% . Similarly, Ziziphora tenuior EO MIC affected the A. baumannii biofilm by 51-84% (Celik et al. 2015). In addition, Salvia glutinosa EO has also shown its antibacterial effectiveness (MIC between 1.25-2.5 ml ml À1 and MBC between 5-10 ml ml À1 and its interesting antibiofilm activity against A. baumannii with a minimal biofilm inhibition concentration of 0.3-2.5 ml ml À1 (Tutar 2016).
Electron scanning showed significant changes caused by L. mairei EO on the biofilm of A. baumannii such as in the study performed by Kerekes et al. (2019) where scanning electron microscopy images demonstrated that the mature structure of the biofilm was largely affected by EOs.
In the present research, L. mairei demonstrated its anti-A. baumannii effectiveness with MIC and MBC of 156 ml ml À1 . This is probably due to the dominance of carvacrol in its chemical composition (79.12%). Moreover, L. mairei EO was able to inhibit adhesion, to detach adherent cells and to eliminate biofilm with percentages of 83.66%, 74.30% and 64.02%, respectively. It can be deduced that the tested EO have a greater effect on adhesion inhibition or detachment of adhered cells than biofilm dispersion. In fact, the cells in the biofilm are more resistant than planktonic cells because of the extracellular matrix that acts as a protective barrier against environmental conditions and antibacterial treatments. Consequently, using the oil as an intervention in the first step of biofilm formation, namely adhesion, is more efficient.

Conclusion
Antimicrobial resistance is one of the most important health problems today. Antibiotic options, that can be used for the treatment of multidrug resistant A. baumannii, have substantially decreased. Consequently, the development of new therapeutic agents for this nosocomial pathogen is significant. It was witnessed in this study that L. mairei EO had quite strong antimicrobial, anti-adhesion and antibiofilm effects on multidrug resistant A. baumannii. According to the present data, L. mairei EO has shown its potential value as a significant phytotherapeutic agent, especially as a preventive approach against A. baumannii infections. Therefore, it will be important to support these results obtained by other studies that consist in identifying the potential action mechanism of this oil against the planktonic or sessile mode of carbapenem resistant A. baumannii. These findings are the beginning of the development of a possible anti-A. baumannii biofilm solution, especially when it consists of multi-resistant strains, and thus, this product should be tested against other pathogenic agents. This EO could be the purpose of a commercialized product to treat surfaces, especially those of the hospital environment.

Disclosure statement
The authors confirm that this article content has no conflict of interest.

Funding
The author(s) reported there is no funding associated with the work featured in this article.