Chemical composition of essential oils of eight Tunisian Eucalyptus species and their antibacterial activity against strains responsible for otitis

Background The chemical composition and biological activity of Eucalyptus essential oils have been studied extensively (EOs). A few of them were tested for antibacterial effectiveness against otitis strains. The chemical composition and antibacterial activity of the EOs of eight Tunisian Eucalyptus species were assessed in the present study. Methods Hydrodistillation was used to extract EOs from the dried leaves of eight Eucalyptus species: Eucalyptus accedens, Eucalyptus punctata, Eucalyptus robusta, Eucalyptus bosistoana, Eucalyptus cladocalyx, Eucalyptus lesouefii, Eucalyptus melliodora and Eucalyptus wandoo. They are assessed by GC/MS and GC/FID and evaluated for antibacterial activity using agar diffusion and broth microdilution techniques against three bacterial isolates (Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae) and three reference bacteria strains (Pseudomonas aeruginosa, ATTC 9027; Staphylococcus aureus, ATCC 6538; and Escherichia coli, ATCC 8739). Furthermore, the selected twenty-one major compounds and all values of the inhibition zone diameters were subjected to further statistical analysis using PCA and HCA. Results The EO yields of the studied Eucalyptus species range from 1.4 ± 0.4% to 5.2 ± 0.3%. Among all the species studied, E. lesouefii had the greatest mean percentage of EOs. The identification of 128 components by GC (RI) and GC/MS allowed for 93.6% – 97.7% of the total oil to be identified. 1,8-cineole was the most abundant component found, followed by α-pinene, p-cymene, and globulol. The chemical components of the eight EOs, extracted from the leaves of Eucalyptus species, were clustered into seven groups using PCA and HCA analyses, with each group forming a chemotype. The PCA and HCA analyses of antibacterial activity, on the other hand, identified five groups. Conclusion The oils of E. melliodora, E. bosistoana, and E. robusta show promise as antibiotic alternatives in the treatment of otitis media. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-021-03379-y.


Background
The genus Eucalyptus L'Herit., native to Australia, belongs to the Myrtaceae family and has around 900 species and subspecies [1]. The leaves of over 300 species in this genus produce volatile oil. The oil yields extracted from Eucalyptus leaves were reported to range from 0.06% to 7.0% [2,3]. The pharmaceutical and cosmetic industries have economically exploited less than 20 species of essential oil (EO) rich in 1,8-cineole (> 70%) [4]. Natural medicine has sparked a surge of interest in recent years, particularly those employed to combat microbial agents, as numerous strains have exhibited resistance to pharmacological chemicals [5,6]. Drug resistance is found in Gram negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as Gram positive bacteria like Staphylococcus aureus [7][8][9][10]. Drug resistance has led researchers to design novel antimicrobial compounds to treat a variety of human infections [9,[11][12][13][14]. Inhalation of EOs extracted from Eucalyptus sp. has traditionally been utilized in Tunisian folk medicine to treat respiratory tract illnesses such as pharyngitis, bronchitis, and sinusitis [15]. The ear is connected to the upper respiratory tract by a mucous membrane that connects the nose and throat. Streptococcus pneumoniae, Haemophilus influenza, Moraxella catarrhalis, Staphylococcus aureus, Haemophilus parainfluenzae, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae have all been found to invade the mucous membrane [16][17][18][19][20][21]. A variety of respiratory diseases have been associated with these bacterial strains, including acute otitis media (AOM), sinusitis, asthma, and pneumonia [17][18][19][20][21]. Furthermore, several of these bacterial strains, including P. aeruginosa and S. aureus, as well as K. pneumoniae and other microorganisms, are responsible for otitis externa [22]. Every year, the AOM affects over 11% of the world's population (about 700 million individuals) [23]. The majority of them (51%) are children under the age of five [24]. It's worth emphasizing that 31 million AOM patients, including more than 7 million children per year, are at risk of developing chronic suppurative otitis media (CSOM) [25]. Hearing loss can occur in more than half of CSOM patients [26,27]. Although EOs derived from numerous Eucalyptus species have been shown to have antibacterial, antiviral, antioxidant, anti-inflammatory, and antiasthmatic activities [28][29][30], Few studies have explored the antibacterial activities of EOs against otitis pathogens. We described and investigated the biological activity of EOs isolated from the leaves of 60 Eucalyptus species collected from six arboreta in Tunisia in earlier works [7,[31][32][33][34][35][36][37][38][39]. The aim of the present study is to determine the variability of the yield, the chemical composition, and the antibacterial activities of EOs extracted from leaves of 8 Eucalyptus species. The antibacterial properties of microbial strains responsible for otitis are of special interest.

Plant material
We used clean mature leaves from eight species of Eucalyptus L'Hér. collected in June, 2017 from the following two regions: i) Eucalyptus accedens Fitzg., Eucalyptus robusta Sm. and Eucalyptus punctata DC. acclimated in Choucha arboretum and located in Sejnane region (37°03′23″N, 9°14′18″E) in the North West of Tunisia, which belongs to the humid inferior bioclimatic stage with mild winter; ii) Eucalyptus melliodora A.Cunn. ex Schauer, Eucalyptus lesouefii Maiden, Eucalyptus cladocalyx F. Muell, Eucalyptus bosistoana F. Muell., and Eucalyptus wandoo Blakeley were collected from the Mjez Elbab arboretum in the North West of Tunisia (36°38′55″N, 9°36′45″E), which belong to the upper semiarid bioclimatic stage with moderate winter.
The leaves were collected from three Eucalyptus trees, dried on an airy basis, protected from light, packed in paper bags, and stored in the shade. Botanical voucher specimens have been deposited at the Herbarium of the Faculty of Pharmacy's Pharmacognosy laboratory (Monastir, Tunisia) under the following numbers: 0173, 0174, 0175, 0176, 0177, 0178, 0179, 180.

Extraction of essential oils
The EOs were extracted using a standard apparatus specified by the European pharmacopoeia [40] by hydrodistilling 100 g of roughly crushed leaves for 4 h. For each sample, hydrodistillation was carried out in triplicate. The EOs were collected and dried with Na 2 SO 4 before being stored at + 4 °C until analysis. The EO yield was calculated as a percentage (%) of the dry weight (v/w).

GC analysis
The EO extracts were analysed subsequently by GC and GC/MS in triplicates. GC analysis was carried out with a Hewlett-Packard 6890 apparatus equipped with FID and apolar HP5 cap. column. The remaining experiment parameters are as follow: the oven temperature (temp.) was programmed at 60 °C for 1 min, rising gradually from 60 °C to 250 °C at 3 °C/min, and then held isothermal at 250° for 3 min; injector temp. at 250 °C; detector temp. at 280 °C, carrier gas, N 2 (1.2 mL/min). For each sample, 1μL (10% EO, in purified hexane) was injected for analysis. The relative concentration was calculated using software HP chemstation, which allows assimilating the percentages of the peak areas to the percentages of the various constituents. Retention indices (RI) were determined relatively to the retention time (t R ) of a series of n alkanes (C 9 -C 28 ).

GC/MS analysis
The EOs were analysed with a Hewlett-Packard 5890 series II apparatus equipped with a 5972 mass selective detector and an apolar HP5 column (30 m × 0.32 mm i.d., film thickness of 0.25 μm). Helium was used as a carrier gas. The mass spectrometer operating conditions were: ionisation voltage, 70 eV; ion source, 230°. The GC analysis was carried out as described above (see GC Analysis).

Compound identification
The identification of the compounds was based on the comparison of their RI (determined relatively to the t R of n-alkanes (C 9 -C 28 )) and their mass spectra with those of authentic compounds by means of NBS75K.L. and Wiley 275 databases, as well as with literature data [41].

Antibacterial testing Bacterial strains
In this study, three clinical bacterial isolates (H. influenzae, H. parainfluenzae, and K. pneumonia) were used, as well as three ATCC bacteria: P. aeruginosa (ATTC 9027), S. aureus (ATCC 6538), and E. coli (ATCC 8739). The Microbiology and Immunology Laboratory (EPS Farhat Hachad, Sousse, Tunisia) generously contributed the clinical strains, whereas the ATCC strains were obtained from the culture collection of the Laboratory of Transmissible Diseases and Biologically Active Substances, Faculty of Pharmacy, Monastir, Tunisia.

Kirby Bauer paper method
Using bacterial inoculums of 0.5 McFarland and Mueller Hinton (MH) enriched with 5% sheep blood, the antibacterial activity of several EOs was assessed using a paper-disc agar diffusion method. The MH medium for P. aeruginosa, E. coli, and S. aureus, on the other hand, was not enriched. Briefly, 10 μL of each EO was impregnated into absorbent discs (Whatman disc N°3, 6 mm diameter) and then deposited on the surface of infected plates (90 mm). Gentamicine ® (10 g/disc) positive control discs were included in each plate. The inhibition zone diameter (izd) was measured and represented in mm after 24 h of incubation at 37 °C.
The results were interpreted as follows: i) not sensitive or no inhibitory effect (-) for izd less than 8 mm; ii) sensitive ( +) or mild inhibitory effect for izd between 8 and 14 mm; iii) very sensitive or moderate inhibitory effect (+ +) for izd between 14 and 20 mm; iv) extremely sensitive or strong inhibitory effect (+ + +) for izd greater than 20 mm [42,43]. All of the tests were carried out in triplicate, and the results were expressed as mean ± standard errors of mean.

Determination of MIC and MBC
The minimum inhibitory concentration (MIC) was determined using the micro-well dilution method according to the National Committee for Clinical Laboratory Standards [44]. An overnight incubated culture (37 °C) of each tested bacterial strain was prepared by adjusting the turbidity of each bacterial culture to reach an optical density of 0.5 McFarland standards. One hundred microliters from each EO diluted in DMSO (10%), initially prepared at a concentration of 931 mg/mL, were added into the third well, followed by two-fold serial dilutions in MH broth medium until the 12 th well. Subsequently, 80 μL of MH, 10 μL of the inoculum, and 10 μL of 0.02% resazurin solution were added into each well. The skipped first and the second wells were reserved for negative and positive controls, respectively. Negative control well contained bacteria in the MH broth medium whereas, positive control well contained bacteria in MH broth medium and 10 μg/ mL of Gentamicin ® antibiotics.
After incubation for 24 h at 37 °C, the bacterial growth was characterized by color change from blue to pink. The MIC was defined as the lowest concentration that completely inhibits visible cell growth after incubation at 37 °C (blue colored well) for 24 h. To determine the minimum bactericidal concentration (MBC), 10 μL of each culture medium with no visible growth were removed and inoculated in MH plates. After incubation for 18-24 h at 37 °C, the number of surviving organisms was determined. MBC was defined as the lowest concentration at which 99.9% of the bacteria culture were killed [7]. As for all analyses, the experiments were performed in triplicate.

Statistical analysis
We carried out the analysis of variance (ANOVA test) to compare: i) the EO yields among different Eucalyptus species; ii) the quantitative content of chemical components among different Eucalyptus species; iii) izd values obtained during the antibacterial analysis among different EOs and among the used bacterial strains. The significance of the difference between means was determined at p < 0.05 using Duncan's multiple range test. To evaluate whether the identified EO constituents are a reflection of the chemical and biological activities, the detected 21 chemical compounds in the EO samples (with contents ≥ 2.1% in at least one species) and all theie izd values were subjected to PCA and HCA analyses using IBM SPSS Statistics for Windows, Version 23.0 (Armonk, NY: IBM Corp).
The EO yields from three distinct trees revealed that they differed considerably (p < 0.05) between species. Four non-overlapping groups of EOs were discovered using the Duncan multiple range test.

Chemical composition of the tested EOs
The EOs were chromatographically analyzed using GC (RI) and GC (MS), resulting in the identification of 128 compounds (Table 2 Suppl.), accounting for 93.6% -97.3% of the total oil content. These compounds were further divided into 15 classes (Table 2 Suppl.).
The aliphatic esters (tr -8.9%), which include methyl amyl acetate, are the seventh significant class. The monterpene aldehydes (0.1% -3.7%) were the eighth main class, with citronellal (tr -3.5%) being a prominent element. Minor compounds having a mean proportion of less than 1.1% made up the rest of the classes.
E. bosistoana EO was relatively rich in 1,8-cineole and α-pinene with comparative mean percentages as those observed in E. pimpiniana.

Principal Component (PCA) and Hierarchical Cluster (HCA) analyses
To evaluate whether the identified EO components may be useful in reflecting the chemotaxonomic relationships of the eight Eucalyptus species, 21 chemical compounds with a yield greater or equal to 2.1% in at least one species (Table 3) were selected for the PCA (Fig. 1) and the HCA analyses (Fig. 2). The concentrations of these  γ-Terpinene γ-ter  21:209 chemical components differed significantly between species (p < 0.05).The HCA analysis identified four groups (A, B, C and D), identified by their EO chemotypes with a dissimilarity of greater than 15%. Group D was further divided into four subgroups (D 1 -D 4 ) with a dissimilarity of greater than 5%. The PCA horizontal axis (axis 1) explained 30.07% of the total variance due to the increasing level of the mean percentage of compounds in group A and C species. The variation along the PCA vertical axis (axis 2) (22.37%) was mainly due to the increase in the mean percentage of compounds in group B and their decreasing level in group C and subgroups D 1 and D 2 , which stand out in both HCA and PCA analyses, forming separate groups and subgroups. Since components of the EOs within the same group were significantly correlated and tend to vary in the same way, we considered each group as a chemotype. Group A is constituted by E. cladocalyx, for which the EO content is distinguished from other groups by the highest percentages of sesquiterpenic alcohols globulol (12.7 ± 2.9%), epiglobulol (2.3 ± 0.5%), viridiflorol (2.6 ± 0.6%), the sesquiterpenic hydrocarbons aromadendrene (8.7 ± 0.7%) and the ester methyl amyl acetate (8.9 ± 1.5%), but by the absence of the monoterpenic alcohol p-cymen-8-ol, monoterpenic hydrocarbons p-cymene and the aldehyde cuminal. On the other hand, E. robusta, constituting Group B, was positively correlated with axis 2 and stood out, forming a separate group in both the HCA and PCA analyses. It was characterized by the highest content in the monoterpenic alcohols trans-pinocarveol (5.3 ± 0.2%), endo-borneol (6.0 ± 0.3%), α-terpineol (6.7 ± 0.3%), aldehyde citronellal (3.5 ± 0.1%), and the sesquiterpenic alcohol rosifoliol (5.2 ± 0.5%). This separation was enhanced further by its poverty in cryptone, β-pinene, and terpinen-4-ol. Group C, constituted by E. pimpiniana, was negatively correlated with axis 1. The EO of E. pimpinianais is characterized by its highest content of cryptone (8.4 ± 1.6%), p-cymen-8-ol (3.0 ± 1.2%), and cumianldehyde (2.1 ± 0.6%). It was also close to E. wandoo of the subgroup D 1 , likely due to its relative richness in p-cymene (28.7 ± 5.7%) and to E. lesouefii of the subgroup D 2 by its richness in β-pinene

Antibacterial testing
The EOs were tested for their putative antibacterial activity against six bacterial strains (  (Fig. 3). The HCA analysis identified two EO groups (A' and B') distinguished by antibacterial activity and a dissimilarity greater than or equal to 20 (Fig. 4). With a dissimilarity of > 5, group A was further subdivided into two subgroups (A'1 and A'2), whereas group B was further subdivided into three subgroups (B'1, B'2, and B'3
The variation in the chemical composition of the EOs could be attributed to environmental factors that affect the biosynthesis of the EOs' compounds in both quantity and quality [75]. To the best of our knowledge, the chemical composition of E. lesouefii EO has not been studied previously.

Antibacterial testing
Altogether, the antibacterial activity of the EOs displayed considerable variation among the different Eucalyptus species oils, but is still much lower than that of the standard antibiotic Gentamicine ® . This variability could be attributed to the chemical composition of the leaf oils [76].
The EO extracted from E. robusta, rich in the monoterpene aldehyde citronellal, the monterpene alcohols endo-borneol, α-terpineol and the sesquiterpene alcohol rosifoliol, showed the highest activity against E. coli and a moderate inhibitory effect  Griffin et al. (1999), that compounds of smaller volume with high hydrogen-bonding capacity interact significantly with water and tend to be active against the Gram negative E. coli [77]. It was also reported by the same author that the aldehyde citronellal has low water solubility and was inactive against the same strain. Therefore, we could deduce that the monoterpene alcohols, endoborneol, α-terpineol, could be the main compounds responsible for the activity against E. coli. E. melliodora oil, characterized by the highest mean percentage of 1,8-cineole, produced the highest antibacterial activity against K. pneumoniae and a medium inhibitory effect against E. coli. In E. bosistoana EO, this activity has decreased, as evidenced by a lower mean percentage of 1,8-cineole and a higher content of spathulenol. Altogether, these findings suggest that the main activity against these strains may be attributed to the richness of the EOs in 1,8-cineole, but the decrease in activity could be due to the presence of a high content in spathulenol. This finding was supported by previous studies [78,79] E. lesouefii EO, characterized by its high levels of β-pinene, terpinen-4-ol and sapthulenol, exhibited the best inhibition activity against both S. aureus and P. aeruginosa. However, it remains less important than other EOs, particularly against K. pneumoniae and E. coli. Comparing the variability of S. aureus sensivity to the oils having less concentration of the previous first three compounds and an equal or superior content of p-cymene, trans-pinocarveol, α-terpineol and citronellal, we could conclude that by antagonism effect, the latter compounds may be responsible for the decrease in activity. However, the increasing level of EOs' effect on the same strain could be due to a synergetic effect between β-pinene, terpinen-4-ol, spathulenol and other minor compounds such as aromadendrene and epiglobulol. Hammer et al. (2003) and Inouye et al. (2001) [82,83], reported that the monoterpene alcohol terpinen-4-ol has strong antifungal and antibacterial activity, especially against S. aureus. However, many studies have reported that minor compounds may have synergetic or additive [84]. The correlation between the chemical composition and the antibacterial activity of the tested oils also showed that the low activity against P. aeruginosa, which was observed with E. lesouefii and E. robusta oils, could be due to a synergetic effect mainly between terpinen-4-ol, β-pinene, citronellal, α-terpineol and other compounds such as spathulenol, rosifoliol, endo-borneol, but the presence of high levels of p-cymene, 1,8-cineole and the presence of other minor components such as aromadendrene, viridiflorol, globulol may considerably reduce the effect of the EO. E. accedens EO, characterized by the highest mean percentage of α-pinene and sharing almost the same mean percentage of p-cymene and 1,8-cineole with E. robusta EO, was relatively more effective against H. parainfluenzae. The EOs extracted from E. melliodora and E. bosistoana, on the other hand, were ineffective against H. parainfluenzae and H. influenzae due to their high content of 1,8-cineole and low content of p-cymene and α-pinene. Similarly, E. cladocalyx EO, which has a nearly identical content of 1,8-cineole as E. accedens EO and a very low content of α -pinene, p-cymene, aromadendrene, globulol, viridiflorol, and methyl amyl acetate, did not show antibacterial activity against the two strains mentioned above.Altogether, α-pinene could be the principal compound responsible for the activity against H. parainfluenzae, whereas p-cymene and α-pinene synergically have an effect on the inhibition of growth of the two Haemophilus strains; 1,8-cineole, aromadendrene, globulol, viridiflorol and methyl amyl acetate could exhibit an antagonism effect causing a significant diminution of the EO activity. This result was confirmed by the correlation analysis of the chemical composition and the antibacterial activity of E. wandoo EO, showing that the activity  [38]. However, both of them were inactive against S. aureus and P. aeruginosa. The difference in activity could be due to the richness of the oils obtained from Mjez Elbab arboretum of 1,8-cineole, globulol, viridiflorol and methyl amyl acetate. It has been reported that most terpenoids have high antimicrobial activity, and that this activity is linked to their hydroxyl group and the presence of delocalised electrons [85]. The MIC results obtained for E. cladocalyx against H. influenzae were in contradiction to the results obtained by the diffusion disc method. This difference could be related to the low diffusion ability of the EO, which in itself is highly dependent on water solubility and the ability of active components to diffuse through the agar [77,81].
In the present study, we used two methods for antibacterial activity: the disc diffusion method and the microbroth dilution method. Each of these methods has its associated advantages and disadvantages. For the disc diffusion method, the interaction between extracts/ bacteria is visually read. However, the inhibition zone could be populated with a minor subpopulation of bacteria, not detected visually; exhibiting increased antibiotic resistance, thus allowing them to grow closer to the disc. Although the disc diffusion test is relatively easy to setup and inexpensive, it does not provide quantitative data. For quantitative data, tests like the microbroth dilution method are available. Therefore, the antibacterial activity procedures depend on the method used as well as the chemical composition of tested compounds [44,86,87], as well as the used bacterial strains [87]. Consequently, results obtained by the disc diffusion and broth dilution methods may show a weak positive correlation or even negative correlation for some natural compounds [88].The effect of many factors on the antibacterial activity response, such as water solubility, diffusion index of the natural compound through the agar medium, and the loss of some molecules by vaporisation mainly for essential oils was reported [77,86]. It was also known that in the case of Gram negative bacteria, the activity was also dependent on the volume and the polarity of the natural components as well as the polarity of bacteria lipopolysaccharide (LPS) layer [89]. In the present study, a difference in results was shown in the antibacterial activity of some compounds. Among them, the essential oils of E. melliodora and E. bosistoana are characterized by their high content of 1,8-cineole, known by its low hydrogenbonding capacity [77,90]. Therefore, their antibacterial activity against K. pneumoniae using the broth microdilution method, which depends on the interaction of compound molecules in solution, showed high MIC values. Additionally, discordant results were shown for E. robusta, E. melliodora and E. wandoo using both discussed methods against E. coli. Although the essential oils of these species had nearly the same inhibition zone diameter as Gentamicine ® , their MIC values were not the same. Aside from the previously mentioned high content of 1,8-cineole, these three species also had a high content of monoterpene hydrocarbons (α-pinene and p-cymene), which are known for their low hydrogenbonding capacity [77]. Altogether, we could confirm that the antibacterial activities by these two methods were not parallel [88]. Indeed, it is more reliable to use the two methods for screening the antimicrobial activity of natural compounds.
Finally, in light of the problems associated with antibiotics, i.e. bacterial resistance, EOs extracted from E. bosistoana, E. robusta, and E. melliodora, could be used as an alternative to treat ear infections.

Conclusion
The chemical PCA and HCA analyses separated the EOs extracted from eight Eucalyptus species into seven groups. Each group constituted a chemotype. On the other hand, PCA and HCA analyses of their antibacterial activity separated them into five subgroups of Eucalyptus species EOs, identified by their levels of antibacterial growth inhibition. E. melliodora and E. bosistoana of the subgroup D 4 were the richest species in 1,8-cineole while the highest mean percentage of α-pinene and p-cymene were detected in E. accedens (Subgroup D 3 ) and E. wandoo (subgroup D 1 ), respectively. The antibacterial activity of the tested Eucalyptus