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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

Mechanism of action of essential oil-antibiotic combinations on bacteria involved in foodborne toxin infections

Paul Sodéré *^1,2, Marius K. Somda ^1,2, Donatien Kaboré ³, Luc Zongo ^4,5, Henriette B. Mihin ¹, Iliassou Mogmenga ^2,6, Agbémébia Y. Akakpo ¹, Mamoudou H. Dicko ²

1 Laboratoire de Microbiologie, Biotechnologie Microbienne, Département de Biochimie Microbiologie, Université Joseph KI-ZERBO, 03 BP : 7021, Ouagadougou 03, Burkina Faso.

2 Laboratoire de Biochimie, Biotechnologie, Technologie Alimentaire et Nutrition, Département de Biochimie Microbiologie, Université Joseph KI-ZERBO, 03 BP : 7021, Ouagadougou 03, Burkina Faso.

3 Département Technologie Alimentaire, Institut de Recherche en Sciences Appliquées et Technologies, Centre National de la Recherche Scientifique et Technologique 03 BP : 7047, Ouagadougou, Burkina Faso.

4 Hôpital Saint Camille de Ouagadougou (HOSCO), 09 BP 444 Ouagadougou 09, Ouagadougou, Burkina Faso.

5 Faculté des Sciences de Santé, Université Saint Thomas d’Aquin (USTA), 06 BP 10212 Ouagadougou 06, Burkina Faso.

6 Université de Banfora Centre, Université NAZI BONI, 01 BP 1091 Bobo-Dsso 01, Burkina Faso.

 

 

The worldwide surge in antibiotic resistance, driven by the rapid spread of innovative resistance pathways, is progressively underming clinical efficacy of a global scale ¹. Pathogenic bacteria pose risks of food poisoning that must be addressed ². Indeed, these bacteria can contaminate various common food products via diverse transmission routes, notably through dairy products such as milk ³, eggs ^4,5, market garden products, meat products, and fish ⁶. 

According to the World Health Organization (WHO), antimicrobial resistance (AMR) is one of the leading public health concerns ⁷. The WHO estimates that nearly 50.000 people die worldwide every day from infectious diseases caused by bacteria and fungi, with  financial burden reaching 380 million euros ⁸.

In recent years, antibiotic-resistant pathogens is one of the major public health issues. In 2019, the ANSSEAT (National Agency for Environmental, Food, Occupational Health and Safety), on biological products (vomitus, diarrheal stools, etc.) has isolated resistant bacterial strains ⁹. Colistin resistance prevalence in Africa has been estimated at 1.2 % and 1.5 % for K. pneumoniae and E. cloacae, respectively ¹⁰. Like other countries, Burkina Faso is facing an alarming frequency of antibiotic resistance. Indeed, Ki Ba et al, Nadembega et al ^9,11 reported that 32 % of S. aureus strains were methicillin-resistant, 98.3% of E. coli and 94.7 % of K. pneumoniae were resistant to amoxicillin + clavulanic acid, and 36.44 % of E. coli and 26.3 % of K. pneumoniae were resistant to third-generation cephalosporins. The resistance rate of beta-lactamase-producing Gram-negative bacteria was 35 %.

Nearly 30 % of antibiotic prescriptions are considered to have little to no clinical efficacy ¹². To address this declining effectiveness, researchers has explored plant-based alternatives to combat bacterial resistance. In this context, the use of essential oils as antimicrobial agents has garnered significant attention due to their broad spectrum of inhibitory effects against various bacterial strains ¹³.

Conventional antibiotic combinations designed to combat antimicrobial resistance are currently reaching their limits due to the emergence of multidrug-resistant bacteria. Nevertheless, the integration of essential oils in conjunction with antibiotics appears to enhance the efficacy of the latter, thereby mitigating therapeutic failures ^14,15.

Consequently, numerous studies have investigated the synergistic use of essential oils in combination with antibiotics to counteract antimicrobial resistance ^8,16-18. However, in Burkina Faso, few studies have addressed the combined effects of essential oils and antibiotics or their underlying mechanisms. 

Antibiotics function by inhibiting the biosynthesis of nucleic acids, with their primary targets being the bacterial cell wall and ribosomes. This high degree of specificity, coupled with the exceptional adaptive capacity of bacteria, represents one of the inherent limitations of antibiotic therapy ¹³. 

Conversely, plants have evolved their own internal mechanisms for biotic control, including defense against microbial infections. These processes favor the synthesis of a diverse array of weakly active molecules, which reduces selective pressure and hinders the development of resistance ¹⁹. Notably, Hyptis suaveolens and Laggera aurita are widely distributed throughout tropical and subtropical regions. In Burkina Faso, these species are employed in traditional medicine ^20-24. Several studies have highlighted the antibacterial and antifungal properties of essential oils derived from H. suaveolens and L. aurita ^20,25-27 underscoring the necessity of exploring their potential in combination with commonly used antibiotics. 

The objective of this study is to evaluate the efficacy of the combined effects of conventional antibiotics and essential oils against antibiotic-resistant bacteria.

2. MATERIALS AND METHODS
3. Essential oils 

A botanist of the National Center for Scientific and Technology Research (CNRST) in Ouagadougou identified the leaves of H. suaveolens and L. aurita. The voucher specimens of L. aurita and H. suaveolens are n° 152A preserved in the National Herbarium of Burkina Faso (HNBU) and n° 110 preserved in the herbarium of University Joseph Ki-Zerbo at the Laboratory of Ecology and plant Biology (LaBEV) respectively.

 

 

Figure 1: Leaves of L. aurita (A) and H. suaveolens (B)

 

 

Amoxicillin + clavulanic acid (Glaxo SmithKline Pharmaceuticals Ltd) and colistin (Coli-4800 ws) obtained from a pharmacy in Burkina Faso were used for this study.

The different bacterial targets of inhibition (wall bacteria, cytoplasmic membrane) and availability in local pharmacy led to the choice of these antibiotics in our study.

The following bacteria were used as test strains : Staphylococcus aureus ATCC 2523, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027 provided by the American Type Culture Collection (ATCC), Enterococcus faecalis 0366 V, Bacillus cereus 0998 V, and Yersinia enterocolitica 0938 V provided by the Food and Nutritional Biological Sciences Research Center (CRSBAN) at University Joseph Ki-Zerbo in Ouagadougou.

The food-borne diseases caused by these bacteria led to the choice of these pathogens for our study.

H. suaveolens and L. aurita plants were harvested in Ouagadougou, specifically in the « Bassinko » (12°25’30.96’’ N, 1°40’21.78’’ O), lowlands and near the dam of « Tanghin » (12°23’40.11’’ N, 1°32’18.10’’ O). Figure 2 shows the location of the harvesting areas.

 

 

Figure 2: Geographical location of the harvest zones

 

 

The plants were transported to Institute for Research in Applied Sciences and Technologies (IRSAT) in « Kossodo/ Ouagadougou » where leaves were removed, washed two times with clean water to remove adhering debris. In the shaded area the leaves were air dried at approximately 37 °C. Essential oils were extracted by hydrodistillation using a modified Clevenger-type apparatus following the protocol described by Bayala ²⁸. Briefly, 10 kg of dried leaves were immersed in a 100-liter still. The mixture was brought to a boil for three hours, and the resulting essential oil-rich vapors were condensed and collected. The organic phase was separated via decantation. The essential oils characterized by densities of 0.7657 g/mL for L. aurita and 0.6493 g/mL for H. suaveolens, were stored in amber bottles wrapped in aluminum foil at 4 °C shielded from UV radiation. Extraction yield was expressed as a percentage (%) and the density in g/mL.

Yield (%) = (Mass of essential oil / Mass of dried leaves) x 100

Density (g/mL) = Mass of essential oil / Volume of essential oil

L. aurita and H. suaveolens concentrations of 0.7657 g/mL and 0.6493 g/mL, respectively, were obtained and placed in bottles wrapped with aluminum foil and stored at 4°C in a refrigerator away from ultraviolet rays. 

All bacterial were cultured in nutritional broth media (India, HiMedia. Laboratories Pvt. Limited) for 24 h. After incubation of 24 h, the bacteria were washed according to Rhayour ¹⁷. The 24h bacterial culture was centrifuged at 400 rpm for 25 min; the resulting supernatant was discarded, and the bacterial pellet was resuspended in a volume of Phosphate Buffered Saline (PBS). The resulting bacterial suspension is hereafter referred to as the washed bacterial suspension. The optical density was read at 625 nm using a spectrophotometer, and the final inoculum was adjusted to 1.5 x 10⁶ CFU/mL according to the standard 0.5 Mac Farland suspension.

Antibiotic solutions were prepared by dissolving 500 mg of each antibiotic in 1 mL of sterile tween 80. The mixture was transferred to a sterile flask containing 199 mL of sterile distilled water to obtain a concentration of 2.5 mg/mL, in accordance with CLSI (29) guidelines.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of the essential oils and antibiotics were determined via the broth microdilution method CLSI ²⁹. Mueller-Hinton broth (MHB) (India, HiMedia. Laboratories Pvt. Limited) supplemented with tween 80 was utilized. A volume of 190 μL was added to first column, and 100 μL to the subsequent wells of 96-well microplate. Ten serials twofold dilutions of each antibacterial agent (L. aurita, H. suaveolens, amoxicillin + clavulanic acid or colistin) were performed from the essential oils and antibiotic concentrations previously prepared to obtain final concentrations ranging from 76.56 to 0.07 mg/mL for H. suaveolens, from 64.92 to 0.06 mg/mL for L. aurita and from 125 to 0.06 μg/mL for each antibiotic. The wells of the broth without antimicrobials served as controls. The 96-well microplates were incubated at 37 °C for 24 h and turbidity or growth was observed. The lowest concentration with no turbidity among the test-wells was recorded as the MIC ³⁰.

The MBC was considered as the lowest antimicrobial concentration that killed 99.9% of the bacterial inocula after 24 h of incubation at 37 °C ³¹. MBC values were determined by subculturing the contents of test-wells without turbidity or no visible growth from MIC determinations to neutral sterile Mueller–Hinton agar plates.

The antibacterial activity was assessed by determining the MIC and the MBC of antibiotics. and essential oil. The bacterial susceptibility to antibiotics was determined via the critical concentrations ³² listed in Table 1. All tests were performed independently in triplicate.

 

 

Table 1: Susceptibility of bacteria to antibiotics

 

Synergistic interactions were evaluated using the checkerboard method ³³ specifically on resistant strains. Serial twofold dilutions of essential oils were prepared horizontally, while antibiotics dilutions were prepared vertically.  The Fractional Inhibitory Concentration (FIC) and the Fractional Inhibitory Concentration Index (FICI) were calculated as follows:

FIC of essential oil = MIC of essential oil in the combination/MIC of essential oil alone; FIC of antibiotic = MIC of antibiotic in the combination/MIC of antibiotic alone.

FICI = FIC of essential oil + FIC of antibiotic as described by Schelz et al. ³⁴.

Synergy (FICI ˂ 0.5); addition (0.5 ≤ FICI ≤ 1); indifference (1 ˂ FICI ≤ 4) and antagonism (FICI > 4). All tests were performed independently in triplicate.

The release of the cytosolic material absorbing at 260 nm at different times of treatment (0h, 12h, and 24 h) were quantified as described by Rhayour ¹⁷ using  Epoch Biotek spectrophotometer. For each bacterial strain (1.5 x 10⁶ CFU/mL), tube containing PBS and an untreated suspension of washed bacteria were performed as control. Tube containing PBS, the washed bacterial suspension, and the antibiotic MIC and tube containing PBS, the washed bacterial suspension, and the synergistic MICs of the essential oil-antibiotic combinations were performed as samples. Tubes were incubated at 37 °C under constant agitation at 200 rpm. At specific time intervals (t = 0, 12h, and 24 h), a 200 µL aliquot was withdrawn from each tube and transferred into a 96-well microplate. Absorbance was subsequently measured at 260 nm. Distilled water served as the blank for the measurements. All assays were performed in triplicate.

The concentration of DNA released into the supernatant was quantified using a BioDrop Cambridge CB4 OFJ spectrophotometer. Following 24 h of incubation under the conditions described above, control and samples were centrifuged at 8000 rpm for 2 min. The resulting supernatant was analyzed to determine DNA content ³⁵. The control was read as blank. All tests were performed independently in triplicate.

Analysis of variance (ANOVA) was used to compare variable means. Fisher’s Least Significant Difference (LSD) test was used for pairwise multiple comparisons of means where there was a significant difference between all means. The difference between means was considered significant when the p-value was less than 0.05. XLSTAT version 7.5.2 2016 software was used for statistical analysis.

3. RESULTS AND DISCUSSION
4. Yield of essential oils

L. aurita and H. suaveolens essential oils yields and density are listed in Table 2.

Table 2: Yields and densities of essential oils

Essential oils	Yield (%)	Density (g/mL)
L. aurita	0.20± 0,01	0.7657± 0,01
H. suaveolens	0.23± 0,01	0.6493± 0,01

 

The essential oil yield obtained from the leaves of L. aurita via hydrodistillation was 0.20 %. This value was lower than the 0.3 % reported by Samaté ³⁶ in Burkina Faso, whose samples were harvested during the flowering period. Conversely, the present yield was higher than the 0.008 % recorded by Kabera et al. ³⁷ for the same species, yet remained comparable to the findings of Mihin et al. ²⁷ in Burkina Faso, who reported a yield of 0.22 %. 

For H. suaveolens, the essential oil yield was 0.23 %. This result aligns closely with yields reported by Mihin et al. ²⁷ in Burkina Faso (0.21 %), Ngom et al. ³⁸ in Senegal (0.22%), and Adjou and Soumanou ³⁹ in Benin (0.23 %). However, it was lower than the yield of 0.34 % observed by Goly et al. ²⁵. When compared with the literature, the analysis of essential oil yields for L. aurita and H. suaveolens reveals both similarities and discrepancies, regardless of whether the studies were conducted in Burkina Faso or other West African countries such as Benin and Senegal. Indeed, factors such as the duration and degree of drying, the date and geographical location of harvest, and the extraction technique employed have a significant impact on both the yield and the chemical profile of essential oils ^27,38,40-42.

The results from the broth microdilution tests for the in vitro antibacterial activity of the antibiotics are presented in Table 3. The MIC of amoxicillin + clavulanic acid ranged from 06 μg/mL to 29.66 μg/mL, while for colistin it ranged from 0.15 μg/mL to 5 μg/mL.

Regarding the MBC, values ranged from 24 μg/mL to 96 μg/mL for amoxicillin + clavulanic acid and from 24 μg/ mL to 60 μg/mL for colistin.

The lowest MIC (0.15 μg/mL) was obtained with colistin on S. aureus and the highest MIC (29.66 μg/mL) was obtained with amoxicillin + clavulanic acid on P. aeruginosa.

The lowest MBC (24 μg/mL) was obtained with amoxicillin + clavulanic acid and colistin on E. faecalis and Y. enterocolitica the highest MBC (96 μg/mL) was obtained with amoxicillin + clavulanic acid on P. aeruginosa.

Amoxicillin + clavulanic acid and colistin revealed 6 bactericidal effects and 6 bacteriostatic effects respectively.

The resistance rates for amoxicillin + clavulanic acid and colistin were 66.66 % and 83.33 %, respectively (Table 3). These findings corroborate the patterns of antimicrobial resistance previously observed in Burkina Faso. Notably, the resistance rate for colistin was higher than that of amoxicillin + clavulanic acid. The elevated resistance to colistin compared to amoxicillin + clavulanic acid may be explained by the relative inaccessibility of the cytoplasmic membrane compared to the bacterial cell wall ⁴³. Furthermore, this high prevalence of colistin resistance could be linked to its extensive use in veterinary medicine. Additionally, reduced antibiotic permeability in certain bacterial strains may contribute to high colistin resistance. Efflux pumps, which actively export antibiotics out of the cell though this mechanism is infrequently observed ⁴⁴ further hinder colistin’s access to its primary target: the plasma membrane ⁴⁵. 

The MIC for P. aeruginosa was significantly higher (ANOVA, P < 0.05) than those of other pathogenic strains. High MIC values for P. aeruginosa have also been reported by Gang-Joon et al. ⁴⁶. Our findings align with those of Lambert et al. ⁴⁷ who reported elevated MIC for P. aeruginosa across 40 bacterial strains. The pronounced resistance of P. aeruginosa to most antibacterial agents is due to its possession of nearly all known enzymatic and mutational mechanisms of bacterial resistance ⁴⁸.

 

 

Table 3 : Antibacterial activity of antibiotics

Minimum Inhibitory Concentration (µg/ml)					MBC/MIC ratio and Capacity
	Amoxicillin + clavulanic acid		Colistin		Amoxicillin + clavulanic acid	Colistin
Bacterial strains
	MIC	MBC	MIC	MBC
E. faecalis 0366V	04±0.00d (S)	24±0.00c	3.75±0.00b (R)	24±0.00c	4*	6**
Y. enterocolitica 0938V	04±0.00d (S)	24±0.00c	3.75±0.00b (R)	24±0.00c	4*	6**
B. cereus 0998V	13±1.00c (R)	48±0.57b	3.33±1.40c (R)	30±0.57b	4*	9**
E. coli ATCC 25922	23.33±0.57b (R)	48±0.00b	5±0.00a (R)	30±0.00b	2*	6**
P. aeruginosa ATCC 9027	29.66±0.57a (R)	96±0.00a	5±0.00a (R)	60±0.00a	3*	12**
S. aureus ATCC 2523	24±0.00b (R)	48±0.00b	0.15±0.06d (S)	30±0.00b	2*	200**

Notes: The values represent the means of three trials ± standard deviations. Values in the same column with the same superscript letters (a, b, c, d) are not significantly different (p<0.05). Values with superscript (*) Bactericidal effects (**) Bacteriostatic effects. (S): Sensitivity, (R): Resistance.

 

 

The MIC of H. suaveolens essential oil ranged from 9.57 mg/mL (against P. aeruginosa and B. cereus) to 38.28 mg/mL (against E. faecalis, Y. enterocolitica, E. coli, and S. aureus). Its MBC ranged from 38.28 mg/mL for S. aureus and B. cereus to 76.56 mg/mL for E. coli and P. aeruginosa (Table 4).

For L. aurita essential oil, the MIC ranged from 1.69 mg/mL against S. aureus to 64.92 mg/mL against E. coli. The MBC values ranged from 30 mg/mL against E. coli to 64.92 mg/mL against E. faecalis and Y. enterocolitica (Table 4). Both H. suaveolens and L. aurita essential oils exhibited five bactericidal effects and one bacteriostatic effect each.

The antibacterial activity of H. suaveolens which is characterized by high concentrations of beta-caryophyllene, 1,8-cineole, sabinene, and beta-pinene (comprising monoterpenes, sesquiterpenes, terpenoids, and sterols) likely functions by damaging bacterial cell structures and stimulating the release of cellular potassium ions, a process that proves lethal to the microorganisms ^49,50,51. In contrast, the antibacterial potency of L. aurita can be attributed to its high content of terpenoid compounds such as carvacrol, thymol, and eugenol, alcohols like linalool, and aldehydes such as cinnamaldehyde ³⁶. 

Statistical analysis (ANOVA, P < 0.05) revealed that the antibacterial activity of L. aurita was significantly more pronounced than that of H. suaveolens. This difference may be explained by the distinct chemical profiles of the two essential oils. Specifically, H. suaveolens essential oil contains fewer oxygenated terpenic compounds ^52,53. It is widely recognized that essential oils rich in these specific molecules tend to exhibit the highest antimicrobial efficacy ^27,54,55.

The most extensively studied essential oils with potent antibacterial properties are those predominantly composed of oxygenated compounds such as eugenol, thymol, and carvacrol. Therefore, the richness of L. aurita in these constituents likely accounts for its superior antibacterial performance ^56,57.

 

 

Table 4: Antibacterial activity of essential oils

Minimum Inhibitory Concentration (mg/ml)					MBC/MIC ratio and Capacity
	Hyptis suaveolens		Laggera aurita		Hyptis suaveolens	Laggera aurita
Bacterial strains
	MIC	MBC	MIC	MBC
E. faecalis 0366V	38.28±0.00a	76.25±0.00a	36.46±0.00b	64.92±0.00a	2*	2*
Y. enterocolitica 0938V	38.28±0.00a	76.25±0.00a	36.46±0.00b	64.92±0.00a	2*	2*
B. cereus 0998V	9.57±0.00c	38.28±0.00b	8.11±0.00c	32.46±0.00b	4*	4*
E. coli ATCC 25922	38.28±0.00a	76.56±0.00a	64.92±0.00a	30±0.00c	2*	2*
P. aeruginosa ATCC 9027	9.57±0.00c	76.56±0.00a	8.11±0.00c	32.46±0.00b	8**	4*
S. aureus ATCC 2523	38.28±0.00a	38.28±0.00b	1.69±0.00d	32.46±0.00b	4*	19**

Notes: The values represent the means of three trials ± standard deviations. Values in the same column with the same superscript letters (a, b, c) are not significantly different (p<0.05). Values with superscript (*) Bactericidal effects. (**) bacteriostatic effects.

 

The FIC indices for the essential oils of H. suaveolens and L. aurita plus antibiotics against pathogen strains was used to determine synergistic MIC of essential oils and antibiotics. 

Out of 20 combinations tested on bacterial strains, 14 Synergistic effects were observed between the tested agents (Table 5).

L. aurita exhibited a higher number of synergistic combinations compared to H. suaveolens, further corroborating its superior antibacterial potency noted previously. The antibacterial efficacy of both colistin and amoxicillin + clavulanic acid against antibiotic-resistant bacteria was significantly enhanced when combined with L. aurita. This essential oil demonstrated the highest frequency of synergism with both antibiotics, a phenomenon likely attributable to its high concentration of terpenic compounds ³⁶. Notably, the combination of amoxicillin + clavulanic acid with L. aurita rendered S. aureus susceptible to the antibiotic, despite its initial resistance. A similar synergistic interaction was reported by Gallucci et al. ⁵⁸ when combining carvone with penicillin against S. aureus.

Most combinations demonstrated synergistic effects against P. aeruginosa, a strain widely recognized as one of the most resistant to antimicrobial agents ⁴⁷.

Oussalah et al. ⁵⁹ reported that monoterpenes particularly the phenolic components of essential oils induce damage to the outer membrane of bacteria. This leads to increased membrane permeability to protons and potassium ions, depletion of intracellular ATP reserves, disruption of the proton motive force, and denaturation of intracellular proteins ^47,60. Similarly, amoxicillin + clavulanic acid inhibits bacterial cell wall synthesis by inactivating key enzymes involved in peptidoglycan assembly ⁶¹. Thus, the observed synergies may be explained by a complementary mechanism of action between the essential oils and the antibiotics.

It should be noted that not all combinations resulted in synergistic effects. This may be due to the fact that essential oil constituents interact differently depending on the specific antibiotic and the target bacterial species ⁶²; Consequently, such combinations must undergo rigorous in vitro testing before their potential application in clinical treatment.

 

 

Table 5: Synergistic effects of essential oils and different antibiotics against pathogens.

Bacterial strains	Antibacterial agents	MICc	MICa	MICa MICC	IFCI	Combination effects
E. faecalis 0366V	Colistin	3.75±0.00	0.93±0.57	0.24	0.49	Synergy
	Hyptis suaveolens	38.28±0.00	9.57±1.00	0.25
	Colistin	3.75±0.00	0.46±0.57	0.12	0.34	Synergy
	Lagerra aurita	36.46±0.00	8.11±1.00	0.22
S. aureus ATCC 2523	Amoxiclav	24.00±0.00	3.75±1.00	0.15	0.27	Synergy
	Hyptis suaveolens	38.28±0.00	4.78±0.57	0.12
	Amoxiclav	24.00±0.00	3.75±1.00	0.15	0.44	Synergy
	Lagerra aurita	1.69±0.00	0.50±0.57	0.20
	Colistin	0.15±0.06	0.12±1.00	0.80	0.93	Addition
	Hyptis suaveolens	38.28±0.00	4.78±0.00	0.13
	Colistin	0.15±0.06	0.12±0.00	0.80	1.00	Addition
	Lagerra aurita	1.69±0.00	0.34±1.00	0.20
B. cereus 0998V	Amoxiclav	13.00±1.00	3.75±0.00	0.29	0.78	Addition
	Hyptis suaveolens	9.57±0.00	4.78±1.00	0.49
	Amoxiclav	13.00±1.00	0.93±1.00	0.07	0.32	Synergy
	Lagerra aurita	8.11±0.00	2.02±0.57	0.25
	Colistin	3.33±1.40	3.75±1.00	1.12	3.12	Indifference
	Hyptis suaveolens	9.57±0.00	19.14±1.00	2.00
	Colistin	3.33±1.40	0.58±1.00	0.17	0.41	Synergy
	Lagerra aurita	8.11±0.00	2.02±0.57	0.24
E. coli ATCC 25922	Amoxiclav	23.33±0.57	3.75±0.00	0.16	0.28	Synergy
	Hyptis suaveolens	38.28±0.00	4.78±1.00	0.12
	Amoxiclav	23.33±0.57	3.75±0.57	0.16	0.28	Synergy
	Lagerra aurita	64.92±0.00	8.11±1.00	0.12
	Colistin	5.00±0.00	9.37±0.00	1.87	3.87	Indifference
	Hyptis suaveolens	38.28±0.00	19.14±0.57	0.50
	Colistin	5.00±0.00	9.37±1.00	1.87	2.12	Indifference
	Lagerra aurita	64.92±0.00	16.23±1.00	0.25
Y. enterocolitica  0938V	Colistin	3.75±0.00	0.78±0.00	0.20	0.22	Synergy
	Lagerra aurita	36.46±0.00	1.01±0.00	0.02
	Colistine	3.75±0.00	1.56±0.57	0.41	0.43	Synergy
	Hyptis suaveolens	38.28±0.00	2.39±0.57	0.06
P. aeruginos a ATCC 9027	Amoxiclav	29.66±0.57	3.75±0.57	0.12	0.37	Synergy
	Hyptis suaveolens	9.57±0.00	2.39±0.00	0.25
	Amoxiclav	29.66±0.57	1.87±1.00	0.06	0.31	Synergy
	Lagerra aurita	8.11±0.00	2.02±0.00	0.25
	Colistin	5.00±0.00	0.78±0.57	0.16	0.41	Synergy
	Hyptis suaveolens	9.57±0.00	2.39±1.00	0.25
	Colistin	5.00±0.00	1.25±0.00	0.25	0.37	Synergy
	Lagerra aurita	8.11±0.00	1.01±0.00	0.12

Notes: The values represent the means of the three trials ± standard deviations. Abbreviations: MICc, MIC of essential oils or antibiotics tested in combination; MICa, MIC of essential oils or antibiotics tested alone; FICI, Fractional Inhibitory Concentration Index.

 

 

To elucidate the mechanism of action underlying the synergism between essential oils and antibiotics, the leakage of intracellular components was evaluated. This investigation sought to determine whether such combinations compromise the structural integrity and permeability of bacterial membranes. Given that intracellular components release occurs almost instantaneously upon plasma membrane rupture, their exudation was quantified via spectrophotometric monitoring at 260 nm.

The mean absorbance of released material following treatment of resistant bacteria with synergistic combinations was markedly higher (0.297) than that observed for antibiotics alone (0.064) or the control (0.038). Kinetic analysis (Figure 3) revealed an initial phase of rapid leakage during the first hour of treatment accounting for 58% of the total 24-hour release followed by a more gradual, sustained efflux. These observations align with findings by Phitaktim et al and Li et al. ^63,64 who reported comparable kinetics when combining Garcinia mangostana L essential oil, which shares a similar chemical profile with L. aurita and H. suaveolens, with oxacillin. Absorbance values for the synergistic combinations were significantly elevated compared to the control (ANOVA, p < 0.05), suggesting that 260 nm-absorbing material release was directly induced by the combined agents rather than occurring as a secondary consequence of cell wall weakening and subsequent osmotic lysis ⁶⁵. Furthermore, the release of absorbing material was significantly more pronounced in Gram-positive bacteria than in Gram-negative bacteria (ANOVA, p < 0.05). This disparity is likely due to the less complex cell wall structure of Gram-positive species ^18,66. However, no significant difference (ANOVA, p > 0.05) was observed between the various synergistic combinations themselves regarding the volume of cytoplasmic leakage.

These results indicate that while intracellular components release depends on the bacterial wall structure, this release varies slightly regardless of the type of essential antibiotic combinations. The substantial loss of membrane integrity and increased permeability likely facilitate the restoration of susceptibility in previously resistant strains. These findings further suggest that L. aurita and H. suaveolens essential oils may inhibit beta-lactamase activity or interfere with lipopolysaccharide (LPS) charge modifications, both pivotal resistance mechanisms. Similar effects were observed by Eumkeb and Chukrathok ⁶⁷ where the combination of ceftazidime and galangin caused ultrastructural damage and restored ceftazidime sensitivity in S. aureus.

 As the mechanisms of colistin and amoxicillin + clavulanic acid are well characterized ⁶⁸, these data strongly suggest that the bacterial cell envelope remains the primary target for L. aurita and H. suaveolens essential oils, consistent with other oils of similar chemical composition  ^{64,65,69,70,71}.

The combination of these oils with antibiotics may result in the independent binding of each agent to the cell membrane, leading to lysis, increased permeability, and subsequent genomic degradation and cell death. Alternatively, these agents may form complexes that subsequently target the membrane. Combinations of L. aurita or H. suaveolens with colistin and amoxicillin + clavulanic acid consistently triggered the release of intracellular contents, mirroring mechanisms observed when similar oils were combined with polymyxin B and piperacillin. ^72,73. In conclusion, the combination of L. aurita and H. suaveolens with colistin and amoxicillin + clavulanic acid possesses the capacity to induce membrane disruption and cytoplasmic leakage ^74,75. Given the heterogeneous nature of these essential oils, it is improbable that a single component or mechanism accounts for the observed antimicrobial efficacy. Consequently, further research is warranted to fully elucidate these complex pathways.

 

 

                                                                               

(A)                                                                                                        (B)

(C)                                                                                                      (D)

 

 

(E)                                                                                               (F)

Figure 3: Loss of 260 nm-absorbing material from the controls and from (A). Y.enterolitica, (B). E. coli, (C). B. cereus, (D). P. aeruginosa (E) E. faecalis (F). S. aureus treated with Col+ Hyptis= Colistin + Hyptis suaveolens, Col+Laggera= Colistin + Laggera aurita, Amc+Hyptis= Amoxicillin + clavulanic acid + Hyptis suaveolens, Amc+Laggera = Amoxicillin + clavulanic acid + Laggera auita, Amc= Amoxicillin + clavulanic acid, Values represent the mean of three trials; vertical bars indicate standard errors.

 

 

 

 

Figure 4 illustrates the quantity of DNA released by bacteria.

The essential oil-antibiotic combinations tested against the bacterial strains resulted in a mean DNA release of approximately 102.28 µg/mL. In contrast, bacteria only treated with antibiotics exhibited a mean release of 32.04 µg/mL. While the control group showed a minimal release of 10.59 µg/mL. The highest concentration of released DNA (270 µg/mL) was recorded during the treatment of S. aureus with the combination of amoxicillin + clavulanic acid and L. aurita. The lowest release among the treated groups (44 µg/mL) occurred during the treatment of P. aeruginosa with the combination of amoxicillin + clavulanic acid and H. suaveolens.

 

 

Figure 4: DNA release from antibiotic-resistant strains treated for 24 hours with Colistin, Amc= Amoxicillin + clavulanic acid, Col+Hyptis= Colistin + Hyptis suaveolens, Col+Laggera = Colistin + Laggera aurita, Amc+Hyptis = Amoxicillin + clavulanique acid + Hyptis suaveolens, Amc+Laggera = Amoxicillin + clavulanique acid + Laggera aurita compared to control. Values represent the mean of three trials; vertical bars indicate standard errors.

 

 

The amount of DNA released by treated bacteria was significantly higher than that of the untreated control (Table 6), though release rates varied by strain. The maximum DNA release rate (93.29 %) was obtained with the amoxicillin + clavulanic acid and L. aurita combination against S. aureus. The lowest rate among synergistic treatments (89.62 %) was observed with colistin and L. aurita against B. cereus.

 

 

Table 6: Amount of DNA released by bacteria treated with synergistic combinations compared to untreated bacteria.

Bacterial strains	DNA (µg/mL)
	Control	Bacteria treated with synergistic MIC	% of DNA release
Y. enterocolitica 0938V	9.33c±0.02	91.66c±0.04	89.82cd±0.02
E. coli ATCC 25922	7.26e±0.01	72.16e±0.01	89.93c±0.02
B. cereus 0998V	8.33d±0.00	80.3d±0.01	89.62d±0.01
P. aeruginosa ATCC 9027	5.33f±0.01	51.46g±0.03	89.64d±0.03
E. faecalis 0366V	16.43b±0.02	189.46b±0.00	91.32b±0.02
S. aureus ATCC 2523	18.10a±0.03	270.1a±0.00	93.29a±0.05

 

 

Statistical analysis (ANOVA, P < 0.05) revealed a significant difference between the DNA quantities released by resistant bacteria treated with antibiotics alone versus those treated with synergistic combinations. This discrepancy is likely attributable to initial antibiotic resistance mechanisms ¹⁷. These results align with those of Rhayour ¹⁷ who noted that E. coli treated with bactericidal doses of polymyxine B showed no detectable nucleic acid bands, unlike those treated with essential oils.

While Ghaly ⁷⁶ observed similar trends testing seven essential oils, their reported maximum release rate was lower (46.94 %) than the rates observed in this study. Consistent with Fawzy ⁷⁷. 

DNA release from treated bacteria significantly (ANOVA, P < 0.05) exceeded that of the controls. Furthermore, a significant difference (ANOVA, P < 0.05) was observed between Gram-positive and Gram-negative bacteria; the former exhibited higher DNA release rates (Table 7), confirming that Gram-positive strains are more susceptible to antibacterial agents ^27,78,79.

A DNA release rate of 93.76 % suggests a mechanism involving the simultaneous disruption of the peptidoglycan layer and the plasma membrane. UV spectroscopy quantification confirms that synergistic combinations successfully damaged the structural integrity of Y. enterocolitica, E. coli, B. cereus, P. aeruginosa, E. faecalis, and S. aureus, leading to substantial DNA loss. These results validate the efficacy of essential oil-antibiotic combinations against multidrug-resistant bacteria.

This study highlights the potential of L. aurita and H. suaveolens essential oils to enhance antibiotic efficacy. These findings also provide a theoretical basis for developing simplified bacterial lysis methods for DNA/RNA extraction that bypass the need for specific enzymes (e.g., lysozymes, lysostaphin) or denaturing detergents like SDS or Triton X100 ⁸⁰.

In conclusion, the findings of this study demonstrate that the essential oils of Laggera aurita and Hyptis suaveolens possess not only intrinsic antibacterial activity against multidrug-resistant bacteria but also potent synergistic properties when combined with conventional antibiotics. The synergistic interaction between these essential oils and beta-lactams or glycopeptides likely involves dual mechanisms of action: the physical disruption of the bacterial cell wall and plasma membrane, alongside the potential inactivation of beta-lactamase activity or the inhibition of charge modifications in the lipopolysaccharide (LPS) layer.

This antimicrobial mechanism induced a bactericidal effect against strains previously resistant to both amoxicillin + clavulanic acid and colistin. Most of these effective essential oil-antibiotic combinations enhanced the antibacterial potency of the tested antibiotics to varying degrees. Collectively, these results provide a theoretical framework for future in vivo studies, which may ultimately lead to the development of novel antimicrobial agents formulated from L. aurita and H. suaveolens essential oils in combination with antibiotics. Such advancements could significantly improve the efficacy of existing clinical treatments in the fight against multidrug-resistant pathogenic bacteria. However, further research is required to identify the specific bioactive compounds responsible for these effects within the complex oil-antibiotic mixtures and to fully elucidate the molecular interactions at play.

Finally, to address the challenges associated with the low solubility and chemical instability of essential oils, future research should focus on encapsulation or nano-emulsion technologies. These delivery systems are essential to optimize bioavailability, ensure targeted release, and minimize potential cytotoxicity.

Authors' contributions: All authors contributed equally in writing the original draft preparation, all authors have read and agreed to the published version of the manuscript. 

Conflict of interest: The authors have no conflicts of interest. 

Financial disclosures: None. 

Acknowledgement: None.

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