Introduction

Microbial contamination is among the main factors related to foodborne diseases and food spoilage which is increasingly presenting a major public health problem and represents significant economic losses1. Since ancient times, diverse chemical compounds, including antibacterial molecules, have been developed, usually known as preservative additives, which extent the life of foods or are used as colouring or sweetening agents2. On the other hand, the overuse of antibiotics in animals, humans, agriculture, and aquaculture to combat pathogenic bacteria has contributed to the emergence of resistant and multidrug-resistant bacteria, such as those resistant to extended-spectrum cephalosporins, carbapenems, and colistin drugs3,4. In Gram -negative bacteria, the main β-lactams resistance mechanism is the production of β-lactamases as extended spectrum β-lactamases (CTX-M, TEM, and SHV) and carbapenemase enzymes (KPC, NDM, VIM and OXA-48). For this reason, a recent trend has emerged of using natural additives as an alternative to common food additives, mainly due to the increasing consumption of raw or minimally processed products. This approach has gained huge popularity nowadays2. One such possibility is the use of edible and medicinal plant derivatives like essential oils (EOs), due to their known immunomodulatory, antioxidant, antimicrobial, and food preservative activities2,5. These activities are dependent on the species, origin, climate, soil conditions, fertilization, cultivation processus, chemical composition, and extraction and purification processing method6,7. These antimicrobial compounds are commonly found in the essential oil part of leaves, flowers or buds, bulbs, rhizomes, seeds, fruits, and other plant parts5.

Due to its geographic location, with a diversified climate and varied landscape, Algeria is considered as a favourable location for rich vegetation. Artemisia campestris L. is an aromatic herb, commonly used as an herbal remedy for a variety of diseases in North Africa8. It is a camephytic plant, systematically classified in the family of Asteraceae, tribe Anthemideae, genus Artemisia L.8,9. Geographically, A. campestris L. is capable of growing in a very wide range of ecological habitats, from the thermo-Mediterranean scrub to mountain belts and from Saharan areas to wetlands. It has also been reported that this species prefers open habitats like grasslands, clearings, the edges of forests, and dry soils, and it predominates in the arid regions of the North African countries such as Algeria10. This plant has many traditional uses, most of them have been pharmacologically demonstrated, including antihypertensive, antihyperlipidaemic, antidiabetic, antivenom, anti-Leishmaniasis, anti-inflammatory, hepatoprotective, and kidney-protective. In addition, it has culinary properties, especially as a food preservative9.

Origanum vulgare, commonly known as oregano, belongs to the Lamiaceae family. It is a plant which grow at altitudes of between 400 m and 1800 m in sunny locations and is a perennial herb distributed across Europe, America, Asia, and North Africa11,12. This plant is widely used in traditional medicine as a lithotriptic, diuretic and antispasmodic, expectorant, stimulant, anticancer, inflammatory, antibacterial, anti- antioxidant, and laxative agent13. In addition, Origanum vulgare has been widely used as a culinary herb and as an aromatic substance in food products, in alcoholic beverages, and in perfumery for its spicy fragrance14.

Brocchia cinerea, also known as medicinal plant, belongs to the Asteraceae family and is an annual woolly herbaceous plant measuring 5 cm to 15 cm in length15. It is widely distributed in the Sahara Desert and represents one of the monotypic genera of Anthemideae, characteristic of the North African flora. It has been used since ancient times for both medicinal and breeding purposes (nutrition). B. cinerea has traditionally been used as a decoction and infusion to treat colic, diarrhoea, headaches, digestive disorders, cough, fever, migraines, rheumatoid arthritis, inflammation, bronchopulmonary cold, and urinary tract and lung infections16.

Thus, the aim of the present study was to evaluate the antibacterial activity of the essential oils of three Algerian medicinal plants (Brocchia cinerea, Artemisia campestris and Origanum vulgare) against multi-drug-resistant Gram-negative bacteria isolates from different food samples, including vegetables, fruits, and fish.

Results

Extraction yield and chemical composition of essential oils

The extraction yields of the studied essential oils from the dry aerial parts of Artemisia campestris, Brocchia cinerea, and Origanum vulgare were 5 ml /kg, 8.75 ml/kg and 20 ml/kg respectively. For B. cinerea, the chemical study by GC/MS showed the presence of different chemical compounds with the dominance of β-thujone (32.4272771%), 1-Methyl-2-(1’-methylethenyl) -3’ ethenylcyclo propylmethanol (15.7176919%), (-)-Camphor (13.3333333%) and limonene-10-ol (12.0553171%). The essential oil of Origanum vulgare, GC-MS analysis revealed that carvacrol (48.270372%) and thymol (34.2162397%) were the major chemical components of this oil. Sixteen constituents were identified from Artemisia campestris EO, of which the major components were 5(6 H)-benzocyclooctenone − 7,8- dihydro-8,8-dimethyl (41.16659756%) followed by 1,2,3,4,4a,9,10,10a-(trans- 4a,10a)-octahydro-9- oxophenanthrene (40.54941257%) and Germacrene D (5.226278405%). The chemical composition of the tested essential oils was presented in Table 1.

Table 1 Chemical composition of the three tested essential oils.
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Antibacterial activity of essential oils

The results of the antibacterial activity assay of the three tested essential oils (Brocchia cinerea, Origanum vulgare, Artemisia campestris) demonstrated that all the tested essential oils showed antibacterial activity against β-lactam and/or colistin-resistant strains harbouring different antibiotic resistance genes.

In the present study, the Brocchia cinerea essential oil was the most active, with lower concentrations than those of the two other tested oils. The MICs range of the tested essential oils were presented in Fig. 1.

Fig. 1
figure 1

The MICs range of the tested essential oils.

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The obtained results also showed that Myroides odoratimimus was very sensitive to the action of the B. cinerea essential oil, with an MIC of 1.333 µg/mL. However, E. coli carried the blaOXA−48 gene, Klebsiella pneumoniae harboured blaCTX−M−15, and TEM-producing C. freundii and E. cloacae resistant to 3GC were the least sensitive with an MIC of 42.68 µg/mL (Table 2).

Table 2 MIC and MBC of Brocchia cinerea essential oil against the tested strains.
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The Artemisia campestris essential oil exerted significant inhibitory activity against all tested resistant strains, with minimum inhibitory concentrations higher than those reported for Brocchia cinerea, as follows: 21.34 µg/mL against Shewanella putrefaciens and 170.72 µg/mL against E. coli, K. pneumoniae, C. freundii and E. cloacae (Table 3).

Table 3 MIC and MBC of Artemisia Campestris essential oil against the tested strains.
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For Origanum vulgare, the obtained results showed that the essential oil of this plant was active against all the tested resistant strains, with higher minimum inhibitory concentrations than those obtained with Brocchia cinerea but lower than those found for Artemisia campestris EO. In our study, we found that Origanum vulgare EO inhibited the growth of Shewanella putrefaciens at an MIC of 18 µg/mL. For OXA-48-producing E. coli, K. pneumoniae positive for the blaCTX−M−15 gene, C. freundii and E. cloacae resistant to 3CG, an MIC of 72 µg/mL was observed (Table 4).

Table 4 MIC and MBC of Origanum vulgare essential oil against the tested strains.
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Discussion

Essential oils are known for their role in protecting plants against micro-organisms. This emphasizes the scientific hypothesis that such oils and their compounds may also present antimicrobial activity against bacterial pathogens of human interest. Indeed, the highest antibacterial activity exerted by the EOs of the three tested plants in the present study can be attributed to their major bioactive compounds as: thujone, 1-Methyl-2-(1’-methylethenyl) -3’ ethenylcyclo propylmethanol, 1,8-Cineole and limonene-10-ol for B. cinerea and to thymol for O. vulgare or it may also be the result of synergies between the different compounds of the same EO.

Brocchia cinerea belongs to the Asteraceae family and has antibacterial activity provided by its main components15. In our study, the main constituents of this oil were beta-thujone, 1-Methyl-2-(1’-methylethenyl)-3’- ethenylcyclopropylmethanol, 1,8-Cineole, and limonene-10-ol. Based on the results of subsequent studies17,18,19, we suggest that the antibacterial activity of the B. cinerea essential oil is strongly related to the presence of beta-thujone, 1,8-cineole, and limonene. Indeed, Dorman and Deans (2000) and Lima et al. (2021) tested a large number of pure constituents of different essential oils against 25 bacterial species and showed that thujone and limonene are compounds with a broad spectrum of antibacterial activity17,18. In this manner, thujone is a natural component of the essential oils of many plants where α- and β-thujone are major constituents and they are biologically active components of essential oils derived from a variety of Asteraceae, Cupressaceae, and Lamiaceae plants19. According to many previous studies, plant species with a thujone chemotype are known for their very potent antibacterial activity20. In the present study, we observed that the Brocchia cinerea EO was the most active compared to the two other tested oils with an MIC of 1.333 µg/mL against Myroides odoratimimus which is considered as a very sensitive bacterial species. In addition, we found that the B. cinerea EO was active against E. coli carrying the mcr-1 gene and against K. pneumoniae producing an ESBL type CTX-M-15, E. coli positive for the blaOXA−48 gene with MICs of 21.34 µg/mL and 42.68 µg/mL, respectively, as well as against P. aeruginosa with an OprD mutation with an MIC equal to 21.34 µg/mL. In this context, our results were different to those reported in Morocco, which noted that the essential oil of the fresh aerial part of the Brocchia cinerea plant exhibits high bactericidal inhibitory activity against K. pneumoniae, and E. coli, with the exception of P. aeruginosa, which was resistant to the high concentrations used. The MIC values mentioned in our study were lower than those reported in the Moroccan study (1.56 mg/mL for K. pneumoniae and 3.12 mg/mL for E. coli). The authors of the Moroccan study suggested that the observed antibacterial activity may be related to the richness of this essential oil in thujone, Santalina Triene, 2-Bornanone and 1,8-Cineol, as well as to the interaction between these different compounds16. Similarly, our results also differed from those observed by Cheriti and Boukhobza (2020), which showed that Brocchia cinerea EO has a better antibacterial activity, where E. coli strain seems to be the most sensitive to the tested oil followed by K. pneumoniae but without any antibacterial effect against P. aeruginosa. The authors associated this antibacterial activity to the oil’s richness in oxygenated monoterpenes such as trans-thujone (36.11%) and camphor (12.08%) known for their broad-spectrum antimicrobial activity21.

In the present study, P. aeruginosa 1117 DSM and that with the OprD mutation were sensitive to the action of B. cinerea EO with an MIC equal to 21.34 µg/mL. This value obtained in our study is higher than that reported by Hamdouch et al., (2022), who found that Pseudomonas aeruginosa was the most sensitive species to B. cinerea EO with an MIC equal to 0.6 µL/mL. The authors associated this activity with the main components of the oil, namely thujone, camphor, and eucalyptol20. Moreover, our results are consistent with those of a Moroccan study which demonstrated a significant inhibitory activity against E. coli and P. aeruginosa strains. The latter species was sensitive to the action of the essential oil with a very low MIC of 0.0018 mg/mL, compared to the MIC of 0.0037 mg/mL against E. coli, which is lower than those found in our study. In the same study, the authors identified 21 constituents representing approximately 99.97% of the tested oil where the main components of which were thujone (24.9%), lyratyl acetate (24.32%), camphor (13.55%), and 1,8-cineole (10.81%)22.

The same observation was noted for Artemisia campestris EO with regards to its antibacterial activity against all tested multi-drug-resistant species. In a Tunisian study, the authors revealed that Pseudomonas aeruginosa and Enterobacter amnigenus were resistant to the action of the Artemisia campestris EO, but it was more effective against E. coli. Indeed, it showed low activity against Klebsiella pneumoniae, Serratia marcescens, and Citrobacter freundii, where the authors linked this activity to two major components: β-pinene (45.8%) and α-pinene (12.5%)23. The results obtained by Akrout et al., (2009) were different from those reported in our study, where we found an effect against P. aeruginosa (MIC = 85.36 µg/ml), E. cloacae (MIC = 170.72 µg/ml), E. coli (MIC = 170.72 µg/ml), and K. pneumoniae (MIC = 170.72 µg/ml). Our results are also different to those obtained by Al Jahid et al., (2017), who revealed that the essential oil extracted from the seeds of the A. campestris L. plant had weak inhibitory activity against P. aeruginosa and had no activity against E. coli24.

In our study, the inhibitory activity of A. campestris EO against P. aeruginosa and E. coli isolates was observed with a MICs of 85.36 µg/mL and 170.72 µg/mL, respectively. These results are lower than those reported by Al Jahid et al., (2016), who showed an inhibitory effect against P. aeruginosa with a minimum inhibitory concentration of 100 µg/mL, and higher than that reported against E. coli (100 µg/mL). They associated this activity with the main constituents of germacrene D (10.20%) and β-pinene (8.90%). In addition, Al Jahid et al., (2016) noted that the mode of action of essential oils containing terpenes still remains unclear, unpredictable, and difficult to characterize due to the variability in the their structures25. This suggestion may explain the activity of the A. campestris EO observed in our study, where germacrene D was one of the main compounds detected.

In the present study, Origanum vulgare EOs also presented high activity against all the bacterial species tested, with higher MICs than those shown for Brocchia cinerea but lower than those found for Artemisia campestris EO. The observed activity may be due to the presence of thymol as a major compound detected in our O. vulgare EO. In this context, Elansary et al., (2018) demonstrated higher antibacterial activity for Origanum vulgare EO against E. coli due to the high proportion of pulegone detected in the tested oil. The MIC value (0.25 mg/ml) obtained by Elansary et al., (2018) is higher than that found in our study for E. coli. The authors observed a high antibacterial activity of pulegone-rich essential oils, suggesting that this compound may have a broad spectrum of antibacterial activity26. In a Brazilian study, Scandorieiro et al., (2016) reported that this EO inhibited the growth of all the bacterial strains tested, including those classified as multidrug-resistant strains. The MIC values of the latter study were 0.596 mg/ml for ESBL- or KPC-producing E. coli, while in our study the MICs observed for OXA-48-producing E. coli was 72 µg/ml and 18 µg/ml for Shewanella putrefaciens positive for the blaVIM−4 gene which are lower than those reported by the Brazilian study27. In addition, in Greece, Fournomiti et al., (2015) showed that the most susceptible bacterial species was K. oxytoca with a mean MIC value of 0.9 mg/mL for oregano essential oil, followed by K. pneumoniae with a mean MIC of 73.5 mg/mL, whereas the E. coli strain was ranked among the most resistant to the antimicrobial action of the oil tested, in contrast to our results28. As mentioned above, the observed activity of O. vulgare in our study may be due to the presence of thymol as a major compound. In accordance with this suggestion, Lambert et al.., (2001) found that the antibacterial activity of the O. vulgare EO results from the high level of thymol, which binds to membrane proteins and increases the permeability of the bacterial cell membrane29. Globally, the activity of the O. vulgare EO may be due to its hydrophobicity, where it is likely to act on lipids of the bacterial plasma membrane, damaging these structures and increasing their proton permeability30.

Materials and methods

Plant material

In March 2016, seed-bearing parts of Brocchia cinerea were collected from the commune of M’lili in the city of Biskra, 396 km from Algiers. It is located between 6° and 42′ 25″east longitude and 34°48′21″ north latitude, at an altitude of 158 m, and extends over an area of 371.80 km2. Nevertheless, the aerial part of oregano (Origanum vulgare) was collected in the early flowering period in February 2020 in a forest located in Hassi Khelifa-Oued Souf, in the south-east of Algeria. Artemisia campestris L. was collected in December 2014 in the commune of Tazoult in the city of Batna, Algeria.

Collection of plant materials complies with all relevant national legislation and regulatory frameworks. A voucher sample of Brocchia cinera (No.003CRSTRA0039) and Origanum vulgare (specimen No. MP23-2022) were deposited at the Research and Technology Center for Arid Zones (CRSTRA), Mohamed Khider university of Biskra and Biophysical Station of Touggourt, Algeria, respectively. However, voucher No. 007PLPD0023 was preserved for the voucher specimen of Artemisia campestris in the Pharmacognosy laboratory, department of pharmacy, Batna, Algeria.

All the harvested plants were cleaned and shade-dried at room temperature before being powdered. The plant materials were identified by Prof. Mohammed Tahar Ben Moussa and Dr. Sonia Meklid from the Pharmacy Department of the University of Batna 2, and Prof. Azzedine Chefrour from the Department of Fundamental Education in Biology, at the University of Souk Ahras, Algeria.

Extraction of essential oils

Essential oils were extracted by hydro-distillation of the aerial parts of the collected plants for three hours using an extraction device following the European Pharmacopoeia guidelines. The distillation flask was filled with the appropriate volume of liquid and a few pieces of porous stone, the condenser was attached, and distilled water was added through the filling tube until it reached the indicated level. The mixture was then heated to boiling and the distillate was collected at a rate of approximately 2–3 mL/min. Hydrodistillation was repeated several times for each sample of dried plant material. The recovered essential oils were stored at 4 °C in tightly sealed dark glass vials, protected from light, until analysis. The essential oil yield was expressed in mL/kg of dry plant material.

Determination of chemical composition of the extracted essential oils (Gas Chromatography/Mass spectrometry (GC-MS) analysis)

The essential oils were diluted in absolute ethanol at a concentration of 1 g/L. The chromatographic analysis of the essential oils was carried out by gas chromatography coupled with a Clarus 600 DMS mass spectrometer (Perkin Elmer, USA). A RESTEK Rtx-5MS® capillary column was used with a length of 30 m, an internal diameter of 0.25 mm, a film thickness of 0.25 μm, and the stationary phase. The injections of samples were performed in splitless mode and helium was used as a carrier gas at a flow rate of 1 mL/min. The injector and transfer line temperatures were increased to 250 °C. The initial temperature was set at 60 °C and maintained for one minute, then increased by 3 °C/min to 200 °C and kept at a constant temperature for 13 min.

The acquisition was made in electronic impact at 70 eV, with a source at 250 °C in Scan mode (from 40 to 600). The compounds were identified by comparing the mass spectra with those provided by the WILEY and NIST libraries. The percentage content of essential oil constituents was determined by the internal standardization method. To monitor the stability of the essential oils during storage at 4 °C in the dark, we routinely performed storage quality control by analyzing their chemical profiles using gas chromatography coupled with electron ionization mass spectrometry (GC–MS) to detect any modifications that might have occurred before performing the antibacterial assays. These quality-control analyses did not reveal any relevant changes in the composition of the oils over the storage period.

Antibacterial activity assay

Tested strains

The antibacterial activity of the essential oils was evaluated against referenced sensitive bacteria (E. coli ATCC 25922 and P. aeruginosa DSM 1117) and antibiotic-resistant bacteria including those exhibiting resistance to extended spectrum cephalosporins, as well as to last resort antibiotics such as carbapenems and colistin isolated from different food types including vegetables, fruits, and fish. These targeted antibiotic-resistant bacterial strains, presented for each tested plant species in Tables 2, 3 and 4, were provided by our research laboratory. They originate from a previous research project conducted by our team that specifically focused on antibiotic resistance, in which their antibiotic resistance profiles and associated genetic determinants had already been determined using phenotypic (antibiogram) and genotypic (PCR) methods31. The antibiotic resistance characteristics of the bacterial strains tested in the present study are summarized in Tables 2, 3 and 4.

Antibacterial activity assay

The antibacterial activity and the minimum inhibitory concentration of the three essential oils was determined by the liquid micro-dilution method in sterile 96-well plates (aligned in eight rows and 12 columns). The oils were diluted in Mueller-Hinton broth with a solution of tween 80 at a final concentration of 0.5%, then sonicated for 30 min and homogenized using a vortex for one minute to obtain a stable emulsion of the essential oils32. The essential oils of Brocchia cinerea, Artemisia campestris and Origanum vulgare were tested by broth microdilution. For B. cinerea and A. campestris essential oils, twofold serial dilutions were prepared in Mueller–Hinton broth to obtain final concentrations of 341.44, 170.72, 85.36, 42.68, 21.34, 10.67, 5.335, 2.6675, 1.33375 and 0.667 µg/mL. For O. vulgare essential oil, the final concentrations tested were 288, 144, 72, 36, 18, 9, 4.5, 2.25, 1.125 and 0.56 µg/mL. For each bacterial strain, growth was evaluated at all ten concentrations. The range of concentrations tested for each oil was defined by referring to the literature data about these medicinal plants. A volume of 100 µL of each concentration was distributed in each well of the plate (columns 1 to 10). For each bacterial strain tested, a 0.5 McFarland suspension diluted to 1/20 was prepared, and then 10 µL of the prepared bacterial suspension was deposited into all wells (except column 11). For each microdilution plate, a negative control well containing Mueller–Hinton broth only and a growth control well containing Mueller–Hinton broth inoculated with the tested bacterial strain were included in columns 11 and 12, respectively, in accordance with EUCAST (2019) recommendations33. Colistin was used as the reference antibacterial agent. Subsequently, the plates were incubated for 24 h at 37 °C. After incubation, growth indicator (iodonitrotetrazolium chloride) was used to interpret the results. The minimum inhibitory concentrations (MICs) represent the lowest essential oil concentration in the dilution series at which no visible bacterial growth was observed after incubation.

The minimum bactericidal concentration (MBC) of the tested oils was evaluated as follows: 10 µL of the wells that showed no visible growth after incubation were inoculated on nutrient agar and then incubated at 37 °C for 24 h. The MBC of essential oils (nano-emulsions) represents the concentration where no colonies were obtained on the agar plates34.

Conclusion

In conclusion, this in vitro study demonstrated that the essential oils of Origanum vulgare, Artemisia campestris and Brocchia cinerea exhibit antibacterial activity against foodborne antibiotic-resistant Gram-negative bacteria isolates, including those resistant to last-resort antibiotics such as carbapenems and colistin. These findings indicate that such essential oils may be considered as promising natural candidates for incorporation into preservation strategies designed to prevent bacterial proliferation in food products. The use of plant-derived essential oils in the food sector could, after appropriate validation, contribute to reducing reliance on synthetic preservatives commonly employed to limit foodborne diseases and spoilage. However, further studies, including toxicological assessments, investigations in real food matrices, are required to define safe and effective application conditions and to optimize their use across a wider range of food products.