Nova Biotechnol Chim (2023) 22(1): e1519 DOI: 10.34135/nbc.1519 1 Nova Biotechnologica et Chimica Zizyphus lotus and Ruta chalepensis essential oils for combating antimicrobial resistance in pathogenic clinical bacteria and fungi Nour El Houda Bekkar1,, Boumediene Meddah1, Bahadir Keskin2, Pascal Sonnet3 1Laboratory of Bioconversion, Microbiological Engineering and Health Safety, Faculty of Life and Nature Sciences, University Mustapha Stambouli of Mascara, Mascara 29000, Algeria 2Department of Chemistry, Faculty of Arts & Science, Yildiz Technical University, Istanbul TR34210, Turkey 3AGIR Laboratory: Infectious Agents, Resistance and Chemotherapy, EA4294 UFR of Pharmacy, Picardie Jules Verne University, Amiens, France  Corresponding author: nourelhouda.bekkar@univ-mascara.dz Article info Article history: Received: 14th October 2021 Accepted: 20th October 2022 Keywords: Antimicrobial activity Essential oil GC-MS Ruta chalepensis Zizyphus lotus Abstract The antimicrobial activities of essential oils (EOs) isolated from Zizyphus lotus (ZL) and Ruta chalepensis (RC) harvested in Oran (north-west Algeria) were assessed against pathogenic clinical bacteria and fungi. The EOs were isolated using the steam distillation process, the phenolic and flavonoid contents were determined using colorimetric methods, and the chemical composition was carried out using GC-MS analysis. The antimicrobial activity was evaluated using agar disc diffusion and microdilution methods. The evaluation of the synergistic effect using the combination of Z. lotus (ZLEO) and R. chalepensis essential oils (RCEO) was done using the checkerboard assay. Effective extraction yields were determined for both plants, with an actual amount in RC than ZL. Concentrations of 8.47 ± 0 mg GAE/g DE and 8.56 ± 0.154 mg CE/g DE of total phenols were determined in ZLEO and RCEO, respectively. Thus, a chemotype of Diisooctyl-phthalate (80.343 %) was determined in ZLEO and the 2-Undecanone (13.236 %) in RC. Both plant EOs exhibited important antimicrobial activity against the selected multidrug-resistant human pathogens. The most potent effect was estimated against Proteus mirabilis, Salmonella enterica subsp. arizonae, and Hafnia alvei with growth inhibition zone diameters of 24.06 ± 0.12, 40.1 ± 0.1 and 40.16 ± 0.15 mm using ZLEO, respectively. Also, essential anti-Candida activity was estimated. ZLEO and RCEO did not exhibit either synergistic or additive effects, with fractional inhibition concentration index values greater than 2. Both plants exhibited significant antimicrobial effects alone, while in combination they did not exhibit a synergistic effect but an antagonistic one. Therefore, ZLEO and RCEO can be developed as natural antimicrobial agents in the medical and food industries to combat antimicrobial resistance. © University of SS. Cyril and Methodius in Trnava Introduction The emergence of antimicrobial resistance in most reanimation services, hospitals, and food-borne pathogens is considered a global health concern. mailto:nourelhouda.bekkar@univ-mascara.dz Nova Biotechnol Chim (2023) 22(1): e1519 2 The bacterial and fungal strains developed increasing resistance to many antibiotics, making the therapeutic application less efficient for treating microbial infections. Besides, the significant side effects of these drugs and the dysbiosis phenomena that appeared after the consumption of antibiotics induce essential consequences for human health. So, this allowed us to search for more safe alternative products, especially in the world of medicinal and aromatic plants. An emergent interest in using natural antibacterial and antifungal molecules, such as plant essential oils, as potent alternative antimicrobials for combating the antimicrobial resistance in various pathogenic clinical strains responsible for severe human microbial infections. The Algerian medicinal plants are known for their contents of bioactive components, and various recent studies have elucidated the antimicrobial effects of Eos and phenolic extracts of a wild variety of medicinal plants (Ayad et al. 2022; Bennacer et al. 2022). Among the medicinal plants of high interest in traditional medicine in Algeria and used by the local population are Zizyphus lotus L. (Rhamnaceae) and Ruta chalepensis L. (Rutaceae) species. Both plants contain an immense variety of bioactive substances with various biological properties. Recent studies have demonstrated that Zizyphus bioactive components exert various biological activities, such as memory enhancement effects (Zhang et al. 2014). Furthermore, the Ziziphus species have mainly been used in traditional medicine to treat severe diseases such as respiratory problems, scabies, pimples, and inflammation of the mouth and gums. The flavonoids, saponins, and fatty acids isolated from Ziziphus species are responsible for the plant's sedative effects (Xie et al. 2012). Moreover, Z. lotus is used in many fields, including nutrition, cosmetics, and healthcare. This plant is widely used in the treatment of urinary tract infections, digestive disorders, and intestinal microbial infections. It is used as a hypoglycemic, hypotensive, antidiarrheal, and anti-ulcer agent in stomach diseases (Bnouham et al. 2002; Borgi et al. 2007). Various studies have reported the anticancer and anti-inflammatory properties of this plant. It also exhibited potent antifungal activity, a gastroprotective effect against Helicobacter pylori and was used as an analgesic, as well as having a potent antibacterial effect (Wahida et al. 2007; Borgi et al. 2008; Benammar et al. 2010; Bakhtaoui et al. 2014; El Hachimi et al. 2016; Bencheikh et al. 2019). Ghalem et al. (2014), Marmouzi et al. (2019), and Bencheikh et al. (2021) elucidated the antioxidant and nephroprotective effects of Z. lotus. Referring to the literature, most of the research is about the biological properties of phenolic extracts of Z. lotus, while limited studies demonstrate the chemical composition and the antimicrobial effects of Z. lotus essential oils, that is why we are interested in selecting this plant for the isolation of potent antimicrobials that can be used as alternatives to antibiotics. R. chalepensis is a common species in Algeria and is intensely interested in international traditional medicine. This plant is known for its richness in therapeutic compounds, especially inessential oils (2-Undecanone) (González-Trujano et al. 2006; Mejri et al. 2010). Recent studies mentioned that R. chalepensis leaf extract is known for its essential antioxidant and hypoglycemic activities (Loizzo et al. 2017). Althaher et al. (2020) demonstrated the cytotoxic effect of R. chalepensis essential oils on human MCF-7, T47D, and Caco-2 cancer cell lines. The essential oils isolated from the aerial parts of this plant exert an antifungal effect against Aspergillus sp., Fusarium culmorum, Fusarium pseudograminearum, Fusarium proliferatum, and Fusarium graminearum (Bouajaj et al. 2014). According to Amdouni et al. (2016), the R. chalepensis EOs also exert an antibacterial effect against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. González-Trujano et al. (2021) have mentioned the multiuse of Ruta chalepensis to treat various disorders such as fever, rheumatism, mental disorders, menstrual problems, anxiety, and epilepsy problems. This is the first time that the antimicrobial effect of the essential oils of Z. lotus and R. chalepensis collected from the Oran-Tafraoui region in western Algeria has been reported. No studies on the chemical composition of ZLEO in volatile bioactive components have been reported. The present study aimed to determine the chemical https://www.google.com/search?client=firefox-b-d&q=Pseudomonas+aeruginosa&spell=1&sa=X&ved=2ahUKEwilg-S504L5AhUP-YUKHXFKDGkQkeECKAB6BAgCEDU Nova Biotechnol Chim (2023) 22(1): e1519 3 composition and chemotypes of EOs isolated from Z. lotus leaves and R. chalepensis aerial parts using GC-MS analysis and their antimicrobial effects against pathogenic clinical bacteria and fungi for combating antimicrobial resistance. Experimental Plant material The wild Z. lotus leaves and R. chalepensis aerial parts were collected in July and April 2017, respectively, during their flowering stages, from the Tafraoui region in Oran (north-west Algeria). Furthermore, both plants were identified by the botanist Pr. Righi K. from the Department of Biology of Mascara University, Algeria. Clinical microbial strains The antimicrobial effect was assessed using pathogenic clinical isolates, including Gram- positive bacteria (Staphylococcus aureus, Streptococcus pyogenes, and Enterococcus faecalis), Gram-negative bacteria (Enteropathogenic Escherichia coli, Salmonella enterica subsp. arizonae, Proteus mirabilis, Hafnia alvei), and pathogenic microscopic fungi (Candida albicans). All these microbial strains were isolated from different clinical samples. Stool specimens from gastroenteritis patients, oral cavity samples from the periodontal pocket of patients with periodontal disease, and urine samples from patients with urinary tract infections. On the other hand, the microbial identification was carried out in the Laboratory of Microbiology of Meslem Taib Hospital in Mascara, Algeria and in the Laboratory of Bioconversion, Microbiological Engineering, and Health Safety Department of Biology, University Mustapha Stambouli of Mascara, Algeria. Antibiotics susceptibility test The antibiotic susceptibility testing was carried out using the agar disc diffusion method, following the CLSI (2015). Susceptibility to various antibiotics was tested: Penicillin family: Penicillin G (10 µg/disc), Amoxycillin (25 µg/disc), Oxacillin (5 µg/disc), Aminoglycosides: Neomycin (30 µg/disc), Polymyxin E: Colistin (10 µg/disc), Macrolide: Spiramycin (100 µg/disc), Streptogramin A: Pristinamycin (15 µg/disc), Nitroquinolone: Nitroxolin (20 µg/disc) for bacteria, and Azole antifungal: Fluconazole (25 µg/disc) for C. albicans strain. According to the FMS-AC (2013) and the FMS- AC/EUCAST (2018) guidelines, the growth inhibition zone diameters were interpreted using the critical diameters mentioned in Table 1. The antibiotic resistance profiles allowed us to qualify the isolated clinical microbial strains as pathogenic, multidrug-resistant strains. Table 1. Antimicrobial resistance profile of clinical isolates. Clinical isolates SP AMX PT NI N OX CT P P-G FCA S. aureus 0 R 12 R 0 R 18 I 15 I 0 R 12 R 10 R 0 R NT S. pyogenes 0 R 15 R 0 R 22 I 15 I 0 R 12 R 0 R 0 R NT E. faecalis 23 S 19 I 20 I 12 I 0 R 0 R 0 R 21 I 0 S NT E. coli 0 R 0 R 0 R 20 I 15 R 0 R 11 R 0 R 0 R NT P. mirabilis 0 R 15 R 0 R 14 I 16 R 0 R 0 R 10 R 0 R NT S. enterica subsp. arizonae 0 R 0 R 0 R 22 I 18 S 0 R 13 R 0 R 0 R NT H. alvei 0 R 0 R 0 R 20 I 20 S 0 R 13 R 0 R 0 R NT C. albicans 0 R 0 R 0 R 0 R 0 R 0 R 0 R 0 R 0 R 0 R SP – Spiramycin; AMX – Amoxycillin; PT – Pristinamycin; NI – Nitroxolin; N – Neomycin; OX – Oxacillin; CT – Colistin; P – Penicillin-G; FCA – Fluconazole; R – Resistant; S – Sensitive; I – Intermediate sensitivity; NT – Not tested. Essential oils extraction The extraction of essential oils (EOs) was carried out using steam distillation. Briefly, each 45 g of the fresh leaves of Z. lotus and the aerial parts of R. chalepensis were subjected to steam distillation in distilled water for three consecutive hours according to the current European Pharmacopoeia Nova Biotechnol Chim (2023) 22(1): e1519 2 (2007). The obtained EOs were collected and stored at 4 °C in brown, sealed glass vials until used. This process was done in triplicate, and the extraction yield ( ) was expressed as the weight of the EO volume (g) on the weight (g) of the plant used. Physicochemical index determination Different physicochemical indices of the EOs were determined: Relative density at 20 °C (NF ISO 279 1999), Refractive index (NF ISO 280 1999), Specific optical rotation (NF ISO 592 1999), Acid index (NF ISO 1242 1999), Ester index (NF ISO 709 2002), and Miscibility with ethanol (NF ISO 875 1999). Determination of total phenolics content (TPC) The total phenolic content (TPC) was determined according to Boizot and Charpentier (2006). In brief, 200 μL of each EO sample (ZLEO and RCEO) at a concentration of 1 mg.mL-1 was mixed with 1 mL of Folin Ciocalteu reagent and 800 µL of sodium carbonate Na2CO3 solution (7.5 %). The mixture was incubated at room temperature in the dark for 10 min, and the absorbance was determined at 735 nm using a spectrophotometer (JENWAY, 6400 spectrophotometer). Gallic acid (GA) (0.05 – 0.2 mg.mL-1) was used as a standard: .The TPC was expressed as gallic acid equivalents (GAE) per gram of dry extract (mg GAE/g DE). Determinations of TPC were performed in triplicate. Results were expressed as mean ±SD. Determination of flavonoids contents (TFC) The total flavonoid content (TFC) was determined according to Samatha et al. (2012). Briefly, 1 mL of 2 % aluminum trichloride (AlCl3) in methanol was mixed with 1 mL of the EO samples. The absorbance values were determined at 430 nm after 40 min against a blank. Quercetin (Q) (0.05 to 0.25 mg.mL-1) was used as a standard: .The TFC of the extracts was expressed as mg of quercetin per gram of dry extract (mg QE/g DE). Determinations of TPC were performed in triplicate. Results were expressed as mean ± SD. Gas chromatography-mass spectroscopy analysis (GC-MS) The identification and quantification of volatile bioactive compounds from Z. lotus and R. chalepensis essential oils was carried out using a Shimadzu gas chromatograph (GC), Agilent GC Model 7890B equipped with the HP 5977A Mass spectrometer. Analytical conditions: Agilent 122- 7062 DB-WAXN capillary column (Dimension: 60m, ID: 250 micrometers, 0.25-micrometer film thickness). Helium was used as a carrier gas with a linear velocity in a column of 19 cm.s-1 and 37.862 psi of pressure. The carrier gas split flow was about 250 mL.min-1, with a split ratio: of 100 : 1, and septum purge flow of 3 mL.min-1. The oven temperature program was between 70 and 260 °C with an equilibration time of 1 min and was then maintained at 250 °C. The injection in split mode of 2 µL of the substance to be analyzed was carried out using a microsyringe of 10 µL. The components were identified by comparing their relative retention times and mass spectra with the data from the library of EO constituents (Wiley, Mass-Finder, and Adams GC/MS libraries). The percentage composition determination was based on peak area normalization without correction factors. Antimicrobial activity The agar disc diffusion method was applied to determine the antimicrobial potency of EOs from Z. lotus and R. chalepensis. Muller-Hinton agar (MHA) plates were spread by adjusted microbial culture suspensions (0.5 McFarland), and therefore, sterile discs were impregnated in 20 μL of each EO solution. The EOs of 200 mg.mL-1 concentration were dissolved in the dimethyl sulfoxide 10 % (DMSO) that was used as a negative control. The discs were aseptically deposited on the inoculated plates. After 2 h at 4°C, plates were incubated at 37 °C for 24 h, and the antimicrobial effect was expressed by measuring the diameters of the microbial growth inhibition zones (ø). Each assay was carried out in triplicate. The EOs effectiveness 4 Nova Biotechnol Chim (2023) 22(1): e1519 3 assessment was determined as follows: Ø < 8mm: Resistant, 9 mm < Ø < 14 mm: Sensitive, 15 mm< Ø < 19 mm: Very sensitive, Ø >20 mm: Extremely sensitive (Ponce et al. 2003). The microdilution method was performed to determine the minimum inhibitory (MIC), bactericidal (MBC), and fungicidal (MFC) concentrations. The assays were done in sterile 96- well microplates and examined according to the method described by Chandrasekaran and Venkatesalu (2004). Briefly, 50 µL of Mueller Hinton and Sabouraud broth (for bacteria and yeast tests, respectively) was distributed aseptically in all the sterile microplates' wells. Subsequently, 50 µL of each EO sample of both tested plants, at a concentration of 200 mg.mL-1 was added to the first well, and then serial dilutions were obtained to achieve a final concentration of 1.56 mg.mL-1. After that, 50 μL of the adjusted microbial suspensions (0.5 McFarland) were inoculated in each microplate well. The Microplates were incubated at 37 °C, and microbial growth kinetics were measured by reading the optical density at 620 nm for bacteria and 450 nm for fungi at 0-4-18 – 48, and 72 h, using a Microplate Absorbance Reader (Tecan Spectra II Microplate Reader). The microbial tests were prepared in triplicate, and the results were expressed as Log germs.mL-1 obtained for each plant extract concentration. Checkerboard method During this study, we evaluated the synergistic, additive, or antagonistic effects using the combinations between both plants’ EOs: ZLEO/RCEO. The synergistic interaction of both antimicrobial drugs was quantified after the determination of the MIC values for each plant EO (previously determined) by calculating the index of fractional inhibitory concentrations (FICI or ∑FIC), which are the lowest concentrations of the antimicrobial drugs in combination, inhibiting completely the growth of the microbial strains tested. A total of 50 µL of sterile Mueller Hinton broth was distributed aseptically in all the sterile cupules of the microplates. The first EO solution of Z. lotus was serially diluted along the abscissa, while the EO of R. chalepensis was diluted along the ordinate. An inoculum equal to 0.5 McFarland turbidity standards was prepared for each culture in sterile saline water. Each suspension cupule was inoculated with 50 µL of the microbial culture, and the microplates were incubated at 37 °C for 18 h. The value of the association was measured using the FIC in the cupules in which the microbial growth is inhibited and considered an effective MIC for the combination (Orhan et al. 2005). The ∑FICs were calculated as follows Eq. 1: (1) where, FIC A = MIC of drug A (PPE or EO of Z. lotus) in the combination/MIC of drug A (Z. lotus extract) alone, and FIC B = MIC of drug B (PPE or EO of R. chalepensis) in the combination/MIC of drug B (R. chalepensis extract) alone. The combination is considered synergistic when the FICI is ≤ 0.5, additive when the 0.5 20 mm). All these results were completed by quantitatively determining an important antimicrobial parameter, the minimum inhibitory concentration (MIC). The results of the MIC values for each clinical strain are mentioned in Table 6. The curves of the microbial growth kinetics in the presence of Z. lotus and R. chalepensis EOs are mentioned in Fig. 1 and 2. An important decrease in microbial cell concentration was detected after the 4th hour, expressed by the decrease in the Log germs/mL number (Fig. 1 and 2). The inhibitory properties of the essential oils of Z. lotus and R. chalepensis against all the microbial strains were determined, with the lowest MIC values of 25 mg.mL-1 against E. faecalis and C. albicans using the ZLEO and against S. enterica subsp. arizonae and C. albicans using the RCEO (Table 6). The bactericidal and fungicidal effects could be explained by the abundant richness of Z. lotus leaves in Diisooctyl- phthalate, the major component in ZLEO, and R. chalepensis aerial part in 2-Undecanone, the major volatile compound in RCEO. Numerous studies have demonstrated the antimicrobial effect of R. chalepensis essential oils with the 2-Undecanone chemotype (Marami et al. 2021; Nahar et al. 2021). Our results agreed with those of other studies (Belkassam et al. 2011; Zellagui et al. 2012; Alloun 2013; Arámbula et al. 2019). Our results also agree with those of Daoudi et al. (2016) on the antibacterial effect exerted by the essential oil of R. chalepensis aerial part on S. aureus and P. mirabilis. However, the results showed that the RCEO has no effect on E. coli, which does not agree with our results. During the current study, an important antibacterial effect was recorded on this bacterial strain by applying the EO of this plant. Thus, our results agree with those mentioned in another study that showed that the R. chalepensis EO has antibacterial activity against Salmonella, E. coli, and S. aureus (Amdouni et al. 2016). The inhibitory effect of Z. lotus and R. chalepensis could be explained by the most abundant richness of these plants EOs in secondary metabolites, in particular antimicrobial compounds, which justified specific uses of these medicinal plants in the treatment of several infectious diseases (García- Lafuente et al. 2009; Ben-Bnina et al. 2010; 9 Nova Biotechnol Chim (2023) 22(1): e1519 2 Haddouchi et al. 2013; Boual et al. 2015; Hammi et al. 2015). Fig. 1. Antimicrobial effect of Zizyphus lotus essential oil on clinical strains isolated from the different biological samples (P <0.05). C1-C8 – Concentrations. C1 – 200 mg.mL-1; C2 – 100 mg.mL-1; C3 – 50 mg.mL- 1; C4 – 25 mg.mL-1; C5 – 12.5 mg.mL-1; C6 – 6.25 mg.mL-1; C7 – 3.13 mg.mL-1; C8 – 1.56 mg.mL-1; T – Control test. A – Staphylococcus aureus; B – Streptococcus pyogenes; C – Enterococcus faecalis; D – Enteropathogenic Escherichia coli; E – Proteus mirabilis; F – Salmonella enterica subsp. arizonae; G – Hafnia alvei; H – Candida albicans. 10 Nova Biotechnol Chim (2023) 22(1): e1519 3 Fig. 2. Antimicrobial effect of Ruta chalepensis essential oil on clinical strains isolated from the different biological samples (P <0.05). C1-C8 – Concentrations; C1 – 200 mg.mL-1; C2 – 100 mg.mL-1; C3 – 50 mg.mL- 1; C4 – 25 mg.mL-1; C5 – 12.5 mg.mL-1; C6 – 6.25 mg.mL-1; C7 – 3.13 mg.mL-1; C8 – 1.56 mg.mL-1; T – Control test. A – Staphylococcus aureus; B – Streptococcus pyogenes; C – Enterococcus faecalis; D – enteropathogenic Escherichia coli; E – Proteus mirabilis; F – Salmonella enterica subsp. arizonae; G – Hafnia alvei; H – Candida albicans. 11 Nova Biotechnol Chim (2023) 22(1): e1519 2 Checkerboard method The individual and combination minimum inhibitory concentrations, the individual fractional inhibitory concentrations, the index inhibitory fractional concentrations, and the FIC index of Z. lotus and R. chalepensis EOs combinations against the different microbial strains tested are mentioned in Table 7. No synergistic effect was determined by applying the combination of Z. lotus and R. chalepensis EOs against the different microbial strains tested. This combination exerted an antagonistic effect against the microbial strains tested, with FICindex values greater than 4 (FICindex˃4). While an indifference interaction was recorded against S. aureus, S. pyogenes, and H. alvei using both plants EOs (Table 7). These results have enabled us to demonstrate the ineffectiveness of plant essential oils used in combination. In some cases, associations between antimicrobial drugs can be used to broaden their action spectrum on pathogenic germs. Various studies have shown the effectiveness of plant extracts and essential oils combinations in the synergistic effect expression against pathogenic bacteria. Amirouche and Belkolai (2013) demonstrated that a combination of sage and tea tree essential oils works synergistically against S. aureus. However, it was determined during this study that the effect of the EO combinations is lower compared to the effect exerted by the EOs of each plant alone. Therefore, Z. lotus and R. chalepensis cannot be used in combination due to the antagonism effect exerted on the different microbial strains tested. Thus, the combination of these EOs can limit and reduce the action spectrum of pathogenic bacteria and yeasts. Table 7. FICindex of the combinations of Z. lotus and R. chalepensis essential oils on the different microbial strains tested. Clinical strains Essential oils: ZLEO/RCEO Individual MICs MICs in combination Individual FIC FICindex S. aureus 100/100 200/200 2/2 4i S. pyogenes 100/100 200/200 2/2 4i E. faecalis 25/100 200/200 8/2 10a E. coli 100/50 200/200 2/4 6a P. mirabilis 100/50 200/200 2/4 6a S. enterica subsp. arizonae 50/50 200/200 4/4 8a H. alvei 50/50 100/100 2/2 4i C. albicans 25/25 200/200 8/8 16a a – antagonism; i – indifference. Conclusion Z. lotus and R. chalepensis EOs are potent antimicrobials with a comprehensive action spectrum against pathogenic microbial strains. However, both plants' EOs combinations were less effective against all the tested microbial strains, with antagonism effects. So, the impact of each plant EO alone is considered more important in inducing the antimicrobial effect than using of EO combinations in both plants. Z. lotus and R. chalepensis essential oils could be used in the medical and food industries as potent antimicrobials for combating antimicrobial resistance. Conflicts of Interest The authors declare that they have no conflict of interest. Acknowledgment The authors thank the University of Mascara (Algeria) for providing financial support to achieve this work, to the Laboratory of Bioconversion, Microbiological Engineering and Health Safety of Mascara University in Algeria for providing support in completing this study. To Pr. 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