key: cord-0274989-oj1lnfoi authors: Joseph Arsene, Mbarga Manga; Irina Viktorovna, Podoprigora; Linda Davares, Anyutoulou Kitio; Mouafo, Hippolyte Tene; Monique Manga, Irma Aurelia; Pavlovna, Smirnova Irina; Sergueïevna, Das Milana title: Screening of antimicrobial activity of aqueous and ethanolic extracts of some medicinal plants from Cameroon and assessment of their synergy with common antibiotics against multidrug-resistant uropathogenic bacteria date: 2021-09-16 journal: bioRxiv DOI: 10.1101/2021.09.10.459100 sha: 56509df985f467a2075498bf46a1ba4c92e93617 doc_id: 274989 cord_uid: oj1lnfoi Background and aim The Cameroonian flora abounds in plants with multidimensional therapeutic virtues which can play an important role in the fight against resistance to antibiotics and the search for new antimicrobials. This study aimed to screen the antimicrobial potential of aqueous and ethanolic extracts of thirteen (13) samples (Bark, leaf, seed) of eight (8) plants from Cameroon against 3 reference pathogens and to evaluate their synergy with conventional antibiotics against eleven (11) multiresistant uropathogenic (MRU) bacteria. Method Bioactive compounds were extracted from leaves of Leucanthemum vulgare, Cymbopogon citratus (DC.) Stapf, Moringa oleifera Lam and Vernonia amygdalina Delile; barks of Cinchona officinalis and Enantia chlorantha Oliv; barks and seeds of Garcinia lucida Vesque and leaves and seeds of Azadirachta indica (Neem) using distilled water and ethanol as solvents. The extracts were tested against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538 and Candida albicans 10231 using the well diffusion method and the microdilution method. The synergistic effect was assessed (using disc diffusion method and the checkerboard method) against MRU bacteria namely Achromobacter xylosoxidans 4892, Citrobacter freundii 426, Enterococcus avium 1669, Escherichia coli 1449, Klebsiella oxytoca 3003, Kocuria rizophilia. 1542, Moraxella catarrhalis 4222, Morganella morganii 1543, Pseudomonas aeruginosa 3057, Staphylococcus aureus 1449 and Streptococcus agalactiae 3984. Results We found that distilled water extracted a more important mass of phytochemical compounds (7.9-21.2%) compared to ethanol (5.8-12.4%). Except C. officinalis and G. lucida leaves, the rest of extracts were active with inhibition diameters (ID) ranging from 5 to 36 mm. Both ethanolic (EE) and aqueous extract (AE) of E. chloranta bark (ECB) were the most active against all pathogens with the mean ID of 17 and 36 mm vs S. aureus ATCC 6538, 23 and 14 mm vs E. coli ATCC 25922 and 36 and 19 mm vs C. albicans ATCC 10231. Only the EE of E. chloranta bark (ECB) had a strong activity against all the microorganisms tested (MIC <2 mg / ml); L. vulgare leaves (LVL) and G. lucida seed (GLS) had moderate (average MIC of 8 mg/ml) while all other extracts showed very weak antimicrobial activity. In addition, the fractional inhibitory concentration (FIC) ranged from 0.125 to 0.750. No antagonism (FIC> 4) or indifference (1≤ FIC≤4) was noted between the extracts and the antibiotics, but the best synergies were found with ECB which well-modulated Kanamycin (FIC = 0.125 against S. aureus and 0.250 against E. coli), nitrofurantoin (FIC = 0.250 against S. aureus and 0.188 against E. coli) and ampicillin (FIC = 0.125 against E. coli). Similarly, compared to other extracts, ECB, LVL and GLS also well-modulated ampicillin, ceftazidime, tetracycline, nitrofurantoin, and trimethoprim against all the above-mentioned resistant uropathogenic bacteria with important increase in fold area (IFA). Conclusion This study show that E. chlorantha bark, L. vulgare leaves G. lucida seed, have good antimicrobial activity against both bacteria (Gram positive and Gram negative) and fungi (C. albicans); and should be more investigated for their possible use to the fight against MDR and MRU microorganisms. typhoid fever, jaundice, wounds, high blood pressure, urinary infection, leprosy spots and convulsions (Etame et al., 2019) . Furthermore, A. indica known as Neem, is a monoecious tree of the Meliaceae family whose oil produced from its seeds is widely used for its medicinal properties in the northern part of Cameroon. It is known that compounds in Neem extracts have antiinflammatory, anti-hyperglycaemic, anti-carcinogenic, antimicrobial, immune-modulator, anti- citratus (lemongrass) is widely used as a tea and is rich in minerals, vitamins, macronutrients (including carbohydrate, protein, and small amounts of fat) and its leaves are a good source of various bioactive compounds such as alkaloids, terpenoids, flavonoids, phenols, saponins and tannins that confer C. citratus leaves pharmacological properties such as anti-cancer, antihypertensive, anti-mutagenicity, anti-diabetic, anti-oxidant, anxiolytic, anti-nociceptive and anti-fungal (Muala et al ., 2021) . Like C. citratus, all plants investigated in this study have various phytocompounds such as terpenoids and xanthones products, alkaloids (such as protoberberines and phenanthrene alkaloids), aporphins, zeatin, quercetin, β-sitosterol, caffeoylquinic acid and kaempferol, saponins, sesquiterpenes, flavonoids, steroid glycosides and lactones (Sylvie et Therefore, the aim of this study was to evaluate the antimicrobial potential of aqueous and hydro ethanolic extracts of thirteen (13) samples (Bark, leaf, seed) of eight (8) above mentioned plants from Cameroon and to assess their synergy with common antibiotics against various multiresistant uropathogenic bacteria. The vegetal materials used in this study were leaves of Leucanthemum University. Plants were grinded and the powders with particle sizes lower than 1 mm were stored in a sterile airtight container until further use. The microorganisms used for the screening of antimicrobial activity consisted of three standard strains. S. Dimethyl sulfoxide (DMSO) was purchased from BDH Laboratories, VWR International Ltd., USA. We also used BHIB (Brain Heart Infusion Broth) (HiMedia™ Laboratories Pvt. Ltd., India), Muller Hinton Agar (MHA HiMedia™ Laboratories Pvt. Ltd., India), Sabouraud Dextrose Broth (SDB, HiMedia™ Laboratories Pvt. Ltd., India) and all other reagents and chemicals used were of analytical grade. Hydro ethanolic solution (80%, v/v) and distilled water were used as solvent for the extraction of bioactive compounds following the method of Mbarga et al.,(2021) . Fifty grams (50g) of vegetal material was weighed and added to 450 ml of the solvent in separate conical flasks. The flasks were covered tightly and were shaken at 200 rpm for 24h and 25°C in a shaker incubator (Heidolph Inkubator 1000 coupled with Heidolph Unimax 1010, Germany). The mixtures were then filtered by vacuum filtration, using Whatman filter paper № 1 then concentrated at 40°C in rotary evaporator (IKA RV8) equipped with a water bath IKA HB10 (IKA Werke, Staufen, Germany) and a vacuum pumping unit IKA MVP10 (IKA Werke, Staufen, Germany). To avoid losses, the extracts were collected when the volumes were small enough and placed in petri dishes previously weighed and then incubated open at 40°C until complete evaporation. The final dried crude extracts were weighed. Extraction volume and mass yield were determined using the following formulas: For each plant extract, the crude extract was dissolved in the required volume of DMSO (5%, v/v) to achieve a concentration of 512 mg/ml. The extracts were sterilized by microfiltration (0.22 μm) and the solution obtained was used to prepare the different concentrations used in the analytical process. Bacteria were cultured for 24 h at 37 °C in 10 ml of BHI broth while the yeast (C. albicans ATCC 10231) was cultured in the same volume of SDB broth and the same conditions. After incubation, the cells were collected by centrifugation (7000 g, 4 °C, 10 min), washed twice with sterile saline, resuspended in 5 mL of sterile saline to achieve a concentration equivalent to McFarland 0.5 using DEN-1 McFarland Densitometer (Grant-bio). The well diffusion method described in our previous investigation (Mbarga et al., 2021) was used to assess the antimicrobial activity of the extracts. Briefly, 15 ml of sterile Muller Hinton Agar (for bacteria) or Sabouraud Dextrose Agar (For C. albicans) were poured into petri dishes and 100 μl of each microorganism were spread. Wells with a capacity of 20 µl were drilled on the culture medium and 20 µl (at 100 mg/ml) of each plant material was added. The sterile DMSO 5% used to prepare the extracts was used as negative control and all the trials was done in triplicate. After incubation at 37 ℃ for 24 h, the inhibition diameters were measured. MIC is the lowest concentration of antibacterial agent that completely inhibits the visible bacterial growth. MBCs were determined by subculturing the wells without visible growth (with concentrations ≥ MIC) on MHA or SDA plates. Inoculated agar plates were incubated at 37℃ for 48h and MBC was considered the lowest concentration that did not yield any microbial growth on agar. Tolerance level of tested bacterial strains against aqueous and ethanolic extract was determined using the following formula: The characteristic of the antibacterial activity of extracts was determined by the tolerance level indicating the bactericidal or bacteriostatic action against the tested strains. When the ratio of MBC/MIC is ≥16, the antibacterial efficacy of the test agent is considered as bacteriostatic, whereas MBC/MIC ≤4 indicates bactericidal activity (Mondal et al.,2020). The modified Kirby-Bauer's disk method described in our previous study (Manga et al.,2021b ) was used to study the antibiotic sensitivity of the tested bacterial strains, and the following eight antibiotics disks After determining the susceptibility of the uropathogenic bacteria to antibiotics, the antibiotics which gave inhibition diameters of less than 20 mm were modulated with 5 mg/ml of each extract. The test was performed as described by Rolta et al. (2018) with slight modifications. Briefly, in the same Petri dish, after having inoculated the test bacteria, a sterile disc paper and an antibiotic disc was placed aseptically. Then the same volume (20 µl) of the considered extract was slowly deposited on each of the two discs. After 24h of incubation at 37°C, the extract inhibition diameter and new antibiotic disc inhibition diameter (Antibiotic + extract) were reported and interpreted as described by Jain et al. (2020) by calculating the increase in fold area (IFA) by the following formula: Where, "A" is the increase in fold area, "Y" the zone of inhibition for extract + antibiotic and "X" is the inhibition zone of antibiotic alone. The checkerboard method, commonly used for the determination of synergy between the antibiotics and natural antibacterial compounds, was used for the antibiotic modulation assay (Mbarga et al.,2021) . Modulations of ampicillin, benzylpenicillin, cefazolin, ciprofloxacin, nitrofurantoin, and kanamycin were performed with extracts whose MIC was successfully determined (Not those with MIC <2 or MIC> 256). The fractional inhibitory concentration (FIC) index was calculated, as described in our previous study The FIC index was interpreted as follows FIC ≤0.5, synergy; 0.5 ≤FIC ≤1, addition of effects; 1≤ FIC≤4, indifference and for FIC >4, Antagonism. Except for the MIC and MBC, all the experiments were carried out at least in triplicate. The statistical significance was set at p≤0,05. T-test was carried out using the statistical software XLSTAT 2020 (Addinsof Inc., New York, USA) and the graphs were plotted by Excel software or SigmaPlot 12.5 (Systat Software, San Jose, CA, USA). The extract yields obtained for the 13 samples from our 8 medicinal plants using ethanolic solution and distilled water are recorded in Table 2 . As observed in Table 2 , volume yields ranged from 78-95% for ethanolic extract (EE) and 75-90% for aqueous extract (AE) while the mass yields ranged from 5.8-12.4% for EE and from 7.9-21,2% for AE. For most of the plant materials, the highest volume yields were observed with EE while the highest mass yields were obtained with AE. The difference in extraction volume yields can be explained by losses during the extraction process. Indeed, we noticed that the filtration of ethanolic extracts was faster (less than 5 minutes for 300 ml) compared to the aqueous extract which took much longer (more than 30 minutes on average for 300 ml) and required in average 4 filter change. Despite the filtration, the EAs still All the extracts have inhibited the pathogens with MIC values which vary significantly from one plant material to another. As previously observed with qualitative tests, the extract of E. chloranta was the most active independently of the solvents or the tested pathogens. As shown in Table 5 The solid-medium modulation test using commercial antibiotic discs is a less complex means for synergy testing (Manga,2021) . In our study, after having observed in liquid medium (checkboard method) that most of the extracts had a synergistic effect with antibiotics on non-resistant bacteria, we undertook to perform modulation tests in solid medium to assess the extent to which the extracts from the plants tested could potentially enhance the performance of conventional antibiotics against a wide range of resistant bacteria. So, we started by determining the effectiveness of antibiotic discs alone. The sensitivity of the eleven (11) uropathogenic bacteria used in this study to eleven (11) antibiotics was determined (Table 6) Table 7) , Ceftazidime (CAZ) ( Table 8) , tetracycline (TE) ( Table 9 ), nitrofurantoin (NIT) ( Table 10 ) and trimethoprim (TR) ( Table 11) . As with the synergy tests using the checkboard method on non-resistant bacteria (Table 6 ), we found that E. chlorantha bark (ECB) had the best modulating properties on most of the antibiotics tested with a significant difference when the IFA compared to those of the other extracts High prevalence of multidrug-resistance uropathogenic Escherichia coli strains, Isfahan, Iran Urinary tract infection: pathogenicity, antibiotic resistance and development of effective vaccines against uropathogenic Escherichia coli Etiologic profile and sensitivity pattern of germs responsible for urinary tract infection among under-five children in Douala, Cameroon: a HospitalBased Study Evaluation of apparent microflora and study of antibiotic resistance of coliforms isolated from the shells of poultry eggs in Moscow-Russia Antibacterial activity of grapefruit peel extracts and green-synthesized silver nanoparticles Urinary tract infections: Virulence factors, resistance to antibiotics, and management of uropathogenic bacteria with medicinal plants-A review Alkaloid group of Cinchona officinalis: structural, synthetic, and medicinal aspects Use and importance of quina (Cinchona spp.) and ipeca (Carapichea ipecacuanha (Brot.) L. Andersson): Plants for medicinal use from the 16th century to the present The Aqueous Extract from the Stem Bark of Garcinia lucida Vesque (Clusiaceae) Exhibits Cardioprotective and Nephroprotective Effects in Adenine-Induced Chronic Kidney Disease in Rats Comparison of in vitro antioxidant properties of extracts from three plants used for medical purpose in Cameroon: Acalypha racemosa, Garcinia lucida and Hymenocardia lyrata Effect of fractioning on antibacterial activity of n-butanol fraction from Enantia chlorantha stem bark methanol extract Antibacterial and cytotoxic effects of Moringa oleifera (Moringa) and Azadirachta indica (Neem) methanolic extracts against strains of Enterococcus faecalis Screening of potential drug from Azadirachta Indica (Neem) extracts for SARS-CoV-2: An insight from molecular docking and MD-simulation studies In vitro digestibility, structural and functional properties of Moringa oleifera seed proteins Moringa oleifera and inflammation: a mini-review of its effects and mechanisms Antiinflammatory potential of ethyl acetate fraction of Moringa oleifera in downregulating the NF-κB signaling pathway in lipopolysaccharide-stimulated macrophages Antioxidant, anti-inflammatory and antiarthritic potential of Moringa oleifera Lam: An ethnomedicinal plant of Moringaceae family The anticancer activities of Vernonia amygdalina Delile. Leaves on 4T1 breast cancer cells through phosphoinositide 3-kinase (PI3K) pathway. Heliyon Anti-cancer effects of Vernonia amygdalina: A systematic review Vernonia amygdalina delile exhibits a potential for the treatment of acute promyelocytic leukemia & Emmanuel, Y. Secondary metabolite contents and antimicrobial activity of leaf extracts reveal genetic variability of Vernonia amygdalina and Vernonia calvoana morphotypes Ethanolic Extract of Ground Vernonia Amygdalina Stem Exhibited Potent Antibacterial Activity and Improved Hematological Bio-Functional Parameters in Normal and Monosodium Glutamate-Intoxicated Rats Simultaneous study of antioxidant activity, DNA protection and anti-inflammatory effect of Vernonia amygdalina leaves extracts Vernonia amygdalina leaf and antioxidant potential Optimization of extraction conditions of phenolic compounds from Cymbopogon citratus and evaluation of phenolics and aroma profiles of extract Annickia affinis and A. chlorantha (Enantia chlorantha)-a review of two closely related medicinal plants from tropical Africa Effect of preservation on two different varieties of Vernonia amygdalina Del.(bitter) leaves Galleria mellonella as a novel eco-friendly in vivo approach for the assessment of the toxicity of medicinal plants Evaluation of changes induced in the probiotic Escherichia coli M17 following recurrent exposure to antimicrobials Prolonged exposure to antimicrobials induces changes in susceptibility to antibiotics, biofilm formation and pathogenicity in staphylococcus aureus Biological and micro-biological factors: Determination of the sensitivity of microorganisms to antibacterial drugs. Federal Center for Sanitary and Epidemiological Surveillance of Ministry of Health of Russia Methanolic extracts of the rhizome of R. emodi act as bioenhancer of antibiotics against bacteria and fungi and antioxidant potential Multi dye degradation and antibacterial potential of Papaya leaf derived silver nanoparticles The antibacterial activity of aqueous and ethanolic leaf extracts of Balanites aegyptiaca (L.) Del plant on some selected clinical human pathogens Anti-fungal study on aqueous and ethanolic leaves extracts of Piper sarmentosum In vitro antimicrobial activity of Millettia laurentii De Wild and Lophira alata Banks ex CF Gaertn on selected foodborne pathogens associated to gastroenteritis Evaluation of the effect of aqueous and ethanolic extraction methods on extraction yield, phenolic compounds, and antioxidant and antimicrobial activity of Stachys schtschegleevii extract Effect of extraction solvent on qualitative and quantitative analysis of major phyto-constituents and in-vitro antioxidant activity evaluation of Cadaba rotundifolia Forssk leaf extracts Influence of extraction solvents on antioxidant and antimicrobial activities of the pulp and seed of Anisophyllea laurina R. Br. ex Sabine fruits In-vitro antimicrobial activities of organic solvent extracts obtained from Dipcadi viride (L.) Moench Antibacterial Activity of Vernonia amygdalina Leaf Extracts against Multidrug Resistant Bacterial Isolates Antimicrobial Studies of Aqueous and Ethanolic Extracts of Enantia chlorantha Leaves and Stem Bark and Their Combined Effect on Selected Bacteria and Fungi Antimicrobial Efficacy, Secondary Metabolite Constituents, Ligand Docking of Enantia chlorantha on Selected Multidrug Resistance Bacteria and Fungi Molecular basis of bacterial outer membrane permeability A repertoire of novel antibacterial diastereomeric peptides with selective cytolytic activity Can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes? In Search of National Substrates and Inhibitors of MDR pumps Antibacterial potentials of aqueous extract of Enantia chlorantha stem bark Potential of Cameroonian plants and derived products against microbial infections: a review Cameroonian medicinal plants: pharmacology and derived natural products Antimicrobial resistance and the alternative resources with special emphasis on plant-based antimicrobials-a review Review on plant antimicrobials: a mechanistic viewpoint Activit_es antibact_eriennes des huiles essentielles de trois plantes aromatiques de C^ote d'Ivoire Bio guided isolation of potential antimicrobial and antioxidant agents from the stem bark of Trilepisium madagascariense Synergy Test for Antibacterial Activity: Towards the Research for a Consensus between the Fractional Inhibitory Concentration (Checkboard Method) and the Increase (Disc Diffusion Method) Methanol extracts from Manilkara zapota with moderate antibacterial activity displayed strong antibiotic-modulating effects against multidrug-resistant phenotypes Synergistic interaction of Helichrysum pedunculatum leaf extracts with antibiotics against wound infection associated bacteria Conformational Response of 30s-Bound IF3 to A-Site Binders Streptomycin and Kanamycin Prescribing pattern for infectious diseases in tertiary care pediatric hospital. Indian journal of research in pharmacy and biotechnology & You, X. The combined antibacterial effects of sodium new houttuyfonate and berberine chloride against growing and persistent methicillin-resistant and vancomycin-intermediate Staphylococcus aureus Pharmacodynamics of nitrofurantoin at different pH levels against pathogens involved in urinary tract infections Studies on total phenolics, total flavonoids and antimicrobial activity from the leaves crude extracts of neem traditionally used for the treatment of cough and nausea & Ismayati, M. Biological activity of local plant extracts from Toba Region as insecticide Biosynthesis of silver nanoparticles using culture supernatant of shewanella sp. Ary1 and their antibacterial activity Antimicrobial resistance and molecular detection of extended-spectrum β-lactamase producing Escherichia coli isolates from raw meat in Greater Accra region A fiveyear retrospective study shows increasing rates of antimicrobial drug resistance in Cabo Verde for both Staphylococcus aureus and Escherichia coli Clinical cases, drug resistance, and virulence genes profiling in uropathogenic Escherichia coli Antibacterials and modulators of bacterial resistance from the immature cones of Chamaecyparis lawsoniana This study has been supported by the RUDN University strategic Academic Leadership Program. The author declares that they have no competing interests.