Vol. 10(31), pp. 1229-1239, 21 August, 2016 DOI: 10.5897/AJMR2016.8186 Article Number: 934391E60068 ISSN 1996-0808 Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR African Journal of Microbiology Research Full Length Research Paper Antimicrobial activity of extracts from Crotalaria bernieri Baill. (Fabaceae) Herizo Lalaina Andriamampianina1, Danielle Aurore Doll Rakoto1, Thomas Petit3,4, Heriniaina Ramanankierana2, Hanitra Ranjana Randrianarivo1 and Victor Louis Jeannoda1* 1 Laboratory of Applied Biochemistry to Medical Sciences, Fundamental and Applied Biochemistry Department, Faculty of Sciences, University of Antananarivo, Antananarivo, Madagascar. 2 Centre National de la Recherche pour l’Environnement (CNRE), Antananarivo, Madagascar. 3 Laboratoire de Chimie des Substances Naturelles et Sciences des Aliments (LCSNSA), Saint Pierre, La Réunion, France. 4 UMR Qualisud, IUT de La Réunion, Saint Pierre, La Réunion, France. Received 27 June, 2016; Accepted 25 July, 2016 This work was designed to study the antimicrobial activity of Crotalaria bernieri Baill. (Fabaceae). Extracts from leaf, root, pod and seed using hexane, ethyl acetate and methanol were tested in vitro for their activity against 17 bacteria, 5 fungi (3 yeasts and 2 molds) using disc diffusion and micro dilution methods. At the concentration of 1 mg/disc, all the extracts exhibited antimicrobial activity depending on the plant part and the extraction method used. The most sensitive germs were Salmonella enteridis, Streptococcus pyogenes and Candida guilliermondii with inhibition zone diameter (IZD) of 11 mm, 15 mm and 13 mm respectively. Most of extracts showed, broad activity spectrum varying from one extract to another. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of all extracts were recorded. Ten extracts displayed an excellent effect (MIC < 100 µg/ml), 8 a moderate effect (MIC from 100 to 500 µg/ml), 5 a weak effect (MIC from 500 to 1000 µg/ml) and the others were ineffective (MIC > 1000 µg/ml). Leaf methanol extracts were the most efficient and Gram positive bacteria the most sensitive. All extracts had bactericidal (MBC/MIC ≤ 4) or fungicidal action (MFC/MIC ≤ 4) in certain microorganisms and bacteriostatic (MBC/MIC > 4) or fungistatic action (MFC/MIC > 4) in others. Antimicrobial activity might be due to tannins, polyphenols, steroids, triterpenes and flavonoids that were present in most of the plant organs, but alkaloids in leaf and pod and saponosides in root might also be involved. C. bernieri with the effectiveness of all its parts, the variety of its secondary metabolites, the great number of sensitive pathogen microorganisms and its ubiquity make this plant species an interesting source of antimicrobial agents. Key words: Crotalaria bernieri, antimicrobial activity, disc diffusion method, microdilution method, minimum inhibitory concentration, minimum bactericidal concentration, minimum fungicidal concentration. INTRODUCTION Antimicrobial resistance is one of the world’s most serious public health problems. There is an urgent need *Corresponding author. E-mail: victor_jeannoda@yahoo.fr. Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 1230 Afr. J. Microbiol. Res b a c Figure 1. Crotalaria bernieri (a) the whole plant; (b) flower; (c) fruits (Source: the authors). to find new disposable and affordable remedies to face this problem (Zongo et al., 2011). Many studies led to systematic screening of plant extracts as a source of antibacterial compounds (Dalmarco et al., 2010; Stefanovic and Comic, 2011). Several Crotalaria species have been reported to display antimicrobial properties. For example, Crotalaria madurensis is active against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Candida albicans (Bhakshu et al., 2008), Crotalaria capensis against Salmonella typhimurium (Dzoyem et al., 2014), Crotalaria burhia against B. subtilis and S. aureus (Sandeep et al., 2010; Mansoor et al., 2011), Crotalaria juncea against S. aureus (Chouhan and Sushil, 2010), Crotalaria pallida against E. coli and Pseudomonas sp (Pelegrini et al., 2009), and Cladophora trichotoma against Alternaria solani (Ravikumar and Rajkumar, 2013). The purpose of this study was to assess the antimicrobial activity of C. bernieri by testing plant part’s extracts obtained in different methods on pathogen bacteria and molds. C. bernieri is one of the 53 Crotalaria species growing in Madagascar, an annual herb which is found in open vegetation, grassy places and roadsides in most regions of Madagascar (Peltier, 1959). It flowers on July to October and December to March (Polhill, 1982; Dupuy et al., 2002). MATERIALS AND METHODS Plants C. bernieri (Figure 1) were harvested in Ibity, District of Antsirabe, Region of Vakinankaratra, 200 km from Antananarivo region. The plant was collected in April and July, 2013 and was identified by Polhill R.M. Voucher specimens (Herizo R. 010) of C. bernieri were deposited in the herbarium of Plant Biology and Ecology Department of the Faculty of Sciences of the University of Antananarivo. Microorganisms strains The microorganisms used in this study consisted of 17 strains of bacteria (10 Gram (-) and 7 Gram (+)), 3 yeasts and 2 molds (Table 1). These strains were obtained from the collections of Laboratoire de Chimie des Substances Naturelles et Sciences des aliments (LCSNSA) of La Réunion University. They were maintained on agar slant at 4°C and cultured on a fresh appropriate agar plate during 24 h prior to antimicrobial tests. Chemicals for antimicrobial assay Commonly used pre-impregnated discs, from Bio-Rad F 92430 Marnes-la-Coquette were chosen as antimicrobial references Andriamampianina et al. 1231 Table 1. Bacterial, yeast and mold strains used to study antimicrobial activities. Microorganisms Strains Campylobacter jejuni Enterobacter aerogenes Enterobacter cloacae Escherichia coli Pseudomonas aeruginosa Salmonella enteridis Shigella flexneri Vibrio parahaemolyticus Yersinia enterocolitica Proteus mirabilis References ATCC 29428 ATCC 13048 ATCC 13047 ATCC 25922 ATCC 10145 ATCC 13076 ATCC 12022 ATCC 17802 ATCC 23715 ATCC 35659 Gram(+) Bacillus cereus Clostridium perfringens Enterococcus faecalis Listeria monocytogenes Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes ATCC 14579 ATCC 13124 ATCC 29121 ATCC 19114 ATCC25923 ATCC 6305 ATCC 19615 Yeasts Candida albicans Candida guilliermondii Candida krusei ATCC 10231 ATCC 6260 ATCC 14243 Molds Aspergillus fumigatus Aspergillus niger ATCC 204305 ATCC 16888 Gram(-) Bacteria Fungi Table 2. Abbreviations designating the different extracts tested. Extracts Leaves Seeds Pods Roots Hexane Extract LHE SHE PHE RHE Ethyl acetate Extract LEA SEA PEA REA Methanol Extract LME SME PME RME hexane, ethyl acetate and sterile distilled water, constituted hexane, ethyl acetate and methanol extracts respectively (Table 2). Phytochemical screening The reactions for the detection of chemical groups were those developed by Fong et al. (1977) and Marini-Bettolo et al. (1981). Antimicrobial assays (Camara et al.,2013; Rakholiya et al., 2014): amoxicillin 25 µg, chloramphenicol 30 µg, penicillin 6 µg as antibiotics and miconazole 50 μg as antifungal. Preparation of extracts The dried leaves, seeds, seed pods, and roots of the plant were grounded into powder. The resulting powder (100 g) was extracted successively with 4x500 mL of hexane, ethyl acetate and methanol for 24 h under stirring at room temperature. After filtration using a Whatman filter paper, each combined extract was evaporated under reduced pressure to dryness. The dry residues, dissolved in Antimicrobial activity test The in vitro antimicrobial activity of the extracts was determined using disc diffusion method described by Pyun and Shin (2006) and Ngameni et al. (2009). Two mL of inoculum corresponding to 0.5 MacFarland (108 CFU/ml) was uniformly spread on the surface of Columbia Agar medium (for Streptococcus); Mueller-Hinton Agar (MHA) for the other bacteria and Potato Dextrose Agar (PDA) for yeasts. Sterilized filter paper discs 6 mm diameter (BioMérieux, REF 54991) were impregnated with 10 μL of each extract to the concentration of 100 mg/mL (1 mg/disc). The soaked discs were then placed on the surface of the agar and incubated at 37°C during 24 h for bacteria, or at 25°C for yeasts. The inhibition zone diameter (IZD) was measured and the results were interpreted by 1232 Afr. J. Microbiol. Res Table 3. Extraction yields of C. bernieri extracts. Extracts Leaf LHE LEA LME Yield % 14.6 22.5 12.0 Seed SHE SEA SME 18.4 12.1 10.0 Pod PHE PEA PME 15.1 11.2 4.2 Root RHE REA RME 13.7 15.3 24.1 MBC and MFC, 5 μl from each well that showed no change in color was transferred on MHA or PDA plate and incubated at 25°C (yeasts and molds) or at 37°C (bacteria) for 24 h. The lowest concentration at which no growth occurred on the agar plates corresponded to the MBC or MFC. The ratios MBC/MIC and MFC/MIC were calculated for each extract, to determine the nature of the effect. The extract is bactericidal or fungicidal for MBC/MIC or MFC/MIC ≤ 4 and bacteriostatic or fungistatic when these ratios are >4 (Djeussi et al., 2013; Bouharb et al., 2014; Chamandi et al., 2015). Statistical analyses Results were expressed as mean values ± standard deviations of three separate determinations. One-way analysis of variance (ANOVA) which was followed by Newman Keuls comparison test with Staticf® software was used for statistical analysis. Statistical estimates were made at confidence interval of 95%. RESULTS Extraction yields means of the scale used by Ponce et al. (2003) and Celikel and Kavas, (2008) stating that bacteria are not sensitive for IZD less than 8 mm, sensitive for IZD of 9 to 14 mm, very sensitive for IZD of 15 to 19 mm and extremely sensitive for IZD larger than 20 mm. Antifungal activity was evaluated by a method described by Favel et al. (1994). One ml of each extract was added to 19 ml of medium culture of PDA and maintained at 45°C. The mixture is then poured into Petri dishes and dried for 15 min at 37°C. 10 µl of each tested microorganism corresponding to 0.5 MacFarland were spread on the medium surface. IZD were measured after incubation at 25°C for 72 h. Negative controls were prepared by using the same solvents employed to dissolve the plant extract samples while the reference antibiotics were used as positive controls. All the experiments were performed in triplicate. The results were expressed as mean values ± standard deviations (mm ± SD). The extractive yield of different parts of C. bernieri with different solvents varied from 4.2 (PME) to 24.1% (RME) (Table 3). Qualitative phytochemical analysis The major secondary metabolites identified in the different organ extracts are presented in Table 4. Tannins, polyphenols, steroids, triterpenes and unsaturated sterols occurred in all the C. bernieri organs. Flavonoids were found in all organs except root. Alkaloids were present only in leaf and pod while saponins only in root. Iridoïds, leucoanthocyanins, and quinones were not detected in all parts of C. bernieri. MIC, MBC and MFC determination Antimicrobial activity For extracts showing antibacterial activity in the disc diffusion method (IZD ≥ 8 mm), MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration) MFC (minimum fungicidal concentration) were determined by microdilution method (Kuete et al., 2009). The concentration of each extract was adjusted to 25 mg/ml. This was serially diluted two-fold to obtain concentration ranges of 0.024 to 25 mg/ml. Each concentration was added in a well (96-well microplate) containing 95 μl of Mueller-Hinton broth (MHB) and 5 μl of inoculum (standardized at 0.5 MacFarland). A positive control containing bacterial culture without the extract and a negative control containing only the medium, were also analyzed. The plates were covered with sterilized aluminum foil, and then incubated at 25°C (yeasts and molds) or at 37°C (bacteria) for 24 h. The MIC of each extract was detected following addition 40 µl of 0.2 mg/ml p-iodonitrotetrazolium chloride and incubation at 25°C (yeasts and molds) or at 37°C (bacteria) for 30 min (Kuete et al., 2009). Viable bacteria reduced the yellow dye to a pink color. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth. For the determination of At 1 mg/disc, a concentration generally used for antimicrobial activity assessment in plants (Sandeep et al., 2010; Govindappa et al., 2011; Linthoingambi and Mutum, 2013; Marimuthu et al., 2014), the large majority of C. bernieri extracts (16 of 22) inhibited the microorganism growth with IZD ranging from 8 to 15 mm (Tables 5 to 7). However, activity depended on the microorganism, the plant parts and extraction method used. The most sensitive germs were S. enteridis (IZD=11 mm), S. pyogenes (IZD=15 mm) and C. guilliermondii (IZD=13 mm) in Gram (-) bacteria, Gram (+) bacteria and yeasts, respectively. Gram (-) strains C. jejuni and E. coli, E. faecalis, Gram (+) L. monocytogenes and the two molds A. fumigatus and A. niger were resistant to all the extracts. REA, with an IZD of 15 mm against S. pyogenes, displayed the highest antibacterial activity. Andriamampianina et al. 1233 Table 4. Phytochemical screening of C. bernieri extracts. Chemical groups Tests Alkaloids Mayer Wagner Dragendorff Confirmatory test (solubility in ethanol) a - Leaf b - c + + + d - Seed e - f - g - Pod h - i + + + j - Root k - - - + - - - - - + - - - l - Foam test Confirmatory test (hemolytic test) - - - - - - - - - - - + Saponins - - - - - - - - - - - + Flavonoids Leucoanthocyanins Willstätter Bate-Smith - + - + - - + - + - - - + - - - - Tannins and Polyphenols Gelatin 1% Gelatin-salt 10% FeCl3 + + - + + + + + - + + - + + + - + - + + + - + + - + + + Quinones Steroids Iridoïds Triterpenes Unsaturated sterols Borntrager Liebermann-Burchard Hot HCl Liebermann-Burchard Salkowsky + + + + - - + + + + + + + + + + + + + + + - + + + + + + - +: positive result; -: negative result a: LHE; b: LEA; c: LME; d: SHE; e: SEA; f: SME; g: PHE; h: PEA; i: PME; j: RHE; k: REA; l: RME. In yeasts, most of leaf extracts were active against the three Candida strains tested, but seed and pod extracts were active only against C. guilliermondii. Antibiotics used as references in this study (amoxicillin 25 µg, chloramphenicol 30 µg, penicillin 6 µg and miconazole 50 µg) were more effective than most of C. bernieri extracts. MIC, MBC, MFC and MBC or MFC/MIC ratio values are presented in Tables 8 to 10. MIC ranged from 0.048 to 25 mg/ml. MIC maximum values registered was 12.5 mg/mL except for RHE on S. pyogenes (MIC=25 mg/ml). Concerning MBC or MFC, maximum values for all extracts were 25 mg/ml except for root extracts on some Gram (+) bacteria and C. guilliermondii (MBC>25 mg/ml). The ratio MBC or MFC/MIC varied from 1 to more than 100. The most sensitive microorganism were P. mirabilis in Gram (-) bacteria (MIC=MBC=0.097 mg/ml), B. cereus (MIC=0.048 mg/ml, MBC=0.195 mg/ml) and S. pyogenes (MIC=MBC=0.048 mg/ml) in Gram (+) bacteria and C. guilliermondii (MIC=MFC=0.048 mg/ml) in yeasts. All methanol extracts were active. This is also the case for ethyl acetate extracts except LEA. As to hexane extracts, PHE and RHE were efficient but not LHE and SHE. Pod extracts had the broadest spectrum of activity with 10 sensitive microorganisms and seed extracts the narrowest ones with 8 sensitive microorganisms. DISCUSSION The present study shows that the C. bernieri extracts inhibited the growth of most tested microorganisms, indicating the presence of antimicrobial compounds in all parts of the plant. Phytochemical screening showed the presence of diverse secondary metabolites, reported to have antimicrobial property. At this stage of the work, results did not yet allow to state whether the same or different compounds are involved in the different parts of the plant. However, they suggested that C. bernieri antimicrobial activity might be mainly due to tannins, polyphenols, steroids, triterpenes and flavonoids, which were present in all or most of the plant organs. Alkaloids might also be concerned in leaves and pods and saponosides in root. C. bernieri extracts showed generally a broad antimicrobial spectrum. They were capable of inhibiting the growth of different Gram (-) and Gram (+) bacterial strains as well as some yeasts. However, each extract 1234 Afr. J. Microbiol. Res Table 5. In vitro Antimicrobial Activity (IZD in mm) of extracts (1 mg/disc) on Gram (-) bacteria. Extracts/ controls Cj Ea Ec Esc Pa Se Sf Vp Ye Pm Leaf LHE LEA LME - 10.00±0.01 - - 9.33±0.47 - - 7.00±0.01 - 8.33±0.47 Seed SHE SEA SME - - 10.00±0.01 - - - - - 7.00±0.01 9.00±0.01 - 8.00±0.01 8.00±0.01 - Pod PHE PEA PME - 9.67±0.47 - 9.00±0.01 - - - 11.00±0.82 8.00±0.01 10.67±0.01 9.00±0.82 8.00±0.01 8.00±0.01 Root RHE REA RME - - - - 10±0.01 - 8.00±0.01 9.00±0.01 - 9.00±0.01 - - 9.00±1.41 PC Amx Chl Pen 45.00 38.00 40.00 25.00 - 25.00 - 23.00 30.00 - 10.00 15.00 - 27.00 32.00 - 25.00 30.00 - - 10.00 38.00 - 25.00 NC Hex EtOAc Sdw - - - - - - - - - - Cj: C. jejuni; Ea: E. aerogenes; Ec: E. cloacae; Esc: E. coli; Pa: P. aeruginosa; Se: S. enteridis; Sf: S. flexneri; Vp: V. parahaemolyticus; Ye: Y. enterocolitica; Pm: P. mirabilis PC: Positive control (Amx: Amoxicillin 25µg; Chlor: Chloramphenicol 30µg; Pen: Penicillin 6µg); NC: Negative control (Hex: Hexane; EtOAc: Ethyl acetate; Sdw: sterile distilled water); −: No activity. displayed a specific activity spectrum that could be due to difference between the chemical nature and concentration of bioactive compounds in extracts. The results obtained with microdilution method were more reliable than those with disc diffusion. That might be due to the fact that bioactive compounds were in direct contact with germs in liquid medium whereas they diffused little or not at all in solid medium. There was no consensus on the acceptable level of inhibition for natural products (Benko and Crovella, 2010). For Dalmarco et al. (2010), for crude extracts and fractions, a MIC lower than 100 µg/mL was considered as an excellent effect, from 100 to 500 µg/ml as moderate, from 500 to 1000 µg/mL as weak, and over 1000 µg/ml as inactive. According to Kouitcheu et al. (2013), when a crude extract was used, the MIC values of 8 mg/mL or below against any microorganism tested was considered as active. If the scale adopted by Dalmarco et al. (2010) was used as a reference, 10 extracts displayed an excellent effect, 8 a moderate effect, 5 a weak effect then the remaining extracts were inactive. Excellent effects were observed on P. mirabilis (RME), S. enteridis (PME), B. cereus (LME, PEA, REA), S. pneumoniae (LME), S. pyogenes (REA), C. albicans (LME) and C. guilliermondii (LME, SEA). Moderate effects, were found against E. aerogenes (SEA), P. mirabilis (LME), P. aeruginosa (LME), C. perfringens (LME), S. aureus (LME), S. pyogenes (RME, PEA, LME) and C. guilliermondii (SME). Weak effects were observed on E. aerogenes (SEA), P. aeruginosa (LME), S. aureus (REA) and S. pneumoniae (REA, RME). The most efficient extracts were RME (MIC=MBC= 0.097 mg/ml) against Y. enterolytica in Gram (-) bacteria, REA (MIC=MBC=0.048 mg/ml) against S. pyogenes in Gram (+) bacteria and LME (MIC=MFC=0.048 mg/ml) against C. guilliermondii. Some of the extracts were very effective against some organisms (LME against B. cereus, S. pneumoniae, C. albicans and C. guilliermondii, REA against B. cereus and S. pyogenes) while others were totally inactive (SME against S. pneumoniae and S. pyogenes). However, if the interpretation of Kouitcheu et al., (2013) was taken into account, only nine extracts had MIC higher than 8 mg/mL on some germs, which means that all the other extracts of C. bernieri used showed Andriamampianina et al. 1235 Table 6. In vitro Antimicrobial Activity (inhibition zone diameter in mm) of extracts (1 mg/disc) on Gram (+) bacteria. Plant parts/controls Extracts Leaf Bc Cp Ef Lm Sa Spn Spy LHE LEA LME 9.00±0.01 8.00±0.01 - - 7.00±0.01 10.00±0.01 12.67±1.25 12.33±1.70 Seed SHE SEA SME - - - - 7.00±0.01 9.00±0.82 - 11.33±0.47 8.33±1.25 9.00±0.01 8.00±0.01 Pod PHE PEA PME 11.33±0.47 - 9.67±1.25 8.33±0.47 - - - 7.00±0.01 10.33±0.47 7.00±0.01 13.00±0.82 8.00±0.01 12.00±1.41 7.00±0.01 Root RHE REA RME 11.33±0.94 7.00±0.01 9.00±1.41 10.00±0.01 - - 11.00±0.01 8.00±0.01 13.00±0.82 13.00±0.83 8.00±0.82 15.00±0.01 12.00±1.41 PC Amx Chl Pen NC Hex EtOAc Sdw 15.00 38.00 15.00 27.00 30.00 - - 30.00 - 37.00 30.00 35.00 26.00 25.00 23.00 32.00 22.00 25.00 - - - - - - - Bc: B. cereus; Cp: C. perfringens; Ef: E. faecalis; Lm: L. monocytogenes; Sa: S. aureus; Spn: S. pneumoniae; Spy: S. pyogenes Amx: Amoxicillin 25 µg; PC: Positive control (Amx: Amoxicillin 25 µg; Chlor: Chloramphenicol 30 µg; Pen: Penicillin 6 µg; NC: Negative control (Hex: Hexane; EtOAc: Ethyl acetate; Sdw: sterile distilled water); −: No activity. Table 7. In vitro Antimicrobial Activity (inhibition zone diameter in mm) of extracts (1mg/disc) on yeasts and molds Leaf LHE LEA LME Ca 8.00±0.01 Yeast Cg 7.00±0.01 7.00±0.01 8,67± 0.94 Ck 7.00±0.01 7.00±0.01 Af - An - Seed SHE SEA SME - 7.00±0.01 13.00±0.82 11.00±0.01 - - - Pod PHE PEA PME - 7.00±0.01 7.00±0.01 7.00±0.01 - - - RHE REA RME Mic 18.00 8.00±0.01 7.00±0.01 8.00±0.82 30.00 33.00 28.00 23.00 - - - - - Plant part Extract Root PC NC Hex EtOAc Sdw Mold Ca: C. albicans; Cg: C. guilliermondii; Ck: C. krusei; Af: A. fumigatus; An: A. niger PC: Positive control (Mic: Miconazole 50µg); NC: Negative control (Hex: Hexane; EtOAc: Ethyl acetate; Sdw: sterile distilled water); −: No activity. 1236 Afr. J. Microbiol. Res Table 8. MIC and MBC values (mg/mL) of C. bernieri extracts (1mg/disc) on Gram(-) bacteria Gram(-) Bacteria Enterobacter aerogenes Extracts LME MIC (mg/ml) 0.195 MBC (mg/ml) 25 MBC/MIC 128.21 Enterobacter cloacae SEA PEA 0.781 6.25 25 25 32.01 4.00 Pseudomonas aeruginosa LME RHE 0.781 0.195 25 25 32.01 128.21 Salmonella enteridis PME RHE RME 0.097 12.5 12.5 12.5 12.5 25 128.87 1.00 2.00 Shigella flexneri PME 3.125 25 8.00 Vibrio parahaemolyticus SEA PEA PME REA 1.562 1.562 3.125 1.562 6.25 25 6.25 1.562 4.00 16.01 2.00 1.00 Yersinia enterolitica SME PME 1.562 1.562 12.5 12.5 8.00 8.00 Proteus mirabilis LME SEA PME RME 0.195 1.562 1.562 0.097 0.781 3.125 0.781 0.097 4.01 2.00 0.50 1.00 antimicrobial activities. All the extracts had bactericidal action (MBC/MIC ≤ 4) in certain bacteria and bacteriostatic action (MBC/MIC >) 4) in other ones. For example LME was bactericidal against B. cereus and C. perfringens but bacteriostatic against S. aureus and S. pneumoniae. The comparison of A. bernieri extract activities to foreign Crotalaria species was not easy because antimicrobial activity was assessed under different conditions (other microorganism strains and extract doses used). Compared to available data, the IZD of C. bernieri extracts were generally of the same order of magnitude as those of leaf ethyl acetate extract from C. madurensis against B. subtilis and S. aureus (IZD=14 mm), M. luteus (IZD=12 mm), E. coli and C. albicans (IZD=10 mm (Bhakshu et al., 2008) and leaf ethanol extract from C. pallida against X. axanopodis (IZD=16 mm), E. coli(IZD=14 mm) and C. michiganensis (IZD=13 mm) (Govindappa et al., 2011). Root methanol extract from C. burhia was more efficient with an IZD of 18 mm against B. subtilis and P. aeruginosa (Sandeep et al., 2010). If comparison was based on antimicrobial indexes, LME (MIC=0.781 mg/ml, MBC=25 mg/ml) and REA (MIC=0.195 mg/ml, MBC=25 mg/ml) were more efficient against P. aeruginosa than the leaf methanol extract from C. quartiniana (MIC=MBC=37.5 mg/ml) (Omori et al., 2011). The leaf hexane extract from C. retusa (MIC=0.125 mg/ml, MBC=37.5 mg/ml) (Maregesi et al., 2008) was less active against B. cereus than LME (MIC=0.097 mg/ml, MBC=0.195 mg/ml), PEA and REA (MIC=0.048 mg/ml, MBC=0.195 mg/ml). By contrast, C. bernieri extracts were less active on P. mirabilis (MIC between 0.097 and 1.56 mg/ml) than a peptide isolated from C. pallida seeds (MIC=0.030 mg/ml) (Pelegrini et al., 2009). Compared to the antibacterial activities from other plant extracts, several C. bernieri extracts were more efficient than methanolic aerial part extracts of Inula viscosa against B. subtilis (MIC=25 mg/ml, MBC=50 mg/ml) and S. aureus (MIC=12.5 mg/ml, MBC=50 mg/ml) (Larbi et al., 2016). By contrast, tuber ethyl acetate extract of Tropaeolum pentaphyllum against E. coli (MIC=0.02 mg/ml, MBC=0.64 mg/ml), P. aeruginosa (MIC=0.04 mg/ml, MBC=0.64 mg/ml) (da Cruz et al., 2016) and organic extract (aerial parts) of Rapanea parvifolia against E. faecalis (MIC=0.03 mg/ml, MBC=0.06 mg/ml) (Suffredini et al., 2006) were more efficient. Andriamampianina et al. 1237 Table 9. MIC and MBC values (mg/ml) of C. bernieri extracts (1mg/disc) on Gram(+) bacteria. Gram (+) Bacteria MIC (mg/ml) 0.097 0.048 0.048 1.562 MBC (mg/mL) 0.195 0.195 0.195 1.562 MBC/MIC 2.01 4.06 4.06 1.00 Clostridium perfringens LME PHE PEA RME 0.195 12.5 6.25 6.25 0.390 25 12.5 ˃25 2.00 2.00 2.00 - Staphylococcus aureus LME SEA PEA REA RME 0.195 6.25 3.125 0.781 12.5 6.25 12.5 25 25 25 32.05 2.00 8.00 32.01 2.00 LME SEA SME Streptococcus pneumoniae PME REA RME 0.097 3.125 12.5 1.562 0.781 0.781 3.125 12.5 25 6.25 ˃25 ˃25 32.22 4.00 2.00 4.00 - LME SEA SME PEA RHE REA RME 0.195 1.562 12.5 0.195 25 0.048 0.195 1.562 12.5 25 0.781 ˃25 0.048 12.5 8.01 8.00 2.00 4.01 1.00 64.10 Bacillus cereus Streptococcus pyogenes Extract LME PEA REA RME Table 10. MIC and MBC values (mg/ml) of C. bernieri extracts (1mg/disc) on yeasts. Yeasts Candida albicans Extraits MIC (mg/ml) MFC (mg/ml) MFC/MIC LME 0.097 0.195 2.01 LME SEA Candida guilliermondii SME RHE RME On fungi, LME (MIC=0.097 mg/ml, MFC=0.195 mg/ml) was more efficient than leaf methanolic extract of Myrtus nivellei against C. albicans (MIC=4.5 mg/ml) (Touaibia and Chaouch, 2015) whereas LME, SEA and SME against C. guilliermondii (MIC=0.08 mg/ml, MFC=0.32 mg/ml) were less efficient than ethyl acetate extract of T. 0.048 0.048 0.195 12.5 12.5 0.048 1.562 25 ˃25 ˃25 1.00 32.54 128.21 - pentaphyllum (da Cruz et al., 2016). Conclusion This study clearly demonstrates the potential of C. bernieri 1238 Afr. J. Microbiol. Res as a source of interesting natural wide spectrum antimicrobial molecules. All its parts were efficient and could be easily found in significant amounts for the plant grows in fields, in the vicinity of homes, on roadsides and can be cultivated. 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