137 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers http://asrjetsjournal.org/ Antibacterial Properties of an Aqueous Extract of Solanum torvum (Solanaceae) on a Few Multidrug Resistant Bacterial Strains to Common Antibiotics N’guessan Koffi Josepha*, Toty Abalé Anatoleb, Irié Bi Jean Sévérinc, Konan Kouadio Ferniqued, Guessennd Nathaliee, Abo Kouakou Jean-Claudef a,c,fLaboratory of animal physiology, faculty of Biosciences, University of Félix Houphouët-Boigny (Abidjan, Côte d’Ivoire) b,d,eInstitut Pasteur of Côte d’Ivoire (Abidjan) aEmail: nguessanphya@gmail.com Abstract This study aimed to evaluate the antibacterial activity of the aqueous extract of Solanum torvum leaves (EASt) on multidrug-resistant bacterial strains to common antibiotics. This plant is used in traditional medicine against diarrhoea. Solid agar diffusion and liquid dilution methods were respectively used to assess the sensitivity of bacterial strains to EASt and to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Tests performed on ten (10) bacterial strains, isolated from various biological products, showed that Staphylococcus aureus 234 UB/17 and Escherichia coli 135 UB/17, were significantly sensitive to EASt, with a higher sensitivity for the latter strain. This sensitivity was shown by an inhibition of bacterial growth in solid agar and liquid medium, by a decrease in turbidity caused by EASt sensitive germs growth as the concentration of this plant extract increased (25 to 100 mg/ml). This demonstrate that EASt is an antibacterial substance. Thus, treated with EASt at a dose of 100 mg/ml, the MICs of S. aureus 234 UB/17 and E. coli 135 UB/17 were 6.25 and 12.5 mg/ml respectively, and the MBCs were 25 and 50 mg/ml respectively. The ratio of these bactericidal parameters (MBC/MIC) equaled to four (4) for these two bacterial strains, indicating that EASt is bactericidal for these bacterial strains. The antibacterial and bactericidal properties of EASt on multiple antibiotic resistant germs could justify the use of this plant in traditional medicine for the treatment of some bacterial infections causing diarrhoea. Keywords: Solanum torvum; antibacterial; bactericidal; multidrug-resistant bacteria. ----------------------------------------------------------------------- * Corresponding author. http://asrjetsjournal.org/ American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 138 1. Introduction Since their discovery a century ago, antibiotics played a key role in medical therapeutic [2].Nowadays, the upsurge and widespread of infections caused by antibiotics resistant in human populations is due to therapeutic failures. Indeed, antibiotic resistance has become a public health issue and a real concern for scientific communities, the challenge is to find new natural antibacterial agents from medicinal plants used for the treatment of infectious diseases [6]. It is the case of Solanum torvum (Solanaceae), used in traditional medicine against diarrhoea [4]. Previous studies showed that the leaf extract of this medicinal plant exhibited an antibacterial and antiviral activities [9] and can be used to relieve anite and shingles [3]. The objective of this work was to evaluate the antibacterial properties of Solanum torvum (Solanaceae). For this purpose, the effects of the aqueous leaf extract of this plant were evaluated on the in vitro growth of bacterial strains resistant to usual antibiotics. 2. Material and methods 2.1. Material 2.1.1. Plant material Fresh leaves of Solanum torvum (Solanaceae) were collected from the district of Cocody (Abidjan, Ivory Coast). Plant material was identified and authenticated by the National Floristic Center of the University of Felix Houphouet-Boigny (Abidjan, Ivory Coast). The voucher specimen number was 8377. 2.1.2. Bacterial strains For this study, seven (7) strains of enterobacteriaceae and three (3) strains of multidrug-resistant Staphylococcus aureus, provided by ASSURMI (Institut Pasteur of Côte d'Ivoire), were used (Table1). [1]) ESBL : Extended-spectrum beta-lactamases (ESBL) KT : Kanamycin-Tobromycin KTG : Kanamycin-Tobromycin-Gentamycin Meti-R : Meticillin-resistant KGNet : Kanamycin-Gentamycin-Netilmycin American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 139 Table 1: Profiles of tested bacteria Code Bacterial strain Phenotypic profiles Origin 076 CO/17 Shigella sp -Fluoroquinolone-resistant strain -Betalactamine-resistant strain Faeces 150 PI/17 Salmonella para typhi B -Phenotype KTGN -Fluoroquinolone resistant strain -Betalactamine-resistant strain Faeces 214 CA/17 Shigella flexneri -Aminioside-resistantstrain -Bêtalactamine-resistantstrain Faeces 118 UB/17 Escherichia coli - ESBL - Fluoroquinolone-resistantstrain Blood 135 UB/17 Escherichia coli ESBL Blood 146 PI/17 Escherichia coli ESBL Blood 238 UB/17 Escherichia coli - ESBL - Fluoroquinolone-resistant strain Blood 129 UB/17 Staphylococcus aureus - Betalactamase - KT Phenotype Blood 234 UB/17 Staphylococcus aureus - Phenotype KTG - Meti-R Phenotype Blood 241 UB/17 Staphylococcus aureus - KTG Phenotype - Meti-R Phenotype Blood 3. Methods 3.1. Preparation of plant extract The preparation of the aqueous extract of Solanum torvum (EASt) was carried out according to the method described by [7]. Two hundred fifty (250) g of fresh leaves of this plant were sliced into small pieces and boiled for 15 minutes in 1.5 L of distilled water. The hot decoction was filtered twice on hydrophilic cotton, then once on Wattman filter paper No. 1. Filtrate was dried in an oven at 50 °C for 72 hours. After drying, the aqueous dry American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 140 m0 m1 X 100 extract of S. torvum (EASt) was obtained. 3.2. Determination of the extraction yield of Solanum torvum (Solanaceae) The dry matter yield was determined by calculating the ratio between the weight of the dry matter and the weight of plant material used for extraction (fresh Solanum torvum leaves), according to the following formula : Yield % = m0 = Weight (g) of evaporated crude extract of S. torvum m1 = Weight (g) of fresh leaves of S. torvum 3.3. Evaluation of the antibacterial activity of plant extract 3.3.1. Preparation of concentration range of the aqueous extract of Solanum torvum The concentration range of the aqueous extract of Solanum torvum(EASt) was prepared in six test tubes numbered from 1 to 6 and one growth control tube (CT) by twofold dilution method, according to the method described by [8]. The prepared concentrations were 100; 50; 25; 25; 12.5; 6.25 and 3.12 mg/ml for EASt. However, for CT, the concentration was 0 mg/ml. 3.3.2. Inoculum preparation For the inoculum preparation, two young colonies were collected from bacterial culture using a platinum loop calibrated at 2 µl and homogenized in 10 ml of Mueller Hinton broth, incubated for 3 hours at 37 °C, to obtain a pre-culture. Then, 0.1 ml of the pre-culture broth was collected for enterobacteriaceae and 0.3 ml for Staphylococci and introduced into a tube containing 10 ml of Mueller Hinton broth. This bacterial suspension was evaluated at about 10-6 cells/ml, representing the pure inoculum (inoculum 100) [15]. 3.3.3. Inoculum count The bacterial inoculum was diluted from 1/10 to 10-4 dilution. Four (4) successive dilutions, from 10-1 to 10-4, were also obtained. The initial bacterial inoculum and the four successive dilutions are inoculated with a 2µl calibrated loop on Mueller Hinton agars with 5 cm long streaks, constituting box A [11] 3.3.4. Sensitivity test Strains sensitivity to EASt was tested by the solid agar diffusion technique. Muëller Hinton agar was swabbed American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 141 as recommended by the Antibiogram Committee of the French Society of Microbiology [1]. Wells of approximately 6 mm in diameter were made in the agar using a sterile punchand each well received 80 µl of EASt at concentrations of 100; 50; 25; 12.5; 6.25 and 3.12 mg/ml. A cup, taken as a control, received 80 µl of sterile distilled water. After 30 minutes of pre-diffusion of the EASt at laboratory temperature (26 °C), the petri dishes were incubated at 37 °C for 24 hours. The presence or absence of an inhibition zone was observed after 24 hours of incubation [11]. The interpretation was made according to the indications of [13,5]. 3.3.5. Inoculation In a series of six (6) hemolysis tubes numbered T1 to T6, and in the growth control tube (CT), 1ml of pure inoculum was introduced. Then 1 ml of EASt was added to the 6 test tubes according to the prepared concentration range (100; 50; 25; 25; 12.5; 6.25 and 3.12 mg/ml). The control tube (CT) corresponding to the control received, instead of plant extract, 1 ml of sterile Mueller Hinton broth. 3.3.6. Determination of the minimum inhibitory concentration (MIC) The MIC is the lowest concentration of the substance for which there is no growth visible to the naked eye after an incubation time of 24 h (Moroh and his colleagues 2008). Its determination is made by observation of the turbidity induced by the growth of organisms studied in each tube (T1 to T6 and CT). 3.3.7. Determination of the minimum bactericidal concentration (MBC) MBC is the lowest concentration of substance that leaves at most 0.01 % of surviving germs [12]. Using a loop calibrated at 2 μl, the contents of the tubes in which no turbidity was observed, were collected and sown on a Mueller Hinton agar starting with the tube containing the MIC, as well as the growth control. Seeding were done by parallel striations 5 cm long on the surface of the agar (Box B). After 24 hours of incubation in an oven at 37 °C, the number of colonies on the striae was compared to those in the inoculum count box (Box A). 4. Statistical analysis Data analyses and graphical representations were carried out using Graph Pad Instat and Graph Pad Prism 4 softwares (Microsoft, San Diego, California, USA). Values were expressed as mean ± standard of deviation. Data analysis was carried out using one way analysis of variance (ANOVA ONE WAY), followed by Tukey-kramer multi-comparison test and significance of difference was observed at p < 0.05. 5. Results 5.1. Yield of dry extract of Solanum torvum (Solanaceae) American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 142 19.66 g of dry aqueous extract (EASt) were obtained from 250 g of fresh leaves of S. torvum, with an extraction yield of 7.86%. 5.2. Sensitivity of bacterial strains to aqueous extract of Solanum torvum (EASt) The sensitivity tests carried out showed that EASt, at concentrations ranging from 3.12 to 100 mg/ml, has a good growth inhibitory activity on Escherichia coli 135 UB/17 and Staphylococcus aureus 234 UB/17. Indeed, compared to control, the inhibition diameters induced by EASt on E. coli 135 UB/17 at concentrations of 50 and 100 mg/ml were 14.00 ± 0.00 mm (P < 0.01) and 18.00 ± 1.16 mm (P < 0.001) respectively and, on S. aureus 234 UB/17, the inhibition diameter was 13.33 ± 0.33 mm (P < 0.01) at a concentration of 100 mg/ml. However, a low sensitivity to EASt was observed at some other concentrations for these two bacterial strains; the growth inhibition diameters were 6 ± 0.00 mm (P > 0.05). Similarly, for the eight (8) other bacterial strains tested (Shigella sp 076 CO/17, S. para typhi B 150 PI/17, S. flexneri 214 CA/17, E. coli 118 UB/17, E. coli 146 PI/17, E. coli 238 UB/17, S. aureus 129 UB/17 and S. aureus 241 UB/17), the growth inhibition diameters were all 6 ± 0.00 mm (P > 0.05)at concentrations ranging from 3.12 to 100 mg/ml (Table 2). Figure 1 shows the growth inhibition diameters of different bacterial strains at a concentration of 100 mg/ml. Table 2: Growth inhibition of bacterial strains by EASt Strains and codes Origin Extract of Solanum torvum From 3,12 to 25 mg/ml 50 mg/ml 100 mg/ml Diameter of bacterial growth inhibition zones (mm) Shigella. sp 076 CO/17 Faeces 6 ± 0,00 6 ± 0,00 6 ± 0,00 S. para typhi B 150 PI/17 Faeces 6 ± 0,00 6 ± 0,00 6 ± 0,00 S. flexneri 214 CA/17 Faeces 6 ± 0,00 6 ± 0,00 6 ± 0,00 E. coli 118 UB/17 Blood 6 ± 0,00 6 ± 0,00 6 ± 0,00 E. coli 135 UB/17 Blood 6 ± 0,00 14,00 ± 0,00** 18,00 ± 1,16*** E. coli 146 PI/17 Blood 6 ± 0,00 6 ± 0,00 6 ± 0,00 E. coli 238 UB/17 Blood 6 ± 0,00 6 ± 0,00 6 ± 0,00 S. aureus 129 UB/17 Blood 6 ± 0,00 6 ± 0,00 6 ± 0,00 S. aureus 234 UB/17 Blood 6 ± 0,00 6 ± 0,00 13,33 ± 0,33** S. aureus 241 UB/17 Blood 6 ± 0,00 6 ± 0,00 6 ± 0,00 n = 3 ** P < 0,01 *** P < 0,001compared to control American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 143 Shig ell a. s p 0 76 S. p ara ty ph i B 15 0 S. fl exn eri 21 4 E. co li 1 18 E. co li 1 35 E. co li 1 46 E. co li 2 38 S. au reu s 1 29 S. au reu s 2 34 S. au reu s 2 41 0 5 10 15 20 *** ** Bacterial strain Di am et er of ba ct er ia l g ro wt h in hi bi tio n (m m ) Figure 1: Sensitivity of bacterial strains to EASt at concentration of 100 mg/ml n = 3 ** P < 0,01 *** P < 0,001compared to control 5.3. Determination of antibacterial parameters (MBC and MIC) of the aqueous extract of Solanum torvum (EASt) For susceptible strains (E. coli 135 UB/17 and S. aureus 234 UB/17), as the concentration of the plant extract increased, a gradual decrease in the intensity of turbidity induced by bacterial growth was observed. S. aureus 234 UB/17 gave the lowest values of MIC and MBC. Indeed, at a concentration of 100 mg/ml S. aureus 234 UB/17 and E. coli 135 UB/17 respectively exhibited MICs of 6.25 and 12.5 mg/ml and CMBs of 25 and 50 mg/ml. Thus, the MBC/MIC ratios for these 2 bacterial strains sensitive to EASt were 4 (Table 3). Table 3: Antibacterial parameters and bactericidal activity of EASt Bacterial strain MIC (mg/ml) MBC (mg/ml) MBC/MIC Activity of EASt Escherichia coli135 UB/17 12,5 50 4 Bactericidal Staphylococcus aureus234 UB/17 6,25 25 4 Bactericidal American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 144 6. Discussion The choice of Solanum torvum (fresh leaves) and the extraction method (decoction) was based on testimonies collected from the population and traditional therapists in Abidjan (Côte d'Ivoire). The extraction yield of S. torvum is similar to that of Justicia secunda (Acanthaceae) [7]. According to the results, this extraction method is satisfactory for the detection of the active ingredients of Solanum torvum and justifies the traditional use of the plant. This study focuses on the antibacterial activity of the aqueous extract of Solanum torvum (EASt) on the in vitro growth of multi-resistant bacterial strains to common antibiotics. The sensitivity test of bacterial strains showed that Escherichia coli 135 UB/17 and Staphylococcus aureus 234 UB/17 were significantly sensitive to EASt. These two bacterial strains growth were significantly inhibited by EASt, their inhibition diameter zones were greater than 10 mm. On the one hand, the growth of eight (8) other bacterial strains studied has an insignificant inhibition diameter zone of 6 mm when tested by EASt, so these 8 bacteria were resistants to EASt. Indeed, according to [13, 5], an extract has an antibacterial activity if its inhibition diameter is greater than 10 mm. On the other hand, when the inhibition diameter is less than 10 mm, the bacterium is said to be resistant. In addition, for the two susceptible bacterial strains (E. coli 135 UB/17 and S. aureus 234 UB/17), compared to the growth control, as the concentration of EASt increased, a decrease in turbidity caused by the growth of EASt susceptible organisms was observed. Inhibition of bacterial growth justifies theantibacterial activity of the aqueous extract of S. torvum. This helped to determine the different antibacterial parameters for susceptible strains, such as MIC and MBC. At a concentration of 100 mg/ml, the lowest values of antibacterial parameters were obtained with the methicillin-resistant (Meti-R) strain of S. aureus 234UB/17, with a MIC of 6.25 mg/ml and a MBC of 25 mg/ml. The highest values were obtained with the extended-spectrum beta-lactamases (ESBL) producing E. coli 135 UB/17 strain, with a MIC of 12.5 mg/ml and a MBC of 50 mg/ml. Thus, for EASt, S. aureus strain was the most sensitive, while E. coli strain was the least sensitive. Comparison of antibacterial properties of this extract with other anti-infective plants showed that the aqueous extract of S. torvum was twice as active on E. coli as the aqueous extract of Harungana madagascariensis (Hypericaceae) [8]. and the aqueous extract of Chromolaena odorata (Asteraceae) [10]. According to these authors, both extracts inhibited the growth of E. coliin vitro with CMBs at concentrations of 100 mg/ml and 117.50 mg/ml respectively, while the CMB for EASt was at a concentration of 50 mg/ml. The CMB/CMI ratios for both bacterial strains susceptible to EASt were four (4). According to [15], when the CMB/CMI ratio value is less than or equal to 4, the activity of the extract studied is qualified as bactericidal. Thus, EASt is a bactericidal substance for S. aureus 234 UB/17 and E. coli 135 UB/17. The antibacterial and bactericidal properties of the aqueous extract of Solanum torvum on multidrug-resistant bacterial strains to common antibiotics justify the use of this plant in traditional medicine for the treatment of some bacterial infections and specifically against diarrhea of bacterial origin. Acknowledgments We are grateful to the Ivorian National Floristic Center (University of Félix Houphouët-Boigny Abidjan, Côte American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2019) Volume 54, No 1, pp 137-146 145 d'Ivoire), for plant authentification, and to Institut Pasteur of Côte d'Ivoire, for providing equipment and technical support for this study. References [1] CASFM/EUCAST, 2017. Comité de l’Antibiogramme de la Société Française de Microbiologie (Recommandation 2017), Edition Mars 2017, Paris (France), 128 p. [2] Coustès T., 2016. Loi d’avenir agricole, règlementation du médicament vétérinaire et lutte contre l’antibiorésistance. Thèse de Doctorat de Vétérinaire, Ecole Nationale Vétérinaire d’Alfort, Faculté de Médecine de Créteil (France), 106 p. [3] Nga E. N., Pouka C. K., Ngo B. G. P. C., Dibong S. D., Mpondo M. E., 2016. 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