Antibacterial Activities of Five Medicinal Plants in Ethiopia against Some Human and Animal Pathogens

Objective To evaluate the in vitro antibacterial activities of five plant extracts which have been used as traditional medicines by local healers against three multidrug resistant bacteria, namely, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Results The highest mean zone of inhibition (4.66 mm) was recorded from methanol extract of Calpurnia aurea (Ait.) Benth. at a concentration of 200 mg/ml against S. aureus, followed by Croton macrostachyus Del. (4.43 mm) at the same dose and solvent for the same bacterial species, while methanol and chloroform extracts of E. brucei Schwein. did not inhibit growth of any bacterial species. The lowest value (100 μg/ml) of minimum inhibitory concentration (MIC) was observed from both methanol and chloroform extracts of C. aurea (Ait.) Benth. against all the three bacteria. The results of the positive control had no statistically significant difference (P > 0.05) when compared with crude extracts of C. aurea (Ait.) Benth. at concentration of 200 mg/ml against S. aureus. Conclusion The results of the present study support the traditional uses of these medicinal plants by the local healers. Except Erythrina brucei Schwein., all the plants investigated in this study exhibited antibacterial activities against the test bacterial species. Further researches are needed to be conducted to evaluate efficacy of these medicinal plant species on other microbes in different agroecological settings and their safety levels as well as their phytochemical compositions.


Introduction
Although plenty of guidelines have been developed and set for application to prevent foodborne diseases and food spoiling microorganisms, it is impossible totally to be free of them. S. aureus and E. coli are among the major causes of foodborne diseases [1,2]. Species of Pseudomonas are also predominant causes of food spoilage [3,4]. The emergence of drug resistance to multiple antibiotics worsens the problem. Nowadays, reports have indicated that Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) are not only multidrug resistant pathogens [5][6][7] but also broadly drug-resistant and pandrug-resistant bacteria [8]. For instance, it has been reported worldwide that S. aureus isolates are increasingly resistant to a greater number of antimicrobial agents as reviewed in Lowy [5]. Multidrug resistance in E. coli increased from 7.2% during the 1950s to 63.6% during the 2000s, and particularly considerable growing trend in resistance was noticed for ampicillin, sulfonamide, and tetracycline [6]. P. aeruginosa has also showed resistance to the most frequently administered antipseudomonal antibiotics: -lactams, aminoglycosides, and fluoroquinolones [7]. S. aureus, E. coli, and P. aeruginosa are also responsible for the majority of nosocomial infections [9]. Antibiotics that show low efficacy in treating human and animal diseases through antibiotic resistance must be replaced with new drugs to combat the burden of these pathogens [10]. Hence, medicinal plants are expected to be the best source of obtaining a variety of drugs [11,12]. In Ethiopia, different communities have extensively been using medicinal plants to treat different diseases for many centuries [13][14][15]. Such plants, however, need to be investigated for better understanding of their properties, safety, and efficiency [16]. Medicinal plants including C. aurea (Ait.) Benth., C. macrostachyus Del., and Withania somnifera (W. somnifera) (L.) Dunal have been tested for their antimicrobial efficacy in vitro and in vivo in different countries [17][18][19][20][21][22][23][24][25]. Previous information of the current study plants is presented in Table 1. However, testing the efficacy of these plants in different agroecological zones can make the evidence more strong since agroecology (e.g., geographic location and soil type) could affect the accumulation of bioactive pharmaceutical ingredients that are found in plants [26].
As reviewed in Mahidol et al. [27], medicinal plants have been used for treating infectious diseases because of their ease of accessibility as well as their lower side effects and toxicity. Medicinal plants may provide an exceptional renewable resource for the discovery of potential novel pharmaceuticals as their constituents have ample structural and biological diversity [27][28][29]. In Ethiopia, many plant species are being used as traditional medicine for the treatment of various human and animal diseases [20,[30][31][32][33][34][35][36][37][38][39]. Hence, more studies related to the use of medicinal plants as remedial agents need to be conducted, especially for plants which are useful to control antibiotic resistant microbes [40]. The aim of this study was to evaluate the efficacy of five plant extracts, which have been used as traditional medicines by local healers for their in vitro antibacterial activities against three multidrug resistant bacteria, namely, S. aureus, E. coli, and P. aeruginosa.

Preparation of Plant Material and Crude Extracts.
Five species of plants (Table 2), which have been widely used by local healers in different areas of southern Ethiopia for the treatment of livestock diseases [39], were collected from different agroecological zones to evaluate their in vitro antibacterial effects. The study was conducted from December 2015 to September 2016 at Dilla University. C. aurea (Ait.) Benth. and W. somnifera (L.) Dunal were collected from Yabello district, Borena zone, Oromia regional state, which is 5-6 km far from Yabello town. C. macrostachyus Del., and E. brucei Schwein. were collected from Dilla University Campus, Gedeo zone, while N. tabacum L. was collected from Seid Hassen Integrated Farm (private farm), Sidama zone, which is located at around 30 km west of Dilla town. Plant leaves and roots (for W. somnifera (L.) Dunal) were collected from their natural habitat and washed with tap water to remove unnecessary particles and dried under shade at a room temperature for about a month.
The procedure used for the preparation of the plant materials was adapted from Eloff [46]. Briefly, the dried plant materials were then ground using a commercial blender.
The powders of each plant were weighed using precision standard electronic balance before maceration ( Table 2). Maceration of each plant was carried out in flasks containing methanol (99.8%) and chloroform (95%) separately in a 1 : 10 (solute : solvents) ratio enough to cover all the plant powders with a continuous shaking with an orbital shaker (Gemmy Industrial Corporation, VRN-480) at 100 rpm and an occasional manual stirring with a glass rod. After 80 hours, the macerates of each plant were filtered in separate flasks using a qualitative filter paper (what man 44). The residues after filtration process were discarded while the filtrate parts were taken to a vacuum rotary evaporator (RE-52A, 220 V/50 Hz) machine for extraction with 35-40 ∘ C temperature adjustment. The methanol and chloroform crude extract products were poured into evaporating dishes and kept in dry oven (DHG-9030) at 40 ∘ C for 3-4 days until the extract materials were concentrated. Finally, the percentage yield of each plant extract was calculated ( Table 2).

Agar Well Diffusion and MICs.
Media preparation and agar well diffusion assay were done as previously described [46]. The American Type Culture Collection of Staphylococcus aureus 25923, Pseudomonas aeruginosa 27853, and Escherichia coli 20922 were obtained from the Ethiopian public health institute and were maintained on Brain Heart Infusion (BHI) agar medium (HiMedia) at 4 ∘ C until the commencement of the experiment. The test suspension was standardized to match 0.5 McFarland turbidity standard which corresponds to approximately 1.5 × 10 8 CFU/ml. A working solution (50 g/ml) of streptomycin (mM EDTA, analytical standard) was prepared from stock solution as a positive control, and 5% DiMethyl Sulphoxide (DMSO) (ACS reagent grade) was used as a negative control.
Three concentrations, 200, 100, and 50 mg/ml, of 5 crude plant extracts were used as a test material. Standardized inocula were swabbed uniformly onto solidified Mueller Hinton agar and the seeded media were allowed to dry for about 5 minutes. Five wells per plate (three wells for extracts, one for each of the positive and negative control) were made in the seeded agar using 6 mm cork borer. With the help of micropipette, 20 l of test solution was poured into the respective wells. The media were kept in the biosafety for 1 hr for proper diffusion and thereafter incubated at 37 ∘ C for 24 hrs. The experiment was repeated three times and the results, zones of inhibition of each dose, were expressed as the mean of the replicates.
The MIC was evaluated using agar dilution method [46], and MHA culture media were used. Serial dilutions of the plant extracts were prepared by dissolving in 5% DMSO to make concentrations of 15.625-1000 g/ml. Two ml aliquot of the plant extract dilution of each concentration to be tested was added to 19 ml of the molten agar. The test was performed in triplicate.    full-factorial ANOVA and expressed with the equation stated below.

Data
= + + + + + + + + , where represents the overall response; , , and stand for plant species, dose, and bacterial species, respectively, while and represent overall mean and common error, respectively. , , , and indicate the interaction effects of the respective variables. Zone of inhibitions were expressed as mean. Whenever significant variation was detected among treatments, means were separated using Tukey HSD at 5% level of significance. Romha et al. [39] based on their preference by local healers for treating animal and human diseases were collected and extracted (see Materials and Methods) using two solvents: methanol and chloroform. The highest (21.3%) and lowest (9.3%) yields were harvested from chloroform extracts of W. somnifera (L.) Dunal and methanol extracts of N. tabacum L., respectively ( Table 2). Table 2 were tested using different concentrations (50, 100, and 200 mg/ml) against three multidrug resistant bacterial species: S. aureus, P. aeruginosa, and E. coli. Of all the plants tested, C. aurea (Ait.) Benth. showed a wide spectrum of antibacterial activities against the investigated bacterial species. Regarding the dosage and solvents, 200 mg/ml showed best antibacterial activities both in solvents and in all plant species. Better antibacterial activities with maximum zone of inhibition (4.66 mm) in C. aurea (Ait.) Benth. were recorded in methanol crude extracts at a concentration of 200 mg/ml against S. aureus followed by C. macrostachyus Del. (4.43 mm) at the same dose and solvent for the same bacterial species (Table 3).

Antibacterial Activities of Plant Extracts. The methanol and chloroform extracts of the plant species listed in
Moreover, good results were observed from methanol and chloroform extracts of C. macrostachyus Del. against P. aeruginosa, E. coli, and S. aureus at a concentration of 200 mg/ml. Methanol extracts of W. somnifera (L.) Dunal showed comparable effect on these bacterial species at the same dose. Methanol and chloroform extracts of E. brucei Schwein. did not inhibit the growth of any bacterial species (Tables 3 and  4). The MIC for crude extracts of each plant for the respective test bacterium was done using the agar dilution method. The lowest value (62.5 g/ml) of MIC was recorded from both methanol and chloroform extracts of C. aurea (Ait.) Benth. against all the test bacteria. For C. macrostachyus Del., W. somnifera (L.) Dunal, and N. tabacum L., the MICs were between 250 g/ml and 1000 g/ml in all the test bacteria in both extractants. E. brucei Schwein. did not inhibit the growth of all bacteria employed in this study.  (Table 2) commonly reported by local healers [39] were evaluated for their efficacy in vitro against three bacteria species: S. aureus, P. aeruginosa, and E. coli. Different zones of inhibition were recorded from each plant extract and from different concentrations for each species of bacterium. The highest zones of inhibition (4.66 mm) were observed from C. aurea (Ait.) Benth. at concentration of 200 mg/ml against S. aureus from methanol extract which had no statistically significant difference ( > 0.05) when compared with the positive control. Consistent with the present study, comparable zones of inhibition with the positive control were reported from methanol extracts of C. aurea against S. aureus, P. aeruginosa, and E. coli in South Africa [17]. Promising antibacterial activities of methanol extracts of C. aurea on these bacterial species have been reported in Ethiopia [21]. Antihypertensive [41], antioxidant [17], and antiparasitic [47] activities of C. aurea have also been reported.

Croton macrostachyus Del.
Promising antibacterial activities were obtained from methanol and chloroform leaf extracts of Croton macrostachyus Del. against the test bacteria at a concentration of 200 mg/ml. The best activity, even more than the positive control, has been reported from methanol extract of C. macrostachyus against S. aureus in 6 Evidence-Based Complementary and Alternative Medicine  a previous study conducted in Ethiopia [20]. The methanol extracts of C. macrostachyus Del. also showed a potential antibacterial activity against E. coli and P. aeruginosa [18]. An interesting activity has been recorded from methanol extracts of leaf C. macrostachyus against wide range of Grampositive and Gram-negative bacterial species including S. aureus, P. aeruginosa, and E. coli [25]. Moderate antibacterial activity result has also been recorded from stem bark extract of C. macrostachyus [24]. In fact, antibacterial secondary metabolites such as polyphenols, triterpenes, and saponins were isolated from the leaf of this plant [25]. Moreover, antimalarial [22] and insecticidal [44] activities of leaf extracts of this plant have also been reported in Ethiopia.

Withania somnifera (L.) Dunal.
Though W. somnifera (L.) Dunal has been used by the Ethiopian local healers for the treatment of different animal and human ailments [34][35][36][37]39], scientific data on its antimicrobial activity is inadequate. Methanol extract of W. somnifera (L.) Dunal (root) revealed a promising antimicrobial activity against the test bacteria while chloroform extract of the plant showed moderate activity. W. somnifera is widely used as traditional medicine in Pakistan and strong antibacterial activities have been reported from methanol extract of W. somnifera (leaf) in the same country as reviewed in Adnan et al. [48]. High and moderate antibacterial activities have been recorded for methanol extract of W. somnifera (L.) Dunal stem against E. coli and P. aeruginosa, respectively, in India [23]. Leaves, fruits, and roots of W. somnifera showed the same antibacterial activities against these two bacterial species and the highest activities were recorded from the plants' leaves [19]. W. somnifera also exhibited antioxidant [19] and anti-inflammatory [49] properties.

Nicotiana tabacum L. and Erythrina brucei Schwein.
Weak antimicrobial activities were observed from methanol and chloroform extracts of N. tabacum L. against all the test bacteria. Moreover, E. brucei Schwein. did not totally inhibit the bacterial growth in the present study. However, good antimicrobial activities from methanol extract of N. tabacum L. have been reported from a previous study [20]. The difference in its medicinal value may possibly be risen due to agroecological difference. The use of these plants as a traditional medicine to treat various diseases of humans and animals is commonly reported in Ethiopia [20,33,36,38,39] and in Pakistan [50]. E. brucei Schwein. did not inhibit the bacterial growth in all concentrations. The plant might be active against parasites which supports the traditional belief documented previously [39]. However, this is preliminary evidence and needs further researches. Additional researches on antimicrobial activities as well as their phytochemical analysis of these plants should be conducted.   that, among a group of bacteria tested for crude extracts of different plant species, S. aureus was found sensitive comparably in all plant extracts [20,[51][52][53]. Likewise, considerable studies indicated that extracts from different plant species exhibited better activities on Gram-positive bacteria than on Gram-negative bacteria [54][55][56][57][58][59][60][61], while some studies reported that P. aeruginosa [61,62] and E. coli [21] were also sensitive to extracts of some medicinal plants. These sensitivity differences between Gram-positive and Gramnegative bacteria to the extract of different medicinal plants might be due to the structural and compositional differences in membranes between the two groups [63]. Indeed, Gramnegative bacteria are more resistant to antibiotics because they possess impermeable outer membrane; consequently, the levels of antibiotics in the cell are reduced [64]. Previous study indicated that plant extracts with MICs up to 100 g/ml have significant antibacterial activity. If the MICs of the plant extracts are between 100 and 625 g/ml and above 625 g/ml, the antibacterial activity of the respective plant extract is considered to be moderate and low, respectively [65]. The present study had its own limitation in that the method we employed for testing the plants' crude extracts was agar well diffusion; thus, nonpolar compounds may not diffuse well into the aqueous agar matrix thereby antibacterial activity of the plants may be underestimated.

Conclusion
The results of the present study support the traditional uses of these plants practiced by the local healers. Except E. brucei Schwein., all the plants investigated in this study exhibited antibacterial activities against the test bacterial species. However, it was reported that E. brucei Schwein. has been used for the treatment of external and internal parasites [39]. Further researches are needed to be conducted to evaluate the efficacy of these medicinal plant species on other microbes in different agroecological settings and their safety levels as well as their phytochemical compositions.

Additional Points
Availability of Data and Materials. All data generated during this study are included in this published article. Plant materials used in this study have been identified at the National Herbarium of Addis Ababa University and voucher specimens are deposited at the College of Agriculture and

Disclosure
The funding institution had no role in designing the study, data collection, analysis and interpretation of data as well as in writing the manuscript.