Abstract

Despite tremendous progress in drug development and treatment, infectious diseases caused by bacteria are still a major threat to human and animal health mainly due to drug resistances. These the ever-increasing threats demand a global effort to search for novel solutions. Different parts of plants and bee’s honey have been used, separately or in combination, as a source of therapeutic agents in traditional medicine since ancient time due to bioactive compounds they contain. Therefore the objective of this study is to determine the antibacterial activity of crude extract of Embelia schimperi leaves and combined with bee’s honey (Apis florae) against some standard and clinical pathogenic bacteria. The In-vitro antibacterial activity of the extracts was tested at different concentrations by using agar well diffusion method and measuring zone of inhibition. The data was analyzed by ANOVA. Both the clinical and standard bacteria showed sensitivity to E. schimperi with inhibition zones ranging from 7.17 ± 0.29 to 28.96 ± 0.25. The ethanol extract of E. schimperi leaves and ethanol extract blended with honey have showed significant inhibition against the tested organisms than the chloroform extract and chloroform extract blended with honey respectively. The inhibition zone was lower against S. aureus during both the Chloroform leave extract and the chloroform blended with honey. The highest inhibition zone was observed against E. coli with the ethanol leave extract and against K. pneumonia with the ethanol mixed with honey. Therefore, the E. schimperi leaves extract and synergy with honey has witnessed broad spectrum antibacterial activity.

Keywords: Antimicrobial activity, Embelia Schimperi, Inhibition zone, Synergy.

Received: 12 April 2019 / Revised: 20 May 2019 / Accepted: 25 June 2019/ Published: 1 August 2019

Contribution/ Originality

This study contributes to the existing literature of the antibacterial effects of leaf extracts of E. schimperi. It is the only study that investigated the synergetic effects of leaves of E. schimperi with honey. Therefore, the synergy of the leaves with honey has shown promising antimicrobial effect.

1. INTRODUCTION

Medicinal plant extracts have been playing a vital role in the treatment of many diseases including microorganism infections since ancient time (Sukanya et al., 2009). It is well-known that native healers not solely use remedies made from one single plantspecies however conjointly prepare specific mixtures of various healthful plants to treat, oral diseases, wounds, and skin disorders. This traditional medical knowledge is useful not only for community healthcare but also for future search and development of effective drugs (Sukanya et al., 2009; Desalegn, 2014).

Microbial diseases continued to be the major threats to the world in spite of the efforts and progress in developing new medications (Cos et al., 2006). The impact of microbial diseases has very substantial problem in developing countries like Ethiopia where there's restricted access to standard medications. The main problem encountered with antibiotics in clinical use is resistances, high toxicity, unaffordable costs and low efficacy. Currently, the ever-increasing threat, urges a world effort to look for novel solutions (Theuretzbacher, 2012; Lee, 2015). Many clinical drugs are derived from natural remedies. Most traditional healers use medicinal plant parts blended with honey or butter. This indicates that blended remedies have synergetic effect in treating microbial infections (Rios and Recio, 2005).

Embelia is a Scandent or climbing shrub or tree, 2-13 m tall; as indicated in Figure 1 appendix part, branches with prominent lenticels, young parts with rust-colored hairs, becoming glabrous leaves alternate or in clusters at end of branches belongs to the family Myrsinaceae (Sebsebe and Hedberg, 2003). In Ethiopia, the bark or fruits from E. schimperi are combined with other species, such as Albizia anthelmintica, Guizotia abyssinica, Glinus lotoides and Hagenia abyssinica, mixed with water and taken as a Taenicide or used as a disinfectant which suggests that the pharmacological effect of this plant can be increased by interaction with other plant ingredients (Doughari et al., 2008; Zafar et al., 2010). The use of honey as a traditional remedy for microbial infections dates back to ancient times. Research has been conducted on honey, which has demonstrated to be effective against several human pathogens, including Escherichia coli (E. coli), Enterobacter aerogenes, Salmonella typhimurium, Staphylococcus aureus (Lusby et al., 2005). In some parts of Ethiopia traditional healers use combinations of honey and parts of E. schimperi to treat microbial infections but it is not investigated scientifically. The present study was, therefore, aimed to see synergic antibacterial effect of leaf extract of E. schimperi and honey against standard and clinical isolated bacteria.

2. MATERIALS AND METHODS

2.1. Study Area

The study was conducted at Microbiology Laboratory, Department of Biology, University of Gondar; Gondar, Ethiopia. Gondar lies between 9° to 13° 45'N latitude and 36° to 40° 30'E longitude. It has variable altitudes that range from 550 m (in western lowland) to 4,620 m (Semen Mountain in North) above sea level and it is the homeland of Walia ibex and Ethiopian wolf (Central Statistical Authority, 2008).

2.2. Plant Material Collection and Identification

The leaves of E. schimperi were collected from University of Gondar garden. The plant species were identified by Taxonomist. Then, the collected plant material was washed with tap water and rinsed with distilled water. The leaves were placed in dark place protected from sunlight and open air at room temperature in Microbiology laboratory until it dries out.

2.3. Preparation of Plant Extracts

The dried leaves were powdered with a mechanical grinder. The powder was stored in a sterile brown bottle at room temperature under dark place (Shahidi, 2004) . The 20g dried leaves powder of E. schimperi was macerated with 100mL chloroform and ethanol, and shacked on shaker (Edmund Buhler GMBH) shown in Figure 2 for 72hr to extract the active compound. Then, the solution was filtered using Whatman no.1 filter paper into previously washed, dried, labeled and weighed beakers (Chulet et al., 2010) . The filtrate was then evaporated on oven at 35ºC. The extract was dissolved in 3% tween eighty to achieve 50% concentration (500mg/mL) prior to the test. The antibacterial activities of the crud extract of the plants were tested against selected organisms (Aladesanmi et al., 2007) .

2.4. Inoculums Preparation for Antimicrobial Test

The tested strains, E. coli ATCC 2592, K. pneumonia ATCC 700603, werestandard strains andS. typhi and S. aureus were clinical isolate. The standard strains as well as the clinical isolates were obtained from stocks of culture collections maintained in microbiology laboratory of department of Medical microbiology, University of Gondar. The bacteria were maintained in triptone Soya broth with 20% glycerol and kept at a temperature bellow 4ºC. Sub-culturing was done on nutrient agar during the experiment. Two to three isolated overnight cultured colonies of each organism were transferred aseptically by sterilized wire loop into 5 ml sterile normal saline in a test tube and mixed thoroughly, using vortex mixer(GEMMY Model: VM-300p), for uniform distribution and compared with 0.5 McFarland standard (Shahidi, 2004).

2.5. Honey Samples

The natural honey used in this experimental study was purchased from local market. The honey was diluted in distilled water to achieve 50% concentration (500mg/mL) prior to the test.

2.6. Preparation of 0.5 Mcfarland Turbidity Standards

The McFarland 0.5 turbidity standard was prepared by adding 50 µL of a 1.175% (wt/v) barium chloride di-hydrate (BaCl2.2H2O) solution to 9.95 mL of 1% (v/v) sulfuric acid. The 0.5 McFarland standards were vigorously agitated to turbidity on a vortex mixer before use. The 0.5 McFarland standards are comparable to a bacterial suspension of 1.5x108 colony-forming units (CFU)/mL (NCCLS, 1993).

2.7. Antibacterial Activity Determination

2.7.1. Agar Well Diffusion

Muller Hinton agar was prepared by dissolving the required amounts of the powder in distilled water and boiled to mix thoroughly and autoclaved. The sterilized media was aseptically poured in to sterile Petri-plates in a laminar air flow and allowed to dry at room temperature for 30 minutes (López et al., 2011). Bacterial suspension was prepared to a density of 1.5x108colony-forming units (CFU)/mL by comparing with 0.5 McFarland standards in sterile normal saline. Aliquots of the organism (100µL) were aseptically transferred to Muller Hinton agar using a micropipette and seeded evenly by using sterilized cotton swab. On each plate, equidistant wells were prepared with a 6 mm diameter sterilized cork borer, 2 mm from the edge of the plate (Shahidi, 2004). Hundred micro liter of plant extract (500 mg/ml) was aseptically introduced into a respective agar wells. Gentamycin were used as positive controls and distilled water was included as negative control. This was followed by allowing the agar plate on the bench for 40 minutes for pre-diffusion followed by incubation at 37°C for 24 hrs. The experiment was performed in triplicate. Those extracts showing any inhibition at all were noted for further tests for the quantitative assessment of their activity.

2.7.2. Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) of the extracts was determined for extracts that showed ≥10 mm diameter inhibition zone according to methods described by Shahidi (2004) and Akinyemi et al. (2005). The test was performed using macro-tube dilution method; the extract solution (500 mg/ml) was serially diluted to 1:2 then 1:4 mg/ mL concentrations with sterilized distilled water. The extract was then aseptically introduced to a tube containing sterilized and specified volumes of Nutrient broth. The inhibition of growth was determined by comparing a tube which contains pure and inoculated nutrient broth after 24 hr incubation at 370C and the minimum concentration that inhibited growth was considered as MIC value of the extract (Parekh et al., 2006) .

Experimental data was collected in biology laboratory and the collected data was analyzed using SPSS version 20 software. Mean and standard deviations of the triplicates analysis were calculated using analysis of variance (ANOVA) to determine the significance difference between the mean followed by Duncan’s multiple range test (p < 0.05). The statistically significant difference was defined as (p < 0.05).

3. RESULT

Ethanol and chloroform extracts of Leaves of E. schimperi were evaluated in vitro for antibacterial activity by agar well diffusion method as it has been shown in appendix part of Figure 3 and its result has been described in Tables below. Variable degree of inhibition zones against E. coli, K. pneumonia, S. thyphi and S. aureus has been shown. As it was presented on Table 3.1 below the antibacterial activities of crud extracts of E. schimperi prepared by ethanol extraction has showed statistically significant difference (p < 0.05) in growth inhibition activity than the chloroform extract against E. coli standard strain but no statistically significant difference was observed against S. thyphi with both extracts. Ethanol extracts of E. schimperi has shown comparable activity against E. coli standard strain when compared with positive control drug gentamycin. E. schimperi ethanol extract showed better activity than chloroform extract against S. aureus standard isolate.

Table-3.1. Antibacterial activities of organic extracts of Embelia schimperi using agar well diffusion method.

Organism	Zone of inhibition (mm) ± standard deviation
	E. schimperi extract		Positive control	Negative control
	Ethanol	Chloroform	Gentamycin	3% tween80
E. coli	15.33 ± 2.082a	9.33 ± 1.53b	16.56 ± 0.51a	00.00
K. pneumonia	14.5 ± 0.5a	11.70 ± 0.61b	28.96 ± 0.25 c	00.00
S. thyphi	10.83 ± 0.76a	10.67 ± 0.58a	15.03 ± 0.40b	00.00
S. aureus	12.67 ± 2.31a	8.83 ± 0.29b	17.33 ± 0.35c	00.00

Values are means of triplicate determinations values within the same row followed by different supper scripts (letters a, b, c) are significantly different at (p < 0.05).

Table 3.2 below shows ethanol and chloroform extracts of E. schimperi blended with honey. E. schimperi extract mixed with honey showed statistically significant difference against the tested organisms when compared with chloroform extract mixed with honey. Statistically significant (p < 0.05) inhibition zones difference was observed on the antimicrobial sensitivity activities by the mixture of chloroform extract with honey compared to positive control drug Gentamycin against the tested organisms but no significant difference was observed with mixture of ethanol extract with honey and Gentamycin (p ≥ 0.05) against E. coli and S. thyphi. When compared to control drug gentamycin, against K. pneumonia greater inhibition zone was seen than ethanol and chloroform extracts mixed with honey.

Table-3.2. Antibacterial activities of organic extracts of Embelia schimperie ethanol and chloroform extracts mixed honey using agar well diffusion method.

Organism	Zone of inhibition (mm) ± standard deviation
	E. schimperi extract		Positive control	Negative control
	Ethanol with honey	Chloroform with honey	Gentamycin	3% tween80
E. coli	15.77 ± 0.25a	7.83 ± 0.29b	16.65 ± 0.61a	00.00
K. pneumonia	21.00 ± 0.00a	10.83 ± 1.04b	28.96 ± 0.25c	00.00
S. thyphi	14.60 ± 0.53a	9.50 ± 0.50b	15.03 ± 0.20a	00.00
S. aureus	11.50 ± 1.32a	7.17 ± 0.76b	17.33 ± 0.35c	00.00

Values are means of triplicate determinations values within the same row followed by different supper scripts (letters a, b, c) are significantly different at (p < 0.05).

4. DISCUSSION

Embelia schimperi is one of the common medicinal plants as reported by local healers (Rondevaldova et al., 2015). In vitro antibacterial activity of E. schimperi had previously been studied in different areas which show antimicrobial effect in comparison with other plants used for the treatment of different diseases (Lulekal et al., 2014). Although the plant parts were used traditionally as an anti-infective preparation in combination with other plant parts (d’Avigdor et al., 2014) to the best of our knowledge there is no report on its in vitro antimicrobial activity in combination with bee’s honey. In this study, the efficacy of the extract and its mixture with honey against different bacterial species was evaluated in vitro. Different zones of inhibition were recorded from each crude extract and synergetic effect of the extract blended with honey at different concentrations for each species of bacterium. Regarding the efficacy of the crude extract, the highest zones of inhibition was observed against E. coli with Ethanol extract and the lowest was against S. aureus with chloroform extract. Concerning the effectiveness of the blend, the highest inhibition zone was witnessed against K. pneumonia with the ethanol extract mixed with honey and the lowest was witnessed against S. aureus with chloroform extract mixed with honey. Thus, the antibacterial activities of the ethanol crude extract and that blended with honey has shown promising potential against the tested bacterium and was found to be almost comparable to the standard control drug gentamycin.

According to Okwori et al. (2007) the formation of clear inhibition zone of >10 mm diameters around the wells was regarded as significant susceptibility of the organisms to the extract. The current study has shown a result that can witness the antibiotic potential of the tested plant crude extract and that blended with honey. The highest mean zone of inhibition against all the bacterial strains tested ranged between 7.17mm and 21.00mm and the positive control produced mean zone of inhibition between 15.03mm and 28.96mm, but the negative control has not produced any zone of inhibition.

The level of antimicrobial activity exhibited by plant material depends on many factors, including the plant part, moisture content, harvesting process, preparation process, strains of the tested bacterium and type of the solvent used to extract the active compound (Ramesh et al., 2001; Wendakoon et al., 2012). For instance, relatively high temperatures that can be generated during tissue grinding can denature chemical constituents. In addition, the extraction solvent, the time of maceration, may affect the level and composition of secondary metabolites extracted from plant tissues (Wendakoon et al., 2012). In our study, there was higher and statistically significant antimicrobial inhibition zone difference. The susceptibility of the tested bacterium was higher to the ethanol crude extract and its blend with honey than that of the chloroform. The extraction solvents used in this study could have caused variation in the antimicrobial activity results. According to Motamedi et al. (2010) report, the solubility of active principles in plant materials varies according to extraction solvent used, which might be related to differences in antimicrobial effect of plant extracts.

Moreover, the sensitivity differences between Gram-positive and Gram negative bacteria to the extract of different medicinal plants might be due to the structural and compositional differences in membranes between the two groups (Hodges, 2002). In contrary of other considerable studies, the current study has indicated that the tested plant extracts has exhibited better activities on Gram-negative bacteria than on Gram-positive bacteria. However, as several studies confirmed, Gram-negative bacteria having an outer phospholipidic membrane carrying the structural lipopolysaccharide components. This makes the cell wall impermeable to antimicrobial chemical substances and act as a diffusion barrier and making them less susceptible to the antimicrobial agents than are Gram-positive bacteria (Nostro et al., 2000; Hodges, 2002). In spite of this permeability differences, however, some of the extracts have still exerted some degree of inhibition against Gram-negative organisms as well. Indeed, Gram negative bacteria are more resistant to antibiotics because they possess impermeable outer membrane; consequently, the levels of antibiotics in the cell are reduced (Bockstael and Aerschot, 2009).

On the other hand, in this study half of the tested bacterium was clinical isolates and the lower inhibition growth was observed during the test. This revealed that, since they have exposure to standard drugs, they might have relatively higher resistance that the standard tested bacterium. As Togan et al. (2014) described possible differences in susceptibility patterns between standard and clinical strains, in which clinical strains may represent current isolates responsible for clinical disease and spread of resistance.

The antimicrobial effects of different Ethiopian honeys were already evaluated and witnessed as it has greater mean inhibition zone against bacteria (Ewnetu et al., 2013). Similarly this study has evaluated the synergetic effect of the plant crude extract blended with honey. As aforementioned, E. schimperi is well known its usage as herbal medicine and most traditional healers prefer to use medicinal plant combined with honey to act synergistically for the treatment of many infections (Nwankwo et al., 2014). The finding of the current research has indicated that ethanol extract of leaves of E. schimperi blended with honey has shown the higher antibacterial activity far better than ethanol crude extract and chloroform extract blended with honey. The higher inhibition zone of ethanol crude extract blended with honey was against K. pneumonia which was standard bacteria.

The inhibitions of ethanol extract of plant leaves were enhanced by mixing with honeys due to their synergistic antibacterial property as already reported (Omoya and Akharaiyi, 2011). The antibacterial action of honey is believed to be due to acidity, osmolarity, hydrogen peroxide generation and phytochemical components (Molan, 1992). Furthermore, the antimicrobial effects of different honeys might be related to Phytochemicals such as Phenolic acids (benzoic and cinnamic acids) and flavonoids (flavanones, flavanols) which were reported for significant contribution of the antibacterial capacity of honey that varies greatly depending on the floral sources (Gheldof et al., 2002). Many, but not all, of the bacterial strains commonly encountered by humans are killed by flavonoids, even though the mechanism is not yet known (Farnesi et al., 2009). These synergistic antimicrobial effects of honey with Ethanol plant extract mixtures might be related chiefly to the increase in volume of these flavonoids in the mixture (Molan, 1992) .

5. CONCLUSION

The tested plant has showed broad spectrum antibacterial activity as confirmed by high zone of inhibition. Both the E. schimperi leaves crude extract with ethanol and chloroform, and blended with honey has witnessed antibacterial potential against the tested pathogens. This finding suggests that there is possibility to isolate potential antibacterial drugs from the medicinal plant and the honey. The antibacterial activity of E. schimperi leaves extract is more effective when it is blended with honey than using the crude extract alone. Therefore, the use of mixture of honeys with E. schimperi extracts for treatment of bacterial infection is recommended. Phyto-chemical screening of the tested plant have to be done to detect secondary metabolites that are responsible for the antibacterial activity and its toxicity and their synergy with honey needs to be tested.

Funding: This study received no specific financial support.

Competing Interests: The authors declare that they have no competing interests.

Acknowledgement: The authors would like to acknowledge Department of Biology, University of Gondar for allowing them to conduct this experiment. Their great appreciation also extended to Medical Microbiology laboratory of the University Hospital for providing them the bacterial strains.

REFERENCES

Akinyemi, K.O., O. Oladapo, C.E. Okwara, C.C. Ibe and K.A. Fasure, 2005. Screening of crude extracts of six medicinal plants used in Southwest Nigerian unorthodox medicine for antimethicillin resistant Staphylococcus aureus activity. Complementary and Alternative Medicine, 5(1): 1-7.

Aladesanmi, A., E. Iwalewa, A. Adebajo, E. Akinkunmi, B. Taiwo, F. Olorunmola and A. Lamikanra, 2007. Antimicrobial and antioxidant activities of some Nigerian medicinal plants. African journal of traditional, complementary and alternative medicines, 4(2): 173-184.

Bockstael, K. and A. Aerschot, 2009. Antimicrobial resistance in bacteria. Open Medicine, 4(2): 141-155.

Central Statistical Authority, 2008. North gondar, agricultural office, statistical report on livestock and farm implement.

Chulet, R., P. Pradhan, K.S. Sharma and K.M. Jhajharia, 2010. Phytochemical screening and antimicrobial activity of Albizzia lebbeck. Journal of Chemical and Pharmaceutical Research, 2(5): 476-484.

Cos, P., A.J. Vlietinck, D.V. Berghe and L. Maes, 2006. Anti-infective potential of natural products: How to develop a stronger in vitro ‘proof-of-concept. Journal of Ethnopharmacology, 106(3): 290-302.Available at: https://doi.org/10.1016/j.jep.2006.04.003.

d’Avigdor, Wohlmuth H., a. Asfaw Z. and Awas T., 2014. The current status of knowledge of herbal medicine E and medicinal plants in Fiche, Ethiopia, Journal of Ethnobiology and Ethnomedicine, 10(1): 38–71.Available at: https://doi.org/10.1186/1746-4269-10-38.

Desalegn, A., 2014. Antimicrobial activity of medicinal plant extracts and their synergistic effect on some selected pathogens. American Journal of Ethnomedicine, 1(1): 18-29.

Doughari, J.H., A.M. El-mahmood and T. I., 2008. Antimicrobial activity of leaf extracts of Senna obtusifolia (L). African Journal of Pharmacy and Pharmacology, 2(1): 007-013.

Ewnetu, Y., W. Lemma and N. Birhane, 2013. Antibacterial effects of Apis mellifera and stingless bees honeys on susceptible and resistant strains of Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae in Gondar, Northwest Ethiopia. BMC Complementary and Alternative Medicine, 13(1): 269.Available at: https://doi.org/10.1186/1472-6882-13-269.

Farnesi, A., R. Aquino-Ferreira, D.D. Jong, J. Bastos and A. Soares, 2009. Effects of stingless bee and honey bee propolis on four species of bacteria. Genetics and Molecular Research, 8(2): 635-640.Available at: https://doi.org/10.4238/vol8-2kerr023.

Gheldof, N., X.-H. Wang and N.J. Engeseth, 2002. Identification and quantification of antioxidant components of honeys from various floral sources. Journal of Agricultural and Food Chemistry, 50(21): 5870-5877.Available at: https://doi.org/10.1021/jf0256135.

Hodges, N., 2002. Pharmaceutical applications of microbiological techniques. 2nd Edn., In: Ed Aulton, M.E., Pharmaceutics: The Science of Dosage Form Design. London: Harcourt Publishers Limited. pp: 606.

Lee, C., 2015. The antibiotic resistance crisis; part 1: Causes and threats. Pharmacy &Therapeutics, 40(4): 277-283.

López, V., A.K. Jäger, S. Akerreta, R.Y. Cavero and M.I. Calvo, 2011. Pharmacological properties of Anagallis arvensis L.(“scarlet pimpernel”) and Anagallis foemina Mill.(“blue pimpernel”) traditionally used as wound healing remedies in Navarra (Spain). Journal of Ethnopharmacology, 134(3): 1014-1017.Available at: https://doi.org/10.1016/j.jep.2010.12.036.

Lulekal, E., J. Rondevaldova, E. Bernaskova, J. Cepkova, Z. Asfaw, E. Kelbessa, L. Kokoska and P. Van Damme, 2014. Antimicrobial activity of traditional medicinal plants from Ankober district, north Shewa zone, Amhara region, Ethiopia. Pharmaceutical biology, 52(5): 614-620.Available at: https://doi.org/10.3109/13880209.2013.858362.

Lusby, P., A. Coombes and J. Wilkinson, 2005. Bactericidal activity of different honeys against pathogenic bacteria. Archives of Medical Research, 36: 464–467.Available at: https://doi.org/10.1016/j.arcmed.2005.03.038.

Molan, P.C., 1992. The antibacterial activity of honey: 1. The nature of the antibacterial activity. Bee World, 73(1): 5-28.Available at: https://doi.org/10.1080/0005772x.1992.11099109.

Motamedi, H., E. Darabpour, M. Gholipour and S.M.S. Nejad, 2010. In vitro assay for the anti-brucella activity of medicinal plants against tetracycline-resistant brucella melitensis. Journal of Zhejiang University Science B, 11(7): 506-511.Available at: https://doi.org/10.1631/jzus.b0900365.

NCCLS, 1993. Performance standard for antimicrobial disc susceptibility test. Approved Standard NCCLS Publication, Villanova, PA, USA, M2- A5.

Nostro, A., M. Germano, V. D’angelo, A. Marino and M. Cannatelli, 2000. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Letters in Applied Microbiology, 30(5): 379-384.Available at: https://doi.org/10.1046/j.1472-765x.2000.00731.x.

Nwankwo, C., C. Ezekoye and S. Igbokwe, 2014. Phytochemical screening and antimicrobial activity of apiary honey produced by honey bee (Apis mellifera) on clinical strains of Staphylococcus aureus, Escherichia coli and Candida albicans. African Journal of Biotechnology, 13(23): 2367-2372.Available at: https://doi.org/10.5897/ajb2013.13570.

Okwori, A., C. Dina, S. Junaid, I. Okeke, J. Adetunji and A. Olabode, 2007. Antibacterial activities of ageratum conyzoides extracts on selected bacterial pathogens. Internet J Microbiol, 4(1): 34-56.Available at: https://doi.org/10.5580/fce.

Omoya, F. and F. Akharaiyi, 2011. Mixture of honey and ginger extract for antibacterial assessment on some clinical isolates. International Journal of Pharmaceutical and Biomedical Research, 2(1): 39-47.

Parekh, J., D. Jadeja and S. Chanda, 2006. Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity. Turkish Journal of Biology, 29(4): 203-210.

Ramesh, N., M. Viswanathan, A. Saraswathy, K. Balakrishna, P. Brindha and P. Lakshmanaperumalsamy, 2001. Phytochemical and antimicrobial studies of bridelia crenulata. Pharmaceutical Biology, 39(6): 460-464.Available at: https://doi.org/10.1076/phbi.39.6.460.5883.

Rios, J. and M. Recio, 2005. Medicinal plants and antimicrobial activity. Journal of Ethnopharmacology, 100(1-2): 80-84.

Rondevaldova, J., O. Leuner, A. Teka, E. Lulekal, J. Havlik, P. Van Damme and L. Kokoska, 2015. In vitro antistaphylococcal effects of Embelia schimperi extracts and their component embelin with oxacillin and tetracycline. Evidence-Based Complementary and Alternative Medicine, 2015: 1-7.Available at: https://doi.org/10.1155/2015/175983.

Sebsebe, D. and I. Hedberg, 2003. Flora of Ethiopia and Eritrea apiaceae to dipsacaceae vol.4 part1;( Editors: Inga Hedberg, Sue Edwards & Sileshi Nemomissa). The National Herbarium, biology department, science faculty, Sweden: Addis Ababa University, Ethiopia and The Department of Systematic Botany Uppsala University. pp: 68-69.

Shahidi, B.G.H., 2004. Evaluation of antibacterial properties of Iranian medicinal plants against micrococcus aureus, Serratia marcescens, Klebsiella pneunomiae and Bordella bronchoseptica. Asian Journal of Sciences, 3(1): 82-86.Available at: https://doi.org/10.3923/ajps.2004.82.86.

Sukanya, S., J. Sudisha, P. Hariprasad, S. Niranjana, H. Prakash and S. Fathima, 2009. Antimicrobial activity of leaf extracts of indian medicinal plants against clinical and phytopathogenic bacteria. African Journal of Biotechnology, 8(23): 6677- 6682.

Theuretzbacher, U., 2012. Accelerating resistance, inadequate antibacterial drug pipelines and international responses. International Journal of Antimicrobial Agents, 39(4): 295-299.Available at: https://doi.org/10.1016/j.ijantimicag.2011.12.006.

Togan, T., E. Evren, Ö. Çiftci, H. Narci, M. Özcan and H. Arslan, 2014. The antibacterial effect of propolis against clinical isolates. Sci-Afric Journal of Scientific Issues, Research and Essays, 2(12): 551–553.

Wendakoon, C., P. Calderon and D. Gagnon, 2012. Evaluation of selected medicinal plants extracted in different ethanol concentrations for antibacterial activity against human pathogens. Journal of Medicinally Active Plants, 1(2): 60-68.

Zafar, A., S.K. Shaukat, K. Mahnaaz, T. Arshiya and A.L. Zahoor, 2010. Synergistic effect of salvadora persica extracts,tetracycline and penicillin against staphylococcus aureus. African Journal of Basic & Applied Sciences, 2(1-2): 25-29.