In-vitro susceptibility of wound bacteria of domestic carnivores to different mixtures of honey and Nigella sativa L. seed extracts

The aim of this study is to evaluate the bactericidal activity of honey alone and honey mixed with different Nigella sativa L. seed extracts against bacterial isolates from chronic wounds of domestic carnivores. The preparation of Nigella sativa L. seed extracts was carried out by macerating the seed powder in increasingly polar solvents (ethyl acetate, ethanol and methanol). The study of the minimum bactericidal concentration of honey alone and honey mixed with different Nigella sativa L. seed extracts was carried out by Broth Dilution Method. The results showed that the addition of different Nigella sativa L. seed extracts decreased the bactericidal activity of honey against Escherichia coli and potentiated this bactericidal activity against Staphylococcus intermedius. Ethanolic and ethyl acetate extracts of Nigella sativa L. seeds did not infl uence the bactericidal activity of honey against Staphylococcus aureus and Enterococcus faecalis. Addition of different Nigella sativa L. seed extracts showed a variable infl uence on the bactericidal activity of honey against Enterobacter sp.. Methanolic extract of Nigella sativa L. seeds potentiated the bactericidal activity of honey against Staphylococcus aureus, Staphylococcus intermedius, Enterococcus faecalis and Enterobacter sp.. These results revealed that combinations of methanolic extract of Nigella sativa L. seeds with honey can be used for the development of potent antibacterial dressing. Research Article In-vitro susceptibility of wound bacteria of domestic carnivores to different mixtures of honey and Nigella sativa L. seed extracts Derbal Saïd1*, Niar Abdellatif2 and Hanfer Mourad3 1Institute of Veterinary and Agricultural Sciences, University of Batna 1, 05000 Algeria 2Faculty of Natural and Life Sciences, University of Tiaret, 14000 Algéria 3Faculty of Natural and Life Sciences, University of Batna 2, 05000 Algéria Received: 04 April, 2019 Accepted: 13 May, 2019 Published: 14 May, 2019 *Corresponding author: Derbal Saïd, Institute of Veterinary and Agricultural Sciences, University of Batna 1, 05000 Algeria. Tel: +213 773258207; Email: https://www.peertechz.com


Introduction
The skin of domestic carnivores is subjected to incessant aggression, trauma causing cutaneous wounds, which is one of the fi rst reasons for consultation in veterinary medicine. The treatment of wounds aims to avoid complications and to obtain a quality healing as fast as possible. The physiological process that allows tissue restoration is wound healing. Infection is one of the factors that hinder this process. Since the use of antibiotics spread more than 50 years ago, bacteria have gradually developed resistance that continues to be a major health problem worldwide [1]. The use of natural products as an alternative treatment in wound healing and treatment has been on the rise in the last few decades [2]. Nigella sativa L. and honey are the most important natural remedies of Arabic Islamic medicine and used for various diseases for over 1500 years. In Islam, Nigella sativa L. and honey are regarded as one of the greatest forms of healing medicine [2]. The ancient Greeks and Egyptians used topical application of honey to treat wounds and burns of the skin, and traditional Persian medicine has documented that honey is effective in treating wounds, eczema and infl ammation [3,4]. Scientifi c interest in honey decreased with the advent of antibiotics in the early 1900 years [5], but today with the emergence of antibiotic resistant microbial strains, honey has again attracted the attention of researchers [5,6]. Honey has long been known to possess antibacterial properties and has established use as a dressing [7,8], but not all honeys are equally effective in wound healing [9]. In human medicine, several published reports indicate the effi cacy of honey in the treatment of various known infected wounds such as venous ulcers in the leg [10,11], burns [12], chronic leg ulcers [13] and pressure ulcers [14]. Nigella sativa L. has traditionally been used for centuries in the Middle East, North Africa, the Far East and Asia for the treatment of various diseases [15], as well as in the treatment of many types of wounds and trauma [16,17]. In this research, the bactericidal activity of Algerian honey against bacterial isolates from chronic wounds of domestic carnivores, was evaluated. Also, infl uence of addition (5% w/w) of different Nigella sativa L. seed extracts on the bactericidal activity of this honey was studied. Honey and Nigella sativa L. seed extracts were combined together for the inhibition of bacterial growth to evaluate the synergistic effect of this combination on these bacteria. Different extracts of Nigella sativa L. seeds were used for this combination to determine the extract capable of giving the most powerful synergistic effect with honey. These extracts were used with a low percentage so as not to alter too much the physical and chemical properties of the honey.

Plant material
The seeds of Nigella sativa L. were purchased from an herbal shop in El-Eulma, Algeria.

Honey sample
The honey used in this study was purchased from an apiary in El-Eulma, Algeria.

Bacterial isolates
Five bacteria were isolated from chronic wounds of dogs and cats, using three selective media (Chapman, MacConkey and BEA). These bacteria were identifi ed by standard and biochemical bacteriological tests and were maintained at 4°C on nutrients agar slants.

Extraction process
The seeds of Nigella sativa L. were cleaned of impurities, washed and dried away from light for a few days, and then ground with a mortar to medium fi ne powder from which different extracts were prepared.
The extraction was carried out by macerating the seed powder in increasingly polar solvents (ethyl acetate, ethanol and methanol) following the method described by Shahid et al. (2013) [18].
Each of 50 g seed powder was macerated in 500 ml of a different solvent, for two weeks at room temperature, with occasional agitation to facilitate extraction. The macerates were fi ltered on fi lter papers. The fi ltrates were evaporated using rotary evaporator (Heidolph ® ) at 50°C. The extracts were stored in sterile glass vials at 4°C until use.

Preparation of natural mixtures
Three natural mixtures were prepared by adding 5% (w/w) of different Nigella sativa L. seed extracts to honey. The composition of these natural mixtures was: · The fi rst natural mixture (NM 1): 95% of honey + 5% of ethyl acetate extract; · The 2 nd natural mixture (NM 2): 95% of honey + 5% of ethanolic extract; · The 3 rd natural mixture (NM 3): 95% of honey + 5% of methanolic extract.
All natural mixtures were freshly prepared and stored in the refrigerator at 4°C in the dark.

Inoculum preparation
The inoculums was prepared following the method described by Majtan et al. (2010) [19] with small changes in the use of nutrient broth instead of PBS.
For each bacterial strain, an active culture on nutrient agar was carried out. Then a few colonies of this culture were suspended in nutrient broth. With a spectrophotometer, the turbidity of the bacterial suspension was adjusted to 10 8 CFU/ ml and diluted with nutrient broth to fi nal concentration of 10 7 CFU/ml.

Study of the bactericidal activity of honey alone and natural mixtures
The study of the bactericidal activity of honey alone and natural mixtures (NMs) was carried out by the Broth Dilution Method following the protocol described by Alzahrani et al. [20], with some modifi cations.
Using a sterile graduated pipette, 1 ml of honey alone and 1 ml of each natural mixture was transferred separately into the fi rst test tubes of the concentration 100% (no dilution). 1 ml of Muller-Hinton broth was transferred into each of the other test tubes for other decreasing concentrations. Then, 1 ml of honey alone and 1 ml of each natural mixture was transferred separately into the fi rst test tubes containing Muller-Hinton broth to obtain a concentration of 50%. The contents of these test tubes were homogenized by a vortex. After homogenization, 1 ml of the contents of each test tube of the concentration 50% was transferred into the next test tube. The same procedures were repeated until the concentration 0.01% from which, after good homogenization, 1 ml of the test tube contents was discarded.
10 μl (10 5 CFU) of each standardized bacterial suspension was poured into the test tubes of different concentrations. A positive control was made by inoculating 10 μl (10 5 CFU) of each standardized bacterial suspension into 1 ml of Muller-Hinton broth. Negative control was made by adding 0.5 ml of Muller-Hinton broth to 0.5 ml of honey alone and 0.5 ml of each natural mixture. All test tubes were incubated at 37°C for 24 hours. All tests were performed in triplicate and were repeated three times to obtain reliable results.

Results and Discussion
The results concerning the standard and biochemical bacteriological tests of isolated bacteria are gathered in table      in honey [21]. The phenolic compounds, particularly the fl avonoids, are responsible for the antimicrobial properties of honey [22][23][24]. Certain types of honey contain other bioactive components with antibacterial activity, including methylglyoxal, lysozyme and defensin-1 [25,26]. In addition, it is suggested that presence of different strains of Lactobacillus acidophilus in honey obtained from different sources may contribute to the antimicrobial properties of honey [27]. Also, hydrogen peroxide plays an important role in the antimicrobial activity of honey [28].
Clinical trials using NM 3 to treat animal wounds could further confi rm the fi ndings of this work. Further research is needed to elucidate and optimize the effective combination of these natural products in clinical practice.
Management of the animal wounds infection should not be limited to local wounds care but may involve other modalities including systemic antibiotics and debridement whenever needed. Neither of these natural products has an adverse effect on tissues, so they can safely be used on animal wounds to clear infection.

Conclusion
The synergism between Algerian honey and methanolic extract of Nigella sativa L. seeds can be exploited for the treatment of infected wounds and other bacterial infection.
This synergistic antibacterial effect can be extremely useful in treatment of infected wounds of domestic carnivores.