Antibacterial Effect of Allium sativum and Zingiber officinale Extracts on Some Clinically Pathogenic Bacteria

S: The ability of some pathogenic bacteria to develop antibiotic resistance has become a global health challenge, and there is an increasing rate of bacteria resistance against the available antibiotic drug. This study aimed to determine the inhibitory effect of Allium sativum (Garlic) and Zingiber officinale (Ginger) extracts on clinical pathogenic bacteria. Four different concentrations (125mg/ml, 250mg/ml, 500mg/ml, and 1000mg/ml) of each extract were tested against the test organisms. The test isolates showed susceptibility to varying concentrations of the crude extracts. For aqueous and ethanolic extracts of Allium sativum, Staphylococcus aureus showed the highest susceptibility (16±0.8mm and 15.7±1.2mm), followed by Escherichia coli (13.7±1.2mm and 14±0.8mm), Pseudomonas aeruginosa (14±0.8mm and 12±0.8mm) while Klebsiella pneumonia showed the least susceptibility of (11±0.8mm and 10±1.3mm). For aqueous and ethanolic extracts of Zingiber officinale, Staphylococcus aureus also showed the highest susceptibility (16±0.8mm and 15±0.8mm), followed by Pseudomonas aeruginosa (14±0.8mm and 12±0.8mm), Escherichia coli (13.7±1.2mm and 13±0.8mm) and the least was Klebsiella pneumonia at (11±0.8mm and 7±0.8mm). However, all the tested organisms were most susceptible to aqueous extracts of garlic and ginger but showed relatively lower susceptibility to ethanolic extracts of ginger. The minimum inhibitory concentration for garlic and ginger extract was 125mg/ml against E. coli , S. aureus

Commercially available antimicrobial drugs have controlled microbial pathogenicity and other infectious diseases for many decades. The therapeutic method of antibiotics has played an essential role in addressing the issue of human infectious diseases. Still, the overuse of these antibiotics has become the primary factor for the emergence and discrimination of drug-resistant strains (Frieri et al., 2017). The increasing drug resistance is the main hindrance to the successful treatment of infectious diseases and the control of microbial pathogenicity (Fu et al., 2007). Therefore, the continuing spread of multi-drug resistant strains and the increased abuse of antibiotics highlight the need for alternative agents. Traditionally, herbs and spices have been used in many communities to manage several ailments, including antimicrobial substances (Dog, 2006;Leja and Czaczyk, 2016;Dini, 2018). Compared to conventional antibiotics, herbs, and spices are generally considered safe for humans owing to their long history of use in food preparation (Akullo et al., 2022). These plants fall under natural products, a significant source of new natural drugs.

BELLO, A.B; SULEIMAN, A; ATTEH, E.O; OLUTIMAYIN, T.A
Their use as an alternative medicine for various diseases has increased in the last few decades (Vuorelaa et al., 2004;Ansari et al., 2006). Compared to formulated drugs, herbs, and spices have fewer side effects. They are inexpensive, show better patient tolerance, and are readily available for low socioeconomic populations (Adeshina et al., 2011;Emmanuel et al., 2021). The antimicrobial activity of spices is due to specific phytochemicals or essential oils (Avato et al., 2000). The main factors that determine the antimicrobial activity are the type and composition of the spice, the amount used, the type of microorganism, the composition of the food, the pH value, and the temperature of the environment (Sagdic, 2003). Several reports have been published that describe the antibacterial and antifungal properties of different herbs and spices. However, still, there is little information about the exact mechanism of their antimicrobial action (Gur et al., 2006;Pattaratanawadee et al., 2006;Yusha'u et al., 2008;Belguith et al., 2010;Poeloengan, 2011;Gull et al., 2012). Ginger (Zingiber officinale) is a member of the family Zingiberaceae. It is widely distributed across the tropics of Asia, Africa, America, and Australia, where it is used as a spice and medicinal plant (Jung Park & Pezzuto, 2002). Several studies have reported on the antimicrobial activity of ginger and Garlic (Akintobi et al., 2013;Indu et al., 2006;Khashan, 2014;Mohammed et al., 2019). There is a broad spectrum of antibacterial activity of ginger rhizome against a range of Gram-negative and Gram-positive bacteria. However, conflicting reports exist about the antibacterial effectiveness of ginger against bacteria from different resources (Abdalla & Abdallah, 2018). Zingiberene in ginger rhizome oils is the most active antibacterial component (El-Baroty et al., 2010), and the therapeutic effectiveness of Garlic is attributed to its oil content and water-soluble organosulfur compounds (Prati et al., 2014). Studies on the diversity of ginger have shown that cultivated ginger exhibits variations in rhizomes and vegetative character, and environmental factors considerably influence the critical bioactive compounds (Kizhakkayil & Sasikumar, 2011;Akullo et al., 2022). Garlic (Allium sativum) belongs to the family Alliaceae, which has been found to help prevent many diseases. Numerous modern studies confirm that Garlic has definite antibiotic properties and is effective against a broad spectrum of bacteria, fungi, and viruses (Shuford et al., 2005;Low et al., 2008). In addition, Garlic's antimicrobial activities are linked to some bioactive compounds (Tsao & Yin, 2001). Due to the high morbidity and mortality burden of infectious diseases, many investigations have focused on understanding, controlling, treating, and preventing these diseases (Murugaiyan et al., 2022). Therefore, natural products are a source of medicine used as an alternative medicine for various diseases. This present study aims to study the inhibitory effect of Garlic (Allium sativum) and Ginger (Zingiber officinale) extract on some clinically pathogenic bacteria.

MATERIALS AND METHODS
Sample collection: Fresh garlic cloves and ginger rhizomes used in this study were obtained from the old market in Bida town. Nigeria. Preparation of Extracts:The aqueous and ethanolic extracts of ginger and Garlic were prepared separately. The fresh garlic cloves and ginger rhizomes were cut into pieces, air-dried at room temperature, pulverized into powder using a clean mortar and pestle, and then packed into clean bottles for further analysis. Fifty grams (50g) of Garlic and ginger powder was soaked in 120ml of distilled water and ethanol separately in a beaker. The beaker was allowed to stand at room temperature for 72 hours with intermittent agitation and filtered using Whatman paper No.11.2. The ethanol extract was evaporated at 40°C while the aqueous extracts were evaporated at 80°C using a rotary evaporator. The extracts were labelled and kept at 4°C to preserve the antibacterial property.
Culture Preparation. The clinical isolates were inoculated into the nutrient broth, allowed to grow overnight at 37°C, and adjusted to 0.5McFarland standard turbidity before performing the antimicrobial assay.
Antibacterial Activity Testing Using Agar Well Diffusion: The bacterial cultures were swabbed onto the surface of solidified Muller Hinton agar using a sterile cotton swab. The agar wells were prepared using a sterile 10mm diameter cork-borer. The wells were filled with different concentrations (125mg/ml, 250mg/ml, 500mg/ml, and 1000mg/ml) of the extracts. The plates were incubated uprightly at 37℃ for 18 hours. The diameters of growth inhibition were measured in millimeter (mm) and recorded. The inhibition zones with a diameter of less than 10 mm are considered to have no antibacterial activity (Dulger & Gonuz, 2004).

Determination of Minimum Inhibitory Concentration (MIC):
The tube dilution method was used to determine the minimum inhibitory concentration. The inoculums were prepared using 24-hour old cultures broth and were adjusted to 0.5McFarland standard turbidity. In the test tubes, 9ml of sterile nutrient broth was dispensed, 1ml of the extract of varying concentrations (125mg/ml, 250mg/ml, 500mg/ml, and 1000mg/ml) was added into different test tubes and 0.1ml of standardized organism was inoculated and incubated for 24 hours at 37oC. The test tube with the least concentration of extract that showed no turbidity was taken as the minimum inhibitory concentration (Mounyr et al., 2016).

RESULT AND DISCUSSION
The Inhibitory activity of Garlic (cloves) and Ginger extracts was investigated against four clinical isolates. Among the clinical isolates, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were more susceptible than Klebsiella. The inhibitory activity of garlic and ginger extracts revealed that Staphylococcus aureus (16mm) and Escherichia coli (15mm) were more susceptible to the aqueous extract of Garlic than the ethanol extract of Garlic (Tables 1 and 2). While in Ginger extract, Staphylococcus aureus (16mm) and Pseudomonas aeruginosa (14mm) were more susceptible to aqueous extract than the ethanol extract of ginger (Tables 3 and  4). The result of activity in this study is similar to previous reports by Gull et al., (2012), Riaz et al., (2015), and Anyamaobi et al. (2020); however, it is at variance with the result of Adeshina et al., (2011), which showed high susceptibility of Escherichia coli and Pseudomonas aeruginosa but no activity against Staphylococcus aureus. Furthermore, a study by Akintobi et al., (2013) reported that Ginger extract was ineffective in Pseudomonas aeruginosa, contrary to this study. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract on the isolates are shown in Table  5. The minimum inhibitory concentration for garlic extract was 125mg/ml against E. coli, S. aureus, and P. aeruginosa. In the case of the minimum inhibitory concentration, ginger recorded 125mg/ml against P. aeruginosa.    This indicates that the crude extracts of Garlic may yield potential molecules in treating infections caused by pathogenic bacteria. The extract's minimum bactericidal concentration (MBC) showed effectiveness at 1000mg/ml for E. coli, S. aureus and P. aeruginosa, and K. pneumoniae for both garlic and ginger extracts. The result is similar to the report by Adeshina et al., (2011), Gull et al., (2012, and Riaz et al., (2015), which stated that the higher the concentration of the extract, the greater the inhibitory effect of extracts. The phytochemical characteristics of garlic and ginger extracts are presented in Table 6. The result revealed the presence of alkaloids, flavonoids, tannins, saponins, and phlobatannins.  The presence of these components may be responsible for the antibacterial activity of the extracts. A study by Kumar et al., (2011), Kamppiah andRajaram (2012), and Riaz et al., (2015) also reported similar phytochemical compounds from garlic and ginger extract identified in this study.
Conclusion: This study revealed the potential antibacterial activity of Garlic and Ginger extracts against some clinically pathogenic bacteria isolates. Findings from this study have shown that aqueous extract of Allium sativum (Garlic) and Zingiber officinale (Ginger) showed the highest antibacterial activity, especially against Staphylococcus aureus. However, for the full potential of the extracts, more studies should be carried out to discover and validate active components responsible for antibacterial activity to serve as an alternative to antibiotics.