In-vitro Antibacterial Activity of crude Garlic (Allium sativum) Extract Against Clinical Isolates of Methicillin Resistant Staphylococcus aureus

1Department of Medical microbiology, Faculty of Medical Laboratory Science, Omdurman Islamic University. 2Department of Parasitology, Faculty of Medical Laboratory Sciences, Al Neelain University, Khartoum, Sudan. 3Department of Medical Laboratory Sciences, Collage of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudia Arabia. *Corresponding Author E-mail: lindarose009@hotmail.com

Plant-derived antimicrobials have extremely large therapeutic effects, and they are efficacious in treating infectious diseases even while reducing many of the problems associated with synthetic antimicrobials 1 . The different pharmacological effects of plant products are generally the result of secondary product combinations derived from plants. Such plant compounds are often secondary metabolites such as alkaloids, steroids, and fatty acid gums that have a certain physiological impact on the body 2, 3, 4 . There are many natural elements and chemicals in garlic, including sulfur compounds, numerous enzymes, different minerals, vitamins, and amino acids. Allicin (diallyl thiosulfinateor diallyldisulfide) is among garlic's greatest crucial biologically vigorous substances 5 . Even though allicin is deemed the prime antioxidant compound, other compounds may represent stronger roles; such as polar compounds of phenolic and steroidal origin, that display diverse pharmacological countenance without odor and are furthermore heat-stable 6 .
The efficacy and broad-spectrum antimicrobial activity of garlic versus huge species of bacteria (Staphylococcus aureus, Streptococcus pneumonia, Streptococcus fecalis, Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Shigella spp, Salmonella spp, Proteus mirabilis, and Helicobacter pylori), viruses, parasites, and fungi have been documented 7 , also it is much more efficacious than industrial antimicrobials and has low toxicity; as a consequence, can be used as an additional option for the treatment of numerous infections 8,9,10,11 . The antibacterial activity of garlic has been fundamentally attributed to the entity of allicin generated by the enzymatic activity of allinase on allicin 12 . Garlic is also efficacious toward antibiotic-resistant microorganisms, and combining garlic extracts with antimicrobial agents results in partial and total synergism 13 . The emergence of multidrug-resistant strains of Gramnegative and Gram-positive bacteria is troubling for humans and animals, as well as the emergence of epidemic MRSA, as a consequence, garlic is a popular alternative substance for the treatment of MRSA 14 . Hence current study aimed to determine the In-Vitro Antibacterial Activity of crude Garlic (Allium Sativum) extract against clinical isolate of Methicillin-Resistant Staphylococcus aureus (MRSA).

study design
A cross-sectional study was carried out during the period from April to June 2018 on a clinical isolate of MRSA from patients who were known to have MRSA after full microbiology identification methods, so specimens were collected from, wound, eye, sputum, and blood. Moreover Standard bacterial strains include American type culture collection (ATCC), S. aureus (ATCC 25923) used as control positive. And Sudanese garlic bulbs with small size and strong odor were obtained from the Khartoum Bahri market to determine their antibacterial activity. Study was approved by the College of High Graduate Studies ethical committee/Shendi University. sample size A hundred Isolates were collected randomly, 73 MRSA isolated from wounds, 3 eye swabs, 21 from blood culture, and 3 isolated from sputum samples. Clinical isolates collected according to availability regardless of their type, and all isolates show resistance patterns to Oxacillin were included in the study.

laboratory methods Plant materials collection
The Garlic bulb (seeds) was collected from Central Sudan, Khartoum Bahri market in April 2018, and it was checked by the Medicinal and Aromatic Plants professional at Traditional Medicine Research Institute (TMRI), Khartoum, Sudan.

Preparation of crude extracts
Extraction was carried out for the seeds of garlic plant 100 g by using maceration techniques after weighting the grounded material by sensitive balance about 34g round material was macerated equal quantity for both aqueous and 70% ethanol extract.
For preparation of aqueous extraction, about 100 g of the garlic was soaking in 100 ml hot distilled water for about four hours with continuous steering. After cooled, extract was filtered using filter paper and the solvents were evaporated using freeze drier, and the yield percentage were calculated as follow: Weight of extract obtained / weight of plant sample X100.
For obtaining 70% extraction of Ethanol, 100 g of garlic was grinding by mortar and impregnating in 70% ethanol for 5 days with liquidation and steaming on daily basis. David calculated the given percentages after the solvent was evaporated under low pressure to arid and the extract was allowed to air dry completely, then multiply the weight of the extract by the weight of the sample. Finally, it was stored at 4°C in a tightly bottled glass flask, ready for use.

Bacteria strains
Under aseptic conditions, 100 clinical isolates of MRSA were obtained from El Ribat University hospital's Microbiology laboratory in Khartoum, Sudan. The specimens were isolated from wound swabs, sputum, eye swabs, and blood culture and cultured in suitable media such as blood agar, chocolate blood agar, and McConkey agar and incubated aerobically at 37 0 C over night.
Identification was achieved depending on colonial morphology, the feature of culture media, gram stain, biochemical reactions (Catalase, coagulase, DNAse test, and Mannitol Salt Agar), and susceptibility to Oxacillin discs (1 µg) using Mueller-Hinton agar MHA; when the zone of inhibition is more than 12 mm were identified as sensitive and 11-12mm identified as intermediate 15,16 .

Catalase test
A pure of 2-3 ml of hydrogen peroxide solution was added in a test tube, by sterile wooden stick several colonies of test organisms were put in hydrogen peroxide solution. The positive results indicated by immediate bubbling 15 Coagulase test a colony of the test organism was emulsified in drop of physiological saline to make thick suspension and a drop of plasma was added to and mixed gently by rotating. The positive results indicated by producing clump within 10 seconds 15 .

deoxyribonuclease (dnase) test
The test organism was cultured on a medium which contains DNA. After overnight incubation, the colonies were tested for DNAse production by flooding the plate with a weak hydrochloric acid solution. The acid precipitated hydrolyzed DNA. DNAse producing colonies were therefore surrounded by clear areas due to DNA hydrolysis 15 .

detection of Methicillin resistance
MRSA identification was carried out using oxacillin screen plates following the guidelines of NCCLS. Briefly, a suspension equivalent to 0.5 McFarland standards, prepared from each strain, was inoculated homogenously on the entire surface of the Mueller-Hinton agar plate (Oxoid-UK) containing 4% NaCl and 6 ìg/mL oxacillin, with the help of sterile swabs. All the plates were incubated at 35 o C for 24 hrs. Indication of growth (>1 colony) identified the isolates as oxacillin/ methicillin-resistant (Genç et al., 2008) ,when zone of inhibition present above 12mm was identified as sensitive and 11-12mm identified as intermediate.

antibacterial susceptibility testing for extracts
The paper disc diffusion method was being used to evaluate the antimicrobial property of garlic extracts using MHA. The dilution factor of Bacterial suspension was done with a sterile physiological solution to 10 8 CFU/ ml (turbidity = McFarland standard 0.5). 100 µl of bacterial suspension swabbed regularly on MHA and was allowed to dry for 5 minutes. Then filter paper discs (Whatman No.1, 6 mm in diameter) was Sterilized in an oven at 80 o C for 30 minutes, and placed on MHA and soaked with 20 µl of a solution of each garlic extract with diluted different serial dilution as follow: 25%, 50%, 100%, 200% (0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml) respectively, next suspended by methanol for 70% ethanolic extract and the same concentration used after re-suspended by sterile distilled water for aqueous extract. The inoculated plates were incubated at 37°C overnight.

data analysis
All data were analyzed using (SPSS) software version 21, descriptive statistics (e.g., frequency and percentage), chi-square test, were used and the threshold for statistical significance was (p < 0.05).

results
A total of 100 clinical isolates were collected from wound swab 73%, eye swab 3%, sputum 3%, and blood for culture 21%. All isolates were positive for catalase, coagulase, Deoxyribonuclease (DNAse) test , and MSA as well as all isolates were showed growth on the Oxacillin disc, which indicates resistance.   Figure  3 display plates (a and c) showed antibacterial activity of 70% ethanol, and aqueous extract of A. sativum extract as well on clinical isolates, plate (b and d) showed antibacterial activity of 70% ethanol, and aqueous extract of A. sativum extract STD ATCC strain.

disCussion
Antibiotics' massive use in the treatment of infectious diseases has resulted in the emergence and development of resistant strains, which is now a significant cause of failure in the treatment of infectious diseases 17 . In this study, 70 % ethanol extract of crude A. sativum showed remarkable antibacterial activity against standard strains of S. aureus and clinical isolates methicillin-resistant that agreed with Janan et al 18 , while aqueous extract showed little activity, these were different from that obtained by Hadir et al 19 , may be referred to using the homogenization aqueous extraction technique. However, the opposite results of extract do not indicate less activity of bioactive constituents because the active ingredient (s) could be existent in inadequate amounts. , but the use of boiled water    Besides that, this could be due to various main: hydrogen bonding of water to the reactive oxygen atom in allicin can minimize its instability; and/or there could be water-soluble elements in minced garlic that destabilize the chemical compound that Lawson mentioned 21 .
The limitation of this strategy is that allicin can develop diallyl di-sulfide once it reacts with water 22, 23 , which may not have the same degree of antimicrobial effect as allicin. The yield percentage of ethanolic extract was noted 7.2 % slightly increased than aqueous 6.8 %, and that due to the time of extract according to the methodology of technique, in aqueous 4 hours only needed and then filtrated the final product in one day, but the ethanolic extract needed 5 days with daily filtration. This effect of time on yield and in sufficient quantity has a role in antibacterial activity. The most probable explanation for these differences between results of zone inhibition mm in this study compared with other studies may be due to the different bacterial strains tested, and the antibacterial activity methods, method of extract, and time, plus the concentration of organo sulfar compounds, which vary greatly between garlic species around the world 24 . A study by Bayan L et al 25 noted that one negative aspect in ascertaining the antimicrobial property of Garlic Extract is the inability to detect Methicillin Resistance results, the scientists used standardization in their methodologies, and this results in significant differences in the outcomes. As a result, it is crucial to establish policies and procedures for all guidelines used to assess the antimicrobial property of garlic extract. One major benefit of garlic is that bacteria need not appear to emerge resistant to it, as they do to several advanced antibiotics: "garlic does not seem to produce such resistant strains" Erdogrul, 26,27 . With greatly increased extract concentration, the mean diameter of the growth inhibition zone of standard and clinical isolates of bacteria enhanced. This outcome is in contact with the report of Mahfuzul Hoque et al. MD 28 . The best inhibition zone obtained by 70% ethanol extract of crude garlic was 20 mm in diameter against clinical isolates of MRSA strains at the concentration of 4 mg\ 2ml, (200% conc). And the minimum inhibition zone was 13 mm in diameter at 0.5 mg/ml (25% conc) on aqueous. Despite the fact that garlic has been postulated to have a synergistic or additive impact with regular antimicrobial agents against E. coli and S. aureus just a few studies have been conducted to demonstrate the effect of garlic when used in combination with standard antimicrobials 29,30 .

limitations of study and Prospective view
The extract's active garlic ingredient (Allicin) compounds are crucial for its antimicrobial properties. More research is required to verify these results and for evaluating the combination of garlic with other commercial antibiotics. so Broad extraction projects for garlic plant components is crucial to determine the best active ingredients by an ideal standard methods. Alternative methods to extract processing by solvents must be more used and tested as a squeezing technique to avoid the loss of bioactivity of components. one of the limitation of current study is that a commercial / clinical antibiotic) to show the efficacy of the garlic extracts were not used in this study.

ConClusion
The findings demonstrate that 70% ethanol extract of crude Allium sativum has significantly inhibitory effect on methicillinresistant Staphylococcus aureus is better than aqueous extract. From this, we can conclude that the potential of using alcohol -related extract of garlic as potential antimicrobial agents. This study does not undermine the value of antibiotic use, but instead the probability of using them in low dosage to minimize their negative consequences.

aCknowledgMent ethical statement
This article does not contain any studies with human participants or animals performed by any of the authors.