Anti-Bacterial Effect of Garlic (Allium sativum) against Clinical Isolates of Staphylococcus aureus and Escherichia coli from Patients Attending Hawassa Referral Hospital, Ethiopia

Garlic (Nechshinkurt in Amharic/Local name/) (Allium sativum L.) is under family Liliacea. It is an erect annual herb with superficial adventitious roots, bulbs composed of a disk like stem [2]. It has long tradition as medicinal plant, started with a direction of preparing a medicinal remedy written in a cuneiform character in about 3000 BC. Scientific investigations of various garlic preparations began in 1939 [3]. There are a number of studies carried out to assess the value of herbal remedies including garlic preparations for treat of illness [1,2,4].


Introduction
The use of higher plants and preparations from them to treat infections is an age-old practice. Interests in plants with antimicrobial properties has come to use again because of emergence of resistance strains against antimicrobials such as penicillin [1].
Garlic (Nech-shinkurt in Amharic/Local name/) (Allium sativum L.) is under family Liliacea. It is an erect annual herb with superficial adventitious roots, bulbs composed of a disk like stem [2]. It has long tradition as medicinal plant, started with a direction of preparing a medicinal remedy written in a cuneiform character in about 3000 BC. Scientific investigations of various garlic preparations began in 1939 [3].
There are a number of studies carried out to assess the value of herbal remedies including garlic preparations for treat of illness [1,2,4].
Garlic is an anti -bacterial agent that can actually inhibits growth of infectious agents and at the same time protect the body from the pathogens. It is known that the most sensitive bacterium to garlic is the deadly Bacillus anthracis which causes the diseases anthrax.
Even the forefather of antibiotic medicine Louis Pasture acknowledged garlic to be an effective antibiotic. Some years later garlic was shown to have similar effect/activity as penicillin. Later studies should similar activity to modern antibiotic including Chloramphenicol. Even the blood of garlic eaters can kill bacteria and it is also reported that the vapor from freshly cut garlic can kill bacteria at a distance of 20 cm! The other, the common and apparently returning diseases tuberculosis was treated with garlic very successfully as invading Mycobacterium tuberculosis is sensitive to several of the sulphur components found in Garlic [6,7].

Allicin
Allicin is the antibacterial component found in Garlic. A molecular mechanism may be the basis for some of garlic's therapeutic effects. The researchers were able to study how garlic works at molecular level using allicin, garlic's main biologically active component [7]. Allicin created when garlic cloves are crushed, protects the plant from soil parasites and fungi and is also responsible for garlic's pungent smell.
It is a natural weapon against infection that disables dysentery causing amoebiasis by blocking two groups of enzymes, cysteine proteinases and alcohol dehydrogenases. Cysteine proteinases enzymes are the main culprits in infection, providing infectious organisms with the means to damage and invade tissues. Alcohol dehydrogenase enzymes play a major role in these harmful organisms' survival and metabolism. Because of these groups of enzymes are found in a wide variety of infectious organisms such as bacteria, fungi and viruses. This research provides scientific bases for the notion that allicin is a broad-spectrum antimicrobial, capable of warding off different types of infections.
It is likely that bacteria would develop resistance to allicin because this would require modifying the very enzymes that make their activity possible scientists found that allicin blocks the enzymes by reacting with one of their important components known as self hydyl (SH) groups, or thiols this finding has important implication because of sulfhydryl groups are also crucial component of some enzymes that participate in the synthesis of cholesterol "Garlic lowers the level of harmful cholesterol'' [7][8][9][10][11][12][13][14].

Review on test organism
Staphylococcus aureus is very important pathogen that causes a variety of diseases including skin infections. Gastro intestinal disease (*staph*) food poisoning, toxic shock syndrome and nosocomial infections acquired during hospitalization.
Staphylococcus aureus is isolated on Mannitol salt agar which is a medium that is high in salt (75% NaCl) and contains Mannitol as a source of carbon and energy. S. aureus ferments the mannitol and causes the medium turn yellow.
Escherichia coli is a bacteria that exists naturally as part of normal gut of healthy humans and mammals. It causes enteric diseases. However, there are relatively few strains of this strains pf this organism, which are pathogenic to humans and are associated with food related illness [10].

Strains of S. aureus (MRSA)
There is a growing medical problem due to increasing frequency of infection caused by penicillin resistant Staphylococci. B-lactamase producing strains of S. aureus that are resistant to penicillin first appeared in clinical specimens in early 1950s. Soon thereafter, multiple antibiotic resistance was detected in chemical isolates of S. aureus; these strains was resistant to macrolide antibiotic, amino glycoside, and tetracycline.
Plasmids and transposons are clearly important in conferring and transferring antibiotic resistance between bacteria.
Although plasmids and transposons are certainly involved, the actual evolutionary mechanisms underlying this phenomenon have yet to be explained. One this consequence is the emergence of epidemics hospitals strains of S. aureus that is resistant to virtually all useful antibiotics, including methicillin and vancomycin. These strains are currently a significance cause of nosocomial (hospital acquired) infections in parts of the world [11].

Ethical clearance
Ethical clearance was obtained from Ethical board of Debub University and support letter were delivered to Ethiopian Public Health Institute and Hawassa Referral Hospital.

Plant materials
Garlic solution was obtained not from the whole part of the plant rather on the bulbs. The bulb of Garlic were peeled, weighted and then ligated using pestle while adding small amount of H 2 O. The extracts then allowed to freeze at -18°C (deep freezing) so as to concentrate the chemical (allicin). Then after freezing, the filtrate was put into lyophilizer till an amorphous powder weighted and then diluted with distilled water and used for the experiment [12].

Test organism
Two test organisms (S. aureus and E. coli) which were clinically isolated from patients were collected from Awasssa Referal Hospital.
Two standard pure cultures were collected from EHNRI (Ethiopian Health and Nutrition Research Institute). Both clinical and standard were sub cultured into subsequent Nutrient broth and definite Medias in which they favored i.e., Mannitol salt agar (MSA) for S. aureus and Mackonkey for E. coli were prepared on a slant and on petri dishes [10].

Inoculum
Cultured of the test organisms were maintained on nutrient both. Briefly, four to six colonies were picked with an inoculating loop and suspended in 5 ml of broth and incubated at 37°C for 24 hours. The turbidity of the broth culture was then equilibrated to match that of 0.5 Macfarlands standards. This provides organisms in the range of 1 × 10⁶ to 5 × 10⁸ cfu/mol which is pathogenic that used for the test [12].

Antibacterial activity test
The antibacterial activity test of the crude extract of Garlic against both standard and clinical isolates were carried out by the Agardiffusion method [13].

Agar diffusion method
The molten agar will mixed with a different concentrations of the test samples at molten state 45-50°C and mixed aseptically with different amounts of garlic extracts to a concentration of 0.25 ml, 0.5 ml, 0.75 ml and 1.5 ml which is equivalent to 5 mg/ml, 10 mg/ml, 15 mg/ml and 25 mg/ml of media. Then, the prepared media were let to solidify. A separate agar plate without sample or drugs was also prepared in order to provide an appropriate growth of organisms. (As the same time as control) Two standard drugs as a positive control were also tested against these microorganisms. These were chloramphenicol 0.30 mg and penicillin 0.30 mg. The negative control used in the cork borer as well as the solvent i.e., distilled water.
Antibacterial effect was determined by direct visual comparison of the growth of the test cultures. All the tests were carried out in triplicate and the results were reported as the averages of these replications.

Cork borker method
In this methods 0.2 ml of garlic extract was mixed with 20 ml of sterile nutrient agar using a mixer (vortex), and then poured into sterile Petri dishes. After congealing, the seeded agar was punched out with a sterile bore (back hole of 10 ml pipette diameter=9 mm) at equally spaced out positions to make four holes. Four of the holes were filled with 0.1 ml of the test sample solution while the fifth with standard antibiotics (chloramphenicol+distilled water) per hole. The plates were then left at room temperature for 2 hours (to favor diffusion over microbial growth) and incubated in an incubator at 37°C for 24 hours. As mentioned before each sample done in triplicate.
The antibacterial activity was evaluated by measuring the diameter of the zone of inhibition using ruler (the media in both methods was prepared according to the instructions that the manufacturer orders (written in the flask that for how many gm of powder how money ml of distilled water is enough).

Results
The results of the susceptibility of the test organisms against the garlic extracts which are stated below in Tables 1a and 1b. Table  2 showed that both clinical isolates of S. aureus and E. coli were sensitive to the concentration of 15 mg/ml (0.75 ml/20 ml of agar media) which is about 80% but about 10% of the organisms were not sensitive for lower concentrations i.e., for 0.25 ml in media.
In addition, larger clear zones were observed at higher concentrations against both microorganisms. Comparatively, Garlic extracts inhibited bacterial growths than Tetracycline and Penicillin.

Discussion
The result showed the both clinical and standard isolates of S. aureus and E. coli were highly sensitive to concentrations of 0.75 ml/20 ml of agar media in using diffusion method and Cork borers. Moreover, unlike clinical isolates of S. aureus, clinical isolate of E. coli was a bit resistant/not sensitive/ at concentration of 0.5 ml/28 ml of media. This could be in regard with the nature permeability of E. coli, which means 20% of membrane of E. coli is made of lipid while that of S. aureus is only made of 2% lipid [2]. Therefore, the garlic extract was more important for the prevention of resistant S. aureus which is currently becoming a challenge developing resistance to many commercially available drugs like penicillin.

Clinical
S. aureus -  In this study we have observed that, as the concentration of the garlic extract increases we have seen efficiency increased and hence inhibition and growth of test bacteria has been diminished. As observed from the above tables, Larger clear zones at higher concentrations and lower clear zones at lower concentrations. This implies that, Garlic has both bacteriostatic and bactericidal effect.

Conclusion
Based on the results of this study which showed garlic to make large clear zones than currently available antibiotics used in the study Garlic could be used as an effective antibacterial agent in Ethiopia where S. aureus is known to be resistant.
It could be made as a tablet in the best concentrations and affordable dosages so that it can be used as medicine to these two pathogenic gastro intestinal enteric. In the era of those drug resistant bacteria, we need to focus on alternative drugs that have long history to avoid such emerging diseases and that could be easily available and affordable.

Recommendations
One can use garlic as a member of the daily diet for better health especially the fresh garlic. This research can be used as a base to well identify the actual allicin minimum inhibition concentration.

Consent to Publish
Not applicable.