IN VITRO ANTIBACTERIAL ACTIVITY OF GARLIC AND SYNERGISM BETWEEN GARLIC AND ANTIBACTERIAL DRUGS

The continuous spread of multidrug-resistant pathogens has become a serious threat to public health and a major concern for infection control practitioners worldwide. In addition to increasing the cost of drug regimens, this scenario has paved way for the reemergence of previously controlled diseases and has contributed substantially to the high frequency of opportunistic and chronic infection cases in developing countries. The slow pace of newer antibiotic development has provided the need to explore nature in search of phytotherapeutic agents with novel targets and mode of actions. The practice of complementary and alternative medicine is now on the increase in developing countries in response to World Health Organization directives, culminating in several pre-clinical and clinical studies of plants used in folk medicine to treat infections (Ako-Nai et al., 2003; Fernandez et al., 2003).


INTRODUCTION
The continuous spread of multidrug-resistant pathogens has become a serious threat to public health and a major concern for infection control practitioners worldwide.In addition to increasing the cost of drug regimens, this scenario has paved way for the reemergence of previously controlled diseases and has contributed substantially to the high frequency of opportunistic and chronic infection cases in developing countries.The slow pace of newer antibiotic development has provided the need to explore nature in search of phytotherapeutic agents with novel targets and mode of actions.The practice of complementary and alternative medicine is now on the increase in developing countries in response to World Health Organization directives, culminating in several pre-clinical and clinical studies of plants used in folk medicine to treat infections (Ako-Nai et al., 2003;Fernandez et al., 2003).
Garlic (Allium sativum L.) is one of the oldest vegetables, and has been used as a medicinal plant (Vijaya and Ananthan, 1997).In particular, because garlic has a strong antimicrobial activity and strong odor, people have used garlic to preserve meat and fish and to mask the smell of their sometimes rotting foods.Historically, the antimicrobial activity of garlic was recognized by Louis Pasteur (1822-1895), who used garlic juice to treat infections, and Albert Sch-weitzer (1875Sch-weitzer ( -1965)), who treated amoebic dysentery only with garlic (Fenwick and Hanley, 1985;Dilhuydy, 2003).Since the discovery of antibiotics, people no longer favor garlic in medical use.However, interest in garlic has been revived by the discovery of its antibacterial principle, alk(en)yl thiosulfinates (allicin), by Cavallito et al (1945).The growth of more than 300 kinds of microbes has been found to be inhibited by allicin (Reuter et al., 1996), and numerous reports have described the antibacterial potential of allicin in comparison with that of clinically effective antibiotic drugs (Lawson et al., 1991;Kivanc and Kunduhoglu, 1997;Okochi et al., 2000).Variations in composition of garlic and genetic disparity among bacteria of the same or different species have been responsible for a few inconsistencies in the antibacterial properties of garlic extract (Ankri and Mirelman, 1999;Cutler and Wilson, 2004), necessitating the need for local antibacterial testing of garlic.
The aim of the present study was to investigate the antibacterial activity of an ethanol garlic extract (EGE) against local multidrug-resistant bacteria.

Garlic and ethanol extract preparation
Fresh bulbs of garlic (Allium sativum L.) were purchased from local markets in Belgrade, Serbia.The cloves were separated and peeled to obtain the edible portion.Fifty grams of the edible portion was chopped and homogenized in 100 mL of 96% ethanol (Zorka Šabac, Serbia).The homogenate was then filtered through a 25-mm pore-size filter to give a crude ethanol extract of 500 mg of garlic/mL.This was collected in a sterile vial and stored at 4ºC until used.

Bacterial isolates
A total of 36 Gram-positive and Gram-negative bacterial isolates were used in this study; four strains per species or serotype of listed bacteria were selected: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus sciuri, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Salmonella enteritidis, Pseudomonas aeruginosa and Acinetobacter baumannii.The isolates were obtained from the Bacteriology Department of Clinical Center of Serbia, Belgrade, Serbia.The isolates were identified using standard procedures (Murray and Baron, 2003).All the isolates were clinical strains recovered from patients with burns, diarrhea, bacteremia, bronchopulmonary disorder, and urinary tract infections.The antibacterial susceptibility profile of the tested organisms was detected by the disk diffusion method according to the Clinical Laboratory Standard Institute (CLSI) (CLSI, 2010).Stock cultures of bacteria were maintained on Tryptone Soya Agar (TSA, bioMerieux, France) at 4°C.Prior to inoculation, all strains were transferred from the stock cultures to TSA and incubated overnight at 35°C.The grown cultures were used for the preparation of suspensions in sterile suspension media (SM, bioMerieux) with densities adjusted to 0.5 McFarland standard.As needed, the obtained suspensions were further diluted in SM to achieve the appropriate number of cells per ml.

Antibacterial activity
The antibacterial activity of garlic extract was determined by two techniques: the agar diffusion method and agar dilution method.
For the agar well diffusion method, all bacterial strains were used.Suspensions of bacteria containing 10 6 CFU/ml were inoculated onto plate surfaces with a sterile cotton swab.Test plates (diameter 10 cm) were prepared with 20 ml of Mueller-Hinton agar (bioMerieux) and holes of 6 mm in diameter were punched in the agar plates.Each hole was filled with 50 μl of EGE or, as the control, with 50 μl of 95% ethanol.The diameters of the growth inhibition zones around the holes were measured after incubation for 24 h at 35°C.The agar dilution method was performed with 18 bacteria, two strains per species or serotype.Test plates were prepared with 19 ml of Mueller-Hinton agar, and 1 ml of 2-fold dilutions of EGE.A control plate was made with 1 ml of 95% ethanol.After cooling and drying, the plates were spot inoculated with 10 μl of the 10 4 CFU/ml suspension.Plates were incubated for 24 h at 35°C, and the minimal inhibitory concentrations (MIC) were defined as the lowest  concentration of EGE that inhibited visible growth of bacterial spots.

Synergy assay
The synergy assay was based on a previously described procedure (Mirzoeva et al., 1997).The synergistic action of EGE with antibiotics was assayed by the disc diffusion method on Mueller-Hinton agar.

Statistical analysis
Results from the synergism assays were subjected to the Wilcoxon nonparametric test to compare the values (mm) of the inhibitory zones obtained by the disk diffusion method (Minitab Statistical Software version 13.32).Results were considered significant when p< 0.05.

RESULTS
Results of the evaluation of antimicrobial activities of EGE obtained by the agar diffusion method showed significant antimicrobial activities against Grampositive bacteria, but far less against Gram-negative bacteria (Table 1).S. aureus was the most susceptible bacteria tested in this study (Fig. 1), and P. aeruginosa was the most resistant analyzed bacteria (Fig. 2).
More precise information on the antimicrobial activities of EGE was obtained by the agar dilution method.The MIC for S. aureus, S. epidermidis, S. sciuri and A. baumannii was 25 mg/ml, and for E. faecalis, E. coli, K. pneumoniae, S. enteritidis, P. aeruginosa, 50 mg/ml.
The results of synergy between the antibacterial agents and EGE on the multiresistant strains tested in this study are presented in Table 2.

DISCUSSION
Garlic has been known for ages to exhibit anti-infective properties against a wide range of microorganisms (Cavallito et al., 1945;Ali Aydin et al., 2007).
The present study has further demonstrated the antibacterial potency of EGE against local multidrug-resistant bacteria isolated in Belgrade, Serbia.The observed zones of growth inhibition on agar of Gram-positive and Gram-negative bacteria show that the isolates exhibited susceptibility.This indicates that EGE has a broad spectrum of antibacterial activity and a wide therapeutic scope.
Although the antibacterial properties of EGE have been the subject of many investigations (Perez-Giraldo et al., 2003;Cutler et al. 2004;Noori et al., 2007), it is difficult to compare the results of different studies due to the different compositions of EGE and/ or different methods used for the evaluation of EGE antibacterial activities (Queiroz et al., 2009).However, it is generally recognized that Gram-positive bacteria are more susceptible to antibacterial action of EGE than Gram-negative bacteria (Cutler and Wilson, 2004;Kim et al., 2004;Ali Adydin, 2007), which is confirmed in this study on various microorganisms.The result of particular interest in this study is that the resistance of the tested bacteria to antibiotics had no influence on susceptibility to EGE.
The isolates tested in this study are responsible for many diseases in Serbia, including nosocomial infections, bacteremia and diarrheal diseases (Wu, 2008).The sensitivity of these isolates to EGE implies that the intrinsic biosubstances in this extract are naive to the various drug resistance factors of the isolates, which include beta-lactamase expression, increased pyrrolidonyl arylamidase activity, aminoglycosidemodifying enzymes, and altered ribosomal binding.
Several studies, including those of Kim et al. (2004) and Lacombe et al. (2010), had previously demonstrated the antibacterial potency of EGE against enteropathogens such as Vibrio parahaemolyticus, E. coli, Klebsiella spp., Proteus spp., and S. aureus.In spite of geographical variation, the MICs of EGE for our isolates are consistent with those of Yin and Cheng (2003) and Yang et al. (2011), but are relatively lower than values obtained by Kim et al. (2004).This antibacterial potency disparity of garlic has been attributed to the different concentrations of individually and synergistically active biosubstances in garlic preparations, coupled with their interactions with sulfhydryl agents in culture media.This phenomenon has been used to explain the stronger antimicrobial effect of allicin than garlic oil disulfides (Fujisawa et al., 2009).
The results of the synergistic action of EGE with antibiotics demonstrated the potential of garlic to enhance antibiotic action.Thus, they support previous findings on the synergistic action between antibiotics and garlic (Betoni et al., 2006;Cai et al., 2007).However, EGE showed different potential to different antibiotics and bacteria.The inhibition zones around the disks of antibiotics, on which EGE had influence, were larger in Gram-positive bacteria then in Gramnegative bacteria.A combination of garlic and antibacterial agents could allow a reduction in the dose of selected antibiotics and potentiation of the antibacterial therapy (Palaksha et al., 2010).
In conclusion, this study has shown that garlic has significant antibacterial potential against bacteria, but the effect is species-dependent.The resistance of the tested bacteria to antibiotics has no influence on susceptibility to EGE.The illustrated ability of garlic to enhance antibiotics is of potential medical interest.Therefore, complementary and alternative medicine practices with plant extracts, including garlic, as a means of decreasing the burden of drug resistance and reducing the cost of the management of disease, could be of clinical and public health importance in this country.

Table 2 .
Synergism between antibiotics and EGE