|Year : 2013 | Volume
| Issue : 2 | Page : 114-118
Studies on in vitro interaction of ampicillin and fresh garlic extract against Staphylococcus aureus by checkerboard method
R Pillai1, Niyati A Trivedi2, Jagat D Bhatt2
1 Department of Pharmacology, College of Dental Sciences and Research Centre, Bopal, Ahemdabad, Gujarat, India
2 Department of Pharmacology, Medical College, Baroda, Vadodara, Gujarat, India
|Date of Web Publication||18-Aug-2014|
Niyati A Trivedi
45, Shree Kunj Kutir, Opp. Yash Complex, 30 m VUDA Road, Gotri, Vadodara - 390 021, Gujarat
Source of Support: The authors acknowledge Alembic Pharmaceuticals
Ltd. for providing ampicillin as a gift sample and Department of
Microbiology for their technical support and guidance to carry out this
work., Conflict of Interest: None
Objective: Staphylococcus aureus has largely developed resistance to the multiple antimicrobial agents. Garlic (Allium sativum) is claimed to retard development of resistance and produce synergy with conventional antimicrobial agents. The aim of the present study was to evaluate the effect of combination of ampicillin with fresh garlic extract (FGE) against S. aureus by using in vitro method.
Materials and Methods: Antimicrobial activity of FGE was studied by well diffusion and broth dilution methods. The effect of combination of ampicillin with FGE was studied by using checkerboard method against the clinical isolates of S. aureus. The organisms were exposed to individual antimicrobial as well the different combinations of the two agents and minimum inhibitory concentration was measured.
Results: Synergistic interaction was observed by the combination of FGE with ampicillin for all the strains of S. aureus. Mean minimum inhibitor concentration (MIC) of ampicillin per se was
24 μg/ml. Addition of 30-60 mg/ml of FGE reduced MIC of ampicillin to <2 μg/ml.
Conclusion: This in vitro study indicates synergism between FGE and ampicillin against S. aureus. Evaluation of these combinations in vivo may be warranted.
Keywords: Ampicillin, checkerboard method, fresh garlic extract, Staphylococcus aureus
|How to cite this article:|
Pillai R, Trivedi NA, Bhatt JD. Studies on in vitro interaction of ampicillin and fresh garlic extract against Staphylococcus aureus by checkerboard method. Ancient Sci Life 2013;33:114-8
| Introduction|| |
Decades after the development of first antimicrobial agent, infectious diseases still remain the major cause of morbidity and mortality of mankind. As almost all antimicrobials are subject to the problem of antimicrobial resistance, we are in a constant need to develop novel therapeutic agents to fight against these microorganisms.
Antimicrobial drug resistance among Gram-positive pathogens is an ongoing worldwide therapeutic challenge. Since last 20 years, there has been an increase in methicillin-resistant Staphylococcus aureus (MRSA).  Vancomycin has remained the only treatment of choice available for many serious MRSA infections. However, recent reports , of unfavorable clinical response to even vancomycin have led to considerable concern about the management of serious infections caused by this pathogen.
Plants have been a valuable source of natural products for maintaining human health. Currently, interest has been focused on exploring antimicrobial properties of plants and herbs. One such botanical is Allium sativum (garlic).
Antimicrobial activities of garlic have been documented since a long time.  The antimicrobial properties of garlic were first described by Pasteur in 1958, and since then, research had demonstrated its effectiveness against bacteria, protozoa, fungi and some viruses. Even modern literature has documented antimicrobial activity of garlic against various Gram-positive and Gram-negative bacteria such as S. aureus, Escherichia coli, Streptococcus pneumonia, and Pseudomonas aeruginosa.
Though garlic has been proposed to exhibit synergistic or additive effect with conventional antimicrobial agents against E. coli and S. aureus, , only few studies have been conducted showing the effect of garlic when used in combination with conventional antimicrobials. Based on this information, this study was planned to evaluate the antimicrobial effect of fresh garlic extract (FGE) in combination with ampicillin using in vitro techniques against S. aureus isolated at SSG Hospital Baroda.
| Materials and methods|| |
Preparation of drugs
Preparation of fresh garlic extract
Garlic was purchased from the local market, garlic bulbs were peeled off and cloves totally weighing 100 g were crushed in a mortar and pestle and allowed to stand for 10 min and then squeezed out using sterile cheesecloth.  This filtrate was considered 100% FGE and was stored at −20°C and was thawed before use. The extract was prepared freshly every week.
The concentration used in this study is the total weight of garlic per milliliter. Ten grams of raw garlic yielded 4 ml of extract (i.e. 2.5 g/ml). This extract was considered as the 100% concentration of the extract. Each time 100% garlic extract was inoculated on nutrient agar media and incubated at 37°C overnight and was found to be sterile.
The stock solution of ampicillin (Alembic Ltd., Baroda, India) was prepared as per manufacturer's instructions.
Staphylococcus aureus were isolated in the microbiology laboratory of Medical College, Baroda from the samples of wound, pus and blood collected from the different clinical wards of the SSG Hospital, Baroda (n = 12). ATCC 25923 strain was kept as standard.
Isolation of pure culture
A nichrome inoculating loop was used to transfer the infective material onto the nutrient agar. The inoculum was spread into four quadrants of the nutrient agar plate with the help of a nichrome inoculation loop. The loop was sterilized between each successive quadrant streak. The purpose of this technique was to dilute the inoculum sufficiently on the surface of agar medium, so that well-defined colonies of bacteria known as colony forming units (CFU) can be obtained. The isolated colonies were sub-cultured individually to obtain pure culture isolates. The pure colonies thus obtained were picked up for confirmatory biochemical tests. Mannitol fermentation test, catalase test, and slide and tube coagulase tests were performed and the bacterium isolated was confirmed as S. aureus.
Preparation of standard inoculum
Pure colonies were picked up with the help of a straight wire and were emulsified in a test tube containing saline and the turbidity was matched with 0.5McFarland standard containing 1 × 10 7 CFU/ml approximately.
Method for antimicrobial susceptibility testing by well diffusion method
Mueller Hinton Agar (Hi Media, India) (pH 7.2-7.4) was poured in plates of 10 cm diameter and depth of agar was 3 mm. Using a sterile cotton swab, the test culture was spread evenly over the plate successively in three directions to obtain an even inoculum. The plates were allowed to dry for 5 min. Wells of 5 mm were cut on the surface of the agar. A volume of 100 μl of 12.5-2500 mg/ml w/v of FGE was added to different wells and in one well normal saline was added. The plates were incubated at 37○C for 24 h. The zone of inhibition was measured by a scale to the nearest mm including disc diameter.
Broth dilution method for determination of minimum inhibitory concentration
Broth dilution method was performed to determine the minimum inhibitory concentration (MIC) of FGE.  Wherein 1 ml of standard inoculum of the microorganism was added to each tube containing different concentrations of the garlic extract (15-360 mg/ml) in the final volume of 2 ml of broth and to a tube of the growth medium without FGE which served as growth control. An uninoculated tube containing medium was incubated to serve as a negative growth control.
After 24 h of incubation, the tubes were examined for turbidity, with the naked eye indicating growth of the microorganism. MIC was determined as the lowest concentration of the drug which prevented the visible growth of test isolates. ,
Checkerboard method for the combination
Antimicrobial effect of combinations consisting of FGE with ampicillin was assessed by checkerboard method test. , Serial dilutions of ampicillin (0.6-32 μg/ml) were mixed in a test tube containing varying concentration of FGE (15-180 mg/ml) in 2 ml of MH broth. A volume of 1 ml of standard inoculum of the microorganism was added to each tube after 24 h of incubation at 37○C.
Minimum inhibitory concentration isobologram
The calculated mean MIC values from the checkerboard assays were utilized to construct MIC isobologram,  which graphically represented the antimicrobial interactions between FGE with ampicillin. The MIC values of ampicillin and FGE were used as coordinate points in two-dimensional graphs. A line was drawn in the graph connecting the highest concentrations of each drug used to produce a reference line. Deviation from linearity relative to this drawn reference line was used as the basis for interpretation of antimicrobial relationship between FGE and ampicillin against S. aureus. A curved deviation of the isobologram to the left of the reference line indicates synergy of the antimicrobial agents, while a right deviation would indicate antagonism. An additive relationship between antimicrobials was represented by an isobologram with points falling proximate to the reference line.
Fractional inhibitory concentration index
To validate the MIC isobologram, we calculated the fractional inhibitory concentration (FIC);  the following formulas were used to calculate the FIC index: FIC FGE = (MIC FGE in combination)/(MIC FGE alone), FIC ampicillin = (MIC ampicillin in combination)/(MIC ampicillin alone), and the FIC index = FIC FGE + FIC ampicillin. FIC indices were used to characterize antibiotic interactions as follows: FIC index value of 1 indicates additivity between the, antimicrobial agents. A value <1 would imply synergism, while a value > 1 suggests antagonism.
One-way ANOVA with Tukey posthoc test was applied for comparison of all pairs of column for analysis of zone of inhibition obtained at different concentration of FGE.
Paired t-test was applied for comparison between MIC of drug alone and in combination. P ≤ 0.05 was considered as statistically significant.
| Results|| |
In well diffusion method, S. aureus showed statistically significant dose dependent increase in the zone of inhibition at FGE concentration 12.5 mg/ml and higher compared with the control (n = 12) Increasing concentration of FGE increases zone of inhibition.
Minimum inhibitor concentration of FGE against S. aureus was determined using broth dilution method. MIC value of FGE ranged from 75 to 120 mg/ml. As per well diffusion method, this concentration of FGE produces mean zone of inhibition of 20-24 mm [Table 1].
|Table 1: Mean zone of inhibition in mm for different concentrations of FGE in 12 clinical isolates of Staphylococcus aureus |
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All 12 S. aureus isolates used in the study were found resistant to ampicillin according to the National Committee for Clinical Laboratory Standards guidelines  with MIC ranging from 8 to 32 μg/ml.
ATCC 25923 standard strain used in the study was sensitive to the lowest concentration of ampicillin (0.06 μg/ml) and FGE (15 mg/ml) used in the study.
[Table 2] shows the results of checkered board method using 12 S. aureus against combination of FGE with ampicillin. In all, 12 clinical isolates of S. aureus, the FIC index produced by combination of FGE with ampicillin was <1.0, which indicates synergism. None of isolates showed antagonism.
Minimum inhibitor concentration isobologram
The MIC isobologram that established the interaction between FGE with ampicillin against S. aureus are presented in [Figure 1]. Maximum incorporation of 90 mg/ml of FGE with 24 μg/ml of ampicillin was used (mean MIC values) [Table 2]
|Table 2: MIC and FIC of various combinations of ampicillin and FGE against Staphylococcus aureus (n=12) |
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|Figure 1: Minimum Inhibitory Concentration (MIC) isobologram representing the combined effects of fresh garlic extract (FGE) and ampicillin on Staphylococcus aureus strains. Straight line represents the reference additive line. Points correspond to the MIC data from 12 checkerboard assays|
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Synergistic interaction was observed between FGE and ampicillin against all the isolates of S. aureus strains with all the MIC points falling leftward to the reference additive line. These findings match with that obtained with FIC index.
| Discussion|| |
In this study, MIC of FGE against S. aureus ranged from 75 to 120 mg/ml producing zone of inhibition of 20-24 mm. Zone of inhibition produced at the MIC value correlates with that obtained with other studies. ,
Very few studies have been conducted to determine the antibacterial effect of garlic in combination with conventional antimicrobials. Synergism has been reported between garlic and vancomycin on all vancomycin resistant enterococci and two vancomycin-sensitive strains. 
All 12 S. aureus isolate used in the study were found resistant to ampicillin  with mean MIC of 24 μg/ml. However, addition of 30-60 mg/ml of FGE reduced MIC of ampicillin to 0.6-1.2 μg/ml.
Thus, combinations of FGE with ampicillin can improve sensitivity of S. aureus to ampicillin thereby can help conserving the usage of vancomycin. This will not only significantly reduce the cost of the therapy but also significantly lower the adverse drug reactions due to potentially toxic drugs (vancomycin in this case).
Several other studies also report synergistic interactions between conventional antimicrobials and biological
Data from this study suggests that FGE based combination regimens may be a useful alternative option in the treatment of resistant S. aureus infections. This can help improve sensitivity of the older less toxic and comparatively cheaper drugs and can reduce the use of newer and costlier drugs like vancomycin.
Therefore, experimental infectious model by MRSA should be developed to evaluate the in vivo efficacy of garlic.
In summary, in vitro data of the present study suggests the possibility of an alternative option in the treatment of MRSA infections that could circumvent the selective pressure of glycopeptides. However, further detailed pharmacokinetic and pharmacodynamic studies are needed to study the effect of combination of FGE with various antimicrobials in vivo.
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[Table 1], [Table 2]
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