Year : 2007 | Volume
: 25 | Issue : 4 | Page : 164--168
Inhibitory activity of garlic (Allium sativum) extract on multidrug-resistant Streptococcus mutans
MM Fani1, J Kohanteb2, M Dayaghi2,
1 Department of Oral Medicine, School of Dentistry, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Medical Microbiology, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
M M Fani
Department of Oral Medicine, School of Dentistry, Shiraz University of Medical Science, Shiraz
Garlic ( Allium sativum ) extract has been known to have inhibitory activity on various pathogenic bacteria, viruses and fungi. The objective of present investigation was to study in vitro inhibitory activity of garlic extract on multidrug-resistant (MDR) strains of Streptococcus mutans isolated from human carious teeth. Filter sterilized aqueous extract of garlic was prepared and used in the present study. For isolation of S. mutans , extracted human carious teeth were cultured in Todd-Hewit broth and Mitis-Salivarius-Bacitracin agar. S. mutans was characterized by colony morphology, biochemical tests and other conventional bacteriological procedures. Disk sensitivity tests and broth dilution methods were used to determine antibiotic sensitivity profile and inhibitory activity of garlic extract on S. mutans isolated from carious teeth. Of 105 carious teeth tested, 92 (87.6%) isolates of S. mutans were recovered, among which 28 (30.4%) were MDR since they were resistant to four or more antibiotics. The highest rate of resistance was observed for tetracycline (30.4%) and least resistance (0%) to teichoplanin and vancomycin while 22.8% and 23.9% of the isolates were resistant to penicillin and amoxicillin, respectively. Chlorhexidine minimum inhibitory concentration (MIC) for MDR and non-MDR S. mutans varied from 2 to 16 µg ml−1 and from 0.25 to 1 µg ml−1 , respectively ( P < 0.05). All isolates, MDR and non-MDR of S. mutans were sensitive to garlic extract with the MIC ranging from 4 to 32 mg ml−1 . Considering in vitro data obtained in the present study, mouthwashes or toothpaste containing optimum concentration of garlic extract could be used for prevention of dental caries.
|How to cite this article:|
Fani M M, Kohanteb J, Dayaghi M. Inhibitory activity of garlic (Allium sativum) extract on multidrug-resistant Streptococcus mutans.J Indian Soc Pedod Prev Dent 2007;25:164-168
|How to cite this URL:|
Fani M M, Kohanteb J, Dayaghi M. Inhibitory activity of garlic (Allium sativum) extract on multidrug-resistant Streptococcus mutans. J Indian Soc Pedod Prev Dent [serial online] 2007 [cited 2020 Jan 27 ];25:164-168
Available from: http://www.jisppd.com/text.asp?2007/25/4/164/37011
Streptococcus mutans are gram-positive cocci, nonmotile facultative anaerobic microorganism which can metabolize carbohydrates and are considered to be the principle etiological agent of dental caries.  The cariogenicity of this bacterium is associated with various factors including dextran, production of high concentration of acid in the plaque and glucosyl transferase activity. Water insoluble glucans are significant constituent of dental plaque biofilms that facilitate adherence and accumulation of S. mutans and other oral bacteria. The biofilm formation is influenced by the amount of glucosyl transferase produced by S. mutans . S. mutans are the most common pathogens isolated from human dental plaque and their prevalence has been reported in several epidemiological studies. ,
Dental caries is considered to be a preventable disease. Prevention requires minimizing the frequency of ingesting simple carbohydrate foods and beverages, regular oral hygiene measures to remove plaque and to introduce topical fluoride in the form of toothpaste. It is recognized that even when the appropriate preventable measures are taken, there are some individuals who have increased susceptibility to dental caries. The discovery of the role of fluoride in dental caries prevention led to significant decrease in caries prevalence in various countries , and as a result, community water fluoridation and widespread use of fluoridated tooth paste were highly considered.
Since the discovery of S. mutans as an etiological agent of dental caries, much attention was focused on this bacterium, as a target for the prevention of disease through the use of antimicrobial agents and vaccine preparation.
Application of antibiotics for prevention and treatment of dental caries would be harmful for the patients and the development of multidrug-resistant (MDR) strains of bacteria is also possible.
Worldwide, hundreds of plants are used in traditional medicine as treatment for bacterial infections. Conventional drugs usually provide effective antibiotic therapy for bacterial infections; however, there is an increasing problem of antibiotic resistance and a continuing need for new solutions. Although natural products are not necessarily safer than synthetic antibiotics, some patients prefer to use herbal medicines. Thus, health care professionals should be aware of the available evidence for herbal antibiotics.
Garlic ( Allium sativum ) is one of the most extensively researched medicinal plants and its typical odor and antibacterial activity depends on allicin produced by enzymatic activity of allinase (a cysteine sulfoxide lyase) on alliin after crushing or cutting garlic clove. , Allicin and other thiosulfinates are believed to be responsible for the range of therapeutic effects reported for garlic. There are extensive literature on antibacterial effects of fresh garlic extract. Garlic extract has been reported to inhibit growth of various gram-positive and gram-negative bacteria including: Micrococcus, Enterobacter, Escherichia, Klebsiella, Lactobacilli, Pseudomonas, Salmonella, Shigella, Proteus and Helicobacter pylori . ,,,,
Garlic extract is also active against MDR organisms such as Pseudomonas aeruginosa , Klebsiella pneumoniae and Mycobacterium tuberculosis . ,, The antifungal  and antiviral  activity of garlic extract has also been reported. The objective of the present investigation was to determine the inhibitory activity of garlic extract on S. mutans using disk-diffusion and broth dilution methods.
Materials and Methods
Garlic extract preparation
Garlic extract was prepared according to method described by Bakri and Douglas.  Briefly, the peeled fresh garlic (80 g) was chopped and homogenized in 100 ml sterile distilled water, centrifuged, filtered through Wattman No 1 filter paper and then sterilized by filtration (0.45 mm). By subtracting the weight of insoluble material from the weight of original cloves, the final concentration of garlic extract in solution was determined to be 64% (w/v). The filtrate was then kept at -70°C until used. The concentration of allicin in each preparation was determined spectrophotometrically by reaction with the 4-mercaptopyridine and was estimated to be 260 mg/ml according to Miron et al . 
Isolation of S. mutans from carious teeth
Extracted carious teeth were used for isolation of S. mutans . The teeth were incubated in 10 ml Todd-Hewit broth (Merk, Germany) at 37°C in the presence of 5% CO 2 for 48 hours. A Mitis-Salivarious-Bacitracin agar (MSBA) medium was then subcultured from Todd-Hewit broth and incubated at 37°C in the presence of 5% CO 2 for 72 h. S. mutans were then identified on the basis of colony morphology (e.g., greenish hemolysis) and biochemical tests including optochin, catalase, voges Proskauer, arginine dihydrolases and hippurate hydrolysis. S. mutans were further identified by positive fermentation of glucose, manitol, raffinose, melbiose and sorbitol.  In the present study, 105 carious teeth were examined and a total of 92 isolates of S. mutans were identified. Pure culture of these isolates were prepared on MSBA and was subjected to susceptibility testing to antibiotics and garlic extract. The carious teeth of those who had received antibiotics within the previous 3 months or with systemic disease were excluded.
Antibiotic sensitivity test by disk diffusion
The antibiotic sensitivity profile of the 92 S. mutans isolates were determined according to the method of Bauer-Kirby  using disks of antibiotics placed on the surface of MSBA medium seeded with the test organism. Inhibition zones were measured after 72 h of incubation at 37°C in the presence of 5% CO 2 . Interpretation of resistance was based on the National Committee for Clinical Laboratory Standards (NCCLS) criteria. The antibiotics used were penicillin, amoxicillin, tetracycline, imipenem, ceftriaxone erythromycin, clindamycin, rifampin, vancomycin and teichoplanin. Twenty-eight isolates of the S. mutans which were resistant to four or more of the above mentioned antibiotics were considered as MDR strains and were used for determination of minimum inhibitory concentration (MIC) of antibiotics and garlic extract.
The lowest concentration of the drug that inhibited the growth of the organism was considered as MIC.  Plate dilution method was used for determination of the MIC's of antibacterial agents on each 28 MDR isolates of S. mutans . The following concentrations of antimicrobial agents expressed as mg/ml were used: penicillin (0.015-16), erythromycin (0.04-64), vancomycin (0.5-8), tetracycline (0.25-32), imipenem (0.03-1), ceftriaxone (0.015-16), amoxicillin (0.03-32), teichoplanin (0.06-32), clindamycin (0.03-8) and rifampin (0.25-128).  Pure culture of each isolates of S. mutans were prepared and 10 4 CFU/spot were seeded on Mueller-Hinton agar supplemented with 5% sheep blood and doubling concentrations of antimicrobial agents.  The plates were incubated at 37°C in the presence of 5% CO 2 for 24 h and results were recorded. Chlorhexidine at concentrations of 0.25-32 µg ml−1 and S. mutans ATCC 25175 were used as control at each time.
The susceptibility breakpoint concentrations were in accordance with the interpretative standards for Streptococci other than Streptococcus pneumoniae recommended by the National Committee for Clinical Laboratory Standards. , Strains were classified for penicillin susceptibility as follow: susceptible (MIC ≤ 0.125 mg/ml); intermediately resistant (MIC = 0.25-2 mg/ml); resistant (MIC ≥ 4 mg/ml).
Antibacterial tests on garlic extract
Disk diffusion assay and broth dilution methods were used for determination of antibacterial activity of garlic extract on 28 MDR and 28 non-MDR isolates of S. mutans .
Disk diffusion assay
Bacterial suspension of each of the S. mutans isolates, equivalent to No 0.5 Macfarland was prepared in Mueller-Hinton broth, diluted by 1:10, and 0.1 ml of this dilution was streaked on a MSBA medium. A 6 mm diameter of sterilized Wattman filter paper No 1 (Rund filter, Macherey-Nagel, D-5160 Doren Germany, Werkstrabe 6-8) was soaked in 0.5 ml garlic extract, placed on the above-mentioned MSBA and incubated at 37°C, in the presence of 5% CO 2 , for 72 h. The diameter of the zone of inhibition around the disk was measured in mm and recorded. The test was repeated three times and the mean diameter of the zone of inhibition was determined. Filter papers soaked in chlorhexidine and saline were used as positive and negative control, respectively.
Broth dilution method
This method was applied for measurement of garlic extract MIC. Briefly, each isolate of S. mutans was grown to stationary phase in cation-adjusted Mueller-Hinton broth, according to NCCLS guide lines.  Each cell suspension was adjusted spectrophotometrically to approximately 10 4 CFU/ml. Garlic extract concentration ranged from 256 to 2 mg/ml, and 25 ml of the bacterial cell suspension was added to the serially diluted garlic extract. All incubations were at 37°C in the presence of 5% CO 2 for 24 h and the highest dilution where there was no growth was recorded as the MIC of garlic extract.
In the present study, out of 105 carious teeth, 92 (87.6%) were positive for S. mutans . [Table 1] represents the resistance rate of these isolates to the antibacterial agents. The least resistance was observed for vancomycin and teichoplanin while the highest rate of resistance was observed for tetracycline. Of 92 isolates, 28 (30.4%) were MDR strains, as they were resistant to at least four antibiotics. [Table 2] represents the resistance patterns of 28 MDR isolates. Five different patterns were identified: 8 isolates were resistant to 5 (pattern #1), 19 to 4 (patterns # 2,4,5) and 1 to 8 antibiotics (pattern#3). None of the isolates were resistant to vancomycin and teichoplanin. Minimum inhibitory concentration of vancomycin and teichoplanin for the 28 MDR strains ranged from 0.125 to 1 mg/ml and 0.015 to 0.06 mg/ml, respectively. Ten (35.7%) isolates of MDR were highly resistant to penicillin (MIC ≥ 0.5-2 mg/ml) and 11 (39.2%) were intermediately resistant (MIC = 0.5-2 mg/ml). All antibiotics except vancomycin and teichoplanin revealed MIC over the break points for resistance. The MIC of chlorhexidine for the 28 MDR and 28 non-MDR of S. mutans varied from 4 to 16 mg/ml and 0.25 to 1 mg/ml, respectively. [Table 3] reveals the inhibitory effect of garlic extract on 28 MDR strains of S. mutans . The diameter of the zones of inhibition around the disks varied from 22 to 44 mm, indicating that all 28 MDR isolates (100%) were sensitive to garlic extract. The MIC of garlic extract for the 28 MDR and 28 non-MDR strains of S. mutans as measured by broth dilution method ranged from 4 to 32 mg/ml (approximately 16-128 mg/ml allicin) among which 25 (90%) showed garlic MIC = 16 mg/ml.
Streptococcus mutans is one of the most important oral bacteria which plays a major role in dental caries, bacteremia and consequently bacterial endocarditis among predisposed patients. , Prevention of dental caries can be achieved by proper and regular tooth brushing and rinsing with mouth rinses containing antibacterial agents such as chlorhexidine. Application of antibiotics for prevention of dental caries is not recommended, since there is risk of development of MDR strains. However, for prevention of bacteremia and endocarditis by S. mutans , antibiotic administration prior to invasive dental procedures is recommended. Chlorhexidine and sodium hypochlorite are widely used as mouthwashes and irrigating agents, respectively. Chlorhexidine has been used as anticariogenic agent in Finland  ; and chlorhexidine gel used twice daily for 2 weeks significantly decreased the caries rate.  Although this antimicrobial agent is widely used, however, immediate hypersensitivity and other side effect reactions to this agent have been reported. Chlorhexidine and sodium hypochlorite were shown to be cytotoxic to human periodontal ligament cells, inhibit protein synthesis and affect mitochondrial activity of these cells , ; and therefore this irrigation fluid may cause detrimental effects on vital tissues. Data obtained in our study revealed that chlorhexidine was active against MDR strains of S. mutans with MIC ranging from 4 to 16 mg/ml while MIC of this agent on non-MDR strains ranged from 0.25 to 1 mg/ml ( P in vitro correlation was observed between the resistance to commonly used antibiotics and chlorhexidine. Our findings are in accordance with other investigators who have also shown chlorhexidine MIC to be ≤1 mg/ml.  Of particular interest is the high rate of penicillin (22.8%) and amoxicillin (23.9%) resistance in this study. These antibiotics are routinely prescribed as prophylaxis to the patients prior to invasive dental procedures. Penicillin resistance of various species of Streptococcus viridans is increasing during the last decade. It has been reported that the introduction of penicillin in prophylactic antibiotic treatment has reduced the incidence of infection, but the long-term use of penicillin could be compromised by the emergence of resistant strains. 
Erythromycin has been recommended as an alternative option for patients who are allergic to penicillin and is also widely used for antibiotic prophylaxis of endocarditis associated with dental procedures. In our study, 23.9% and 14.1% of S. mutans were resistant to erythromycin and clindamycin, respectively, while no resistant isolates of S. mutans to these agents were reported from other regions.  The widespread use of erythromycin in our country may contribute to its relatively high resistance rate. Considering the development of MDR strains of bacteria, there is a need for more agents with marked antimicrobial activity, greater sensitivity and less toxicity. The in vitro antibacterial, antifungal and antiviral activities of garlic extract have been widely recognized. ,,,,,,, Moreover, in vivo studies on experimental animals also documented the inhibitory activity of garlic extract on various infectious agents such as methicillin resistant Staphylococcus aureus ,  Cytomegalovirus  and Shigella spp.  However, there is limited number of papers concerning the therapeutic activity of garlic extract in human being. Young, in 1959,  has treated 17 cases of pulmonary tuberculosis by the intrabronchial administration of garlic extract. Garlic extract has also been used intravenously as a therapeutic agent on patients with cryptococcal and viral meningitis.  In 2002, the Center for Disease Control (CDC) encouraged pregnant women with positive rectal/vaginal culture of Group B Streptococci (GBS) at 36 weeks of pregnancy, to use garlic to prevent new born GBS infection.  In vitro data obtained in this study revealed that garlic extract could significantly inhibit the growth of MDR S. mutans at MIC range of 4-32 mg/ml. It is thought that tooth paste or mouth wash containing optimum concentration of garlic extract might be useful for prevention of dental caries. Concerning the possible side effects of garlic on human, it has been shown that patients could tolerate i.v. extract of garlic daily for at least 1 month without apparent major toxic damage to liver, kidney or bone marrow  . Oral or i.v. administration causes minor side effects such as vomiting, diarrhea and nausea. However, lack of sufficient in vivo studies precludes its recommendation for the clinical practice at present. Work is in progress at the medical center for standardization and preparation of toothpaste and mouthwash containing this antimicrobial agent for prevention of dental caries.
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