Microbial Ecology of Dental Plaques of Jordanian Patients and Inhibitory Effects of Allium sativum and Allium cepa L. Extracts
Hani A. Masaadeh ,
Wail A. Hayajneh
Natheer M. Momani
The present research aimed to study the inhibitory effects of garlic (Allium sativum) and onion (Allium cepa L.) extracts on microorganism isolated from dental caries plaques of Jordanian patients. Dental caries plaques were collected in a private clinic then processed and worked upon in a university laboratory. Dental plaques from 91 patients were obtained and grown on plates following standard protocols. A total of 116 isolates were grown. E. coli, Lactobacillus acidophilus, Candida albicans, Streptococcus mitis, Actinomyces naeslandii, Enterococcus faecalis and Streptococcus oralis were decreasingly the most frequent organisms. Standard and different dilutions of garlic and onion extracts were tested against those isolated microorganisms. Median values for zones of inhibitions were calculated and blotted. Garlic extracts consistently showed significantly higher inhibitory effects than onion extracts. This study concludes that Garlic extracts do have significant inhibitory effects against microorganisms associated with dental caries and further studies are needed to explore potential therapeutic uses for such extracts. It also showed that microorganisms associated with dental caries of Jordanian patients are similar to those encountered in other different areas of the world.
Dental caries is a very common problem that affects all age groups. It is a
process in which the enamel and the dentine, are demineralized by acids produced
by bacterial fermentation of carbohydrates (Soet and Graaff, 1998). In real
life, it is the most common infectious disease affecting human beings (Balakrishnan
et al., 2000). Mutans streptococci (Streptococcus mutans and Streptococcus
sobrinus) are the most common organisms that are associated with dental
caries (Hanada, 2000). Lactobacilli are also common important players
in the pathogenesis of dental caries (Soet and Graaff, 1998). Nikawa et al.
(1998) have also shown that Candida albicans colonizing the oral cavity
can also cause dental caries. Many studies have been conducted trying to modulate
the interactions of different factors that facilitate the effect of above and
other organisms in the pathogenesis of dental caries (Balakrishnan et al.,
2000; Hanada, 2000; Marsh, 1999; Michalek et al., 2001; National Institutes
of Health (US) 2001; Simmonds et al., 2000).
Many other studies have focused on studying the microbial ecology of dental plaques trying to find measurable risk factors and establish suggested mechanisms for dental caries (Bowden, 1997; Marsh and Bradshaw, 1995; Marsh, 1994).
There has been a renewal of interest in antimicrobial activity of medical plants including Allium species. Garlic extracts have been studied extensively for different purposes including inhibitory effects against a variety of microorganisms. McNulty et al. (2001) have tested garlic oil against Helicobacter pylori in dyspeptic patients. Sasaki et al. (1999) tested successfully garlic powder made from fresh garlic pulps against Escherichia coli O-157, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enteritidis and Candida albicans. Many studies have shown variable to significant effects of garlic and garlic-derived extracts on common cold (Josling, 2001), fungi, (Wang and Ng, 2001), herpes viruses, Mycobacterium tuberculosis (Uchida et al., 1975) and parasites (Lun et al., 1994).
Onion extracts have also been studied, but less extensively than garlic (Farbman et al., 1993). It showed bactericidal effects on oral Streptococcus mutans and Streptococcus sobrinus (Kim, 1997). Zohri et al. (1995) demonstrated inhibitory effects of onion oil against four gram positive bacteria, four gram negative bacteria and dermatophytic fungi. Recently, Mantawy (2001) showed that exposure of snails (the intermediate host of Schistosoma mansoni) to water containing either garlic or onion caused snail toxicity.
MATERIALS AND METHODS
Patients and samples: A total of 91 samples were collected from patients attending a private dental clinic in Irbid city in northern Jordan who are suffering from dental caries plaques on their teeth. This work was done between July 2003 and September 2003. Specimens were transported to our laboratory with thioglycolate transport medium and grown on blood agar, nutrient agar, MacConkey agar, chocolate agar and sabouraud dextrose agar (Difco) at 37°C.
Preparations of aqueous garlic extract and aqueous onion extract: Fresh
peeled garlic cloves and onion (100 g) were chopped separately into small pieces
and were homogenized at high speed in 200 mL of sterile distilled water in a
blender for 5 to 7 min. The homogenate was filtered through a gauze cloth. The
filtrate was passed through 0.45 Millipore filter and was immediately used.
The plant materials used in this study were purchased from local markets in
Susceptibility testing: The clinical isolates were identified and approved by biochemical tests. Five mm diameter filter paper discs were impregnated with 50 μg of the tested plant and placed in duplicates onto Muller-Hinton agar, the surface was then spread with 0.2 mL of microorganism culture (ca. 108 cells/mL) and the plates were incubated for 24 h at 37°C for bacteria and 48 h at 28°C for Candida albicans. Experiments were carried out in duplicates and the results (mean of three experiments) were recorded by measuring the zones of inhibition surrounding the discs. Control discs were used and contained sterile transport broth medium only. Dilutions of extracts used were 1:2, 1:4, 1:8 and 1:16.
Calculations: The median values for zones of inhibitions for both garlic and onion extracts at different dilutions for different isolated microorganisms were calculated and blotted (Fig. 1 and 2).
RESULTS AND DISCUSSION
The present study trying to find out the microorganisms and their frequency
in association with dental caries in Jordanian patients. A total of 116 isolates
were grown from 91 patients with dental caries (Table 1).
E. coli, Lactobacillus acidophilus, Candida albicans, Streptococcus mitis,
Actinomyces naeslandii, Enterococcus faecalis and Streptococcus
oralis were decreasingly the most frequent organisms. These results are
more or less consistent with already accumulated literature regarding pathogens
associated with dental caries.
||Frequency and percentage of microorganisms isolated from patients
with dental caries
||Median values of zones of inhibitions measured at different
garlic (Allium sativum) extracts against mentioned microorganisms
These results show that garlic extracts have significantly higher inhibitory
effects than onion extracts even though most microorganisms showed same patterns
of inhibition to both garlic and onion extracts. (apart from Candida albicans,
Streptococcus mitis and Veillonella spp.) (Fig. 1
and 2). In both onion and garlic groups, Klebsiella and
Pseudomonas aeruginosa isolates were the least and most resistant organisms,
||Median values of zones of inhibitions measured at different
onion (Allium cepa L.) extracts against mentioned microorganisms
However, both microorganisms were of no statistical significance in ecology
of dental caries. Klebsiella species, E. coli, Streptococcus mutans
and Enterobacter species were consistently the least resistant organisms
for both extracts. On the other end, Pseudomonas aeruginosa, Streptococcus
sanguis and Streptococcus anginosus were consistently the most resistant
organisms for both garlic and onion extracts. It is interesting that E. coli
which is the most common microorganism associated with dental caries, is also
very sensitive to both onion and garlic extracts. The weaker antimicrobial activity
of onion extract might be possibly explained by the presence of component that
are less active than allicin which is found in garlic. Toxicity of garlic has
been reported by several studies and garlic-sensitive patients have shown positive
reactions to allyl propyl disulfide, allyl mercaptan and allicin.
Garlic extracts with different dilutions were of significant inhibitory effects
against microorganisms associated with dental caries which might predict potential
beneficial future uses of garlic extracts. On the contrary, onion extracts showed
significantly weaker inhibitory effects against those microorganisms. Microorganisms
associated with dental caries of Jordanian patients are similar in types and
frequency to those encountered in other different areas of the world. Based
on the in vitro results of this study and on the known organic chemistry
of garlic, future studies should focus on finding the concentration of the active
constituents that can be tolerated by humans in order to try using them for
topical treatment. Other studies may investigate the effect of human intestinal
proteolytic enzymes on the active components of Allium species
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