Microbiological
Communication
Biosci. Biotech. Res. Comm. 9(1):
Studies on ampicillin resistant plasmid of Streptococcus mutans isolated from dental caries patients
S. Karikalan and A. Mohankumar*
Division of Microbial Technology, Post Graduate Research Department of Zoology, Chikkanna Government Arts College, Tirupur - 641 602, Tamilnadu, India
ABSTRACT
Dental caries is a well known major oral health problem in most countries. The multifactorial etiology of the disease includes multiple bacterial species, S. mutans is the main pathogen associated with the disease. Streptococcus mutans is recognized as the main pathogen of dental caries in human beings. One of the important virulence properties of S. mutans is their ability to form biofilms on tooth surfaces which make them a primary an etiological agent in dental caries. This bacterium allows the colonization of other microorganisms resulting in dental plaque. Recently multi drug resistant species of S. mutans were identified from the dental caries patients against many commercial antibiotics. Several therapeutic agents are available to treat or prevent tooth decay, but none of them showed complete sensitivity and have significantly influenced the disease’s global burden. Totally, fifty extracted tooth samples with chronic dental caries were selected for this study. The effectivity of caries was determined by the caries susceptibility test. S. mutans, the predomi- nant cariogens were isolated from dental caries patients. Ten antibiotics
KEY WORDS: DENTAL CARIES, STREPTOCOCCUS MUTANS, ANTIBIOTIC SUSCEPTIBILITY, R- PLASMID
INTRODUCTION
Dental caries and periodontal diseases are the leading cause of tooth loss, and while dental caries leads to
ARTICLE INFORMATION:
*Corresponding Author:
Received 1st March, 2016
Accepted after revision 31st March, 2016 BBRC Print ISSN:
Online ISSN:
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©A Society of Science and Nature Publication, 2016. All rights reserved.
Online Contents Available at: http//www.bbrc.in/
odontogenic infections, which may be primary or sec- ondary to periodontal, pericoronal, traumatic or postsur- gical infections. Dental plaque is an organized mass of bacteria already present in the oral cavity, on the surface
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of the teeth as a result of their accumulation in the face of poor oral hygiene and poor diet. Caries result when bacteria in the plaque, via a process called fermentation, break down carbohydrate for their energy requirement and produce end products, such as acids, including lactic acid, which causes the pH of plaque to fall in a mat- ter of few minutes to 4.0 to 4.5, thus overcoming the saturation of the saliva and plaque with calcium and phosphate, and demineralization occurs.
The understanding of mechanisms involved in the formation, virulence and drug resistance of dental bio- films is very important and have showed the impor- tance of dental caries disease, a critical disease to human beings. Streptococcus mutans is commonly found in the human oral cavity, is a significant contributor to tooth decay and has the ability to cause bacteria endocar- ditis. It is a member of the viridans Streptococci and occurs in chains. The mixture of organisms regularly found at any anatomical site is referred to as the normal flora. At about
The conventional antibiotic therapy is effective in treating bacterial infections. However, with increas- ing antibiotic resistance, there is a need for newer approaches in the management of these infections. In recent years, some isolates of S.mutans have begun to show considerable resistance to commonly used antibi- otics. The characterization of these drug resistant iso- lates and their heterogeneity studies may provide more information on their genetic makeup and also be useful in the management and control of drug resistance to at least that region (Dhamodhar et al., 2012).
Hasan et al., (2014) have used quercitrin in combi- nation with deoxynojirimycin is synergistic across the range of cariogenic mechanisms of S.mutans compared to their individual effect combination to suppress the cariogenic pathways of S. mutans but negative results of inhibition of S.mutans still aroused. One of the recent investigation showed the (MDR) Multidrug resistant spe- cies of S.mutans can thrive at any fluctuating condi- tions and different concentrations of growth parameters like pH, Temperature and NaCl tolerance in oral cavity and exhibited the resistance ability of S.mutans against all natural stipulations of oral crater of human beings (Karikalan and Mohankumar, 2016a).
mid (pSC101) that proved instrumental to the genera- tion of the first recombinant DNA molecule, (Kennath 2008). This study focuses on isolate the
MATERIAL AND METHODS
Extracted tooth samples were collected from endocardi- tis patients in critical condition from a reputed Hospital. Decayed tooth from the patient was collected with a ster- ile forceps and emptied into Mitis Salivarius Bacitracin agar,
The isolates were identified on the basis of their mor- phological, physiological and biochemical characteris- tics. Biochemical tests included, Gram staining, Motility, Catalase test,
The disc diffusion test by
RESULTS AND DISCUSSION
The collected samples were processed, from 20 - 60 year old male patients and female patients (Table 1). The iso- lated colonies of Streptococcus mutans on
Table 1: Sample collection from Caries patients
Vancomycin (30mcg), Tetracycline (30mcg), Ampicillin (5mcg), Kanamycin (10mcg) and Amoxicillin (30mcg).
Most of the (100%) S.mutans isolates were resistant to Ampicillin and (70%) were
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(63%) Amoxicillin and lowest were (50%) to Erythro- mycin. All the 10 isolates of S. mutans were found to be multidrug resistant and were resistant to a mini- mum of 4 antibiotics and maximum of 100% of isolates were resistant to Ampicillin antibiotic. Sensitivity was observed for the antibiotics Ciprofloxacin, and Chlo- ramphenicol. The isolates showed decreasing sensitivity towards above two antibiotics. While 50% of the isolates showed intermediate sensitivity to Erythromycin, 50% showed complete resistance. The isolates were showed complete resistance to
Viridans group Streptococci resistant to antibiotics have increasingly been reported over the past decade, while studies on antibiotic resistance of Streptococcus mutans group are few. Streptococcus mutans, being
Table 3: Antibiotic Susceptibility of Streptococcus mutans
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FIGURE 1: Antibiotics showed resistant to all the tested strains
FIGURE 2: Ampicillin, Amoxicillin and Penicillin antibiotics showed resistance
FIGURE 3: Migration of plasmid DNA fragments in agarose gel electrophoresis (0.7%) at 50volts and Lane 1 to 5 – S. mutans strains and Lane 6
A highly resistant percentage of 80% to cipro- floxacin observed against S. mutans isolates (Gamal, 2014). Two important antibiotics were showed the 50% by Amoxicillin and Ciprofloxacin resistant against S.mutans isolates were documented (Karikalan and Mohankumar, 2016). 80% of the recovered isolates exhibited the sensitive activity against Amoxicillin, Ampicillin, Chloramphenicol, Erythromycin, Penicillin, Clindamycin, Tetracycline, and resistant activity against Ciprofloxacin against Streptocoocus spp (Devi et al., 2011) and these reports differed from this study.
This antibiotic is routinely prescribed as prophylaxis to the patients prior to massive dental procedures. It has been reported that the introduction of penicillin in the prophylactic treatment has reduced the infection, but the
A new recent study proved that 50% of resist- ant was observed in 6 antibiotics among 14 antibiotics, above, particularly Penicillin, Ceftriaxone, Cephipime, Cephotaxime, Ampicillin and Cefaclor and these drugs were prescribed in clinical trials against dental prac- tices (Karikalan and Mohankumar, 2016 b, c).But in this study antibiotics totally varied and totally resistance to all the S. mutans isolates from above study. These anti- biotics have developed resistance against the strains of S. mutans as revealed by the zone of inhibition in the present study.
Thus the present study revealed the emergence of multi drug resistance in Streptococcus mutans in the urban Tirupur region, which has actually not only made general prophylaxis a very cumbersome affair but also has further intricated the antibiotic resistance pattern of this region.
The presence of plasmids in the dental caries bacterial isolates was determined using a modification of boiling preparation method (Holmes and Quigley, 1981). Plas- mid DNA was separated by electrophoresis on a 0.7% agarose gel (w/v) at 50 volts overnight. The gel was stained with
Only Ampicillin resistant strains are selected to iso- late the
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plasmid of Ampicillin resistant strains, were isolated in this study.
CONCLUSION
Only 50 dental caries culture samples were obtained and processed. Out of 50 patients aged from
ACKNOWLEDGEMENT
The authors wish to thank Dr. K. Shunmugasund- haram, Principal, Chikkanna Government Arts College, and Tirupur for providing facilities to conduct the entire research investigation.
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