Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 3(ISC-2013), 218-223 (2014) Res. J. Recent. Sci. International Science Congress Association 218 In-vitro evaluation of Antimicrobial potency of commercially available drugs against Dentinal Caries microbes Arul A Sri Kennath J. and Palanivelu Peramachi Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, INDIAAvailable online at: www.isca.in, www.isca.me Received 27th December 2013, revised 21st January 2014, accepted 31st March 2014 AbstractBacterial infections are common in dental practice and use of antibiotics for their treatment is also frequent. But their rampant use leads to antimicrobial resistance; a global growing issue affecting both developed and developing countries. Determining the susceptibility profile of potential pathogens is therefore necessary. Efficacy of natural phyto-chemicals isolated from plants and oral hygiene products against Streptococcus mutans; the principal dental pathogen associated with dental caries and other cariogenic bacteria isolated using the method of pure culturing has been evaluated. However, the literature lacks studies evaluating the efficacy of antibiotics against the microflora responsible for dental caries. Therefore, commercially used drugs were screened for their in-vitro antimicrobial potency against the microbial community obtained from dentinal caries lesion using Kirby-Bauer disc diffusion method. The polymicrobial growth showed susceptibility towards all the tested antibiotics except in few samples where Ampicillin/Cloxacillin, Cefixime/Clavulanic acid, Cephalexin, Cefixime and Ampicillin/ Sulbactam showed no zone of inhibition; suggesting possible resistance to such antibiotics in these patients. The comprehensive data obtained may support the polymicrobial etiology of dental caries. Such study may further allow investigation on the spatial distribution of pathogenic, antibiotic resistant bacteria among patients suffering from dental caries. Keywords: Dental caries, antibiotic susceptibility, Kirby-Bauer disc diffusion method. Introduction The preference of micro-organisms to live as surface adherent communities forms the basis of what is called a biofilm Compared to their planktonic counterparts, such microbial communities are self- sustainable, resistant to immune defense mechanisms of host and have increased tolerance to antimicrobial agents; the root cause of several bacterial infections perceived in day-to-day clinical practice. The oral cavity is a habitat to diverse microflora that colonizes various mucosal surfaces including teeth. Certain bacterial species have been implicated in oral diseases such as dental caries and periodontitis, which are among the most common biofilm (dental plaque) dependent bacterial infections in humans. Although Streptococcus mutans S. mutans) is established as the key pathogen responsible for dental caries; other species by their ability to produce acids can also be implicated in the disease; therefore pointing towards the polymicrobial nature of dental caries. Odontogenic infections resulting from widespread caries and its sequelae are common in dental practice; thus antibiotic use prescribed for their treatment is also frequent that may lead to antimicrobial resistance. Determination of susceptibility pattern of microbes towards the antimicrobials used is essential; guiding the clinicians in selecting the best drug to combat such infections. Many studies have been undertaken to evaluate the efficacy of commercially available drugs, natural phyto-chemicals isolated from plants used in traditional medicine and oral hygiene products against S. mutans; the principal dental pathogen associated with dental caries and other cariogenic bacteria isolated using the method of pure culturing4-10. However,there is lack of literature on studies evaluating the efficacy of antibiotics against the microflora responsible for dental caries. With this aforementioned problem in mind, commercially used drugs in dentistry were screened for their in-vitro antimicrobial potency against the microbial community obtained from dentinal caries lesion using Kirby-Bauer disc diffusion method.Material and Method The following antibiotic discs were purchased from HiMedia Laboratories, Mumbai, India: Ampicillin/ Cloxacillin (Ax10), Norfloxacin (Nx10), Doxycycline Hydrochloride (DO30), Ofloxacin (OF), Gatifloxacin (GAF), Cefixime/Clavulanic acid (CMC5/10), Amoxycillin/Clavulanic acid (AMC30), Gentamicin (GEN10), Cephalexin (CN30), Tetracycline (TE30), Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 218-223 (2014) Res. J. Recent. Sci. International Science Congress Association 219 Ceftriaxone (CTR30), Cefixime (CFM), Amikacin (AK30), Lomefloxacin (LOM10), Ampicillin/Sulbactam (A/S10/10), Ampicillin (AMP10), Erythromycin (E15), Ciprofloxacin (CIF) and Levofloxacin (LE). All the other chemicals used were of analytical grade and manufactured in India. The study was approved by the Institutional Review Board of Madurai Kamaraj University at Madurai, Tamil Nadu (India) on activities involving human subjects. Thirty adult patients were enrolled in the study with their informed consent. Patients were included if they had a carious lesion that had spread into the dentine (confirmed radiographically) with cavitation. The superficial, necrotic layer was removed and discarded, and the samples were sequentially hand excavated with sterile, spoon excavator and collected at a level that represented the middle of the dentine lesion. The thirty dentinal caries samples were collected and subjected to further analysis. Lesion samples from dentinal caries were inoculated on Luria-Bertani (LB) broth. Following overnight incubation at 37C on shaker at 180 rpm, the presence of turbidity in LB broth indicated bacterial growth. As per routine procedure in microbiology, the obtained bacterial growth was neither isolated further to get individual micro-organisms using pure culturing method nor characterized on the basis of biochemical tests. Antimicrobial potency of commercially available drugs against the obtained polymicrobial growth was evaluated using Kirby-Bauer disc diffusion method11. About 25 ml of the Mueller Hinton agar medium (HiMedia M173-500G) was cooled to 45C and poured into sterile petri plates and allowed to solidify completely. The entire surface of the agar plate was seeded with the polymicrobial growth using sterilized spreader. The plates were allowed to dry before antibiotic disc application. To prevent condensation, antibiotic discs were allowed to warm to ambient temperature before application. Within 15 min of inoculation, the antibiotic discs were placed flat side down on the seeded agar surfaces using sterile forceps, followed by incubating agar plates for 24 h at 37C. The relative susceptibility of the polymicrobial growth to each antibiotic was shown by a clear zone of inhibition measured in mm. For control, polymicrobial growth-free agar plates were incubated with antibiotic discs. Results and Discussion Results: The susceptibility pattern of the dentinal caries microbes against 19 commercially used antimicrobial drugs in dentistry was assayed by Kirby-Bauer disc diffusion method. The polymicrobial growth showed susceptibility towards all the tested antibiotics except in few samples where Ampicillin/Cloxacillin, Cefixime/Clavulanic acid, Cephalexin, Cefixime and Ampicillin/Sulbactam showed no zone of inhibition; suggesting possible resistance to such antibiotics in these patients. Among the fluoroquinolone group (tables 1 and 2) ; the susceptibility of the polymicrobial growth from the dentine samples were tested against both the first and second generation antibiotics, viz., Norfloxacin (Nx), Ofloxacin (OF), Ciprofloxacin (CIF), Gatifloxacin (GAF), Levofloxacin (LE) and Lomefloxacin (LOM). Nx exhibited largest inhibition zone and thus appears to be the most effective among the first generation fluoroquinolones, with zone of inhibition of 42 mm followed by OF (35 mm) and CIF (30 mm); while GAF ranked highest with 40 mm zone of inhibition followed by LOM (34 mm) and LE (30 mm). As a whole, the second generation fluoroquinolones were found to be more effective than the first generation antibiotics; since the additional fluoro and other substitutions in these drugs exhibited higher potency and had further extended their antimicrobial activity from Gram-positive cocci to anaerobes (figure 1). Among the -Lactam antibiotics group (tables 3 and 4); the susceptibility of the polymicrobial growth was tested against cephalosporins and extended spectrum penicillins. Cephalosporins tested included both the first and third generation antibiotics, viz., Cephalexin (CN), Cefixime (CFM) and Cefatrixone (CTR). CN exhibited greatest potency with zone of inhibition of 45 mm followed by CTR (33 mm) and CFM (25 mm). 5/30 samples of CN and 2/30 samples of CFM exhibited no clear zone of inhibition. The extended spectrum penicillin i.e., ampicillin (AMP) was also tested and showed an inhibition zone of 35 mm. Among the antimicrobial combinations used: the greatest potency was shown by Amoxycillin / Clavulanic acid (AMC) (40 mm) followed by Cefixime / Clavulanic acid (CMC) (39 mm), Ampicillin / Sulbactam (A/S) (39 mm) and Ampicillin / Cloxacillin (Ax) (38 mm). Combinations were used with an objective to broaden the spectrum of antimicrobial action. It is interesting to note that two samples each of Ax and CMC; and one sample of A/S exhibited no clear zone of inhibition against the polymicrobial growth (figure 2). The other groups selected for the present study were aminoglycosides, macrolide and broad spectrum antibiotics (tables 5 and 6). Among the aminoglycosides, two antibiotics were selected, viz., Gentamicin (GEN) and Amikacin (AK); showing almost similar susceptibility pattern with maximum zone of inhibition of 32 mm and 31 mm respectively. From the macrolide antibiotics group, Erythromycin (E) was selected for the present study since the drug is known to be highly active against S. pyogenes. From the broad spectrum antibiotic group, Tetracycline (TE) and Doxycycline Hydrochloride (DO) were tested against the polymicrobial growth. TE (37 mm) was found to exhibit greater zone of inhibition than DO (32 mm) (figure 3). Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 218-223 (2014) Res. J. Recent. Sci. International Science Congress Association 220 Tables-1 and 2 Antibiotic susceptibility pattern of the polymicrobial growth samples (1 – 30) towards fluoroquinolones. The numbers in the column indicates zone of inhibition (mm) Fluoroquinolones Samples Antibiotics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Nx 30 35 21 12 19 19 25 11 21 20 19 20 35 34 09 OF 26 35 20 23 20 21 21 17 18 18 20 24 28 33 17 GAF 30 35 30 22 30 29 38 22 29 28 23 25 34 40 21 LOM 23 27 22 22 20 22 20 10 14 17 18 20 25 28 11 CIF 29 26 30 08 25 25 17 15 22 24 18.5 25 28 11 23 LE 26 27 29 13 26 25 30 18 20 22 21 19 14 25 16 Fluoroquinolones Samples Antibiotics 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Nx 32 18 35 26 42 12 10 28 20 10 19 31 19 12 22 OF 33 18 25 29 34 23 17 25 20 18 21 24 19 21 25 GAF 39 26 32 34 44 22 24 30 29 22 27 33 23 25 30 LOM 30 18 15 23 34 22 11 25 22 16 20 28 12 26 27 CIF 09 21 11 22 14 10 25 26 11 28 23 24 17 23 18 LE 18 14 25 20 24 15 26 19 17 14 25 28 23 25 27 Figure-1 Zones of inhibition exhibited by Nx [a], LOM [b] and CIF [c] on Mueller Hinton agar media seeded with the polymicrobial growth from dental caries samples. Tables-3 and 4 Antibiotic susceptibility pattern of the polymicrobial growth samples (1 – 30) towards -Lactam Antibiotics. Column number indicates zone of inhibition (mm) -Lactam Group Samples Antibiotics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ax 34 06 24 15 11 11 21 15 38 23 08 06 - 28 07 CMC 28 17 25 20 15 15 39 16 23 23 12 10 - 30 11 AMC 36 06 25 16 18 19 40 20 24 23 12 13 09 25 14 CN 29 - 33 16 18 19 12 12 25 23 18 06 - 34 - CTR 26 12 30 22 28 27 30 17 21 20 15 13 22 33 18 CFM 20 06 08 20 25 25 10.5 12 15 31 - 11 13 15 13 A/S 36 07 33 18 20 20 39 20 30 29 12 12 12 30 15 AMP 35 06 15 17 14.5 12 21 14 15 13 20 16 23 18 22 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 218-223 (2014) Res. J. Recent. Sci. International Science Congress Association 221 -Lactam Group Samples Antibiotics 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ax 29 12 35 26 - 15 18 08 25 17 13 16 23 20 10 CMC 29 19 19 25 - 20 16 09 22 13 15 09 10 18 14 AMC 28 18 15 28 07 16 20 11 23 16 19 10 09 25 10 CN 45 - 16 31 - 16 12 07 30 08 19 14 25 13 09 CTR 33 24 22 30 16 22 17 22 15 19 27 20 13 26 14 CFM 12 13 06 20 - 20 12 13 11.5 13 24 17 10 07 08 A/S 34 14 30 33 - 18 20 14 33 21 16 18 11 23 09 AMP 16 23 18 22 11 17 14 20 15 16 12 20 22 13 08 Figure-2 Samples exhibiting no clear zone of inhibition on Mueller Hinton agar media seeded with the polymicrobial growth from dental caries samplesTables-5 and 6 Antibiotic susceptibility pattern of the polymicrobial growth samples (1 – 30) towards other antibiotics selected. The numbers in the column indicates zone of inhibition (mm). Other Group Samples Antibiotics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 DO 32 27 29 16 19 19 32 25 29 28 19.5 17 12 20 22 GEN 23 32 26 15 19 20 24 19 19 20 18 19 16 26 20 TE 29 23 32 23 23 24 29 25 28 31 15 22 19 27 20 AK 24 31 26 18 20 20 25 25 23 24 18 20 20 30 19 E 23 27 30 11 21 18 30 24 24 14 19 23 26 20 19 Other Group Samples Antibiotics 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 DO 29 20 22 32 12 16 25 14 17 17 19 11 15.5 16 24 GEN 30 18 21 21 20 16 19 16 26 23 20 18 16 24 23 TE 29 23 37 31 15 23 25 19 18 20 24 27 21 27 25 AK 31 23 26 25 21 19 25 20 25 19 21 22 20 21 13 E 23 26 21 19 20 19 22 24 20 19 19 21 23 22 21 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 218-223 (2014) Res. J. Recent. Sci. International Science Congress Association 222 Figure-3 Zones of inhibition exhibited by GEN [a], AK [b] and DO [c] on Mueller Hinton agar media seeded with the polymicrobial growth from dental caries samplesDiscussion: Dental caries is a worldwide disease known to affect every population throughout life. Due to its prevalence and high cost of treatment; it has emerged as key health predicament in developed countries. Furthermore, determining the bacterial species implicated directly in the disease or recognizing the mere by-standers, makes the etiology of dental caries challenging3,12. Utilization of antimicrobials in treating dental infections has gained much attention worldwide. But the widespread concern about the increasing problem of antimicrobial resistance has emphasized the need for rationalization of antibiotic use. Therefore, in context to lowering down the antimicrobial resistance rate; it is imperative to consider routine susceptibility testing. Such tests could be useful in improving the treatment efficacy13. Susceptibility testing is the determination of the bacterial pattern of resistance to a number of antibiotics. Antimicrobial susceptibility testing can be done by several different methods. Kirby-Bauer disc diffusion method was utilized in the present study, since the method employed is easy to perform and reliable. The antibiogram of the isolated polymicrobial growth, in the present study indicated a wide range of susceptibility to different antibiotics and therefore, seems that the use of these antimicrobial drugs may reduce the incidence and severity of the disease per se and its sequelae. Fluoroquinolones are quinolone antimicrobials having one or more fluorine substitutions; that further expand its antimicrobial activity, potency and /or confer metabolic stability. These are also active against many -lactam resistant bacteria. -lactams such as cephalosporins and penicillins are bactericidal drugs that act by inhibiting the bacterial cell wall synthesis. -lactams are non-toxic and have minimum concentration-dependent adverse effects; but allergic reaction mainly with penicillins is the most significant undesirable effect14. Amoxycillin/clavulanic acid, combination of a -lactam antibiotic (amoxicillin trihydrate) and a -lactamase inhibitor (potassium clavulanate) has broad antimicrobial spectrum and effective against amoxicillin-resistant bacteria that produce lactamase15. Such antimicrobial agent may prove valuable for managing dental infections. The tetracyclines are broad spectrum antibiotics that act by inhibiting bacterial protein synthesis. They are relatively inexpensive drugs and have been extensively used in the prophylactic and therapeutic regimens. These antibiotics have selective affinity for deposition in tooth substance, possibly through formation of a complex with calcium ions of hydroxyapatite crystals; leading to tooth discolouration16. Therefore, pertaining to dental infections, such antibiotics must be used with caution. Selection of an antibiotic regimen should be based on knowledge of the efficacy of an antibiotic for the bacteria, most often associated with severe infections. It should also be remembered that dental caries represent an ecosystem of bacteria in which by-products of one species of bacteria may be nutrients for other species of bacteria. Thus, if an antibiotic is effective against some species of bacteria in a polymicrobial infection, it may indirectly affect other bacteria in that ecosystem. Dental caries, though a disease of great antiquity, is not a disease of the past since it is reappearing in many countries as a public health crisis. It is therefore, important that the public health be taught on prevention, early recognition and reporting to dental clinics for proper prognosis. This underscores the need for laboratory diagnosis, confirmation of dental caries and the antibiograms of incriminating micro-organisms for proper management of patients and to reduce the development of resistant strains or multiple resistances to antibiotics. Since antibiotic sensitivity tests aids in determining the targets for therapeutic intervention; further studies using larger samples are necessary to assess the level of protection against dental caries offered by the use of these antibiotics. ConclusionThe comprehensive data obtained from the present study may support the polymicrobial etiology of the disease. Continue surveillance in the form of routine susceptibility tests is an Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 218-223 (2014) Res. J. Recent. Sci. International Science Congress Association 223 excellent source of information on the prevalence of resistant pathogens within the dental caries. Further, since the results from the present investigation reveals that patients differ in their antibiotic sensitivities; therefore such studies may prove an invaluable aid to form a sound policy on antibiotics usage in the clinical practice. 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