Comparative antimicrobial efficacy of Metapex, Metronidazole, BioPure MTAD, Aztreonam on Bacteroides fragilis and Propionibacterium acne.

AIM: The purpose of this study was to evaluate the comparative antibacterial efficacy of Biopure MTAD, Metapex, Metronidazole, and Aztreonam against two obligate anerobic bacteria.
MATERIALS AND METHODS: Antimicrobial efficacy of selected medicaments against two obligate anaerobic bacteria Bacteroides fragilis and Propionibacterium acnes was done by Agar disc-diffusion method. Pre-sterilized Whatman paper discs, 6 mm in diameter and soaked with the test solution, were prepared and placed onto the previously seeded agar Petri plates. Each plate was incubated in anaerobic jar for anerobic environment at 37°C for 48 hours. A zone of inhibition was recorded for each plate and the results were analysed statistically. Saline and ethanol used as control group in this study.
RESULTS: Biopure MTAD, Metapex and Metronidazole were effective against all the selected microorganisms. Aztreonam was effective against Bacteroides fragilis. Saline and ethanol used as control were ineffective.
CONCLUSIONS: Metronidazole showed the superior antibacterial property amongst the tested medicaments.

INTRODUCTION

Microorganisms play a key role in pulpitis, infection-mediated necrosis and their extension to the periapical tissue.[1] The necrotic pulp presents a polymicrobial flora characterized by a wide variety of combinations of bacteria, averaging 4-7 species per canal, predominantly anerobic, with approximately equal proportions of Gram-negative and Gram-positive bacteria.[2]

The ultimate goals of endodontic treatment are complete removal of bacteria, their byproducts, and pulpal remnants from infected root canals.[3] Endodontic medicaments have been thought to be an essential step in killing the bacteria in root canals. Calcium hydroxide is the most widely used medication because of its well-documented antibacterial activity which can be use in various combinations. Among these combinations Metapex (Meta Biomed Co. Ltd, Cheongju, Korea), a silicone oil-based calcium hydroxide paste containing 38% iodoform is very commonly used.[4]

Metronidazole has a wide spectrum of bactericidal action against oral obligate anaerobes, even against isolates from infected necrotic pulps, in fact, more than 99% of the bacteria found in carious lesions and infected root dentine.[5]

BioPure MTAD (Dentsply Tulsa Dental, Tulsa, OK) is a mixture of a tetracycline isomer, an acetic acid, and Tween 80 detergent (MTAD) – was designed to be used as a final root canal rinse before obturation.[6] It represents an innovative approach in simultaneous removal of endodontic smear layers and disinfection of root canals. BioPure MTAD has been shown to be a clinically effective[7] and biocompatible[8] endodontic irrigant with potential antibacterial substantivity.[9]

Aztreonam, a monobactem, has been successfully used in medical field since long time as an antibacterial due to its high affinity against Gram-negative bacteria.

This study was undertaken to evaluate the antimicrobial efficacy of Metapex, Metronidazole, BioPure MTAD and Azetronam on Bacteroides fragilis (Gram-negative strict anerobe) and Propionibacterium acne (Gram-positive strict anerobe).

MATERIALS AND METHODS

The micro-organism employed in this study were two obligate anerobic bacteria i.e., B. fragilis (ATCC 25285) and P. acnes (ATCC 6921). These strains were allowed to reactivate in media which contains Brain Heart Infusion broth (BHI) (Difco Co, Becton Dickinson, Sparks, MD) supplemented with hemin (5 mg/l) and menadione (0.5 mg/l).

Antibiotic preparation

BioPure MTAD (Dentsply Tulsa Dental, Tulsa, OK) prepared after mixing BioPure MTAD powder with the diluents as manufactures instruction. Metapex (Meta Biomed Co. Ltd, Cheongju, Korea) solution prepared by dissolving Metapex in ethanol (99.9%), by heating until it completely dissolved (0.22 mg/dl conc). Control group used in this study were normal saline and ethanol (99.9%).

Antibiotic discs preparation

Antibiotic discs of 6-mm diameter were prepared from Whatman paper No. 1 which was sterilized by hot air oven. These discs were then saturated with 50 μl of each medicament. Other antibiotic discs used were of Aztreonam and Metronidazole from BD diagnostic.

After revival of the organisms in about 48 hours, inoculum for each bacterial strain was prepared by picking up four to five colonies with the help of a circular, previously sterilized loop and dissolving them into respective test tube containing 5 ml of 0.85% saline solution-to produce a turbidity of 0.5 on McFarland scale (1.5 × 108 CFU/ml). These organisms were streak on Wilkins-Chalgren agar plate (Difco, Becton-Dickinson and Co.) in a form that lawn growth can be observed.

Antibiotic discs and control discs were then aseptically transferred to the agar plate previously inoculated with bacteria. Plates were than incubated for 48 hours at 37°C in an anerobic jar with mixture of gas (N2, H2, and CO2 used). All assays were repeated four times to ensure reproducibility. Microbial zones of inhibition were measured in millimetres.

Data analysis

Results are expressed as the mean ± one standard deviation. The values were statistically evaluated with one-way ANOVA and the intergroup comparison was performed with the Bonferroni multiple comparison test. Values of P < 0.05 were considered statistically significant. Statistical analysis was performed with SPSS PC version 10 software.

RESULTS

Tables 1 and 2 show the diameters (means and standard deviations) of the zones of microbial growth inhibition (in mm) for the tested materials against B. fragilis [Figure 1] and P. acne, respectively.

Table 1

Comparision of the antimicrobial effect of endodontic medicaments against Bacteroides fragilis

Table 2

Comparision of the antimicrobial effect of endodontic medicaments against Propiobacterium acne

Figure 1

Zone of inhibition against B. fragilis by aztreonam, Metronidazole and Metapex

Metronidazole show the largest zone of inhibition size against both B. fragilis and P. acne. According to Bonferroni test significant differences in zone of inhibition size against B. fragilis were found between BioPure MTAD and Metronidazole (t = 4.83, P < 0.01), zone of inhibition was non-significant for BioPure MTAD and Metapex (t = 0.37, P > 0.05) and BioPure MTAD and Aztreonam (t = 2.23, P > 0.05).

The evaluation showed that Aztreonam is not active against P. acne. According to Bonferroni test significant differences in zone of inhibition size against P. acne were found between BioPure MTAD and Aztreonam (t = 58.44, P < 0.001), BioPure MTAD and Metronidazole (t = 7.31, P < 0.001).

DISCUSSION

The selection of the test endopathogens for this study was arbitrary and not a random sampling from necrotic teeth. Nevertheless, an attempt was made to select representative bacteria that have been commonly isolated from necrotic canals.

Agar disc-diffusion test method is a well-established technique for antibacterial study.[1011] Advantage of this method is that chemical properties of the medicaments are not changed and the antimicrobial resistance can be detected by challenging bacterial isolates with antimicrobial discs.[12] Moreover, this is an easy and less technique-sensitive method.

In the present study, antimicrobial efficacy of medicaments were tested against B. fragilis because it is a Gram-negative anerobic bacilli, commonly isolated bacteria in endodontic infections.[1314] In previous studies, P. acnes has been identified among the microfiora of endodontic infections[151617] which is the cause of its selection in the present study.

Metapex in this study, showed antimicrobial effect against both B. fragilis and P. acnes which is an agreement with previous studies.[18] The antimicrobial effects of Metapex may be due to the combination with iodoform and the viscous and oily vehicle, which may prolong the action of the medicament. Accordingly, Gomes et al.,[19] showed that oily vehicles increase the antimicrobial effects of calcium hydroxide which might be the reason of effect on P. acne. Calcium hydroxide usually increases pH and allowed an unfavorable microenvironment to the growth of B. fragilis strains.[20]

Metronidazole has shown effect against both B. Fragilis[2122] and P. acne,[22] which is in accordance with previous studies. Metronidazole a 5-nitroimidazole drugs exerts antimicrobial actions via inhibition of DNA synthesis.[23] This action requires intracellular reduction of the nitro group of these prodrug to produce reactive radical species.

BioPure MTAD in this study has shown significant zone of inhibition against both B. fragilis and P. acne which might be due to presence of doxycycline (broad-spectrum antibiotic), as Shabahang et al.,[24] concluded that Doxycycline in MTAD is responsible for its antibacterial effect. Previous literatures have proved the effect of doxycyline on B. fragilis and P. acne,[25] by inhibiting the protein synthesis.

Aztreonam is a novel monocyclic 1-lactam antibiotic which is highly active against aerobic Gram-negative bacteria in vitro and in vivo. It is relatively inactive against Gram-positive organisms, such as Staphylococcus aureus but it is little effective against B. Fragilis[2627] which is in accordance with the present study.

Aztreonam has shown significant effect on B. fragilis but it has low efficacy than Metronidazole but higher efficacy than other endodontic medicaments. P. acne was not affected by Aztreonam.

CONCLUSIONS

Metronidazole showed maximum antimicrobial efficacy against B. fragilis followed by Azetronam, Metapex, BioPure MTAD in descending order. Antimicrobial efficacy against P. acnes was again shown maximum by Metronidazole followed by BioPure MTAD and Metapex.