Antibacterial effect of resin based root canal sealer antibiotic combination against enterococcus faecalis - an invitro study

INTRODUCTION

One of the key goals of endodontic therapy is to obtain 3-dimensional obturation of the entire root canal system. Chemo-mechanical preparation supplemented with irrigation and antimicrobial medication followed by adequate filling of root canal space with an inert filling material did not result in complete elimination of these microorganisms.1,2 Micro-organisms remained deep inside the dentinal tubules, lateral canals and apical ramifications leading to failure of Endodontic treatment.3

Gram positive facultative anaerobes like E.faecalis, Lactobacilli, PeptoStreptococci, Streptococci and Staphylococci usually predominate in failed RCT cases. Enterococcus faecalis is a fastidious organism that has many virulence factors capable of withstanding nutritional deprivation, resistant to most of the antimicrobial agents used during cleaning and shaping procedures.4 Hence, it would be better to use a root canal filling material or the sealer that has antimicrobial property capable of reducing or halting the growth of these residual micro-organisms.5

ZOE sealers have been the most frequently used sealer with a good track record and antimicrobial property despite a few disadvantages.6 Resin based root canal sealers aim to create a monoblock effect by allowing the adhesion of polymer core to root canal wall. AH plus has low solubility, good adhesion to dentine besides satisfactory biological response and antimicrobial property. Resilon/ Epiphany is another dual-cure hydrophilic methacrylate resin based root canal sealer with good antimicrobial property which decreased with time.7 Hence, powdered antibiotics were added to these sealers to see if this will extend their antimicrobial property.

For years, antibiotics have been prescribed widely for treating chronic endodontic/ retreatment cases. However, these teeth are usually pulpless with no blood supply, hence the drug may not reach the required concentration at the local site.8 Also, administration of the drug at the local site prevents untoward systemic complications and higher drug concentration can be attained at the intended site. With this intention, powdered Amoxicillin and Doxycycline was incorporated while mixing these resin sealers and checked for antibacterial efficacy. Systemic Amoxicillin and Doxycycline have been routinely prescribed during dental treatment. The susceptibility of E.faecalis to antibiotics such as Amoxicillin, Vancomycin, Erythromycin, Benzylpenicillin and Doxycycline is already proved.9 Hence, these antibiotics in the powdered form have been mixed with the resin sealers to evaluate and compare their antibacterial property at 1st, 3rd, 5th and 7th day intervals.

METHODOLOGY

A Suspension of Enterococcus faecalis (ATCC 29212) was obtained and streaked onto blood agar plate, incubated under aerobic conditions at 370C for 48 hours to obtain pure colonies of E.faecalis. Inoculum of the organism was then transferred to BHI broth and incubated for 24 hours. A standard concentration of 1.5×108 cells/ ml was obtained using McFarland tube which was then collected over cotton swab and streaked over 60 blood agar plates. A 6mm well was created using a sterile pastern pipette of the same diameter into which, sealer/ sealer – antibiotic paste was placed.

A known weight of AH plus and Epiphany Sealer was dispensed on a mixing pad according to manufacturer’s instructions and divided equally into 3 portions. To the 2nd and 3rd portion, powdered antibiotics-Amoxicillin and Doxycycline-10% by weight of the sealer was added individually and all the three portions of the sealers were mixed separately using a cement spatula. These freshly mixed sealer/ sealerantibiotic combination were then approximately divided into 10 equal parts to be immediately transferred to the cut wells in the blood agar plates using an applicator tip. Thus, we obtain 6 groups with 10 samples in each group. All the plates were incubated for 24hrs at 37°C under aerobic condition and zone of growth inhibition (fig 1) which is the shortest distance from the outer margin of sealer to the initial point of microbial growth was measured using a scale on 1st, 3rd, 5th and 7th day intervals. Hence, the groups I-VI are: Group I - AH Plus Sealer Group II - AH Plus Sealer + Amoxicillin Group III - AH Plus Sealer + Doxycycline Group IV - Epiphany Group V - Epiphany + Amoxicillin Group VI - Epiphany + Doxycycline The groups were then subjected to Kruskal Wallis and Mann Whitney statistical analysis to see if there is any significant difference between the groups tested.

RESULTS

Antibacterial property of AH Plus sealer was better than Epiphany with/without antibiotics at all time intervals. The antibacterial action of AH Plus – Doxycycline combination was twice and AH Plus – Amoxicillin combination was thrice compared to AH plus sealer alone. The zone of inhibition around Epiphany – Doxycycline combination was thrice and Epiphany – Amoxicillin combination was four times greater compared to Epiphany sealer group mentioned in Table 1, graph 1.

DISCUSSION

Endodontic sealers mainly fill up the gap between the master cone and accessory cones thus decreasing the leakage in obturated canals 5–20 folds.10 Freshly mixed sealers showed antimicrobial property which decreased with time. Hence, antibiotic powder was incorporated to the sealer while mixing to see if it will enhance or prolong the antimicrobial action of these resin sealers, as they disrupt the local microbial environment rather than merely entombing them and maintains bactericidal properties beyond its setting time, thus providing an extended therapeutic effect.11

Amoxicillin is a broad spectrum beta lactam antibiotic that acts by inhibiting the cell wall synthesis and Doxycycline is the alternative drug usually prescribed for patients allergic to Penicillin. Hence, these powdered antibiotics were added to the sealer during mixing. Antimicrobial property was tested using agar diffusion method instead of direct contact test as it depended on the chemical property and diffusibility of the tested material. In Direct Contact test, only the freshly mixed sealer showed significant antimicrobial action, whereas a 24hr – 7 day old sample did not.12

Antibacterial property of AH plus sealer can be attributed to the presence of epoxy resin, amines, calcium and formaldehyde released during polymerization process. Freshly mixed AH plus sealer exhibited some antibacterial effect on the 1st day which reached maximum by 3rd day, remained stable and consistent upto 7th day. On adding Amoxicillin to AH plus, its antibacterial property increased three times and twice on adding Doxycycline and the effect remained the same when measured at 7th day. Epiphany sealer also showed antibacterial action lasting for 7 days and this is in accordance with the study done by Resende A &Vineeta Nikhil.13,14 Its antibacterial property increased by four times on adding Amoxicillin and thrice on adding Doxycycline. The antibacterial action of both the sealers was more with addition of powdered amoxicillin compared to Doxycycline. This could be because Doxycycline chelated with calcium present in AH Plus and Epiphany sealer.

Although there was a significant difference in the antibacterial action seen when AH plus and Epiphany sealers were compared, addition of Doxycycline to AH plus sealer did not proportionately increase the antibacterial action as was expected. Addition of antibiotics to both the sealers did not seem to alter the mixing time, handling property, consistency, setting time of the tested sealers. But further studies needs to be done in this regards to check whether the addition of antibiotics would bring about any change in the sealing ability, flow rate, solubility, film thickness and other properties of the sealers.

CONCLUSION

Within the limitations of the present study, it can be concluded that:

1. The antibacterial effect of AH Plus sealer was better compared to Epiphany sealer at all time intervals.

2. Measuring the zone of inhibition in each group, the antibacterial effect was seen to decrease in the following order AH plus + Amoxicillin> Epiphany + Amoxicillin> AH plus + Doxycycline ≥ Epiphany + Doxycycline > AH plus > Epiphany.

3. The zone of inhibition seen with AH PlusDoxycycline was twice and AH plus– Amoxicillin was thrice that of AH plus sealer alone at all the time intervals.

4. The antibacterial action of Epiphany – Doxycycline was thrice and Epiphany – Amoxicillin was four times the Epiphany sealer alone at all the time intervals.

5. Sealer–Amoxicillin combination showed better antibacterial action compared to sealer - Doxycycline combination in both the groups tested at all the time intervals.