Evaluation of the Antibacterial Efficacy of Daptomycin, Gentamicin, and Calcium Hydroxide—Antibiotic Combinations on Enterococcus faecalis Dentinal Biofilm: An In Vitro Study

Preparation of the Test Solutions

Commercially available CH paste (Dentocal™, Anabond Stedman Pharma Research Pvt Ltd, Chennai, India) was used.

G (Gentalab, Laborate Pvt Ltd, Panipat, Haryana, India) was procured in the injectable form at a concentration of 80 mg/2 mL, which was diluted to obtain a concentration of 40 mg/mL for the present study.

D was procured in powder form (Cubicin, OSO Biopharmaceuticals Ltd, Albuquerque, New Mexico, USA). A stock solution was prepared in sterile distilled water at a concentration of 60 mg/mL for this study.

Screening for Antibacterial Activity and Determination of Minimum Bactericidal Concentration (MBC)

The antibacterial ability of the test solutions against E. faecalis a standard strain, American Type Culture Collection (ATCC 29212), and a clinical strain (SBEF 2) isolated from a post-treatment root canal infection maintained in the Department of Microbiology was assessed by agar well diffusion assay and disc-diffusion assay.

For the agar well diffusion text, lawn cultures of the bacterial isolates were prepared on separate Mueller Hinton Agar (MHA) (Hi media Laboratories Pvt Ltd, Mumbai, India) plates. The assay was performed in triplicate (three MHA plates). Wells of 8-mm diameter were punched using sterile cork borer and 50 µL of each test solution (CH; CH+G; CH+D; CH+G+D) was added in the labeled wells and the plates were incubated at 37 °C for 24 hours. For CH alone and CH combined with G and D, four wells per plate were used separately for ATCC strain (Fig. 1A) and clinical strain (Fig. 1B).

Disk-diffusion method was adopted to determine the antibacterial efficacy of the antibiotics; G (without CH) and D (without CH) impregnated on sterile filter paper disc (Hi media Laboratories Pvt Ltd, Mumbai, India). For G and D, two discs per plate were used (Fig. 1). After overnight incubation, the diameter of the zone of inhibition around the wells/discs was measured and recorded in millimeters (mm). The assay was performed in triplicates. The minimum inhibitory concentration (MIC) was determined by broth microdilution method in sterile disposable 96-well microtiter plates. The minimum bactericidal concentration (MBC) that inhibits approximately 99% of bacterial growth was determined by spot inoculation of 5 µL of the culture from each well of the microtiter plate onto an MHA plate and incubated for 18 hours at 37 °C.¹⁰,¹¹

Selection and Preparation of Tooth Samples

Sixty intact, non-carious, single-rooted human mandibular premolars with fully formed apices freshly extracted for orthodontic treatment were selected for the study. The teeth were cleaned and stored in normal saline to prevent dehydration until the analysis. Both mesiodistal and buccolingual digital radiographs of all teeth were obtained using intraoral periapical radiography photostimuable phosphor plate system (Kavo Scan Exam One, Kavo Dental Ltd, Mi Healthcare, Knowsley, England, UK) to confirm the presence of single canal. All the specimens were decoronated to 2–3 mm below the cemento-enamel junction (CEJ) using a safe-sided diamond disc to standardize root length to 10 mm. A size 10 K-file (Mani Inc, Tochigi, Japan) was used to scout and establish patency till the tip of the file was seen at the apical foramen. The working length was corrected by reducing 1 mm from the radiographic apex.¹² The coronal third was prepared by Gates-Glidden drills (sizes—1, 2, 3, and 4) (Mani Inc, Tochigi, Japan). The samples were then instrumented with ProTaper Universal rotary system (Dentsply, Maillefer, Switzerland, USA) in a crown-down technique.

Two mL of 3% NaOCl (Prime Dental Products Pvt Ltd, Bhiwandi, Maharashtra, India) for 1 minute and 17% EDTA (Pulpdent Corporation, Massachusetts, USA) for 1 minute was used as the working solution during instrumentation, delivered by a 30-gauge side-vented needle placed as far apically as possible into the canal without binding. The apical third of each canal was prepared up to size F5 (5% taper 50 size) to allow adequate flushing and penetration of the irrigating solution.

After instrumentation, the enlarged apical foramen was sealed with epoxy resin to prevent bacterial leakage and the tooth samples were mounted vertically in acrylic blocks to simulate the position of the tooth in the oral cavity.

Sterilization of Teeth Samples

The mounted samples were placed in clean glass test tubes (5 tooth/test tube) containing 5 mL of sterile distilled water and autoclaved at 121 °C at 15 lbs pressure for 20 minutes. After sterilization, the tubes were placed in an incubator at 37 °C for 48 hours and checked for turbidity.

Biofilm Formation

The sterilized samples mounted on acrylic blocks (1 mounted teeth/well) were transferred aseptically into the wells of sterile 24-well tissue culture plates (DSMZ- German collection of micro-organisms and cell cultures, Zellkulter, Germany). They were randomly divided into two groups, A and B (n = 30 in each group). Group A was inoculated with E. faecalis ATCC 29212, while Group B with E. faecalis clinical isolate (10 µL of bacterial culture/tooth) followed by incubation at 37 °C to form a 3-week dentinal biofilm.¹³

Initial Microbiological Sampling and Determination of Initial Bacterial Count

At the end of the third week, the initial microbiological sampling was performed by flooding the canals with 5 mL sterile saline with a double-vented close-ended 27-gauge needle to full length of the canal followed by the insertion of a size 50 Hedstrom file (Mani Inc, Tochigi, Japan) into the canal to scrape the bacteria adherent to the dentin. The file was manually agitated in microfuge tubes (Tarsons India Pvt Ltd, Kolkata, India) containing 1 mL of sterile saline to dissolve the debris. Three sterile absorbent paper points were placed in the lumen of each canal for 60 seconds and transferred into same microfuge tube and vortexed for 60 seconds in a vortex mixer (Spinx, Tarsons India Pvt Ltd, Kolkata, India).

Colony count was performed for all 60 samples to establish the level of contamination before application of medicament by spread plate method. Briefly, 10 µL of the sample was transferred onto sterile MHA plates using micropipettes. The inoculum was spread uniformly using sterile disposable plastic loops (Hi-FlexiLoop4, HiMedia Laboratories, Mumbai, India). The plates were incubated aerobically for 24 hours at 37 °C. After incubation, the numbers of colonies on the agar plate were counted using a digital colony counter (Deep vision, India), and the colony-forming units per mL was calculated using the formula, cfu/mL = No of colonies × 100.

Placement of Intracanal Medicaments

The final concentration of the antibiotics, G and D admixed with the intra-canal medicament (CH), was adjusted to six times the MIC of the planktonic cells. The tooth samples of group A, E. faecalis (ATCC 29212), and group B, E. faecalis clinical isolate, were randomly divided into three subgroups of (n = 10) in each subgroup: 1 A – CH; 2 A – CH+G; 3 A – CH+D; 1 B – CH; 2 B – CH+G; 3 B – CH+D.

The respective medicament was placed inside the root canals using lentulo spiral size 30 and condensed with hand pluggers. Cavit G (3 M ESPE, Minnesota, USA) was placed at a thickness of 2 mm and kept undisturbed for 7 days.

Final Microbiological Sampling and Determination of Final Bacterial Count

After 7 days, the final microbiological sampling was done as described for the initial sampling and for all the 60 samples, final colony count was determined by spread plate method. The antibacterial activity of the intra-canal medicaments was measured by comparing the reduction in colony counts before and after intra-canal medication.¹⁴