Pulpal and periapical diseases are the result of bacterial contamination that includes microorganisms that normally inhabit the oral cavity (Fouad, 2017). Bacterial infections are a major cause of pulp necrosis and periapical lesions and the main cause of endodontic failure (Shabahang, 2005). Successful endodontic therapy involves the elimination of bacteria and their products from root canals.

Together with instrumentation, irrigation is essential for cleansing and shaping the root canal system before three-dimensional sealing. Endodontic infections are polymicrobial and include strict anaerobes, facultative anaerobes, and microaerophiles (Rocas et al., 2008; Shabahang, 2005; Haapasalo et al., 2005). Bacteria located in the apical  portion of  the root canals potentially participate in the pathogenesis of periapical lesions. The microbiota is usually complex and includes different bacterial species Porphyromonas endodontalis in 65% (Rôças et al., 2010).

CatDex is a guanidine conjugate prepared by reacting aminoguanidine or agmatine with periodate oxidized dextran followed by reductive amination. It has a cationic charge, a wide pH range and it is hydrophilic. Also, CatDex has shown effect on tumor cell growth in prostate, breast, bladder and renal cancer cell lines (Meurling et al., 2009; Escamilla-Garcia et al., 2017). There are similarities in the proliferation, growth and development of tumor cells and bacteria as well as in the electrostatic condition of the cell-membrane wall relationship. A recent study demonstrated the antimicrobial activity of CatDex against oral cariogenic (Streptococcus mutans) and periodontal (Porphyromonas gingivalis) pathogens (Escamilla-Garcia et al., 2017). The proposed method of action in tumour cells is an electrostatic interaction with an anionic cell membrane, internalisation by the polyamine uptake system, and electrostatic binding of anionic structures in the cytoplasm, which kills the cell (Deka et al., 2015; Marquez et al., 2002). Similar to CatDex, but as a hydrogel, cationic synthetic dextran demonstrated antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 (O’Connoret al., 2015). Therefore, this study was performed to determine if CatDex has a similar effect against P. endodontalis compared to sodium hypochlorite (NaClO).

Bacterial culture

P. endodontalis ATCC35406 strain was used in this study and was obtained from the American Type Culture Collection (ATCC). The microorganism was activated and cultured under the growth conditions recommended in the technical specifications of ATCC. P. endodontalis was subcultured at 37°C for 48 h on agar plates with a brain-heart infusion medium (BHI) (BD Bioxon, Becton Dickinson, Mexico City) in anaerobic atmosphere using an anaerobic gas chamber (Plas-Labs, Lansing, MI). The preculture was washed with 0.9% NaCl (w/v), then inoculated at an optical density at a wavelength of 600 nm (OD_600nm) between 0.08 to 0.1 corresponding to 0.5 McFarland (Genesys 10S UV-Vis Spectrophotometer, Thermo Fisher Scientific Inc., Waltham MA) in a 250 ml Erlenmeyer flask with 100 ml of BHI broth. The bacterium was incubated at 37°C for 18 h until it reached the logarithmic phase of bacterial growth.

Growth kinetics

An absorbance of 0.5 McFarland was used for this study at a wavelength of 600 nm. The growth curve was performed in duplicate in 250 ml Erlenmeyer flasks with a volume of 100 ml of culture medium. Bacterial growth was monitored at time intervals of 2 h to reach the most representative kinetic  points. The  culture  pH was monitored using an Ultrabasic UB-5 PH Meter (Denver Instrument Co., Bohemia, NY); also, the bacterial concentration (CFU/ml) and OD_600nm were determined.

Minimum inhibitory concentration (MIC)

The MIC was used to determine the lowest concentration of CatDex which inhibits growth. For this, CatDex was diluted in BHI broth. Next, 500 μl of each concentration 0.05% were transferred to 5 ml assay tubes. The positive control for this study was NaClO (Cloralex^®). A concentration of 5.25, 2.5 and 1.25% was used. The negative control was saline solution. 500 µl of P. endodontalis inoculum was cultured for 18 h at a final bacterial concentration of 1.5×10⁸ CFU/ml with a 0.5 McFarland turbidity for a final volume of 1 ml, as previously described by Wanger et al. (1995). P. endodontalis was incubated in an anaerobic atmosphere of H₂ (10%), CO₂ (5%), and N₂ (85%) (Praxair Mexico, Monterrey, Mexico) at 37°C for 24 h. After the incubation, each tube was analyzed qualitatively by observing the presence or absence of turbidity and its optical density was measured.

Disk diffusion method

The sensitivity of P. endodontalis to CatDex was determined by the Kirby-Bauer method. An aliquot of 100 μl of each bacteria on agar plates was seeded in BHI agar. A no. 40 Whatman filter paper disc with a diameter of 6 mm (GE Healthcare, Boston, MA) was placed in 20 ml of CatDex at different concentrations. Saline solution was the negative control. Petri dishes were marked with external dividing lines. The seeded plates with discs were incubated at 37°C for 24 h. The inhibition or lack of inhibition around the discs was measured and the results are given in millimeters. Each concentration was done in triplicate. Culture media and material were pre-sterilized for 15 min at 120°C (ALL-AMERICAN^® Sterilizer, Hillsville, VA). All procedures were performed under sterile conditions in class II, type A2 biosafety cabinets (Thermo Fisher Scientific Inc.). The results represent the mean of three repetitions of three independent experiments.

Statistical analysis

Three independent replicates of each experiment were performed, and their results were expressed as mean diameter ± standard deviation (SD). We used data analysis, and the significances of the inhibitory effect of CatDex against P. endodontalis were evaluated with unequal variance analysis based on the t test and the Mann–Whitney (P<0.05) test and calculated with statistical software SPSS v22.0.

Growth kinetics

The culture of P. endodontalis presented an exponential bacterial growth phase from 12 h until reaching a maximum peak at 17 h, during which time OD₆₀₀ was 2.16±0.14, corresponding to 10.8×10⁸±7.07 CFU/mL and pH 7.0 ±0.0 (Figure 1).

Minimal inhibitory concentration

The greatest reduction in growth was observed with 5.25% NaClO, 0.145±0.092×10⁸ CFU/ml, OD₆₀₀ 0.029±0.0, pH 8±0.0. The growth reduction of 2.6±4.38×10⁸ CFU/ml, OD_600nm 1.1 ± 0.376 and pH 7±0.6 was achieved at concentration of 0.145% CatDex (Table 1). For the control of P. endodontalis, a bacterial concentration of 5.78±0.685×10⁸ CFU/ml, OD₆₀₀ 1.36±0.42 and pH 8.0±0.0 was obtained.

In summary, the percentage of bacterial growth reduction of P. endodontalis using 5.25, 2.5 and 1.25% of NaClO was 97.8, 78.73 and 57.9%, respectively. The MIC of CatDex was 0.145% with a 55±0.2% cell reduction. For other CatDex concentrations (0.17, 0.12, 0.08 and 0.05%), cell reduction reached ~35.26 ± 2.62% (Table 1).

Inhibitory effect

NaClO had a greater inhibitory effect against P. endodontalis. The diameter of the inhibition halo for 5.25, 2.5  and  1.5%  of  NaClO  was  9.5±0.70 mm, 7.5±0 mm and 6.5±0 mm, respectively. In the case of CatDex, the dose with the greatest antimicrobial effect was 0.17% with a diameter of 9 ± 0.7 mm. The inhibition halo of 0.145% CatDex was 7.5±0.58 mm, 0.12% was 6.75±0.96 mm and 0.075% was 6.25±0.50 mm.

Based on our results of CatDex and NaClO against P. endodontalis, the antimicrobial effect of NaClO was greater than CatDex at all the concentrations tested except for 0.145% (P<0.05). CatDex is a molecule that has not been previously reported in irrigation studies in endodontics. However, Escamilla-Garcia et al. (2017) showed that CatDex is effective against oral bacteria such as S. mutans and P. gingivalis. The MIC for P. endodontalis with CatDex was 0.145%. This is in contrast with Escamilla-Garcia et al. (2017) who mention that for S. mutans and P. gingivalis, the MIC was 0.05 mM with CatDex. In this study, P. endodontalis showed sensitivity to the positive control (NaClO). NaClO has been used as an irrigant at different concentrations (from 5.25 to 1.5%) in other studies (Haapasaloet al., 2014; Yamashita et al., 2003).

The most effective concentration of NaClO to inhibit the bacterial growth was 5.25%. The antimicrobial potential of 2.5% NaClO has also been analyzed at different concentrations against P. endodontalis where its ability to significantly reduce the presence of bacterial colonies in root canals is notable (Rocas and Siqueira, 2011). In this investigation, 2.5% NaClO achieved a 60% cell reduction of P. endodontalis, unlike the study by Sena et al. (2006) where 100% of bacterial colonies of P. endodontalis were eliminated with NaClO.

The third NaClO concentration tested was 1.25% where results for P. endodontalis showed a reduction of 80%. Results that are similar to those recently published in which the mean number of bacterial cells recovered from 1% NaClO group was significantly higher than that of 4% NaClO (Christo et al., 2016). The logarithmic growth phase identified by growth kinetics for P. endodontalis was 17 h. It was possible to observe an antimicrobial effect on P. endodontalis with CatDex using the disc susceptibility technique by the Kirby-Bauer method at a concentration of 0.17%. The percentage of P. endodontalis inhibition for 5.25% NaClO was 97.8%, while for CatDex was 55%.

NaClO is a frequently used irrigant and disinfectant fluid. CatDex showed an antimicrobial efficacy against oral pathogenic P. endodontalis similar to NaClO. Consequently, CatDex could be considered as a possible alternative for irrigation in root canal therapy. However, more studies are needed to verify its effect and to determine its correct clinical use.

The authors have not declared any conflict of interests.

Authors thank Sergio Lozano-Rodriguez, M.D. for his help in translating the manuscript. We also thank Dr. Marcela Marquez for providing the CatDex molecule used for this study.