Effect of duration and dilution on antimicrobial efficacy of octenidine hydrochloride as an intracanal medicament with chitosan carrier against Enterococcus faecalis - A modified direct contact test.

BACKGROUND: The ambiguity in key influential factors such as minimal time for effective action, dilution, and need of drug carrier for intracanal medicaments necessitates a microbial analysis that aids in the potential selection of an intracanal medicament for ensuring optimal root canal disinfection. AIMS: This study aims to evaluate the antimicrobial efficacy of octenidine hydrochloride (OHC) and gold standard calcium hydroxide (Ca(OH) 2) as intracanal medicaments, both independently and along with chitosan (CTS) as medicament vehicle against the common resistant endopathogen - Enterococcus faecalis.
MATERIALS AND METHODS: A modified direct contact microbial test was used to evaluate the amount of surviving bacteria after predetermined contact time (2, 5, 20, and 60 min) and 5-fold serial dilution of the intracanal medicaments. The experiment was carried out under aseptic conditions and performed in triplicate to ensure reproducibility. STATISTICAL ANALYSIS USED: The results were analyzed by Kruskal-Wallis analysis of variance followed by pairwise comparisons by Mann-Whitney U-test. RESULTS AND
CONCLUSIONS: The results showed that all the four medicament groups were able to show the maximum antimicrobial efficacy against E. faecalis at 60 min time interval and that the antimicrobial efficacy of OHC and Ca(OH) 2 was at its peak when used alone. The study thereby concluded that the addition of CTS as a carrier did not enhance the antimicrobial efficacy of OHC or Ca(OH) 2 against E. faecalis. Copyright:

INTRODUCTION

The main objective in the field of endodontics remains the elimination of microorganisms from the root canal systems and prevention of treatment failure.[1] Complexity of the root canal system, invasion of the dentinal tubules by microorganisms, smear layer formation during canal instrumentation, and presence of dentin as tissue are the major hindrances for effective root canal disinfection.[2]

When pathological changes occur in the dental pulp, the root canal space acquires the ability to harbor various bacterial irritants along with their toxins and byproducts. Enterococcus faecalis apart from being small enough to competently invade dentinal tubules is also resistant to low concentrations of sodium hypochlorite irrigant.[3] Hence, the use of intracanal medicament is vital to eliminate any residual bacteria in a root canal, after instrumentation and irrigation.

The most routinely used intracanal medicament, calcium hydroxide (Ca(OH) 2) is believed to possess many of the ideal properties, mainly due to its alkaline pH. It has been demonstrated that Candida albicans and E. faecalis are resistant to its antimicrobial effect and can survive within dentinal tubules even with Ca(OH) 2 as an intracanal dressing.[34]

However, a newer intracanal medicament octenidine hydrochloride (OHC) is a potential antimicrobial agent and appears to be more effective than chlorhexidine.[4] Its antimicrobial action is by interfering with the bacterial cell wall and cell membrane.[5]

To ensure complete canal disinfection, an effective antimicrobial agent is required for a predetermined time period and in a particular concentration. Furthermore, to increase the intracanal medicament stability, insolubility, and controlled sustained release, a drug carrier or vehicle like chitosan (CTS) has been suggested, which is a natural copolymer of glucosamine and N-acetyl glucosamine, present in some crustaceans, insects, and fungi.[6]

Therefore, this study was designed to compare the antimicrobial efficacy of OHC and Ca(OH) 2 as intracanal medicaments, both independently and along with CTS as a carrier molecule, and to evaluate the significance of duration and concentration of the same against E. faecalis.

MATERIALS AND METHODS

Modified direct contact test

For this study, intracanal medicaments used were divided into four groups:

Octenidine – OHC (TCI, Tokyo Chemical Industry, Japan)

Octenidine with CTS – OHC + CTS (TCI, Tokyo Chemical Industry, Japan)

Ca(OH) 2 with CTS – Ca(OH) 2 + CTS

Ca(OH) 2 alone – Ca(OH) 2 (Prevest DenPro, India).

A 96-well microtiter plate was held vertically, and an area of fixed size on the side walls of the wells was coated with an equal amount of each material using a cavity liner applicator and allowed to set. A 10 μL of bacterial suspension was then carefully placed in each well. Bacterial suspensions placed on the wall of uncoated wells were used as control.

After incubation in 100% humidity at 37°C for 2, 5, 20, and 60 min, 240 μL of Tryptic soy broth (TSB) was added to each well. After gently mixing with a pipette for 1 min, the bacterial suspension from each well was then transferred and serially diluted in TSB [Figure 1]. The survival of the bacteria was then assessed by culturing aliquots of 20 μl onto Tryptic soy agar plates after 5-fold serial dilutions. After incubation for 24 h at 37°C, colonies on the plate were counted and the colony-forming unit (CFU) was calculated (CFU/ml) [Figure 2].

Figure 1

The bacterial suspension from each well was transferred and serially diluted in Tryptic soya broth

Figure 2

After incubation for 24 h at 37°C, colonies on the plate were counted and the colony-forming unit/ml was calculated

The entire experiment was carried out under aseptic conditions and performed in triplicate to ensure reproducibility. The mean values of log10 CFU/mL and the standard deviation of bacteria were calculated. The results were analyzed by Kruskal–Wallis analysis of variance followed by pairwise comparisons by Mann–Whitney U-test. The level of significance was set at 95%. Statistical analysis was performed with the statistical software SPSS v. 21.0 (SPSS for Windows; SPSS Inc., Chicago, IL, USA).

RESULTS

Based on the values obtained from the test, the following results were deciphered:

Irrespective of the dilutions, all the four medicament groups were able to show the maximum antimicrobial efficacy against E. faecalis at 60 min time interval with a progressive reduction in the counts of CFU from 2 to 60 min [Table 1].

Table 1

Comparison of four groups with respect to colony-forming unit counts of Enterococcus faecalis at different time points by Kruskal-Wallis analysis of variance (total irrespective of dilutions)

Time (min)	Mean				P
	OHC	OHC + CTS	Ca (OH)2 + CTS	Ca (OH)2
2	15.6	12	82.9	13.4	<0.0001
5	1.3	9.9	76.5	8	<0.0001
20	5.07	4.27	38.4	5.33	<0.0001
60	0	1.6	9.2	1.4	0.03

OHC: Octenidine hydrochloride, CTS: Chitosan

Irrespective of the medicament used, the antimicrobial efficacy showed progressive reduction as test solution was serially diluted. At dilutions I, II, and III, all the groups of medicaments were able to inhibit E. faecalis growth at all the time intervals, except the Ca(OH) 2 + CTS group [Table 2].

Table 2

Comparison of four groups with respect to colony-forming unit counts of Enterococcus faecalis at different time points in dilution I-V by Kruskal-Wallis analysis of variance

Time (min)	Dilutions	Mean				P
		OHC	OHC + CTS	Ca (OH)2 + CTS	Ca (OH)2
2	I	0	0	98.3	0	0.01
	II	0	0	83.3	0	0.01
	III	6.3	0	73	3	0.01
	IV	34	22.6	76.6	17.6	0.01
	V	38	37.6	83.3	46.3	0.02
5	I	0	0	63.3	0	0.01
	II	0	0	71.3	0	0.01
	III	0	0	78.0	0	0.01
	IV	0	6.3	82.3	3.3	0.01
	V	6.6	43.3	87.6	36.6	0.01
20	I	0	0	78	0	0.01
	II	0	0	55.6	0	0.01
	III	0	0	9.3	0	0.01
	IV	0	3.3	34.6	0	0.01
	V	25.3	18	14.3	26.6	0.02
60	I	0	0	0	0	1.00
	II	0	0	18	0	0.01
	III	0	0	28	0	0.01
	IV	0	0	0	0	1.00
	V	0	8	0	7.3	0.02

OHC: Octenidine hydrochloride, CTS: Chitosan

DISCUSSION

There are many reasons as to why failed endodontically treated teeth may not respond to retreatment successfully. Undoubtedly, the major factor associated is the persistence of microbial infection in the root canal system and/or the periradicular area even after treatment. This includes Gram-positive bacteria such as E. faecalis which have been demonstrated to be extremely resistant to several medicaments, including Ca(OH) 2.[7] Therefore, the present investigation used E. faecalis as the test microorganism against which the antimicrobial action of intracanal medicaments – Ca(OH) 2 and OHC – was evaluated. The most commonly used intracanal medicament Ca(OH) 2 produces antibacterial damage by protein denaturation and damage to DNA.[8] A systematic review disclosed that Ca(OH) 2 has limited effectiveness in eliminating bacteria from human root canal when assessed by routine culture techniques.[9] The study tests OHC as well which demonstrates broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, fungi, and several viral species.[5]

Moreover, most enterococci are naturally resistant to various antimicrobials including β-lactams, clindamycin, low concentrations of aminoglycosides, and fluoroquinolones or may acquire resistance to these antibiotics after exposure either through the acquisition of resistance genes on plasmids or transposons from other organisms. They even can secrete pheromones and stimulate the synthesis of the surface aggregation substance that contributes to the exchange of plasmids carrying resistance.[910]

Thus, the combination of medicaments is advantageous as it decreases the development of resistant bacterial strains and helps produce a synergistic antimicrobial effect.[2] Therefore, CTS was added to OHC and Ca(OH) 2 in an attempt to test if it had any potential additive or synergistic effects on the viability of E. faecalis.

The modified direct contact test employed in this study has many advantages over the routinely used agar diffusion test. Direct contact test is a quantitative assay which allows water-insoluble materials to be tested. It relies on direct and intimate contact between the test microorganisms and the tested medicaments and is virtually independent of the diffusion properties of both the tested material and the media unlike in the agar diffusion test. It is reproducible and is insensitive to the size of the inoculum brought in contact with the tested material.[11]

In this study, the discrete cosine transform method was modified in such a way that plating was done immediately after each interval of contact time. This modification makes it possible to measure the material's bactericidal effect by calculating the number of surviving bacteria after each time duration. This is clinically relevant as surviving bacteria can continue to grow after removal or loss of activity of the medicament. Moreover, lack of growth on the culture plates could be a result of bacteria changing into a so called viable but nonculturable (VBNC) state because of the stress caused by the antimicrobial components of the medicament. However, the development of VBNC bacteria typically requires several days of continuous stress and is therefore unlikely to be an error factor in this study.[12]

The results obtained from the modified direct contact test showed that as test solution was serially diluted, the antimicrobial efficacy of all medicaments groups in the study showed a progressive decline, and also, as time progressed from 2 to 60 min, all the medicament groups, in general, were able to completely inhibit the test microorganisms within 60 min up to the fourth serial dilution. These results suggest that elevated concentration of drug compound is required in direct contact with the microorganism, and if used in a lesser concentration, they should be placed as an intracanal dressing for a longer duration of time from 20 to 60 min to produce maximum antimicrobial efficiency against E. faecalis.

The results of the present study showed that Ca(OH) 2 when used alone exhibited maximum antimicrobial efficacy against E. faecalis, whose efficacy has been previously displayed in a study by Ooi et al.[13] However, it is noteworthy that except the Ca (OH)2 and CTS combination group (Ca(OH) 2 + CTS), all the other groups of medicaments showed maximum antibacterial efficacy when used at higher concentrations till the third dilution against E. faecalis. The results of the modified direct contact test are comparable to that obtained in a study, where it was found that Octenisept gel showed the modest antimicrobial activity against E. faecalis in the root canal after 1 min and more pronounced effective dentin disinfection after incubation for 10 min and 7 days. The authors also reported that the potency of OHC against various microorganisms with a higher phenoxyethanol concentration was not more effective than the 1:1 gel. Therefore, it can be assumed that octenidine itself is the active agent and that the synergistic antimicrobial effect with other combinations is limited.[141516]

Another microbiological study using broth dilution method confirmed that the combination of OHC + CTS and Ca(OH)2 + CTS produced inferior results than that of the medicaments used alone, suggesting that the addition of CTS as a carrier was not able to produce any additive or synergistic antimicrobial effect, but instead its addition reduced the efficacy of the pure compound against the test microorganisms.[17] Hence, it can be inferred that to maximize the antimicrobial effect of the intracanal medicament, the determining crucial factors are necessarily the quantity and the duration for which the medicaments are placed.[18]

Further in vivo studies are required to use these medicament combinations with CTS as a drug carrier. The depth of penetration of the medicament combinations into dentinal tubules, their duration of action, the concentration of the medicaments, and the volume of the medicament to be given are to be investigated and compared. However, preclinical and clinical trials are required for the evaluation of biocompatibility and safety to recommend these intracanal medicament combinations for clinical usage.

CONCLUSIONS

Within the limitations of the study, the test results concluded that:

The antimicrobial efficacy against E. faecalis of these two medicaments: octenidine and Ca(OH) 2– is maximum when used alone and for a period of at least 60 min to ensure complete root canal disinfection

The highest efficacy of the medicament was seen till when it was serially diluted three times

The addition of CTS as a carrier did not enhance the antimicrobial efficacy of octenidine and especially Ca(OH) 2 against E. faecalis. However, the combination of CTS with Ca(OH) 2 drastically reduced its antimicrobial efficiency

Among all the groups compared, the highest antibacterial efficacy against E. faecalis was shown by the Ca(OH) 2 alone group.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.