Comparison of antibacterial activity of alexidine alone or as a final irrigant with sodium hypochlorite and chlorhexidine

ABSTRACT AIMS: To compare the antibacterial activity of alexidine (ALX) alone or as a final irrigant in combination with sodium hypochlorite (NaOCl), with the most common canal irrigants,

NaOCl and chlorhexidine (CHX). MATERIALS AND METHODS: Ninety-four root fragments from extracted human teeth were infected with _Enterococcus faecalis_ for 24 h and then distributed into 4

groups of 20 fragments each. The NaOCl, CHX and ALX groups were immersed in 1 ml of 2.5% NaOCl, 2% CHX, and 1% ALX for 10 min, respectively. The samples of the NaOCl+ALX group were immersed

in 1 ml of 2.5% NaOCl for 10 min followed by 1% ALX for 10 min. Bacteriological samples were taken, cultured, and the colony-forming units were counted. RESULTS: There was no significant

differences among the experimental groups (_P_>0.05) except for the comparisons CHX versus ALX and NaOCl+ALX versus ALX (_P_=0.004). ALX alone was the worst irrigant. CHX and NaOCl+ALX

eradicated all bacteria. All experimental groups were significantly more effective than the control group immersed in saline (_P_<0.05). CONCLUSIONS: The antibacterial effect of ALX alone

was inferior to 2% CHX and 2.5% NaOCl. However, the combination of NaOCl with ALX as a final irrigant eradicated the biofilms. SIMILAR CONTENT BEING VIEWED BY OTHERS A MATCHED IRRIGATION

AND OBTURATION STRATEGY FOR ROOT CANAL THERAPY Article Open access 25 February 2021 CLEANING AND DISINFECTION OF THE ROOT CANAL SYSTEM PROVIDED BY FOUR ACTIVE SUPPLEMENTARY IRRIGATION

METHODS Article Open access 15 February 2024 COMPARISON OF CONVENTIONAL AND CONTEMPORARY ROOT CANAL DISINFECTION PROTOCOLS AGAINST BACTERIA, LIPOTEICHOIC ACID (LTA), AND LIPOPOLYSACCHARIDE

(LPS) Article Open access 21 January 2023 INTRODUCTION The aim of treatment in infected root canals is to eliminate microorganisms from the root canal system and to prevent its reinfection.

Bacteria are the main microorganism implicated in the apical periodontitis.1 Among them, _Enterococcus faecalis_ deserves attention because of its high prevalence in the different types of

endodontic infection, especially in persistent infections.2,3 The inherent ability of _E. faecalis_ to adhere and invade dentinal tubules and form communities in an organized biofilm may

contribute to its resistance to irrigant solutions and intracanal medicaments.4 Consequently, this microorganism is often chosen to induce _ex vivo_ bacterial biofilms in assays comparing

antimicrobial solutions. During the root canal treatment, mechanical debridement is of utmost importance to remove microorganisms and organic content that might serve as nutrients for

residual bacteria. Nonetheless, studies have demonstrated that although instrumentation and irrigation are effective in substantially reducing the number of bacteria in infected canals, in

many cases bacteria remain in the main root canal even when sodium hypochlorite (NaOCl) is used as the irrigant.5 NaOCl is the most common root canal irrigant due to its tissue-dissolving

capability, its broad antimicrobial action, as well as its ability to neutralize toxic products.6,7 However, NaOCl has many disadvantages, including cytotoxicity, reduced efficacy in the

presence of organic matter, and interference with pulp regeneration procedures.8–10 These limitations stimulate the search for safer and more effective irrigants. An alternative to NaOCl is

chlorhexidine digluconate (CHX). This irrigant is a bisbiguanide disinfectant that has high antimicrobial activity, substantivity, and biocompatibility. However, CHX has been shown to have

no tissue-dissolving activity and, when combined with NaOCl, produces para-chloroaniline, a toxic precipitate.11–13 The search for the ideal root canal irrigant revealed another candidate -

alexidine (ALX). This substance is a bisbiguanide disinfectant similar to CHX, it contains two hydrophobic ethylhexyl groups in its structure and it has a higher affinity for major bacterial

virulence factors such as bacterial lipopolysaccharide and lipoteichoic acid than CHX.14,15 Alexidine is used as a disinfectant in contact lens solutions16,17 and as an antiseptic in

mouthwashes.18–20 A recent study showed that the antibacterial activity of alexidine against _E. faecalis_ infecting dentin blocks was superior to CHX.21 Also, while there are many reports

of allergic reactions, including anaphylaxis, following exposure to chlorhexidine, there is a lack of reports for ALX.22–24 Another important advantage of alexidine is that its combination

with NaOCl does not produce any precipitate or para-chloroaniline.25 Therefore, the combination of NaOCl as the main irrigant with ALX as the final irrigant may be of great utility for the

treatment of endodontic infections. The purpose of this study was to compare the efficacy of ALX alone or as a final irrigant in combination with NaOCl with the most common canal irrigants,

NaOCl, and chlorhexidine MATERIALS AND METHODS PREPARATION OF DENTIN BLOCKS Forty-seven upper canines were obtained from the Tooth Bank of the Estácio de Sá University, Rio de Janeiro, RJ,

Brazil. The teeth were extracted for orthodontic or prosthetic reasons. The study was approved by the Ethical Committee at Estácio de Sá University (approval number: 34551214.2.0000.5284).

The coronals and the apical thirds of the teeth were removed using diamond disks (KG Sorensen Ind. Com. Ltda, Barueri, Brazil). Thereafter, the middle thirds of the roots were split along

the long axis and cut into 25 mm2 fragments. The 94 specimens generated were immersed in 2.5% NaOCl solution for 5 min and then in 17% EDTA (Biodinâmica, Ibiporã, PR, Brazil) for 5 min,

followed by washing with 2.5% NaOCl for 5 min to remove the smear layer formed by the cutting action of the disks and any pulp tissue remaining. During these procedures, all solutions were

agitated in an ultrasonic bath at a frequency of 50 Hz (Cristófoli, Campo Mourão, Brazil). Finally, the root fragments were washed with distilled water and sterilized by autoclaving. _E.

FAECALIS_ BIOFILM FORMATION The root fragments were infected with _E. faecalis_ (ATCC 29212) using an apparatus described by Luppens26 and specially adapted by the authors for the present

study (Figure 1). The apparatus is composed of an acrylic chamber, a peristaltic pump (Exatta, Palhoça, SC, Brazil) and two 9-liter glass containers. The three components were connected by

silicone tubing to have a constant flow of the culture medium. All components and supports were cleaned with 70% ethanol and autoclaved before use. Before inoculation, the cementum surfaces

of the 94 root fragments were bonded onto the internal acrylic base of the apparatus. Afterwards, the medium Tryptic Soy Broth (TSB, Difco, Detroit, USA) supplemented with 10% glucose

(Merck, Whitehouse Station, USA). was pumped through the system for 30 min after which it was removed. Then a 24 h culture (20 ml) of _E. faecalis_ was introduced into the device and was

maintained in contact with the root fragments for 30 min. After this period, the pump was restarted, and samples were allowed to develop biofilm for 24 h at 37 °C in the presence of a

constant TSB flow of 6.25 ml/min. At the end of this 24 h period, the root fragments were removed from the device and placed into cell culture wells (1 dentin block per well) of a 24-well

plate (Nest Biotechnology, Wuxi, China). The manipulation of root fragments during the experiment was performed aseptically in a laminar flow hood (Nuaire, Plymouth, MN, USA). The quality

control of the materials sterilization process was attested by the Institutional Sterilization Center. Two samples were used to confirm the biofilm formation. On removal from the device,

they were immediately fixed in freshly prepared 2% glutaraldehyde (Merck, Whitehouse Station, NJ, USA) and then dried in ascending ethanol concentrations. They were then dehydrated to their

critical point in CO2 and sputter-coated with gold under vacuum and analyzed in a scanning electronic microscope at 10.00 Kv and at 5000 magnification (Inspect F-50, FEI, Hillsboro, OR,

USA). DENTIN DISINFECTION ASSAY The root fragments were divided randomly into 4 groups (NaOCl, CHX, ALX and NaOCl+ALX) of 20 blocks each and 12 samples were separated for the control group.

The root fragments of the NaOCl, CHX, and ALX groups were immersed in 1 ml of 2.5% NaOCl, 2% CHX and 1% ALX for 10 min, respectively. The 1% solution of ALX was prepared by dissolving ALX

dihydrochloride powder (Sigma-Aldrich, St Louis, MO, USA) in sterile distilled water (1 g/100 ml). The samples of the NaOCl+ALX group were immersed in 1 ml of 2.5% NaOCl for 10 min followed

by 1% ALX for 10 min. In all groups, except the control group, a neutralizer solution was used for 5 min after the action of the irrigants. This solution was composed of 3% Tween 80, 0.3%

lecithin, 0.1% histidine and 0.5% sodium thiosulfate. In the control group, the root fragments were immersed in 1 ml of sterile saline for 10 min. Microbial samples were obtained from root

fragments by agitation in ultrasound for 3 min. Tenfold serial dilutions were carried out in saline. Then, aliquots of 20 μl of each dilution were plated onto Mitis-Salivarius agar (Difco,

Detroit, MI, USA) plates, and incubated at 37° C for 24 h. The colony-forming units (CFU) that grew were counted and then transformed into actual counts based on the known dilution factors.

Bacterial counts were analyzed via Kruskal–Wallis and Mann–Whitney tests. The significance level was established at _P_<0.05. The statistical analysis was performed using SPSS 17.0

computer software (IBM, New York, NY, USA). RESULTS An _E. faecalis_ biofilm was observed by electron microscopy on both fragments analyzed (Figure 2). Intergroup analysis revealed no

significant difference among the experimental groups (_P_>0.05) except for the comparisons CHX versus ALX, and NaOCl+ALX versus ALX (_P_=0.004). ALX alone was the less effective irrigant.

CHX and NaOCl+ALX eradicated all bacterial cells in all samples. The NaOCl group showed bacterial growth only in one of the 20 samples while ALX showed bacterial growth in seven of the 20

samples (Table 1). All experimental groups were significantly more effective than the control group (_P_<0.05). DISCUSSION Biomechanical cleaning with files and antibacterial irrigants

reduces the bacteria load in infected root canals; however, microbial communities grown in biofilms are remarkably difficult to eradicate with antimicrobial agents.27 There are reports

showing that microorganisms grown in biofilms could be 1000–1500 times more resistant to antimicrobials than planktonically grown bacteria.27,28 This _in vitro_ study compared the

antibacterial effect of ALX, a promising root canal irrigant, alone or as a final irrigant in combination with NaOCl, with the most common root canal irrigants: NaOCl and CHX. _E. faecalis_

was chosen as a bacterial marker since its resistance to many intracanal disinfectants is well documented2,4,29,30 Gram-positive facultative anaerobe bacterium is commonly found in

endodontically treated root canals that failed2. The persistence of _E. faecalis_ may stem, in part, from its ability to form biofilms in root canals and its capability to invade dentinal

tubules.31,32 Additionally, this bacterium possesses a plethora of virulence factors, highlighting: aggregation substances, surface adhesins, sex pheromones, lipoteichoic acid, extracellular

superoxide, gelatinase, hyaluronidase, and cytolysin (hemolysin).4 In the present study, the inoculation apparatus allowed the formation of the biofilm under a slow turbulent flow to

facilitate the adhesion of cells. When a tooth undergoes pulpal necrosis and subsequently develops periradicular periodontitis, exudates may cycle in and out of the canal. However, the exact

flow rate that occurs _in vivo_ has not been determined. This fluid exchange provides proteins, glycoproteins and other nutrients to the bacteria growing as a biofilm. This not only

provides a sustainable nutrient source but also exerts a shear force on the bacterial biofilm.33 Contrary to expectations ALX alone was the less effective irrigant, but its combination with

NaOCl was similar to CHX. Two previous studies compared the antibacterial activity of ALX and CHX, in the same concentration, and neither study found any significant difference. The first

tested the canal irrigants against _E. faecalis_ infected bovine dentin34 and the second compared these irrigants against _Streptococcus mutans_ biofilm cultivated on human dentin blocks.35

Methodological differences such as the substrate and the bacterium tested could have influenced these results. Contrary to these results, another study21 found a better antibacterial

substantivity against _E. faecalis_ using 1% ALX in comparison to 2% CHX. However, it is important to emphasize that in this substantivity assay, the antimicrobial action was evaluated over

a period of 80 days. Also, the dentin fragments were immersed in the antimicrobial solution first and after transferred to the bacterial suspension, which is the opposite sequence from the

other studies. In the present and previous studies, the antibacterial action was analyzed only once, immediately after the irrigant contact time. The results from the present study are in

accordance with a recent study36, which found that 5.25% NaOCl was highly effective against _E. faecalis_ compared with CHX and ALX. There was no significant difference between 1% ALX and 2%

CHX. Despite both studies used different concentrations of NaOCl, it is not expected significant differences in the antimicrobial activity of NaOCl varying its concentration.37–39 The best

results were obtained with 2% CHX and with the combination of 2.5% NaOCl+1% ALX as a final irrigant. In fact, both substances completely destroyed the bacterial biofilms. However, CHX in not

able to dissolve organic tissues. Thus, the combination of NaOCl+ALX has a good potential for endodontic treatment to eliminate biofilms: the solvent capability of NaOCl, the high

biocompatibility of ALX, the advantage that it does not form any precipitate when in combination with NaOCl and now, the confirmed antibacterial efficacy of the tested protocol, compatible

with CHX and NaOCl alone, justify this potential. However, it is important to highlight that the group NaOCl+ALX was privileged by a higher contact time between the root fragments and

irrigant solutions (20 min) in comparison with the other groups (10 min). This difference was necessary since ALX was used in this group as a final irrigant. Certainly, further studies are

required to compare this final irrigation protocol with others. Under the conditions of the present study, it was concluded that 1% ALX alone should not be indicated as an intracanal

irrigant since its antibacterial effect against _E. faecalis_ was inferior to 2% CHX and 2.5% NaOCl. However, the combination of NaOCl with ALX as a final irrigant has potential to be used

in endodontic treatment to eliminate biofilms. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), a Brazilian Governmental Institution. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Estácio de Sá University, Endodontics

580, Alfredo Baltazar da Silveira, Rio de Janeiro, Brazil Thaís M da Silva * Endodontics, Rio de Janeiro, Brazil Flávio RF Alves * Microbiology, Rio de Janeiro, Brazil Márcia TS Lutterbach *

Electronic Microscopy, Rio de Janeiro, Brazil Maurício M Paiva * Department of Medical Microbiology, Institute of Microbiology, Rio de Janeiro, Brazil Dennis de Carvalho Ferreira Authors *

Thaís M da Silva View author publications You can also search for this author inPubMed Google Scholar * Flávio RF Alves View author publications You can also search for this author inPubMed 

Google Scholar * Márcia TS Lutterbach View author publications You can also search for this author inPubMed Google Scholar * Maurício M Paiva View author publications You can also search for

this author inPubMed Google Scholar * Dennis de Carvalho Ferreira View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to

Flávio RF Alves. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution 4.0

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