Influence of Irrigation Regimens on the Adherence of Enterococcus faecalis to Root Canal Dentin Anil Kishen, BDS, MDS, PhD,* Chee-Peng Sum, BDS, MSc,* Shibi Mathew, BSc, MSc,* and Chwee-Teck Lim, PhD † Abstract Enterococcus faecalis is frequently associated with post-treatment endodontic infections. Because adher- ence of bacteria to a substrate is the earliest stage in biofilm formation, eliciting the factors that links adher- ence of this bacterium to dentin would help in under- standing its association with treatment-failed root ca- nals. This investigation aimed to study the effects of endodontic irrigants on the adherence of E. faecalis to dentin. The bacteria adherence assay was conducted by using fluorescence microscopy, and the adhesion force was measured by using atomic force microscopy. There were significant increases in adherence and adhesion force after irrigation of dentin with ethylenediaminetet- raacetic acid (EDTA), whereas sodium hypochlorite (NaOCl) reduced it. With the use of chlorhexidine (CHX), the force of adhesion increased, but the adher- ence assay showed a reduction in the number of ad- hering bacteria. The irrigation regimen of EDTA, NaOCl, and CHX resulted in the least number of adhering E. faecalis cells. This study highlighted that chemicals that alter the physicochemical properties of dentin will in- fluence the nature of adherence, adhesion force, and subsequent biofilm formation of E. faecalis to dentin. (J Endod 2008;34:850 – 854) Key Words Adhesion force, bacterial adherence, biofilm, dentin, irrigation E nterococcus faecalis has been increasingly associated with refractory apical peri- odontitis. Although this species of bacteria is not commonly found in the oral cavity without the history of endodontic treatment, it is frequently found as a single species in patients with post-treatment endodontic infection (1–4). One possibility of high pro- portion of E. faecalis in root-filled teeth could be because of the ability of this bacterium to survive chemomechanical preparations during endodontic treatment and retreat- ment, the ability of this bacterium to enter the canal in the process of treatment (during or between treatment sessions), or the ability of this bacterium to enter after root filling. In addition to the inherent capacity of this bacterium to resist many antimicrobials and invade tissues, the ability to form distinct biofilm under difficult growth conditions is considered as the major factor attributing to their survival in postendodontic environ- ment (5–8). Adherence of bacteria to hard tissues or artificial biomaterials is the first step toward biofilm-mediated infections. E. faecalis possess different virulence factors that enable them to adhere to dentin and invade dentinal tubules (9). Enterococci also express factors that aid their adhesion to host cells and extracellular matrix, which in turn facilitates tissue invasion, causes immunomodulation, and produces toxin-medi- ated damage (10). Biofilm-mediated infections are not resolved by the host immune system, whereas the bacteria residing in a biofilm are difficult target to treat with local or systemic antimicrobials (11–13). Thus, mechanical removal of biofilm is indicated to be the ideal approach to treat biofilm-mediated infections. However, this procedure is influenced by the location of biofilm in an endodontic infection. Although intracanal biofilms might be removed to certain extent by rigorous chemomechanical preparation (14), extraradicular or periradicular biofilms might necessitate endodontic surgical intervention (15–17). Sodium hypochlorite (NaOCl) is one of the most commonly used endodontic irrigants because of its ability to destroy a broad spectrum of microbes and dissolve organic materials. NaOCl dissolves necrotic pulp tissue and denatures collagen. Ethyl- enediaminetetraacetic acid (EDTA) is often used with NaOCl to remove the smear layer (18). Removal of the smear layer has been reported to reduce microleakage after root filling, although it increases dentin permeability (19). EDTA denatures proteins rich in cysteine and therefore can denature human Type I collagen in dentin. Chlorhexidine (CHX) contains 1, 6-bis-p-chlorophenylbiguanidohexane and has also been used for endodontic disinfection (20). Although studies in the past have examined the antimi- crobial efficacy of different endodontic irrigants, very few studies have investigated the influence of different irrigation regimens on the adherence of bacteria or, more spe- cifically, E. faecalis to root canal dentin (5, 18, 21, 22). Chemical and physical factors that determine the adherence of E. faecalis to dentin are not fully understood. Measuring force of adhesion between E. faecalis and chem- ically treated dentin would contribute to further our understanding of how this bacte- rium can persist in a post-treatment endodontic environment. Once bacteria adhere to a substrate and form biofilm, they gain exceptional ability to resist antimicrobials and survive tough growth conditions. The atomic force microscope (AFM) has been used re- cently to study the forces of interaction between bacteria cells and between bacteria cell and substrate (23, 24). The bacteria cell or substrate particle can be attached onto an AFM tip, and the forces of interaction between bacterial cells and between the bacterial cell and substrate can be determined. Basically, as the AFM tip approaches the substrate and the gap between the 2 interacting bodies closes to nanometer range, the interacting forces developed From the *Department of Restorative Dentistry, Faculty of Dentistry, and † Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore. Address requests for reprints to Dr A. Kishen, Department of Restorative Dentistry, Faculty of Dentistry, National Univer- sity of Singapore, 5 Lower Kent Ridge Rd, Republic of Singa- pore 119074. E-mail address: rsdak@nus.edu.sg. 0099-2399/$0 - see front matter Copyright © 2008 American Association of Endodontists. doi:10.1016/j.joen.2008.04.006 Basic Research—Technology 850 Kishen et al. JOE — Volume 34, Number 7, July 2008