Characterization of Enterococcus faecalis associated with root canal failures: Virulence and resistance profile

Abstract

Background:

Enterococcus faecalis is a facultative anaerobe frequently associated with chronic root canal infections. Its virulence factors and antimicrobial resistance complicate its elimination. This research aims to analyze the virulence factors and antibiotic resistance profiles of E. faecalis isolates derived from unsuccessful root canal procedures.

Materials and Methods:

A cross-sectional study was conducted with 50 patients who required nonsurgical retreatment due to previous root canal therapy failures. The samples were assessed for biofilm formation and antibiotic resistance, while the production of gelatinase and hemolysin was evaluated through the standard microbiological methods. The correlation between biofilm production and antimicrobial resistance was analyzed using Pearson’s correlation coefficient.

Results:

Among the 50 E. faecalis isolates, 66% demonstrated biofilm formation, with 24% categorized as strong biofilm producers. Antibiotic susceptibility testing indicated complete resistance to metronidazole, whereas all isolates were sensitive to amoxicillin-clavulanic acid, vancomycin, and piperacillin-tazobactam. Significant resistance rates were observed for ampicillin (44%), erythromycin (60%), tetracycline (64%), and ciprofloxacin (66%). A strong negative correlation (r = −0.88) was found between biofilm formation and antibiotic susceptibility, suggesting that strains capable of biofilm production exhibited greater resistance to antibiotics.

Conclusion:

The research underscores the critical importance of the pathogenic traits of E. faecalis, especially its biofilm production potential, in the persistent nature of root canal infections and its association with antibiotic resistance. These results highlight the necessity for enhanced disinfection methods and the exploration of alternative treatment strategies aimed at biofilms to improve the therapeutic outcomes and reduce the risk of reinfection after root canal procedures.

INTRODUCTION

Enterococcus faecalis is a Gram-positive, facultative anaerobe that intrinsically resides in the human alimentary canal as a component of its indigenous microbiota. Although typically benign in its native environment, it is also an opportunistic pathogen with the potential to cause various infections, including urinary tract infections, wound infections, bacteremia, endocarditis, and intra-abdominal infections. It’s remarkable resilience and adaptability enable it to survive in challenging environments, such as the nutrient-depleted and alkaline conditions found in treated root canals.[1]

Root canal treatment is a widely performed endodontic procedure aimed at eliminating infections and preserving natural teeth. Despite its high success rate, failures can occur, often due to persistent or secondary infections. Several factors contribute to root canal treatment failure, including microbial persistence, incomplete removal of bacterial biofilms, inadequate disinfection, and procedural errors.[2] Among the microorganisms implicated in these failures, E. faecalis is among the most commonly isolated species due to its resilience in surviving the hostile conditions within the root canal system.[1] Furthermore, the complexity of root canal anatomy, small apical diameter, ineffective penetration of disinfectants, and procedural mishaps – such as uncleaned accessory canals, instrument fractures, or perforations – can leave residual bacteria.[3,4,5] Addressing these challenges requires the adoption of improved disinfection strategies, meticulous instrumentation techniques, and greater precision technologies such as dynamic navigation system or use of artificial intelligence during root canal therapy.[6]

In the field of endodontics, E. faecalis is a primary pathogen linked to persistent apical periodontitis and recurrent root canal infections.[1] Studies have revealed its frequent association with secondary infections, where it can survive for prolonged periods within the endodontic space, even following the implementation of conventional decontamination protocols. Its pathogenicity is largely attributed to multiple virulence factors, most notably its ability to form biofilms that provide protection against antimicrobial agents and host immune defenses.[7] In addition, E. faecalis produces gelatinase and hemolysins, enzymes that facilitate tissue destruction and promote infection establishment. A major challenge in managing E. faecalis infections is its inherent and acquired resilience to a wide range of antibiotics, including aminoglycosides, tetracyclines, and beta-lactams. This resistance is conferred through multiple mechanisms, such as the activity of efflux pumps, the integration of transferable genetic materials like plasmids and transposons, and horizontal gene transfer. Such traits not only complicate the eradication of the bacterium but also increase the likelihood of reinfection following root canal therapy.[8]

The persistence and adaptability of E. faecalis in endodontic infections underscore the need for a comprehensive understanding of its virulence determinants and resistance mechanisms. These insights are crucial for advancing disinfection protocols, refining therapeutic approaches, and ultimately improving the prognosis of root canal treatments by minimizing the risk of reinfection.

MATERIALS AND METHODS

Patient selection

A cross-sectional study was conducted adhering to the Strengthening the Reporting of Observational Studies in Epidemiology directives and checklist. The study cohort consisted of patients who returned to the conservative and endodontic outpatient department for nonsurgical retreatment following the failure of root canal procedures. A total of 50 patients were enrolled in the study, the study was granted ethical clearance by the Panjab University Institutional Ethical Committee, Panjab University, Chandigarh (PUIEC/2018/120/A-1/29/10). An ethically approved consent form was secured from all participants who authorized their participation in the research. The selected patients were within the age range of 18–50 years, encompassing a broad spectrum from young adults to middle-aged individuals. All teeth involved in the study were single-rooted, characterized by a single main canal structure, and had previously undergone endodontic treatment that resulted in failure.

Sample collection and processing

The samples were collected using the previously described method.[9] Following irrigation, the canal was parched with absorbent sterile paper points to eliminate moisture. Subsequently, a sterile moistened paper point (Dentsply Maillefer, Ballaigues, Switzerland) was inserted to the apical extent of the root canal and retained in place for 60 s to capture the microbial content. The paper point was then placed into a sterile vial containing thioglycolate broth transport media, and the sample was promptly transported to the microbiology laboratory for the microbial analysis. Aseptic techniques were strictly adhered to during the entire sample collection process. The samples were cultured and presence of E. faecalis was confirmed by the presence of black colonies on bile esculin agar [Figure 1a].

Figure 1.

Virulence factors of Enterococcus faecalis (a) E. faecalis on Bile esculin agar (b) Hemolysin production by E. faecalis (c) Gelatinase production by E. faecalis (d) Biofilm assay

Assessment of enterococcal virulence factors

Biofilm formation

The evaluation of biofilm formation was conducted using the crystal violet assay as described by Christensen et al.,[10] a widely recognized technique for quantifying biofilm development. In this procedure, E. faecalis cultures were inoculated into a 96-well plate containing trypticase soy broth and incubated at 37°C for a duration of 24–48 h to allow biofilm formation. After incubation, the wells were rinsed with phosphate-buffered saline to remove planktonic cells, thereby retaining only the biofilm adhered to the well surfaces. The surface-bound cells were subsequently stained with 200 μl of 2% crystal violet reagent, incubated for 10–15 min, and subsequently rinsed to remove any excess stain [Figure 1d]. The dye that binds to the biofilm was solubilized using acetic acid, and the absorbance was measured at 545 nm using an ELISA absorbance reader to quantify the biofilm. Based on the OD readings, the bacteria were categorized as strong, moderate, or weak biofilm formers according to the strength of the biofilms.

Gelatinase production

The assessment of gelatinase activity was performed following the method described by Coque et al.[11] This involved inoculating E. faecalis isolates onto Todd Hewitt agar containing gelatin, followed by incubation at 37°C for 24–48 h. The presence of gelatinase activity was indicated by the formation of a turbidity zone around the colonies, resulting from the degradation of gelatin [Figure 1c].

Hemolysin production

To evaluate hemolytic activity,[11] E. faecalis cultures were streaked onto Todd Hewitt agar plates enriched with defibrinated sheep blood. The plates were incubated at 37°C for 24–48 h. Hemolysin activity was determined by the appearance of a clear halo surrounding the colonies [Figure 1b].

Assessment of enterococcal resistance profile

The antibiotic sensitivity testing was performed using the disk-diffusion method following the standard protocol issued by the Clinical Laboratory Standards Institute (CLSI).[12] The bacterial susceptibility was evaluated against a range of antibiotics, including ampicillin, amoxiclav, erythromycin, tetracycline, ciprofloxacin, metronidazole, vancomycin, and piperacillin plus tazobactam. For susceptibility testing, the plates of Mueller-Hinton agar were inoculated with standardized bacterial suspension of E. faecalis, and then antibiotic disks were applied on to the plates. Plates were then incubated at 37°C for 18–24 h. Postincubation, the extent of zone of inhibition around every respective disk was measured. The results were interpreted as susceptible, intermediate, and resistant as per the measurements to CLSI interpretive charts.

RESULTS

Enterococcal virulence factors

Biofilm formation

The findings from the crystal violet biofilm assay revealed that 33 out of 50 Enterococcal strains, representing 66%, were capable of producing biofilm, while 17 strains did not show any biofilm formation. Among the 33 biofilm producers, 12 strains (24%) were classified as strong producers, 10 strains (20%) as moderate producers, and 11 strains (22%) as weak producers. For further details regarding the classification criteria based on the absorbance values obtained from the ELISA reader, please refer to Table 1.

Table 1.

Criteria for evaluation of biofilm strength among Enterococcus faecalis isolated from root canal treatment failure cases

Cut off OD value	Biofilm class	Assay result n (%)
OD >4 × ODC	Strong	12 (24)
2 × ODC <OD ≤4 × ODC	Medium	10 (20)
ODC < OD ≤2 × ODC	Weak	11 (22)
OD ≤ ODC	Nil	17 (34)

OD: Optical density

Gelatinase and hemolysin production

Among the 50 bacterial strains tested, 12 (24%) demonstrated gelatinase activity, as evidenced by the presence of a turbidity zone, while 38 strains (76%) exhibited no gelatin degradation. In addition, the production of hemolysins by E. faecalis isolates sourced from root canals was found to be relatively low, with only three strains (6%) exhibiting hemolysin activity, whereas the majority, 47 strains (94%), did not exhibit hemolytic activity.

Enterococcal resistance profile

The findings of the antibiotic susceptibility testing are summarized in Table 2. All strains exhibited complete sensitivity to amoxicillin-clavulanic acid, vancomycin, and piperacillin-tazobactam, with no resistance observed to these three antimicrobial agents. Conversely, all strains demonstrated total resistance to metronidazole, with none showing sensitivity to this antibiotic. Among the strains, 28 (56%) were susceptible to ampicillin, while 22 (44%) displayed resistance. For erythromycin, 20 (40%) of the bacterial strains were sensitive, whereas 30 (60%) were resistant. Sensitivity to tetracycline was detected in 18 (36%) strains, with resistance noted in 32 (64%) strains. Ciprofloxacin susceptibility was observed in 17 (34%) strains, whereas 33 (66%) strains were resistant.

Table 2.

Antibiotic susceptibility test results of Enterococcus faecalis isolated from root canal treatment failure cases

Antibiotic	Resistant, n (%)	Sensitive, n (%)
Ampicillin	22 (44)	28 (56)
Amoxycillin-clavulanic acid	Nil	50 (100)
Erythromycin	30 (60)	20 (40)
Tetracycline	32 (64)	18 (36)
Ciprofloxacin	33 (66)	17 (34)
Metronidazole	50 (100)	Nil
Vancomycin	Nil	50 (100)
Piperacillin-tazobactam	Nil	50 (100)

Correlation between drug resistance and virulence factors

A statistical computation was carried out to evaluate the association between biofilm formation and antibiotic sensitivity in E. faecalis isolates from the study, using the Pearson Correlation coefficient formula. The resulting Pearson correlation coefficient of r = −0.88 indicates a strong negative linear relationship between these two variables. This indicates that as the optical density value of biofilm-forming E. faecalis strains increases, the diameter of the antimicrobial inhibition zone decreases, demonstrating an inverse relationship between biofilm formation and antibiotic susceptibility. Therefore, higher biofilm-forming capacity correlates positively with increased antibiotic resistance.

DISCUSSION

This study underscores the virulence attributes and antimicrobial resistance profiles of E. faecalis isolates recovered from failed root canal treatments. The findings highlight the critical role of biofilm formation, along with the production of gelatinase and hemolysin, in contributing to the persistence and pathogenic potential of these bacterial strains within the root canal environment.

A substantial proportion (66%) of the E. faecalis isolates in the present study demonstrated the capacity to form biofilms of varying intensities. These findings align with those reported by Saffari et al.,[13] who observed that approximately 60%–70% of E. faecalis isolates from endodontic infections exhibited biofilm-forming ability. Similar to our results, their study highlighted that isolates capable of strong biofilm production displayed increased resistance to antimicrobial agents, underscoring the pivotal role of biofilm formation in the persistence and resilience of these infections. Moreover, Al-Ahmad et al.[14] corroborated these observations, reporting that biofilm-associated infections within the root canal system contribute significantly to higher rates of endodontic treatment failure, further supporting the conclusions drawn from the present investigation. Gelatinase activity was detected in 24% of the E. faecalis isolates in this study, which is consistent with the findings of Akbari Aghdam et al.,[15] who reported gelatinase production in 20%–30% of E. faecalis strains associated with dental infections. Furthermore, Salah et al.[16] emphasized the importance of gelatinase in promoting bacterial adhesion, tissue invasion, and biofilm maturation – all of which may contribute to the chronicity and persistence of endodontic infections. In contrast, hemolysin production was observed in only 6% of the isolates, which is lower than the 10%–15% prevalence reported by other investigators, including Sedgley et al.[17] Such discrepancies may reflect strain-specific variability, geographic differences, or variations in the clinical source of isolates.

Regarding antibiotic susceptibility, the present study demonstrated complete resistance of all E. faecalis isolates to metronidazole, aligning with the findings of Hollenbeck and Rice,[8] who confirmed the intrinsic resistance of E. faecalis to this antimicrobial agent. Importantly, none of the isolates exhibited resistance to amoxicillin-clavulanic acid, vancomycin, or piperacillin-tazobactam. These observations are consistent with those reported by Lins et al.,[18] who emphasized the sustained efficacy of vancomycin against the majority of E. faecalis strains, supporting its continued use in clinical practice for managing resistant infections. However, the resistance rates identified in this study for ampicillin (44%), erythromycin (60%), tetracycline (64%), and ciprofloxacin (66%) highlight a concerning trend of increasing antimicrobial resistance, similarly noted by Lins et al.[18] These findings underscore the pressing need for regular surveillance of antibiotic susceptibility profiles and the prudent use of antimicrobials to guide effective and evidence-based endodontic treatment protocols.

The Pearson correlation analysis conducted in the present study demonstrated a strong negative correlation (r = −0.88) between biofilm formation and antibiotic susceptibility in E. faecalis isolates. This finding aligns with the observations of Kouidhi et al.,[19] who reported a similar inverse relationship, indicating that biofilm-producing strains exhibit heightened resistance to antimicrobial agents. These results further support the established concept that biofilm formation serves as a protective strategy, enabling bacterial survival under antimicrobial stress, and underscore the necessity of developing therapeutic approaches targeting biofilm disruption. Notably, our study did not detect the presence of vancomycin-resistant enterococci (VRE); however, the potential emergence of such strains in root canal infections cannot be overlooked. In endodontic cases, biofilm-associated infections pose significant treatment challenges due to the resilience and persistence of these bacterial communities. Moreover, virulence factors such as gelatinase, hemolysins, and aggregation substances contribute to the enhanced pathogenicity of VRE within the oral environment, as reported by Mohamed et al.[20]

Our findings suggest that the persistence of E. faecalis in cases of unsuccessful root canal treatment is multifactorial, being strongly associated with the expression of key virulence factors such as biofilm formation, gelatinase production, and hemolysin production. These factors play a critical role in facilitating bacterial survival within the complex and hostile environment of the root canal system, thereby contributing to endodontic treatment failure. The collective action of these virulence determinants underscores the need for innovative therapeutic approaches that go beyond conventional antimicrobial protocols. Future treatment strategies should focus on targeting biofilm disruption and exploring alternative antimicrobial modalities, including antimicrobial peptides,[21] nanoparticles,[22] and nano-curcumin,[23] to improve the management and clinical outcomes of endodontic infections.

CONCLUSION

This study demonstrates that E. faecalis isolates from failed root canal treatments exhibit significant biofilm formation, virulence factor expression, and antibiotic resistance. The strong inverse correlation between biofilm formation and antibiotic susceptibility highlights the challenge of eliminating these infections with antibiotics alone. Future research should focus on developing strategies to disrupt biofilms and enhance antimicrobial effectiveness. Clinically, the use of biofilm-targeting agents and combination therapies may improve the treatment outcomes in endodontic infections.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was financially supported by DST (UT Chandigarh).