Evaluation of Enterococcus faecalis adhesion, penetration, and method to prevent the penetration of Enterococcus faecalis into root cementum: Confocal laser scanning microscope and scanning electron microscope analysis.

AIM: To ascertain the role of Enterococcus faecalis in persistent infection and a possible method to prevent the penetration of E. faecalis into root cementum.
METHODOLOGY: One hundred and twenty human single-rooted extracted teeth divided into five groups. Group I (control): intact teeth, Group II: no apical treatment done, Group III divided into two subgroups. In Groups IIIa and IIIb, root apex treated with lactic acid of acidic and neutral pH, respectively. Group IV: apical root cementum exposed to lactic acid and roughened to mimic the apical resorption. Group V: apical treatment done same as Group IV and root-end filling done using mineral trioxide aggregate (MTA). Apical one-third of all samples immersed in E. faecalis broth for 8 weeks followed by bone morphogenetic protein and obturation and again immersed into broth for 8 weeks. Teeth split into two halves and observed under confocal laser scanning microscope and scanning electron microscope, organism identified by culture and polymerase chain reaction techniques.
RESULTS: Adhesion and penetration was observed in Group IIIa and Group IV. Only adhesion in Group II and IIIB and no adhesion and penetration in Group I and V.
CONCLUSION: Adhesion and penetration of E. faecalis into root cementum providing a long-term nidus for subsequent infection are the possible reason for persistent infection and root-end filling with MTA prevents the adhesion and penetration.

INTRODUCTION

Apical periodontitis, a sequel of endodontic infection, occurs because of the dynamic encounter between microbial challenge and the host defense mechanism at the interface between infected radicular pulp and periodontal ligament resulting in local inflammation and leading to periapical lesions. Most of these lesions regress, but some persist as asymptomatic periapical lesions.[1]

Enterococcus faecalis is the most commonly detected microorganism from persistent and endodontic reinfections,[2] prevalence ranges from 24% to 77%.[3] E. faecalis possesses several survival and virulence factors that make it a resistant bacteria.[456] Most cited virulence factors are adhesion substance (AS), surface adhesins, sex pheromones, lipoteichoic acid, extracellular superoxide products, the lytic enzymes gelatinase, hyaluronidase, and the toxin cytolysin. Angiotensin-converting enzyme (ACE) and serine protease (Spr) help in binding the tooth structure. Each of them may be associated with various stages of pulpal and periapical infections.[45] Many studies have been conducted to show penetration of E. faecalis into root dentin.[78910] Bacteria and bacterial by-products penetrate into root cementum in periodontally diseased teeth.[1112] However, literature is scarce regarding the microorganisms and in particular the E. faecalis penetration and adherence into root cementum in endodontic infections.

Mineral trioxide aggregate (MTA) is a biocompatible, bacteriostatic agent with better-sealing properties when used as root-end filling material. It has cement-conductive properties and therefore forms new bone and periodontal ligament in direct contact with it, MTA is ideal to form an apical barrier against which gutta-percha can be condensed three dimensionally.[1314]

The present study aims to evaluate the adhesion and penetration of E. faecalis into root cementum in different conditions at root apex, its role in causing persistent infection and also to evaluate the adhesion and penetration of E. faecalis when MTA is used as root-end filling material.

METHODOLOGY

The present in vitro study was conducted in the Department of Conservative Dentistry and Endodontics (A B Shetty Memorial Institute of Dental Sciences). Teeth sterilization (gamma irradiation) at Microtrol, Bengaluru. Confocal laser scanning microscope (CLSM) (ZEISS LSM 510 META GmbH, Mannheim, Germany) and scanning electron microscope (SEM) (FEI Quanta 200, Oregon, USA) at the Indian Institute of Science, Bengaluru. The microorganism invading the root cementum was identified by cultural characteristics, biochemical tests at Gulbarga University, Kalaburagi and further confirmed by real-time polymerase chain reaction (PCR) technique at Rajarajeswari Dental College and Research Institute, Bengaluru, India.

One hundred and twenty human single-rooted teeth recently extracted for orthodontic reasons were collected for the study and stored in chlorhexidine solution until use. The presence of single canal was confirmed by radiograph and observation under microscope (×20) to rule out the presence of any cracks, fractures, and the craze lines. All the specimens were randomly divided into five groups as follows:

Group I (n = 20): (Control) intact teeth; root apex sealed using varnish

Group II (n = 20): Access cavity prepared and no apical treatment done

Group III (n = 40): After access cavity, preparation divided into two subgroups (n = 20 each)

Group IIIa: Apical one-third of the root was exposed to lactic acid (organic acid) at acidic pH

Group IIIb: Apical one-third of the root was exposed to lactic acid at neutral pH

Group IV (n = 20): Access opening done, 1 mm of the root apex exposed to lactic acid and also the root apex was roughened using diamond point to mimic demineralization and resorption

Group V (n = 20): Same procedure as in Group IV followed by root-end filling using MTA after gamma irradiation.

In Group I (control group), no access preparation was done, and apical 1 mm of the teeth were sealed using varnish. In Groups II, III, IV, and V, access opening and canal debridement were done. All the groups were subjected to gamma irradiation.

Culturing procedure

Vancomycin-sensitive strains of E. faecalis (ATCC-29212) were cultured in tryptone soya bean agar broth prepared by mixing 1.8 g powder in 60 ml of distilled water and sterilized in an autoclave. The E. faecalis strain was inoculated in the broth and incubated at 37°C for 24–48 h for bacterial growth and confirmed by Gram's stain. To mimic the primary endodontic infection, the E. faecalis broth was inoculated into the root canals of all the samples except Group I (control) with a micropipette and the apical one-third of all the teeth were submerged into the broth for 8 weeks with alternate day refreshment.

The specimens of Groups II, III, IV, and V were subjected to biomechanical preparation using ProTaper nickel–titanium rotary instruments in a contra-angle gear reduction handpiece (X-Smart, Dentsply Maillefer, Ballaigues, Switzerland) up to size F3 followed by obturation up to the working length (Root ZX II, J. Morita, Japan) using AH Plus sealer and gutta-percha (single-cone technique) and coronal sealing using glass ionomer cement. In Group V, apical 3 mm of the root canal was filled using MTA [Figure 1] and finally obturated using gutta-percha.

Figure 1

Scanning electron microscope image shows mineral trioxide aggregate as root-end filling material (×60)

Apical one-third of all the teeth were immersed in the E. faecalis broth for another 8 weeks with alternate day refreshment for secondary infection; subsequently, the samples were washed using 1 ml phosphate-buffered saline to remove any nonadherent bacteria that might have been attached. Vertical grooves were made using a tapered fissure diamond point on buccal and lingual surfaces, and each tooth was split into two halves with the help of a chisel. Samples were stained with 50 μL fluorescein diacetate (FDA) (Himedia, India) and 50 μL of propidium iodide (PI) (Sigma) to facilitate observation under CLSM. For SEM analysis, gold sputtering was done and observed under SEM bacterial growth and organism invading the root cementum was identified by Gram's staining, culture characteristics, biochemical tests, and real-time PCR technique.

RESULTS

Statistical analysis was done using Chi-square test and Student's t-test (SPSS-20, IBM Analytics, New York, United States). Scoring criteria of “0” for no adhesion and “1” for adhesion were used. Penetration was measured using depth measuring tool from the CLSM software. Figure 2 shows live bacteria (green color) and dead bacteria (red color). Figure 3 shows depth of penetration of E. faecalis in root cementum.

Figure 2

Red color shows dead Enterococcus faecalis and green shows live Enterococcus faecalis under confocal laser scanning microscope

Figure 3

Depth measuring scale of confocal image showing - score 0 for no adhesion and score 1 given for adhesion, Group I (control group): absence of Enterococcus faecalis Group II and IIIb samples showing adhesion of Enterococcus faecalis to the root cementum. Group IIIa samples showed penetration of Enterococcus faecalis up to 140 μm deep into the root cementum, and Group IV shows penetration of Enterococcus faecalis up to 160 μm into the root cementum under confocal laser scanning microscope

In Group I and V, no adhesion and no penetration of E. faecalis into the root cementum were seen [Tables 1–3, Figure 3 and Graph 1].

Table 1

Data of all the samples

Table 3

Analysis of residues for E. faecalis adhesion to root cementum in all the groups

Graph 1

Frequency of Enterococcus faecalis adhesion to root cementum in all groups

In Group II, few samples showed adhesion with the mean of adhesion being 0.55 and no penetration [Tables 1–4, Figure 3 and Graph 1].

In Group III A, all the samples showed adhesion and penetration with a mean of penetration being 132.5 μm. In Group IIIb, few samples showed adhesion and the mean was 0.6 and no penetration [Tables 1–4, Figure 3 and Graphs 1, 2].

Table 4

Students-t test for comparison of the group IIIA and IV penetration of E. faecalis into root cementum

Figure 4

Scanning electron microscope image showing colonies of Enterococcus faecalis into the root cementum (×10,000)

In Group IV, all the samples showed adhesion and penetration were seen up to 160 μm into root cementum with the highest mean of penetration being 150.05 [Tables 1–4, Figures 3, 4 and Graphs 1, 2].

Graph 2

Mean penetration of Enterococcus faecalis into root cementum in Group IIIa and IV

In Group V, no adhesion or penetration was seen [Figures 1, 5 and Table 1].

Figure 5

Scanning electron microscope image shows no adhesion and penetration of E. faecalis into the root cementum (×5000)

Chi-square test showed a significant difference (P < 0.001) among all the groups and the adhesion progressively increased from Group I–IV being lowest in Group I and V and highest in Group IIIa and Group IV [Table 2].

Table 2

Comparison of the all groups using Chi-square test

Student's t-test for comparison of the penetration in Group IIIa and IV showed a significant difference between the two groups (P < 0.001) with significantly higher values in Group IV [Table 4 and Graph 2].

DISCUSSION

The primary goal of root canal treatment is elimination and prevention of the infection which includes proper shaping and cleaning, root canal disinfection, three-dimensional obturation, and proper coronal restoration. The overall success depends on successful infection control.[15]

Even after rapid development in the root canal treatment procedures, even when highest standards and most careful root canal procedures were followed, failures still occur[16] because the microbial infection persists in critical root canal regions which cannot be cleaned, obturated, and also the factors located in the periapical region interferes with posttreatment healing of the lesion.[117] Current literature suggests the persistent infection or reinfection as the major causes of failure of endodontically treated teeth.[18]

In the present study, E. faecalis was chosen as the test organism because it is commonly detected species from persistent and reinfection.[2] It has the unique properties such as production of collagen-binding proteins such as ACE and Spr, can survive in alkaline pH,[19] long starvation periods,[6] can derive the nutrients from hyaluronan converted by enzyme hyaluronidase and also from dentinal fluid even in a well-sealed root canal system, and can become viable in the presence of serum.[59] Cytolysin, AS-48, and bacteriocin inhibit other bacterial growth. Cytolysin destroys the cells such as erythrocytes, peripheral nerve myelin cells, and macrophages.[5] All these factors may be associated with various stages of an endodontic infection as well as with periapical inflammation.[45] While some products of the bacteria may be directly linked to periradicular tissue damage, a large part of the tissue damage is mediated by host immune response to the bacteria.[5] The incidence of E. faecalis was seen in both asymptomatic primary infections and in asymptomatic persistent infections when evaluated by nested PCR technique. However, its prevalence is higher in secondary infections.[20] Continued research on E. faecalis, its characteristics and its ability to invade tooth structures and occurrence in persistent infections will help to eradicate this “too robust” microorganism from the dental apparatus may well define the future of the endodontics.[3]

ACE and Spr produced by E. faecalis help for strong adherence mainly to Type I collagen.[5] Nallapareddy et al. showed E. faecalis adhesin (ACE) mediates the attachment to extracellular matrix proteins, i.e. collagen Types I, IV and laminin.[21] Hubble et al. showed ACE and Spr play a significant role in binding E. faecalis strains to the root canal walls.[8] Cementum is composed of organic substance mainly consisting of more than 90% of Type I collagen and 5% of Type III collagen.[11] The results of the present study showed the adhesion and penetration of E. faecalis into the root cementum. Therefore, the ability of the E. faecalis to bind to the collagen Type I and the biofilm formation might be the contributing factors for adhesion and penetration to the root cementum.[321]

In Group I, there was no adhesion or penetration, as all the teeth were intact and root-apex was sealed. In Group II, few samples showed adhesion and no penetration, samples in this group were not subjected to any apical treatment. In Group IIIa, all the samples showed adhesion and penetration; this was because an acidic environment created by immersing the teeth samples in organic acid (lactic acid) to mimic apical demineralization as seen in the initial stages of periapical infections. In Group IIIb, the root apex was immersed in lactic acid with neutral pH; the results showed only adhesion and no penetration.[22] In Group IV, all the samples showed adhesion and penetration with a mean penetration up to 160 μm into the root cementum; the apical root cementum was exposed to lactic acid and also the root apex was roughened to mimic apical resorption.[23]

The results of the present study indicate that if the treatment is done at the early stages of primary infection as in the case of Group II, there are fewer chances of penetration of E. faecalis and reinfection or persistent infection. Whereas in Group IIIa and IV, there was a delay in treatment leading to changes in the apical environment such as demineralization or resorption of root cementum as seen in periapical lesions which provides favorable conditions for E. faecalis to penetrate the root cementum thereby increasing the chances of persistent infection or reinfection. In Group V, no adhesion and no penetration were seen irrespective of the apical changes because the root apex was sealed with MTA which acts as a barrier and prevents the adhesion or penetration of E. faecalis.

Daly et al. showed the penetration of bacteria and its by-products such as lipids of about 10 μm (micron meter) into cementum of periodontally diseased teeth,[24] endotoxin of Escherichia coli penetrated into root cementum,[25] and Bosshardt and Selvig showed bacterial by-products penetrating 40–70 μm deep into the diseased root cementum.[11] The results of the present study are in accordance with these studies showing the adhesion and penetration of E. faecalis into the root cementum. The difference in the depth of penetration might be because of the difference in the methods used to evaluate the data and the environmental conditions affecting the microorganism. In the present study, data were collected using CLSM and SEM confirmative test for organism identification into root cementum on cultured tooth specimens was done by Gram's stain, cultural characteristics, biochemical tests, and further, the specific analysis was done by PCR technique[Graphs 3 and 4]. All these methods detected the presence of E. faecalis into the root cementum.

Graph 3

Polymerase chain reaction graph shows melt curve, confirms Enterococcus faecalis penetration into root cementum in the culture tooth specimen

Graph 4

Polymerase chain reaction graph shows amplification plot, confirms Enterococcus faecalis penetration into root cementum in the culture tooth specimen

CLSM has several advantages over other methods. It can easily differentiate between live and dead bacteria, shows the metabolic activity of the bacteria, measures the depth of bacterial penetration into the tooth structures, and also has the ability to represent a three-dimensional picture of infection.[26] FDA and PI facilitate the observation under CLSM. FDA is a nonfluorescent, cell-permeable dye which allows the viable cells appears green. In viable cells, the dye crosses the cell membrane and gets metabolized by intracellular esterase and causes the fluorescein (green). PI is a noncell permeable, red fluorescent dye which adheres to ruptured cell membranes, and therefore, the dead bacteria appear red in color.

PCR techniques are more specific, sensitive, and accurate than culturing methods can detect uncultivable and fastidious microorganisms.[2728] Therefore, the organism infecting the root cementum was finally confirmed by real-time PCR technique.

Teeth were sterilized using gamma irradiation as it does not alter collagen characteristics of the teeth.[29] Chivatxaranukul et al. showed the adherence of E. faecalis was less to autoclaved teeth compared with gamma irradiated teeth and stated less adherence of E. faecalis to autoclaved teeth might have been caused by alteration of collagen strands by autoclaving.[7]

Eight weeks of culturing was done twice because, the first 8 weeks showed primary infection followed by bone morphogenetic protein and obturation of the teeth samples, giving the impression of routine root canal treatment for primary infection. The next 8 weeks of E. faecalis culturing was done to show secondary infection or reinfection. The present study shows that irrespective of the primary or the secondary infection, the apical changes in the environment such as apical demineralization or apical resorption helps E. faecalis to penetrate the root cementum and cause reinfection or persistent infection. Therefore, the severity of the infection plays a significant role in the adhesion and penetration of E. faecalis and with the use of advanced materials such as MTA as root-end filling material prevents the adhesion or penetration of E. faecalis into the root cementum.

Lactic acid was used for demineralization of root cementum because E. faecalis is Gram-positive lactic acid producing bacteria,[30] and the end product of anaerobic glycolysis is lactic acid.[31] Nekoofar et al. showed that pH at periapex is generally acidic, it is reported that pH dropped to 3.5–4.5 within 7–15 min of ingestion of microorganisms.[32] Kato et al. stated the importance of acidic extracellular pH, which is a microenvironmental factor that causes tumor progression. Acidic metabolite such as lactic acid, a by-product of anaerobic glycolysis which occurs in hypoxia is a major cause for such lesions. It is seen that acidic pH activates protease activity, induces gene expression, signals the transconduction pathway, and stimulates the destruction of adherence junctions between the cells.[31] The study highlights the role of lactic acid in causing inflammatory lesions.

Numerous studies have been conducted on E. faecalis. However, this single microorganism still poses to be an endodontic challenge. In the emerging periods of time, with the advent of the sophisticated, innovative techniques, the occurrence of the bacterial combinations particularly in persistent infections indicates the need of research for the identification of the microorganisms and their role in disease process. The use of advanced materials such as MTA as root-end filling material might be helpful in controlling the endodontic infections and to achieve endodontic success.

CONCLUSION

Adhesion and penetration of E. faecalis into root cementum provide a long-term nidus for subsequent infection, therefore, causing the persistent infection or reinfection. Early intervention at initial stages of infection will increase the success rate of root canal treatment and use of MTA as root-end filling material showed promising results indicating its routine application for root canal treatment to prevent reinfection or persistent infection. However, further studies should be done regarding this aspect using other materials and methods so as to prevent persistent infection or reinfection after endodontic treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.