Enterococcus Faecalisa Formidable Foe

INTRODUCTION

Among the Enterocci isolated from root canal Enterococcus faecalis (E.faecalis) is the most common microorganism.¹ E.faecalis is the most common species associated with root canal failure. Thus it is abundantly found in post treatment failure cases. It is facultative anaerobe and gram positive cocci. It can grow in the presence as well as absence of oxygen. It can survive as a single organism or as a major component of a flora. E faecalis can survive and grow in harsh conditions. Extreme alkaline pH and salt concentrations do not harm them. Growth is possible between 10 to 45 degrees temperature. They can survive in 60 degrees of heat up to 30 minutes.²

Enterococcus species live in vast quantities in the human intestinal lumen and under most circumstances cause no harm to their hosts. They are also present in human female genital tracts and the oral cavity in lesser numbers.2They catabolise a variety of energy sourcesincluding carbohydrates, glycerol, lactate, malate, citrate, arginine, agmatine, and many keto acids.²

At present there are 23 Enterococci species. These are divided into five groups based on their interaction with mannitol, sorbose, and arginine. E. faecalis belongs to the same group as E. faecium, E. casseliflavus, E.mundtii, and E. gallinarum. These five species form acid in mannitol broth and hydrolyze arginine; however, they fail toform acid in sorbose broth. E. faecalis can normally be identified byfurther testing with arabinose, tellurite, and pyruvate. E. faecalisis arabinose negative and except for some atypical variants, is theonly member of the group to utilize pyruvate and to tolerate tellurite. More recently, molecular techniques have been developed that havethe capability to rapidly and accurately identify the Enterococcusspecies. Techniques involving DNA-DNA hybridization, sequencing of the 16S rRNA genes, whole-cell protein (WCP) analysis and gas

liquid chromatography of fatty acids have been used for taxonomic purposes.²

Laser confocal scanning microscopic study and scanning microscopic study has shown that E.faecalis can penetrate up to a depth of 1483.33 micrometres(nutrient rich aerobic condition),1166.66 micrometres (nutrition rich anaerobic condition), and 620micrometers (nutrition deprived anaerobic condition). The coloniesare mushroom shaped.^3,4

For the past few years E.faecalis has been mentioned as being prominent in teeth with post treatment disease (PTD). The prominence of E.faecalis in root canals of teeth with apical
periodontitis has given it much attention as an etiological factor in PTD. It has been speculated that the presence of E. faecalis is encouraged by the conventional endodontic techniques, mainly the use of calcium hydroxide for root canal dressing. Enterococci pose

ever increasing problems not only in dentistry but also in medicine, food engineering and environmental control.⁵

Characteristics of E.faecalis and virulence factors

In failed root canals they have been found nine times higher when compared to primary endodontic infections.1There are several reasons for its survival and virulence which gives it therapeutic resistance. Few of them are

• Altering host response.
• Extracellular superoxide production⁵
• Aggregation substance.⁵
• Suppresses the action of lymphocytes¹
• It can endure nutritional deprivation for a very long period. Once nutrition is available the starved cells are able to recover by utilising serum as a nutritional source.
• It invades the dentinal tubules very efficiently and proficiently.
• It possesses lytic enzymes, cytolysin, pheromones, and lipoteicholic acid.
• It can share its virulence factors among the species.
• It has been found to exhibit genetic polymorphisms.
• It passively maintains homeostasis and thus an intracanal medicament like calcium hydroxide is made inactive. This homeostasis is due to cytoplasm’s buffering capacity as well as because of the ions penetrating the cell membrane. Proton pump in the microorganism is an additional help in maintaining the homeostasis. Proton pump helps in pumping helps by pumping protons into the cell to lower the internal pH.
• E. faecalis is unable to survive at 11.5 pH. But the dentin has a buffering capacity and so such a pH won’t be maintained in the dentinal tubules with current techniques.^2,6

Calcium hydroxide, a very commonly used intracanal medicament is thus found ineffective in killing E.faecalis, especially when a high pH is not maintained.^{7, 8, 9}

AGGREGATION SUBSTANCE

Aggregation substance (AS) may serve as a virulence determinant of E.faecalis. It may also protect against polymorphonuclear leukocyte (PMN) or macrophage killing by promoting phagocytosis of bacteria in a manner that activates PMNs or macrophages, but does not result in microbial killing. Esp is a large chromosome encoded surface protein. The esp encoding gene has been sequenced in infection-derived E.faecalis strains and is shown to be often absent in less pathogenic species. This observation suggested a role for
esp as a virulence factor in E.faecalis. A study has demonstrated the relationship of the presence of the esp and the biofilms formation capacity in E.faecalis. None of the esp-defective E. faecalis strain was able to form a biofilms. This indicated the genetic association between esp and the presence of adhesions. Another study indicated that the presence of esp could increase hydrophobicity, biofilms formation and adherence to biotic surfaces.⁵

Aggregation substance, surface carbohydrates or fibronectin- binding moieties promote the adherence of organism to host collagen type-1 and extra cellular matrix proteins present in the dentin. Extra cellular toxins such as cytolysin may induce tissue damage, while bacteriocins like AS-48 inhibit growth of other organisms. Lipoteicholic acid, superoxide, or pheromones or corresponding peptide inhibitors may modulate local inflammatory process and contribute to periradicular damage.¹⁰

AS is a pheromone responsive, plasmid encoded bacterial adhesin that mediates efficient contact between donor and recipient bacterium, facilitating plasmid exchange. While the AS is expressed by the donor cell, the bacterial conjugation process requires that ‘binding substance’ (BS, the chromosomally encoded cognate ligand for AS) be expressed on the surface of the recipient cell. AS is proteinaceous and appears as a hair-like structure on the cell surface. It is incorporated into the older parts of the cell wall. Its expression on the cell surface may be induced by serum .AS is also reported to promote opsonin independent adherence to and phagocytosis of E.faecalis by human macrophages as well, facilitating intracellular survival time in macrophages. Super antigens are molecules produced by viruses, bacteria, parasites and yeast. They can induce inflammation through stimulation of T-lymphocytes. This is followed by massive release of inflammatory cytokines resulting in tissue damage. AS in combination with BS was also reported to possess super antigen activity ¹¹

Important characteristics 11

Sex pheromones are chromosomally encoded, small, hydrophobic peptides, 7 or 8 amino acids long, which function as signalling peptides in E. faecalis . The transfer frequency of certain conjugative plasmids in E. faecalis is known to be increased several-fold by the sex pheromone system. Formerly called haemolysin, cytolysin, as expressed by various isolates of E. faecalis, is most frequently a plasmid-encoded toxin, but it may also be chromosomally encoded. Among the target cells of cytolysin are the erythrocytes, PMNs and
macrophages , and a broad range of Gram-positive, but not Gramnegative, organisms .AS-48 is a plasmid encoded peptide antibiotic originally isolated from E.faecalis S-48. According to studies AS-48 has showed lytic activity toward a broad range of Gram-positive and Gram-negative bacteria. Gelatinase is an extra cellular zinc containing metalloproteinase from E.faecalis. It can hydrolyze gelatine, collagen, fibrinogen, casein, haemoglobin, insulin, certain E.faecalis sex pheromone related peptides and some other
bioactive peptides. Production of extracellular superoxide has been reported in strains of E.faecalis. The superoxide anion is very highly reactive oxygen radical. It can damage cells, tissues and can be involved in inflammatory diseases. This superoxide anion and other oxygen radicals exert a destructive effect on a wide variety of biological compounds such as nucleic acids, proteins and lipids.¹¹

Biofilm formation 12

Biofilms are sessile microbial communities composed of cells irreversibly attached to a substratum and interface to each other. The slow metabolic rate of microorganisms in biofilms as well as the extra cellular matrix of the biofilms can impede the effectiveness of any antimicrobials. The inhibitions of E.faecalis biofilms require very high concentrations of antibiotics such as ampicillin, vancomycin and linezolid. Biofilms have been detected in undebrided parts of root canal system of surgically resected root apices.^{12, 13} The biofilm forming capability and characteristics of clinical isolates of E.faecalis recovered from root canal have been less studied.¹⁴ A more recent study concluded that in contrast to strains from other clinical sources, in particular endocarditis strains, E.faecalis strains from oral and endodontic sources have a lower inherent capacity to form biofilms. This is of interest because prophylactic antibiotic coverage is provided for patients at risk although its value and efficacy is difficult to confirm.¹⁵

Antimicrobial resistance 10

The resistance acquired by the microorganism can be intrinsic or acquired via gene transfer. The intrinsic resistance of enterococci to many commonly used antimicrobial agents may have allowed them a cumulative advantage for further acquisition of genes encoding high level resistance to aminoglycosides, penicillin ,tetracycline, Chloramphenicol, and vancomycin.¹⁰

Some studies have shown Chlorhexidine to be very effective against E.faecalis. Whereas few other studies have shown iodinepotassium iodide to be more potent than sodium hypochlorite or Chlorhexidine.¹⁰ A 2-5 minute exposure of E.faecalis to MTAD has proven to be effective in killing the microorganism up to 200 x dilutions.¹⁶

FASTER WAYS TO MONITOR E.FA ECALIS

Polymerase chain reaction is fast, sensitive and accurate when compared to other culturing methods. High specificity and rapid identification are its features. Real time quantitative PCR (qPCR) and reverse transcription PCR (RT-PCR) are very sensitive in detecting and quantifying E.faecalis in endodontic infections.

E.faecalis activity can be detected quantitatively and qualitatively with the help of Optical spectroscopy in conjunction with specific enzymesynthetic chromogenic substrate based medium. Here there is no need for additional laboratory based culturing and plating for cell counting.^{3, 17}

ERADICATION METHODS

In many studies sodium hypochlorite has been successful against E.faecalis. Still when infected dentin is exposed to sodium hypochlorite some cells of E.faecalis may survive. Many studies prove the ability of E.faecalis to survive harsh conditions and bounce back to activity. The sodium hypochlorite solution may not reach the remote accesses where the microorganisms hide and thus escape the lethal effect of such agents even though the concentration used is higher than required lethal concentration. The dentin is a good buffer of sodium hypochlorite. Thus eradication of E.faecalis is still a challenge for modern endodontic treatment.¹⁸

E.faecalis can gain entry into the root canal system during treatment, between appointments and even post treatment.¹⁹ Many studies have been directed towards finding an effective way to block this entry. Treatment regimen must consider in eliminating or preventing the infection during every phase of endodontic treatment. Canals can be prepared to larger size instruments and this helps the antimicrobial agents to reach inaccessible areas. Larger apical sizes facilitate removal of pulpal dentin. This helps in better removal of intratubular bacteria.²

EDTA has little antibacterial activity, but it removes the inorganic portion of the smear layer and this gives a better access to the irrigating solutions to the dentinal tubules.¹⁶ MTAD has shown the ability to destroy E.faecalis in initial studies.^{16, 19} Its effective action is due to the anticollagenase activity, low pH, and gradual release over a period of time.¹⁹ 1.3% sodium hypochlorite when used as an irrigant the effects of MTAD are enhanced.²

2 % Chlorhexidine liquid or gel is found to be effective in reducing or eliminating E.faecalis from root canal space and dentinal tubules.20 When heated to 46°C, both 0.12% Chlorhexidine and 10% calcium hydroxide have greatest antimicrobial effects against E. faecalis than at normal body temperature ²

Ozonated water and stannous fluoride have also proved to be potent against E.faecalis. Metapex is a calcium hydroxide paste which contains 3% iodoform and has been found more effective than calcium hydroxide alone.²¹ The combination of calcium hydroxide and stannous fluoride also was found to be better than the calcium hydroxide alone. Chlorhexidine combined with calcium hydroxide will result in a greater ability to kill E. faecalis than calcium hydroxide mixed with water. Until further studies have been conducted, an
intracanal dressing of 2% Chlorhexidine placed for 7 days may be the best way to eradicate E. faecalis from dentinal tubules and the root canal space.^{20, 2}

Roth 811 (Roth International Ltd., Chicago, IL), a zinc-oxide eugenol based sealer, has been shown to exhibit the greatest antimicrobial activity against E. faecalis when compared toother sealers.22 AH Plus and Grossman’s sealer are effective in killing E. faecalis within infected dentinal tubules.² Based on these studies it can be concluded that a combination of adequate instrumentation and appropriate use of irrigants, medicaments, and sealer will optimize the chances of eradicating E. faecalis during retreatment of failed root canal cases.

Additional precautions like making the patient rinse with Chlorhexidine before treatment, disinfecting the tooth and rubber dam with Chlorhexidine or sodium hypochlorite, and disinfecting gutta-percha points with sodium hypochlorite before insertion in the canal can be done .² Newer obturation systems like Epiphany have been designed to bond to the root canal walls. Thus it may provide more effective seal and prevent microleakage of E.faecalis than gutta percha filled canals.²³ A well-sealed coronal restoration and root canal filling are important steps in preventing bacteria from entering the canal space.²

CONCLUSION

The common recovery of E. Faecalis in root canals of teeth where endodontic treatment has failed implies that this species is intimately involved in the pathogenesis and maintenance of persisting apical periodontitis. One feature that may enable E. faecalis to persist in the root canal is ability for it to survive conventional antimicrobial agents used during endodontic treatment, such as the alkaline pH of calcium hydroxide. Clinically E.faecalis still poses a formidable challenge. In order to develop methods of eradicating E.faecalis from persistent root canal infections, the mechanism through which this organism maintains these infections must be understood.

REFERENCES