Endodontic Microbiology PDF

# Endodontic Microbiology ## Introduction * Microbes virtually cause all pathoses of pulp and peri apical tissues. * To effectively treat endodontic infections, clinicians must recognize the cause of microbial invasion. * Knowledge of microbes associated with endodontic disease helps develop a bas...

# Endodontic Microbiology ## Introduction * Microbes virtually cause all pathoses of pulp and peri apical tissues. * To effectively treat endodontic infections, clinicians must recognize the cause of microbial invasion. * Knowledge of microbes associated with endodontic disease helps develop a basic understanding of the disease process, sound rationale for the effective management of patients. ## Can Normal Flora Cause Disease? * The oral microbiome is similar in all humans, although each individual has a characteristic "fingerprint" that is unique to himself or herself. * This commensal flora exists in symbiotic harmony with the host, but disease conditions supervene when the microbial equilibrium is broken(dysbiosis). * This transient flora cannot get a foothold in the oral environment because of the colonization resistance exerted by the resident flora. ## Stages of Root Canal Infection The image shows a diagram representing the four stages of root canal infection: 1. **Microbes enter pulp** 2. **Pulp becomes necrotic** 3. **Pulp loses blood supply** 4. **Reservoir of microbes & their end products** 5. **Climax Community** ## Rate of Bacterial Invasion * Nagaoka found that the rate of bacterial invasion increased with the passage of time. It was approximately 1.6 µm/day during the first 25 days and 14 µm/day by 120 days. * The deepest bacterial invasion was 3.0 mm after 210 postoperative days. ## A Historic View * **Antonie Van Leeuwenhoek (17th century)** * First to describe oral microflora. * Observed dental plaque from exposed cavity. * He reported that the root canals were stuffed with a soft matter, and the whole matter seemed to be alive. * **W.D. Miller (1890)** * Father of oral microbiology. * Wrote "Microorganisms of the human mouth." * First to associate bacteria with pulpal disease. ## Etiological Factors * **1965: Kakehashi* et al found that bacteria are the main etiological factors in the development of pulpal and periradicular diseases. Kakehashi *et al proved that without bacterial involvement, only minor inflammation occurred in exposed pulp. * **1976: Sundqvist** used different culturing techniques for the identification of both aerobic and anaerobic organisms and concluded that root canal infections are multibacterial. ## Portals of Entry for Microorganisms * Under normal conditions, the pulpodentin complex is sterile and isolated from oral microbiota by the overlying enamel, dentin, and cementum. * The pulpodentin complex is then challenged by microorganisms present in caries lesions, saliva bathing the exposed area, or dental plaque formed on the exposed area. * Microorganisms from subgingival biofilms associated with periodontal disease may also have access to the pulp via dentinal tubules at the cervical region of the tooth and lateral or apical foramina. * Whenever dentin is exposed, the pulp is at risk of infection as a consequence of the permeability of normal dentin dictated by its tubular structure. * Dentinal tubules traverse the entire width of the dentin and have a conical conformation, with the largest diameter located near the pulp (mean, 2.5 µm) and the smallest diameter in the periphery, near the enamel or cementum (mean, 0.9 µm). * The smallest tubule diameter is entirely compatible with the cell diameter of most oral bacterial species, which usually ranges from 0.2 to 0.7 µm. ## Routes of Root Canal Infection The image shows a diagram representing the different routes of root canal infection: * **Caries, open cavity** * **Trauma-induced cracks, fractures** * **Faulty Restorations** * **Wasting diseases** * **Scaling** * **Root planing** * **Subgingival plaque biofilms** * **Apical, Lateral, Accessory foramina** * **Root perforations** * **Anachoresis** * If pulp is vital → protection from bacterial invasion * If pulp is necrotic → Easily infected (Host defenses do not function) ## Microbial Penetration in Canal | During Treatment | Between Appointments | After Obturation | | :--------------------------------: | :-----------------------: | :----------------------------: | | Remnants of biofilm, calculus. | Leakage through temporary restoration | Leakage through temporary/permanent restoration | | Caries on tooth crown | Fracture of tooth | Delay in placement of permanent restoration | | Leaking rubber dam | Open dressing | Recurrent decay | | *Contamination of endodontic instruments, irrigant solutions* | | | ## Sources of Nutrients for Bacteria Colonizing in Root Canal * Necrotic pulp tissue * Proteins and glycoproteins from tissue fluids, and exudate that seep into Root canal via apical and lateral foramen * Salivary components * Products of metabolism of other bacteria ## Classification of Microorganisms Microbial flora can be classified based on: * **Gram Stain Technique** * **Gram-positive organisms:** *e.g*, Streptococcus, Enterococcus, Treponema, Candida, Actinomyces, Lactobacillus, etc. * **Gram-negative organisms:** *e.g*, Fusobacterium, Campylobacter, Bacteroides, Veillonella, Neisseria, etc. * **Oxygen Requirements** * **Obligate aerobes:** The organisms which require oxygen for their growth, *e.g*, tubercle bacilli. * **Facultative anaerobes:** These organisms can grow in the presence or absence of oxygen, *e.g*, Staphylococcus. * **Microaerophilic:** They grow in an oxygen environment but derive their energy only from fermentative pathways that occur in the absence of oxygen, *e.g*, Streptococcus, etc. * **Obligate anaerobes:** These bacteria can grow only in the absence of oxygen, *e.g*, Bacteroides, Fusobacterium. ## Microbial Ecosystem of the Root Canal * Most commonly gram-positive organisms are found in the root canals, but gram-negative and obligate anaerobes have also been found in the root canals. * In necrotic pulp, there is a lack of circulation with a compromised host defense mechanism. This makes pulp a reservoir for invading microbes. ## Organisms Most Commonly Encountered in Endodontic Infections * Bacteroides → B.endodontalis, B. gingivalis * **Streptococci** → S.milleri * Enterococci * Lactobacilli * Actinomyces * Yeasts * Tannerella forsythia * Fusobacterium nucleatum ## Microflora of Traumatized But Intact Teeth with Necrotic Pulps * B.melaninogenicus, Eubacterium yurii subsps, as well as key "bridging oral species" F. nucleatum sp., Corynebacterium were highly associated with traumatized teeth. ## Microflora of Acute Infections of Endodontic Origin * Bacteroides in association with Porphyromonas. endodontalis, P. gingivalis, and Prevotella intermedius. ## Microflora of Endodontic Flare-Up Infections * **Obligate Anaerobes:** Veillonella Capnocytophaga Eiknella Bacteroides Fusobacterium Treponema. ## Microflora on Refractory Endodontic Cases * E. faecalis * Candida albicans * Actinomyces israelii ## Microflora of Infected And Untreated Necrotic Pulp * Fusobacterium * Porphyromonas * Prevotella * Eubacterium * Peptostreptococcus ## Endodontic Flora in Previously Root Filled Teeth with Apical Periodontitis * Actinomyces * Enterococcus * Propionibacterium ## Mechanisms of Microbial Pathogenicity and Virulence Factors * The ability of a microorganism to cause disease is regarded as its pathogenicity. * Virulence denotes the degree of pathogenicity of a microorganism, and virulence factors are the microbial products, structural cellular components, or strategies that contribute to pathogenicity. * Some microorganisms routinely cause disease in a given host and are called primary pathogens. Other microorganisms cause disease only when host defenses are impaired and are called opportunistic pathogens. ## Virulent Factors * **Lipopolysaccharides (LPS)**: These are present on the surface of gram-negative bacteria. LPS have nonspecific antigens which are not neutralized by antibodies. * **Extracellular Vesicles**: Extracellular vesicles are produced by gram-negative bacteria in the form of endotoxins, outer membrane fragments or blebs. * **Enzymes**: Enzymes produced by bacteria have numerous activities like they help in the spread of the infection, neutralization of immunoglobulin, and the complement components. * **Fatty Acids**: Various short-chain fatty acids like propionic acid, butyric acid are produced by anaerobic bacteria. These cause neutrophil chemotaxis, degranulation, phagocytosis, and stimulate interleukin-1 production, which further causes bone resorption and periradicular diseases. * **Polyamines**: These are biologically active chemicals found in the infected canals. They help in the regulation of the cell growth, regeneration of tissues, and modulation of inflammation. * **Pili**: May play an important role in the attachment of bacteria to surfaces and interaction with other bacteria. * **Capsule**: Present in gram-negative black-pigmented bacteria, enable them to avoid phagocytosis. ## Types Of Endodontic Infections The image shows a diagram representing the different types of Endodontic Infections * **Intraradicular Infections** * **Primary/Initial/Virgin:** Caused by microorganisms that initially invade and colonize the necrotic pulp tissue (initial or "virgin" infection). * **Secondary:** Caused by microorganisms not present in the primary infection but introduced in the root canal at some time after professional intervention (i.e., secondary to intervention). * **Persistent/Recurrent:** Caused by microorganisms that were members of a primary or secondary infection and in some way resisted intracanal antimicrobial procedures and were able to endure periods of nutrient deprivation in treated canals. * **Extraradicular Infections** * **Sequalae of Intraradicular**: Caused by microorganisms that initially invade and colonize the necrotic pulp tissue (initial or "virgin" infection). * **Independent of Intraradicular:** Caused by microorganisms that initially invade and colonize the necrotic pulp tissue (initial or "virgin" infection). * **Primary Intraradicular Infections:** * Caused by microorganisms that initially invade and colonize the necrotic pulp tissue. * Gram-negative anaerobes, such as Prevotella, Fusobacterium, Tannerella, Dialister, Porphyromonas, Campylobacter, and Treponema have been frequently isolated from primary endodontic infections. * Gram-positive anaerobes from genera Peptostreptococcus, Eubacterium, Actinomyces, and facultative or microaerophilic streptococci can also be commonly found in primary intraradicular infections. ## Symptomatic Infections * No strong evidence discloses specific involvement of a single species with any particular form of endodontic disease. * Factors other than mere presence of putative pathogens may play a role in the etiology of symptomatic infections (no. of bacterial cells, environmental cues, bacterial interactions, difference in virulence ability). * Bacterial succession occurs before symptoms rise. * At a given moment in the endodontic disease, the microbiota reaches a certain degree of pathogenicity and elicits acute inflammation at the peri apical tissues. This causes Pain + Swelling. ## Asymptomatic to Symptomatic The image shows a diagram summarizing the process of asymptomatic to symptomatic infection: * Asymptomatic → Symptomatic * Shift in the structure of the microbial community before the appearance of symptoms (Arrival of new pathogens/ variations/rearrangement in bacterial species). * **Detected by** * Difference in dominant species in communities and a larger number of species in asymptomatic cases * No key pathogen but occurrence of certain bacterial combinations in infected root canals → decisive factors in causing symptoms ## Persisters After Intracanal Disinfection * Persistent intraradicular infections are caused by microorganisms that resisted the intracanal antimicrobial procedures. These microbes endure periods of nutrient deprivation in a prepared canal. * However, fewer species are present than primary infections. * Higher frequencies of fungi (Candida species) are present than in primary infections. * **Common organisms found in this scenario** * S.mitis * S.gordonii * **E.fecalis** * Staphylococci * Actinomyces * Bifidobacterium ## Enterococcus Faecalis * E. faecalis is a gram-positive, facultative anaerobic coccus that is strongly associated with endodontic infections. * Found in a high percentage of root canal failures. * Able to survive in the root canal as a single organism or as a major component of the flora. * Endures prolonged periods of nutritional deprivation. * Binds to dentin and invades dentinal tubules. * Suppresses the action of lymphocytes. * Utilizes serum as a nutritional source. * Resists intra canal medicaments. * Forms a biofilm. * It is commonly found in a high percentage of root canal failures and is able to survive in the root canal as a single organism or as a major component of the flora. * They can grow in extremely alkaline pH, a salt-concentrated environment, in a temperature range of 10-45°C, and survive a temperature of 60°C for 30 minutes. * The prevalence of E. faecalis is 40% in primary endodontic infection and 24%-77% in persistent endodontic infection. * E. faecalis biofilm is 1000 times more resistant to phagocytosis, antibodies, and antimicrobials (intracanal dressings like calcium hydroxide). * Rocas et al. have also suggested that E. faecalis was more frequently detected in asymptomatic cases than in symptomatic ones. ## Extra Radicular Infections If microorganisms invade the periradicular tissues overcoming the defense mechanisms of the body, then extraradicular infection occurs. The most common species are Actinomyces, Streptococcus, and P. propionicum. * **Extraradicular Infection Dependent on Intraradicular Infection:** This infection occurs if microorganisms invade the periradicular tissues overcoming the defense mechanisms of the body or debris extrusion due to over-instrumentation. *e.g.*, Acute alveolar abscess. * **Extraradicular Infection Independent of Intraradicular Infection:** Actinomyces species have been found in association with unhealed periapical lesions *e.g.*, periapical actinomycosis. ## Biofilms * Biofilm can be defined as a sessile multicellular microbial community characterized by cells that are firmly attached to a surface and enmeshed in a self-produced matrix of extracellular polymeric substances. * Bacterial biofilms are very prevalent in the apical root canals of teeth with primary and post-treatment apical periodontitis. ## Endodontic Biofilms * Endodontic micro flora less diverse than oral micro flora * Persist in anatomical +geometrical complexities (Isthmuses/deltas/apical portion of the root canal system) * Shelter adhering bacteria from cleaning and shaping procedures * Bacterial activities are not confined to intracanal spaces. They also access regions beyond the apical foramen. * The root canal environment favors biofilm formation (Both primary, and post-treatment) ## Development of Biofilm The image shows a diagram representing the development of a biofilm: 1. **Conditioning layer** 2. **Planktonic bacterial cell attachment** 3. **Bacterial growth and biofilm expansion** 4. **Detachment of biofilm microorganisms** ## Biofilm Location | Intracanal | Extraradicular | Periapical | | :------------: | :----------------: | :-------------: | | Formed on root canal dentin of endodontically treated tooth | Root surface biofilms | Isolated biofilms in peri apical region of endodontically infected tooth | | *Documented by Nair (1987)* | Formed on cementum Surface adjacent to root apex of endodontically treated teeth | Actinomyces P. Propionicum | | Present as both loose collection and biofilm structure | Asymptomatic apical periodontitis and chronic abscess | Sunde *et al*, reported occurrence of sulphur granules in 9 refractory P/A lesions → A. viscosus A. naeslundii A. israelii | | E. fecalis resists starvation, develops biofilms under different environmental and nutritional conditions | Clinical evidence: Ricucci *et al*- Reported presence of calculus-like deposits on apex. | | ## Biomaterial Centered Infection * Biomaterial centered infection (BCI) occurs when bacteria adheres to an artificial biomaterial surface, such as root canal obturating materials, and forms biofilms. * The presence of a biomaterial in close proximity to the host immune system can increase the susceptibility to BCI. * BCI usually reveals opportunistic invasion by nosocomial organisms. Coagulase-negative Staphylococcus, S. aureus, enterococci, P. aeruginosa, and fungi are commonly isolated from infected biomaterial surfaces. * There are three phases of bacterial adhesion to the biomaterial surface: * Phase 1: Transport of bacteria to the biomaterial surface * Phase 2: Initial, nonspecific adhesion phase * Phase 3: Specific adhesion phase ## Microbiological Diagnostic Techniques The image shows four different types of microbiological diagnostic techniques: * **Culture** * **Microscopy** * **Immunology** * **Molecular methods** ## Microbial Sampling from the Root Canal * **Types** * Initial or Diagnostic * Intermediate * Final * **Methods** * Through the canal ## Through the Canal The image shows a diagram representing a method to collect samples through the canal: 1. **Isolate tooth with rubber dam** 2. **If drainage is present, collect with sterile paper points** 3. **Air vented from syringe** 4. **Surface of tooth disinfected** 5. **Access to canal with sterile burs** 6. **Aspirate placed in anaerobic medium** ## How to Combat Microbes in the Endodontic Therapy? * Thorough cleaning and shaping of the root canal system, with three-dimensional obturation of the root canals, have shown to produce complete healing of periradicular tissue. * Complete debridement of the canal should be done with the adjunctive use of irrigants, like sodium hypochlorite, which efficiently remove bacteria, as well as their substrate from the irregularities of the canal system, where instruments cannot reach (fins, indentations, culde-sacs, etc.). * Oxygenating a canal simply by opening it is detrimental to anaerobes. The use of oxygenating agents like glyoxide can be of great help, but care should be taken to avoid the inoculation of these oxygenating agents into the periapical tissues. ## Measures to Take During Endodontic Treatment * A tooth with serous or purulent or hemorrhagic exudate should be allowed to drain with rubber dam in place for a time under supervision. * An abscess, which is a potent irritant, has an elevated osmotic pressure. This attracts more tissue fluid, and thus, more edema and pain. Drainage by canal or by soft tissues decrease discomfort caused by inflammatory mediators. * Antibiotics should also be considered as adjunctive in severe infections. The choice of antibiotic agent should be done based on the knowledge of microorganisms associated with endodontic infections. * Intracanal medicaments play an important role in combating the microorganisms. The use of calcium hydroxide in canals with necrotic pulps after instrumentation has shown to provide beneficial effects. ## Review of Literature * Most authors highlight E. faecalis as the main microorganism associated with endodontic failure, nevertheless, there are recent studies that isolate, to a greater extent, other bacteria, such as Fusobacterium nucleatum and Propionibacterium. ## Studies and Research * **Assessment of Microbiota in Root Canals with Pulp Necrosis by Means of Gram Test** The study aimed to evaluate the type of microbiota present in root canals with pulp necrosis, with and without a periapical lesion. * **Method:** Absorbent paper cones were introduced in the canal for 20 s. Contaminated paper cones were then inoculated into a Brain Heart Infusion Agar-BHI culture medium and incubated in an oven for 48 h at 37°C. * **Results:** The results revealed that there was no statistically significant difference between the amount of Gram-positive and negative bacteria in endodontic infections, indicating that the microbiota of these endodontic infections is mixed. * **Evaluation of Antibacterial Efficacy of Chitosan, Chlorhexidine, Propolis and Sodium Hypochlorite on Enterococcus Faecalis Biofilm: An In Vitro Study** This study evaluated the efficacy of different irrigating solutions against E. faecalis biofilm. * **Results:** Chlorhexidine is equally efficacious as a combination of 1% chitosan + 1% chlorhexidine against E. faecalis biofilm. NaOCl performed equally well as a combination of 2% chitosan + 2% chlorhexidine. Propolis also exhibited significant antimicrobial activity. Thus, the study suggests that all three combinations (1% chitosan+1% chlorhexidine, 0.2 chitosan+2% chlorhexidine, and 2% chitosan+ 2% chlorhexidine) could be used as an alternative to NaOCl for endodontic infections. * **Comparative Efficacy of Endodontic Medicaments and Sodium Hypochlorite Against Enterococcus Faecalis Biofilms** This study compared the efficacy of commonly used medicaments against E. faecalis, cultured as a biofilm, on dentine substrate. * **Results:** Sodium hypochlorite achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis biofilm. The 50:50 combinations of Ca(OH)2/Ledermix or Ca(OH)2/Odontopaste reduced the viability by more than 99%, while 0.2% chlorhexidine reduced bacterial numbers by 97%. The study concludes that Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. However, Ca(OH)2, in isolation and combined with Ledermix, and Odontopaste was highly effective in reducing bacterial viability. ## Conclusion **ORAL FLORA - friend or foe?** * Microorganisms have been living as commensals in the oral cavity, but any change in the environment can render them pathogenic. This means that a simple change in the environment can cause good bacteria to become harmful. * Maintaining the ecology of both the microbes and the host is important to smoothly continue this commensalism. Basically, treating bacteria with respect and helping them thrive in the host's mouth can stop them from doing harm. # Thank You The image shows an illustration of individuals with different kinds of bacteria living in their bodies.