Endodontic microbiology /certified fixed orthodontic courses by Indian dental academy

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Endodontic microbiology /certified fixed orthodontic courses by Indian dental academy

  1. 1. ENDODONTICMICROBIOLOGY INDIAN DENTAL ACADEMY Leader in Continuing Dental Education www.indiandentalacademy.com www.indiandentalacademy.com
  2. 2. Contents Introduction History Gram +ve and Gram –ve organisms Aerobic and Anaerobic microorganisms Microflora of oral cavity Portals of root canal infection Bacterial pathogenicity Bacterial defence Host response Endodontic microflora www.indiandentalacademy.com
  3. 3.  Microorganisms of persistent infections Identification of microorganisms culture tests DNA probes PCR Immunoassays Immunofluorescence Treatment of endodontic infection www.indiandentalacademy.com
  4. 4. Introduction  Over three centuries that have passed since Leeuwenhoek first observed bacteria and protozoa with his primitive microscope , a vast amount of knowledge ,has been accumulated about the “small animals” ,now known as microorganisms. www.indiandentalacademy.com
  5. 5. Microorganisms cause virtually all pathoses of the pulp and the periradicular tissues. To effectively treat endodontic infections, clinicians must recognize the cause and effect of microbial invasion of the dental pulp space and the surrounding periradicular tissues.A thorough understanding of these organisms including their growth and destructive potential, sensitivity to pharmacotherapeutic agents and their relationship to clinical symptoms is necessary to formulate a sound approach to root canal therapy. www.indiandentalacademy.com
  6. 6. History 1890 W.D .Miller  Authored a book called : “Microorganisms of Human Mouth”  First researcher to identify bacteria in the diseased pulp 1909 E. C. Rosenow, “Theory of Focal Infection”  localized or generalized infection caused by bacteria traveling through the bloodstream from a distant focus of infection. www.indiandentalacademy.com
  7. 7. 1939Winfred Fish  Fish Zones of reaction  Fish related these bone changes to infections from the dental pulp and theorized that removal of the nidus of infection would lead to resolution of the infection Schuller  Anachoresis : Process by which blood borne bacteria,dyes ,pigments,metallic substances,foreign proteins are attracted to and fixed in, circumscribed areas of inflammation www.indiandentalacademy.com
  8. 8.  1941 Anachorectic pulpitis Robinson and Boiling cites the movement of systemic bacteria into inflamed pulps, a major concern in dental related bacterial endocarditis. 1981 Moller and associates illustrated the importance of bacteria in the development of pulp and periradicular disease www.indiandentalacademy.com
  9. 9.  Sundquist Studied the significance of bacteria and their association with periradicular disease. Analysis of aerobic and anaerobic bacteria Positive cultures for teeth with radiographically exhibited periradicular disease. Noted –Bacteriodes melaninogenicus was sessential for the development of periradicular destruction. www.indiandentalacademy.com
  10. 10. Gram +ve and Gram-ve Cell wall The cell wall of a bacterium is an essential structure that protects the delicate cell protoplast from osmotic lysis. The cell wall of Bacteria consists of a polymer of disaccharides cross-linked by short chains of amino acids (peptides). This molecule is a type of peptidoglycan called murein. www.indiandentalacademy.com
  11. 11.  In the Gram-positive bacteria, the cell wall is thick (15-80 nanometers), consisting of several layers of peptidoglycan complexed with molecules called teichoic acids . In the Gram-negative bacteria, the cell wall is relatively thin (10 nanometers) and is composed of a single layer of peptidoglycan surrounded by a membranous structure called the outer membrane. www.indiandentalacademy.com
  12. 12.  Gram staining by Christian Gram Gram +ve bacteria retain the primary stain and resist decolorisation,and appear violet Gram –ve bacteria get decolorised and take up the counter stain and appear red. www.indiandentalacademy.com
  13. 13. Oxygen requirement  Assimilation of glucose results in terminal generation of free oxygen radical superoxide(O2-)  Reduced to oxygen gas and H2O2- superoxide dismutase  H2O2 is converted into water and oxygen- Catalase www.indiandentalacademy.com
  14. 14. Oxygen requirementAnaerobic bacteria grow only in the absence of oxygen but vary in their sensitivity to oxygen. They function at low oxidation- reduction potentials lack the enzymes superoxide dismutase and catalase.Microaerophilic bacteria can grow in an environment with oxygen but predominantly derive their energy from anaerobic energy pathways. www.indiandentalacademy.com
  15. 15. Facultative anaerobes grow in the presence or absence of oxygen and usually have the enzymes superoxide dismutase and catalase.Obligate aerobes require oxygen for growth and possess both superoxide dismutase and catalase. www.indiandentalacademy.com
  16. 16. BACTERIA COMMONLY FOUND IN ORALCAVITY Staphylococcus epidermidis  Streptococcus pneumoniae Staphylococcus aureus  Streptococcus pyogenes Streptococcus mitis  Neisseria sp Streptococcus salivarius  Neisseria meningitidis Streptococcus mutans  Veillonellae sp Enterococcus faecalis  Enterobacteriaceae (Escherichia coli)  Proteus sp www.indiandentalacademy.com
  17. 17.  Pseudomonas aeruginosa Haemophilus influenzae Corynebacteria Bacteroides sp Mycobacteria Bifidobacterium bifidum  Actinomycetes  Spirochetes Lactobacillus sp Mycoplasmas Clostridium sp Clostridium tetani www.indiandentalacademy.com
  18. 18. PULPAL INFECTION Endodontic infections are polymicrobial The number of CFU is usually between 102 to 108. Majority of the microbes associated with endodontic infections are ANAEROBIC. The anaerobic bacteria that constitute endodontic infections are, Obligate Anaerobes 60-63%, Facultative Anaerobes 34-36%. www.indiandentalacademy.com
  19. 19.  Anaerobic Bacteria causing endodontic infections can be grouped into, Gram Positive Rods:  Actinomyces sp.,  Lactobacillus,  Eubacterium sp. Gram Positive Cocci:  Peptostreptococcus sp.,  Streptococcus sp.,  Staphylococcus sp. www.indiandentalacademy.com
  20. 20.  Gram Negative Rods:  Bacteroides sp.,  Fusobacterium sp.,  Campylobacter,  Prevotella sp.,  Porphyromonas sp. www.indiandentalacademy.com
  21. 21. PORTALS OF ROOT CANAL INFECTION Openings in the dental hard tissue wall—resulting from caries, clinical procedures, or trauma-induced fractures and cracks Gingival sulci or periodontal pockets through severed periodontal blood vessels Through exposed dentinal tubules at the cervical root surface, due to gaps in the cemental coating Anachoresis www.indiandentalacademy.com
  22. 22. Dental CariesInvasion of the pulp cavity by bacteria is most oftenassociated with dental caries. Bacteria invade and multiplywithin the dentinal tubules . Dentinal tubules range in size from 1 to 4 ∝m in diameter,whereas the majority of bacteria are less than 1 µm indiameter. If enamel or cementum is missing, microbesmay invade the pulp through the exposed tubules.   www.indiandentalacademy.com
  23. 23. TraumaFollowing trauma and direct exposure of the pulp,inflammation, necrosis, and bacterial penetration areno more than 2 mm into the pulp after 2 weeks. Incontrast, a necrotic pulp is rapidly invaded and colonized. The “dead tracts” of empty dentinal tubules followingdissolution of the odontoblastic processes may leavevirtual highways for the microbes’ passage to the pulpcavity.Microbes may reach the pulp via direct exposureof the pulp from restorative procedures or traumainjury and from pathways associated with anomalous tooth development. www.indiandentalacademy.com
  24. 24. Gingival sulci or Periodontal ligament Possible sources- Hard tissue communication Lateral canals in the furcation area and in the apical third of tooth roots Lymphatic and hematogenous routes Root planing Trope and Tronstad Theorized that spirochete count from the exudate of periradicular lesions ,differentiate an abscess of endodontic origin from one of periodontal origin. 0 – 10 % -endodontial origin 30 -58 % - periodontal origin www.indiandentalacademy.com
  25. 25. AnachoresisAnachoresis is a process by which microbes may be transported in the blood or lymph to an area of inflammation such as a tooth with pulpitis, where they may establish an infection. Anachoresis may be the mechanism through whichtraumatized teeth with intact crowns become infected. The process of anachoresis has been especiallyassociated with bacteremias and infective endocarditis. www.indiandentalacademy.com
  26. 26. Endodontic microflora of a human tooth with apical periodontitis (GR). The areas between the upper two and the lower two arrowheads in are magnified in and , respectively. Dense bacterial aggregates (BA) sticking to the dentinal (D) wall .A transmission electron microscopic view (d) of the pulpo-dentinal interface shows bacterial condensation on the surface of the dentinal wall, forming thick, layered biofilm.www.indiandentalacademy.com
  27. 27. Well-entrenched biofilm at the apical foramen of a tooth affected with apical periodontitis . The canal ramifications on the left and right in (b) are magnified in (c) and (d), respectively. Note the strategic location of the bacterial clusters (BA) at the apical foramina. The bacterial mass appears to be held back by a wall of neutrophilic granulocytes (NG).www.indiandentalacademy.com
  28. 28. Pathogenicity/Infection Microbial pathogenicity has been defined as the structural and biochemical mechanisms whereby microorganisms cause disease. Pathogenicity in bacteria may be associated with unique structural components of the cells (e.g. capsules, fimbriae, LPS or other cell wall components) or active secretion of substances that either damage host tissues or protect the bacteria against host defenses. Infection may imply colonization, multiplication, invasion or persistence of a pathogen on or within a host, Disease is used to describe an infection that causes significant overt damage to the host. www.indiandentalacademy.com
  29. 29. Colonization is the establishment of microbes in a hostif appropriate biochemical and physical conditions areavailable for growth. Normal oral flora is the result of a permanent microbial colonization in a symbiotic relationship with the host. Although the microbes in the normal oral flora participate in many beneficial relationships, they are opportunistic pathogens if they gain access to a normally sterile area of the body such as the dental pulp or periradicular tissues and produce disease. www.indiandentalacademy.com
  30. 30.  Invasiveness is the ability to invade tissues. This encompasses mechanisms for adherence and initial multiplication, ability to bypass or overcome host defense mechanisms, and the production of extracellular substances ("invasins") which facilitate the actual invasive process. Toxigenesis is the ability to produce toxins.Toxic substances, both soluble and cell-associated, may be transported by blood and lymph and cause cytotoxic effects at tissue sites remote from the original point of invasion or growth www.indiandentalacademy.com
  31. 31. PATHOGENICITY OF ENDODONTICFLORA Interactions with other micro-organisms in the root canal, to develop synergistically beneficial partners; ability to interfere with and evade host defenses; Action of virulence factors www.indiandentalacademy.com
  32. 32.  Interactions with other micro-organisms Microbial interactions that influence the ecology of the endodontic flora may be positive (synergistic) or negative associations, as a result of certain organisms influencing the respiratory and nutritional environments of the entire root canal flora Sundqvist et al., 1979; www.indiandentalacademy.com
  33. 33.  Microbial interference Certain microbes have the ability to shirk and interfere with the host defenses Bacterial toxins can effectively interfere with the various mechanisms of host immune system A. israelii aggregate to form large cohesive colonies that cannot be killed by host phagocytes www.indiandentalacademy.com
  34. 34. Virulence factors Capsules-Present external to the outer layer of the cell wall-Composed of polysaccharide,rarely polypeptide-Functions : Protects the cell from dessication and from toxic materials in the environment Promotes the concentration of nutrients at the bacterial cell surface Adherence of bacteria to host cells Resistance to bactericidal action of complement and serum antibodies www.indiandentalacademy.com
  35. 35.  Glucan capsule of streptococcus mutans Practical importance of virulence factor as it forms the matrix of dental plaque Adherence of bacteria to this matrix and subsequent formation of acids from dietary sucrose leads to initiation of caries www.indiandentalacademy.com
  36. 36.  Fimbriae(pili) They are small appendages fond on the surface of many gram-ve bacteria. Although the terms “pili” and “fimbriae” are used interchangeably, Fimbriae- non flagellar hair like appendages Pili- fimbriae of gram –ve bacteria that function specifically in the transfer of DNA from one cell to other during process of conjugation(sex pili) www.indiandentalacademy.com
  37. 37. Functions participates in the aggregation of bacteria or attachment to tissues. Pili may extend from one bacterium to another during conjugation and exchange DNA for virulence factors, including resistance to antibiotics. www.indiandentalacademy.com
  38. 38. Enzymes as spreading factors Hyaluronidase. is the original spreading factor It is produced by streptococci. Staphylococci. The enzyme attacks the interstitial cement ("ground substance") of connective tissue by depolymerizing hyaluronic acid. Collagenase is produced by Clostridium histolyticum and Clostridium perfringens. It breaks down collagen, the framework of muscles, which facilitates gas gangrene due to these organisms. www.indiandentalacademy.com
  39. 39. Neuraminidase It degrades neuraminic acid (also called sialic acid), an intercellular cement of the epithelial cellsStreptokinase and Staphylokinase produced by streptococci and staphylococci, respectively. Kinase enzymes convert inactive plasminogen to plasmin which digests fibrin and prevents clotting of the blood. The relative absence of fibrin in spreading bacterial lesions allows more rapid diffusion of the infectious bacteria www.indiandentalacademy.com
  40. 40.  Enzymes Porphyromonas and Prevotella species to break down plasma proteins—particularly IgG, IgM and the complement factor C3 is of particular significance, since these molecules are opsonins necessary for both humoral and phagocytic host defenses. In abscesses, neutrophils lyse and release their enzymes to the surrounding milieu to form purulent exudates. This enzyme-rich exudate has an adverse affect on the surrounding tissues. www.indiandentalacademy.com
  41. 41. Endotoxins and Exotoxins Exotoxins are proteins formed by Gram +ve bacteria and are highly potent in minute quantities. Endotoxins are polysacharide protein complexes which form an integral part of the cell wall of Gram –ve bacteria. They are less potent than the exotoxins www.indiandentalacademy.com
  42. 42. Lipopolysaccharides (LPS) Endotoxins consists primarily of Lipopolysaccharide.It is the structural component of Gram –ve outer membrane. It has been shown that the concentration of endotoxin in the canals of symptomatic teeth is higher than that in the canals of asymptomatic teeth. www.indiandentalacademy.com
  43. 43. Functions of LPS It is a permeability barrier to toxic molecules Barrier to lysozyme and many antimicrobial agents Impedes destruction of the bacterial cells by serum components and phagocytic cells. Important role as a surface structure in the interaction of the pathogen with its host. LPS may be involved in adherence (colonization), or resistance to phagocytosis, or antigenic shifts that determine the course and outcome of an infection. www.indiandentalacademy.com
  44. 44. Extracellular vesicles  The vesicles are formed from the outer membrane of gram- negative bacteria and have a trilaminar structure similar to the parent bacteria. These vesicles may contain same antigens which neutralizes antibodies against parent organism. The vesicles may contain  Enzymes, or  Toxic agents. These vesicles are involved in,  Hemagglutination,  Hemolysis,  Bacterial adhesion, and  Proteolytic action on host tissues. www.indiandentalacademy.com
  45. 45. Fatty acids The short-chain fatty acids most commonly produced by bacteria in infected root canals are Propionic, Butyric, and Isobutyric acids. Short-chain fatty acids affect neutrophils chemotaxis, degranulation, chemiluminescence, and phagocytosis. Butyric acid has the greatest inhibition of T-cell growth and stimulates the production of interleukin-1 which is associated with bone resorption. www.indiandentalacademy.com
  46. 46. Polyamines Polyamines are biologically active compounds involved in regulation of growth, regeneration of tissues, and modulation of inflammation. Teeth that are painful to percussion or have spontaneous pain have been shown to have higher concentration of total polyamines in necrotic pulps.  Ammonia,  Hydrogen sulphide. www.indiandentalacademy.com
  47. 47. Host Response CELLULAR ELEMENTS PMN Though PMN are essentially protective cells, they cause severe damage to the host tissues Their cytoplasmic granules contain several enzymes that, on release, degrade the structural elements of tissue cells and extracellular matrices. Because they are short-lived cells, PMN die in great numbers at acute inflammatory sites. Therefore, the accumulation and massive death of neutrophils are a major cause for tissue breakdown in acute phases of apical periodontitis. www.indiandentalacademy.com
  48. 48.  Lymphocytes Among the three major classes of lymphocytes—T- lymphocytes, B-lymphocytes, and the natural killer (NK) cells.They are involve din antibody production. Macrophages cytokines IL-1, TNF- , interferons (IFN), and growth factors that are of particular importance periadicular infection. They also contribute serum components and metabolites, such as prostaglandins and leukotrienes, that are important in inflammation. www.indiandentalacademy.com
  49. 49.  Osteoclasts A major pathological event of peri apicalinfection is the osteoclastic destruction of bone and dental hard tissues the pro-osteoclasts migrate through blood as monocytes to the periradicular tissues and attach themselves to the surface of bone. Several daughter cells fuse to form multinucleated osteoclasts that spread over injured and exposed bone surfaces. www.indiandentalacademy.com
  50. 50.  Bone resorption takes place beneath the ruffled border, facing the bone surface The bone destruction happens extracellularly at the osteoclast/bone interface and involves: (i) demineralization of the bone by solubilizing the mineral phase in the resorption compartment, as a result of ionic lowering of pH in the micro-environment; and (ii) enzymatic dissolution of the organic matrix. Root cementum and dentin are also resorbed by fusion macrophages designated as ‘odontoclasts’ www.indiandentalacademy.com
  51. 51.  Epithelial cells During periapical inflammation, the epithelial cell rests are believed to be stimulated by cytokines and growth factors to undergo division and proliferation, a process commonly described as ‘inflammatory hyperplasia’. These cells participate in the pathogenesis of radicular cysts by serving as the source of epithelium. However, ciliated epithelial cells are also found in periapical lesions ,particularly in lesions affecting maxillary molars. The maxillary sinus-epithelium was suggested to be a source of those cells www.indiandentalacademy.com
  52. 52. MOLECULAR MEDIATORS 1. Pro-inflammatory & chemotactic cytokines 2. IFN 3.Colony-stimulating factors (CSF) 4.Growth factors 5.Eicosanoids a-Prostaglandins rapid bone loss Apical hard-tissue resorption can be suppressed by parenteral administration of indomethacin, an inhibitor of cyclo-oxygenase ( Torabinejad et al., 1979).b-Leukotrienes 6.Antibodies IgG have been shown to b epresent in plasma cells residing in the periapical cyst wall and in the cyst fluid www.indiandentalacademy.com
  53. 53. Bacterial Defense Against Phagocytes Most successful pathogens, however, possess additional structural or biochemical features that allow them to resist the host cellular defense against them, i.e., the phagocytic and immune responses. If a pathogen breaches the hosts surface defenses, it must then overcome the hosts phagocytic response to succeed in an infection. www.indiandentalacademy.com
  54. 54.  Bacteria can avoid the attention of phagocytes in a number of ways. remain confined in regions inaccessible to phagocytes avoid provoking an overwhelming inflammatory response inhibit phagocyte chemotaxis Some pathogens can cover the surface of the bacterial cell with a component which is seen as "self" by the host phagocytes and immune system. Such a strategy hides the antigenic surface of the bacterial cell. www.indiandentalacademy.com
  55. 55.  Inhibition of Phagocytic Engulfment Survival Inside of Phagocytes Products of Bacteria that Kill or Damage Phagocytes www.indiandentalacademy.com
  56. 56. The seriousness of an infection beyond the apex of a tooth depends on the number and virulence of the organisms, host resistance, and anatomic structures associated with the infectionOnce the infection has spread beyond the tooth socket, it may localize or continue to spread through the bone and soft tissue as a diffuse abscess or cellulitis. www.indiandentalacademy.com
  57. 57. Abscess is a cavity containing pus (purulent exudate) consisting of bacteria, bacterial by-products, inflammatory cells,numerous lysed cells, and the contents of those cells.Cellulitis is a diffuse, erythematous, mucosal, or cutaneous infection that may rapidly spread into deep facial spaces and become life threatening www.indiandentalacademy.com
  58. 58. The periapical inflammatory responses that occurfollowing bacterial infection of the root canal system result in the formation of granulomas and cysts with the resorption of surrounding bone. Interleukin-1 and prostaglandins have been especially associated with periapical bone resorption. www.indiandentalacademy.com
  59. 59. Bacteria From The Root Canals Of Teeth With Apical Rarefactions Eubacterium lentum Fusobacterium nucleatum Fusobacterium sp Streptococcus sp Campylobacter sp Bacteroides sp Peptostreptococcus sp Prevotella intermedia Actinomyces sp Peptostreptococcus micros Eubacterium timidum Eubacterium alactolyticum Peptostreptococcus Capnocytophaga anaerobius ochracea Lactobacillus sp Eubacterium brachy Selenomonas sputigena Veillonella parvula www.indiandentalacademy.com
  60. 60.  Porphyromonas endodontalis Prevotella buccae Prevotella oralis Proprionibacterium propionicum Prevotella denticola Prevotella loescheii Eubacterium nodatumIt was shown that Black pigmented Bacteriodes were the bacteria most reactive with IgG produced by explant cultures of periapical lesions www.indiandentalacademy.com
  61. 61.  Recent Taxonomic Changes for Previous Bacteroides SpeciesPorphyromonas—black-pigmented (asaccharolyticBacteroides species)Porphyromonas asaccharolyticasPorphyromonas gingivalisPorphyromonas endodontalis www.indiandentalacademy.com
  62. 62.  Prevotella—black-pigmented (saccharolytic Bacteroidesspecies)Prevotella melaninogenicaPrevotella denticolaPrevotella loescheiiPrevotella intermediaPrevotella nigrescensPrevotella corporisPrevotella tannerae www.indiandentalacademy.com
  63. 63.  Prevotella—nonpigmented (saccharolytic Bacteroides species)Prevotella buccaePrevotella biviaPrevotella oralisPrevotella orisPrevotella oulorumPrevotella ruminicola www.indiandentalacademy.com
  64. 64.  Bacteroides, a group of Gram-negative, anaerobic, non- sporeforming bacteria www.indiandentalacademy.com
  65. 65. Microorganisms isolated from periapical lesionswith refractory apical periodontitis Actinomyces sp-A.israelli Bacillus Bacteriodes Candida sp-C.albicans Clostridium sp E.faecalis Hemofillus sp Streptococcus sp Staphylococcus sp Veillonella sp www.indiandentalacademy.com
  66. 66. A microbial biofilm at the root-tip of a failed root canal. The mixed bacterial flora consists of numerous dividing cocci, rods ,filaments (FI), and spirochetes (S).Rods often reveal a Gram-negative cell wallwww.indiandentalacademy.com
  67. 67. Axial sections through the surgically removed apical portion of the root with a therapy- resistant apical periodontitis. visible cluster of bacteria (BA ) in the root canal. emerging and gradually widening profiles of an accessory root canal (AC) that is clogged with bacteria (BA )www.indiandentalacademy.com
  68. 68. Actinomyces sp Species of Actinomyces have been associated with endodontic treatment that failed to heal A. viscosus A. israelii A. naeslundii were detected in clinical samples from infected root canals, and abscesses,and few were associated with cellulitis. www.indiandentalacademy.com
  69. 69. Actinomyces- infected periapical pocket cyst typical ‘ray-fungus’ type of actinomycotic colonywww.indiandentalacademy.com
  70. 70. Fungi in endodontic infections Species C.albicans –dentinophilic organism C.glabrata C.guillermondii C.inconspicua Geotrichium candidum Dentin colonisation by fungi Invasion of fungi into dentinal tubules protect it from intracanal procedures Availability of Ca ions help in growth and adhesion www.indiandentalacademy.com
  71. 71. Fungi as a potential cause of endodontic failures axial section of a root-filled (RF) tooth with a persisting apical periodontitis lesion showing microbial clusters and budding formswww.indiandentalacademy.com
  72. 72.  Susceptibility to antimicrobial endodontic medicaments Nystatin Sodium carylate Sen et al, EDTA –most effective –reduces adhesion decreases metabolic activity Nystatin,ketaconazole,1.5%chorhexidine gluconate Resistant to Ca(OH)2 It can survive wide range of pH Combination with CPMC/glycerin Chlorhexidine gluconate and zinc oxide Chlorhexidine iodine Iodine potassium iodide OOO 2004 www.indiandentalacademy.com
  73. 73. Enterococcus faecalis  Invasion into dentinal tubules and remain viable within the tubule.  Adherence to collagen  Regulates internal pH with an efficient proton pump  Withstands high pH upto 11.5,resisting effects of Ca(OH)2  E. faecalis can survive prolonged starvation.  It can grow as a mono-infection in treated canals in the absence of synergistic support from other bacteria JOE 2001 www.indiandentalacademy.com
  74. 74. Enterococcus faecaliswww.indiandentalacademy.com
  75. 75. Viruses in Periapical pathosis Herpes Virus-Human cytomegalo virus Epstein Barr virus FIV –Feline immunodeficiency virus antiviral medications for Rx in periapical infection. root canal irrigants- NaOcl and iodine JOE 2004 IEJ 2004,IEJ 2001 www.indiandentalacademy.com
  76. 76. ISOLATION AND DETECTION OF MICROBES  Clinically identification of bacteria provides a more specific targeting of the microbes. Identification of microbe through laboratory support is necessary especially in, Patients who are immunosuppressed Patients with progressive/ persistent infection High risk of developing an infection (e.g., history of infective endocarditis) www.indiandentalacademy.com
  77. 77. CULTURE TESTS: Clinical reasons for culturing root canals 1.To determine the bacteriologic status of the root canal system before obturation and assess the efficacy of debridement procedure. 2.To isolate microbial flora for antibiotic sensitivity and resistance profiles in cases of persistent infections. www.indiandentalacademy.com
  78. 78. When culturing is done a proper media must be used that will support the growth of aerobic and anaerobic organisms.Common endodontic culture media Thioglycollate Trypticase soy broth(with 1% agar) Dextrose broth Brain heart infusion broth Serum dextrose broth www.indiandentalacademy.com
  79. 79.  SPECIMEN COLLECTION: Specimen collection in involved root canal or periradicular region should be under strict aseptic conditions without any error. Fundamental consideration in specimen collection: 1. Specimen collection should be from the actual infection site with minimal contamination, 2. Optimal timing: Culturing should be done before and after cleaning and shaping of the root canal system. 3. Sufficient quantity: adequate quantity of specimen is required to grow the microbes. 4. Appropriate collection devices, specimen containers and culture media www.indiandentalacademy.com
  80. 80. Steps in Specimen Collection: Specimen collection from root canal:1) The tooth must be isolated with a rubber dam to obtain aseptic sample2) The surface of the tooth and surrounding field must be disinfected with Sodium hypochlorite or other disinfectants3) Access to the canal is made with sterile burs and instruments,4) If there is drainage the sample is collected using sterile needle and syringe or sterile paper points, www.indiandentalacademy.com
  81. 81.  When samples are taken for anaerobic bacteria,it is important that the canal orifice be free from atmospheric oxygen by the proper use of nitrogen gas flow over the canal orifice before the samples are taken.The anaerobic environment is thus maintained. www.indiandentalacademy.com
  82. 82.  Specimen collection from surgical site or mucosal swelling: 1) A surgical soap scrub is first done on the collection site, 2) Apply 70% ethyl alcohol and tincture iodine to disinfect the surface layer of mucosal site, 3) Removal of iodine with alcohol, 4) Sample from mucosal swelling is best acquired by needle aspiration with a 16 to 20 gauge needle, 5) The aspirate is then injected into an anaerobic transport media. www.indiandentalacademy.com
  83. 83. CULTURE REVERSAL Grossman found that 2٪ of the cultures were negative after 48 hours of incubation, but they turned positive when incubated for 10 days. It is advisable to allow more than 48 hours between taking the culture and filling the root canal, preferable 96 or more hours, and it is recommended that the culture tube be re-examined immediately before obturating a canal to make certain that no evidence of growth is present. www.indiandentalacademy.com
  84. 84. False-positive culture: 1. Failure in sterilization of the operating field, and instruments 2. Rubber dam leaks 3. Use of unsterile paper points and cotton tube plugs; 4. Air or hand contamination during collection or transport www.indiandentalacademy.com
  85. 85.  A false-negative culture can occur with the following, 1. Incomplete penetration of a paper point ; 2. Use of a paper point that is too narrow; 3. Undetectable microbes ( hidden in dentinal tubules, accessory or lateral canals, or cementum lacunae); 4. Inadequate amount of specimen www.indiandentalacademy.com
  86. 86. 6. Presence of antimicrobial materials in the canal; 7. Insufficient incubation time; 8. Failure to consider culture reversal (negative cultureat the obturation visit becoming positive after theobturation visit); and 9. Use of a single culture medium that fails to allow forgrowth of obligate anaerobic microbes and other hard-to-culture species. www.indiandentalacademy.com
  87. 87. DNA PROBES The advances in the field of immunology and molecular biology have affected the field of diagnosis and detection of microorganisms based upon unique sequences of DNA or RNA. With the development of gene cloning technique almost any nucleic acid sequence can be prepared in large quantities for use as a probe. www.indiandentalacademy.com
  88. 88. APPLICATION OF NUCLEIC ACID PROBES • Detection of organisms difficult to culture • Detection of organisms which do not have diagnostic antigens • Differentiation of a virulent strains from pathogenic ones • Identification of antibiotic resistance genes • Detection of latent virus infection • Rapid confirmation of cultured organisms www.indiandentalacademy.com
  89. 89.  DNA probes provide reliable results in a short time (usually less than one day) on a large number of specimens. The identification and production of a nucleotide sequence is highly sophisticated and expensive. www.indiandentalacademy.com
  90. 90. POLYMERASE CHAIN REACTION The development of the polymerase chain reaction (PCR) in 1983 was major methodological break-through in molecular biology. www.indiandentalacademy.com
  91. 91. PCR is an in vitro method for producing largeamounts of specific DNA fragment of defined length andsequence from small amounts of complex template. By exponentially amplifying a target sequence, PCRsignificantly enhances the probability of detecting raresequences in a heterologous mixture of DNA. www.indiandentalacademy.com
  92. 92. Applications: 1. The PCR amplification permits the detection of as few as 100 microbes per 100 gm sample, 2. PCR Is useful for measuring gene expression by viable microorganisms as well as detecting specific populations based upon diagnostic gene sequence. 3. Also useful for cloning genes. www.indiandentalacademy.com
  93. 93. LIGASE CHAIN REACION (LCR) The LCR, or ligase amplification reaction, was first described in 1989 and modified in 1991. The principal advantage of is its ability to detect single base-pair mismatches between target DNAs. LCR based probe amplification is used detection of M. tuberculosis, Borrelia burgdorferi and N. gonorrhoeae. www.indiandentalacademy.com
  94. 94.  Immunoassays demonstrating antigen antibody reactions –EIA RIA Immunofluoresence method detecting a specific antigen of the pathogen using fluorescent antibody www.indiandentalacademy.com
  95. 95. Treatment of endodontic infectionThe goal of clinical treatment is to completely disruptand destroy the bacteria involved in the endodonticinfection. Endodontic disease will persist until thesource of the irritation is removed. www.indiandentalacademy.com
  96. 96. Root Canal Debridementand antisepsis Root canal débridement includes the removal of the microorganisms and their substrates required for growth. Cleaning and shaping of the root canal system remove a great deal of the irritants, but total débridement is impeded because of the complex root canal systems with accessory canals, fins, cul-desacs, and communications between the main canals. Irrigants and intra canal medicaments support mechanical instrumentation of root canal system for complete asepsis. www.indiandentalacademy.com
  97. 97. TREATMENT OF ENDODONTICABSCESSES/CELLULITISVast majority of infections of endodontic origin can be effectively managed without the use of antibiotics.Systemically administered antibiotics are not a substitute for proper endodontic treatment. Chemomechanical débridement of the infected root canal system with drainage through the root canal or by incision and drainage of soft tissue will help in normal healing process. www.indiandentalacademy.com
  98. 98. Because of the lack of circulation, systemically administered antibiotics are not effective against a reservoir of microorganisms within an infected root canal system. A minimum inhibitory concentration of an antibiotic may not reach a space filled with pus because of poor circulation. Incision for drainage will allow drainage of the purulent material and improve circulation to the area.. www.indiandentalacademy.com
  99. 99.  Indications for Adjunctive Antibiotics(Antimicrobial Therapy) Systemic involvementFever > 100°FMalaiseLymphadenopathyTrismus Progressive infectionsIncreasing swellingCellulitisOsteomyelitis Persistent infections www.indiandentalacademy.com
  100. 100. Selection of antimicrobial agent Empirical selection of an antibiotic (antimicrobial agent) must be based on one’s knowledge of which bacteria are most commonly associated with endodontic infections and their antibiotic susceptibility. The antibiotic should generally be continued for 2 to 3 days following resolution of the major clinical signs and symptoms of the infection. www.indiandentalacademy.com
  101. 101. Antibiotic senstivity test Pathogenic bacteria exhibit great strain variations in susceptibility to antibiotics. It is, therefore, essential to determine the susceptibility of isolates to antibiotics that are likely to be used in the treatment. Diffusion tests Stokes disc diffusion method; Kirby – Bauer disc diffusion method. Dilution tests Broth dilution method ; Agar dilution method. www.indiandentalacademy.com
  102. 102. Diffusion method The ‘disc diffusion’ method uses filter paper discs charged withappropriate concentration of the drugs. The test bacterium isinoculated on the medium and antibiotic discs are applied.Sensitivity to the drug is determined from the inhibition of bacterialgrowth around the disc.Dilution tests Serial dilutions of the drug in broth are taken in tubes and astandardized suspension of the test bacterium is inoculated.An organism of known sensitivity should also be titrated andIncubated. The minimum inhibitory concentration (MIC) is read by noting thelowest concentration of the drug at which there is no visible growth www.indiandentalacademy.com
  103. 103.  Antibiotics For Medically Compromised Patients Standard general prophylaxis Amoxicillin Adults: 2.0g Children: 50mg/kg orally 1hr. before procedure Unable to take oral medications Ampicillin Adults: 2.0g IM or IV Children: 50mg/kg orally 30 min. before procedure www.indiandentalacademy.com
  104. 104. Allergic to penicillin Clindamycin or Adults: 600mg Children: 20mg/kg orally 1hr. before procedure Cephalexin or Adults: 2.0g Children: 50mg/kg orally 1hr. before procedure Cefadroxil or Adults: 2.0g Children: 50mg/kg orally 1hr. before procedure Azithromycin Adults: 500mg Children: 15mg/kg orally 1hr. before procedure www.indiandentalacademy.com
  105. 105.  Allergic to penicillin unable to take oral medications Clindamycin or Adults: 600mg Children: 20mg/kg IV 30 min. before procedure Cefazolin Adults: 1.0g Children: 25mg/kg IM or IV 30 min. before procedure www.indiandentalacademy.com
  106. 106. Conclusion Knowledge of the microorganismsassociated with endodontic disease is necessaryto develop a basic understanding of the diseaseprocess and a sound rationale for effective managementof endodontic infections. www.indiandentalacademy.com
  107. 107. Thank Youwww.indiandentalacademy.com
  108. 108. Thank Youwww.indiandentalacademy.com
  109. 109. www.indiandentalacademy.com
  110. 110.  Edwardson’s study on the bacterial composition of dental caries Predominantly Gram + ve Actinomyces Bifidobacterium Arachina Eubacterium www.indiandentalacademy.com
  111. 111.  Even as the irritants approach the pulp, new protective layers of reparative dentin may be laid down to avert exposure which rarely can prevent microorganism entry without intervention by some type of caries excavation www.indiandentalacademy.com
  112. 112.  Traumatic injuries or operative procedures also may remove the protective dentin barrier and allow access to pulp. To prevent contamination, only materials with good sealing ability should be used following operative procedures when any possibility of pulp exposure is present. www.indiandentalacademy.com
  113. 113.  Traumatic injuries Predominantly gram +ve organisms Mode of entry is through communication of the fracture with gingival sulcus. Or from the canal space itself www.indiandentalacademy.com
  114. 114. Through The Dentinal Tubules Dentinal tubules range from 1 to 4 µm, whereas the majority of bacteria are less than 1µm in diameter. These invaders may enter the tubules from salivary contamination during operative procedures or thorough adjacent carious lesions. www.indiandentalacademy.com
  115. 115.  The microorganisms able to penetrate after cavity preparation are usually low in number and virulence and rarely cause clinical symptoms of pulpitis. The pressure of impression materials, temporary restorative materials, acids, and cements may drive microorganisms from the surface of a preparation the defense cells of the pulp can frequently remove these invaders and retain a healthful environment. www.indiandentalacademy.com
  116. 116.  Even though irritation of this dimension may not cause clinical symptoms, protection of the pulp is available. Tubule sealants such as varnishes, sedative bases, or sedative cements should be used over exposed dentin in proximity to the pulp immediately after the completion of cavity or crown preparation. www.indiandentalacademy.com
  117. 117.  However, when a deep carious lesion brings high numbers of microorganisms to tubules in proximity to the pulp, it has been shown that bacteria will penetrate to the pulp well in advance of the carious process. The pulpitis that may result occurs without direct pulp exposure. www.indiandentalacademy.com
  118. 118. Through The Ginigival Sulcus Or Periodontal Ligament. Microorganisms and other irritants from the periodontal ligament may reach the pulp through the vessels in the apical foramen or any auxiliary foramina present. Also, in some teeth auxiliary canals may be present some distance from the apex of the root, toward the crown of the tooth. If periodontal disease destroys the protecting bone and soft tissues to a sufficient degree, the canal may be exposed to microorganisms present in the gingival sulcus. www.indiandentalacademy.com
  119. 119.  www.indiandentalacademy.com
  120. 120.  Predominantly in periodontal disease Strptococcus Peptostreptococcus Eubacterium Bacteriodes Fusobacterium www.indiandentalacademy.com
  121. 121. Through The Bloodstream: ANACHORESIS may be defined as the transportation of microbes through the blood or lymph to an area of inflammation, such as tooth with pulpitis. ANACHORESIS may be mechanism by which some traumatized teeth may become infected. ANACHORESIS could not be demonstrated in instrumented but unfilled canals. www.indiandentalacademy.com
  122. 122. Through A Broken Occlusal Seal Or Faulty RestorationOf Tooth Previously Treated By Endodontic Therapy: Controlled studies by Torabine Jad et al have proved that salivary contamination from the occlusal aspect reach the periapical area in less than six weeks in canals obturated with gutta-percha and sealer. If there is a delay in restorative procedures following endodontic therapy and the temporary seal is broken, if the tooth structure fractures before final restoration, or if the final restoration is inadequate or becomes inadequate due to subsequent decay, bacteria may gain access to the periapical tissues and result in infection. www.indiandentalacademy.com
  123. 123.  This problem is often compounded by hydrophilic composite cores placed under cast restorations that begin to leak with time, absorb contaminants, and serve as a reservoir for bacteria. Unused post space further complicates the situation in a leaking restorative system acting as an incubator for anaerobic bacteria, inviting them to travel to the periradicular tissues through the apex. Microbes traveling around only 4 to 5 millimeters of gutta-percha and sealer-or through the much shorter route of contamination, are causing an increasing number of endodontic failures over extended periods of time. www.indiandentalacademy.com
  124. 124. Through Extension Of Periapical Infection From AdjacentInfected Teeth There is considerable question whether or not bacteria from a periapical area will enter an adjacent, noninfected tooth. Large periapical radiolucency may appear to encompass the roots of multiple teeth, yet be caused by pulp necrosis of only one tooth. This occurs with greatest frequency in the lower anterior teeth. Only the causative tooth is treated endodontically, and the entire radiolucency heals. Despite the presence of the granulomatous tissue and multiple colonies of microorganisms, the nerves and blood vessels can safely penetrate and course through the lesion. www.indiandentalacademy.com
  125. 125.  If a pulpitis or trauma severely affects a tooth and if its neighbor has an infected periapical area, the microorganisms may easily reach the newer problem by the interlacing blood and lymph systems, by physical extension, or by pressure. In a process similar to the anachoretic effect, the injured pulp is invaded, and the proximity of the source of microorganisms may yield a high number of organisms. www.indiandentalacademy.com
  126. 126. Microorganisms found in root canals Mostly gram negative anaerobic microorganisms www.indiandentalacademy.com
  127. 127. Significance of microorganisms inendodontic therapy Hobson’s equationNumber of MO x Virulence of MO Severity Resistance of host of disease All the factors,except for the number of microorganisms,are qualitative in nature. Host resistance depends on many factors and can even varyprocess significantly from host to host. Disease severity can be quantitatively compared on factors suuh as pain,swelling and tissue destruction. www.indiandentalacademy.com
  128. 128. Bacteria From The Root Canals Of Teeth With Apical Rarefactions Percentage of incidence Eubacterium lentum 31 Fusobacterium nucleatum 48 Streptococcus sp. 40 Bacteroides sp. 35 Prevotella intermedi 34 Peptostreptococcus micros 34 Lactobacillus sp. 32 Fusobacterium sp. 29 Actinomyces sp. 15 Eubacterium timidium 11 Eubacterium brachy 9 Campylobacter sp. 25 Veillonella parvula 9 Prevotella buccae 8 Prevotella oralis 8 Peptostreptococcus sp. 15 Prevotella loescheii 6 Eubacterium alactolyticum 34 Prevotella denticola 6 Peptostreptococcus anaerobius 31 Porphyromonas endodontalis 9 Propionibacterium propionicum 8 Eubacterium nodatum 6 www.indiandentalacademy.com
  129. 129.  The presence of microorganisms does not ensure endodontic failure nor does the absence of microroganisms guarantee success.However,the presence of microorganisms,particularly those of certain types,provides an additional source of irritation that the body must overcome to gain optimum results.Therefore ,the control of microorganisms and possible substrate must be an objective in every endodontic case . www.indiandentalacademy.com
  130. 130. Bacterial culturing Common endodontic culture media Thioglycollate Trypticase soy broth(with 1% agar) Dextrose broth Brain heart infusion broth Serum dextrose broth When samples are taken ,it is important that the canal orifice be free from atmospheric oxygen by the proper use of nitrogen gas flow over the canal orifice before the samples are taken.The anaerobic environment is thus maintained. www.indiandentalacademy.com
  131. 131.  Carbon di oxide Some bacteria use atmospheric CO2 as a principle source of carbon for biosynthetic reactions. CO 2 is required for certain macromolecular synthetic pathways,such as fatty acid biosynthesis. www.indiandentalacademy.com
  132. 132.  Because apical periodontitis is essentially a disease of root canal infection, the logical treatment has been to eliminate infection from the root canal and exclude further infection of the canal. Since the essential role of root canal microbes in both primary and post- treatment apical periodontitis has been well-recognized, the major thrust of treatment procedures should be with the clinical management of problems associated with the control and elimination of infection. In recent years, there has been a trend to focus on the purely mechanical aspects of treating the disease. While those are important, a clear understanding of the etio- pathogenic factors involved is necessary for the therapeutic application of intelligent solutions to solve the problem. www.indiandentalacademy.com

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