* Corynebacteria
* Gram positive rodsSpore-forming AEROBIC Genus: Bacillus B. anthracis B. cereus B. subtilis ANAEROBIC Genus: Clostridium C. tetani C. botulinum C. difficile C. perfringens
Non spore-forming AEROBIC Corynebacteria C. diphtheriae diphtheroids C. jeikeium Listeria monocytogenes Erysipelothrix rusiopathiae ANAEROBIC Lactobacillus spp.
* Corynebacteria - Overview
Gram positive, non motile bacilli with irregularly stained segmentsFrequently show club shaped swellings – corynebacteria (coryne = club)C. diphtheriae : most important member of this genus, causes diphtheriaDiphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva.
Corynebacterium spp
Gram positive bacilli, with characteristic morphology (club shaped and beaded)Non motile Non spore formingNon capsulatedNon--hemolytic on sheep blood agarFacultative anaerobicC. diphtheriae is fastidious while diphtheriods are non-fastidiousCatalase positiveOxidase negativeSpecies of Corynebacteria
Normal flora of RT, urethra, vagina, SkinOther Significant Corynebacterium species C. xerosis C. pseudodiphtheriticum C. pseudotuberculosis C. jeikeium, (skin) C. ulcerans
Corynebacterium diphtheriae
* Introduction – C. diphtheriae Diphtheros – leather (tough, leathery pseudomembrane)Also known as Klebs–Loeffler bacillusCauses Diphtheria
* Important features of C. diphtheriae
Slender Gram positive bacilli Pleomorphic, non motile, non sporingChinese letter or Cuneiform arrangement Stains irregularly, tends to get easily decolorised May show clubbing at one or both ends - Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granulesMetachromatic Granules:made up of polymetaphosphateBluish purple color with Loeffler’s Methylene blueSpecial stains* Virulence factor
Exotoxin – Diphtheria toxin:Protein in naturevery powerful toxinResponsible for all pathogenic effects of the bacilliProduced by all the virulent strainsTwo fragments A & B* Diphtheria toxin – Mechanism of action DT - Acts by inhibition of protein synthesis
Fragment A – inhibits polypeptide chain elongation by inactivating the Elongation factor EF 2 in the presence of NADMechanism of Action of Diphtheria Toxin: Inhibition of Protein Synthesis
Molecular Structure of Diphtheria ToxinCatalytic Region
Receptor-Binding Region
Translocation Region
A Subunit
B Subunit
* Diphtheria Toxin
Toxigenicity can be induced by Lysogenic or phage conversion – corynephages (tox+ phage) or beta phagesCan be toxoided by -Prolonged storageIncubation at 37°C for 4 - 6 weeksTreatment with 0.2 – 0.4 % formalin orAcid pH.Antibodies to fragment B - protective* Epidemiology
Habitat – nose, throat, nasopharynx & skin of carriers and patientsSpread by respiratory droplets, usually by convalescent or asymptomatic carriersNasal carriers harbour the bacilli for longer time than pharyngeal carriersLocal infection of throat - toxemiaIncubation period of diphtheria – 3 to 4 daysIn tropics, cutaneous infection is more common than respiratory infection* Diphtheria
Site of infection Faucial (palatine tonsil) – commonest typeLaryngealNasalOtiticConjunctivalGenital – vulval, vaginal, prepucialCutaneous – usually a secondary infection on pre-existing lesion, caused by non toxigenic strains* Pathogenesis & Clinical Manifestations
Human Disease Usually begins in respiratory tract Virulent diphtheria bacilli lodge in throat of susceptible individual Multiply in superficial layers of mucous membrane Elaborate toxin which causes necrosis of neighboring tissue cells Inflammatory response eventually results in pseudomembrane (fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria) Usually appears first on tonsils or posterior pharynx and spreads upward or down In laryngeal diphtheria, mechanical obstruction may cause suffocation Regional lymphnodes in neck often enlarged (bull neck)*
* Diphtheria - Clinical Classification
Based on the severity of clinical presentation:Malignant or hypertoxic – severe toxemia with marked adenitisSeptic – ulceration, cellulitis, & gangrene around the pseudomembraneHemorrhagic – bleeding from the edge of membrane, epistaxis, conjunctival hemorrahge, purpura & generalized bleeding tendency.* Complications of diphtheria
Mechanical complications are due to the pseudomembrane, while the systemic effects are due to the toxin.Asphyxia – due to obstruction of respiratory passageAcute circulatory failure Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, palatine & ciliary, spontaneous recoverySepsis – pneumonia & otitis media*
Diphtheria
Acute, Toxin mediatedChildhood disease affect upper respiratory tract
Transmitted by droplet infection
2-6 days I.P. Sore throat, Pharyngitis
Cased by C. diphtheriae
2-3 days, Bluish white adherent pseudo membrane
Respiratory obstruction due to extensive membrane formation
Recovery or complication & death (if more toxin absorbed)
Mycocarditis, neuroitis,
* Laboratory Diagnosis
Specimen – swab from the lesionsMicroscopy Gram stain: Gram +ve bacilli, chinese letter patternImmunofluorescenceAlbert’s stain for metachromatic granulesMorphology Gram +ve, nonspore forming nonmotile bacilli Club-shaped (Coryne= club) arranged at acute angles or parallel to each other (Chinese letters appearance) Beaded (metachromatic granules) Gram stain: C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped
Biochemical Reaction
All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)* Laboratory Diagnosis
Culture – isolation of bacilli requires media enriched with blood, serum or eggBlood agar Loeffler’s serum slope – rapid growth, 6 to 8 hrsTellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the coloniesHoyle’s media modifications of TBAMcLeod’s media* Growth of diphtheria bacilli
Blood agarLoeffler’s serum slope Tellurite blood agar
3 biotypes of C. diphtheriae are characterized on BTA i.e. Gravis, mitis and intermedius biotypes The most severe disease is associated with the gravis biotype Colony of gravis biotype is large, non-hemolytic & grey. Colonies of mitis biotype are small, hemolytic and black Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.
* Features
1. Gravis2. Intermedius
3. Mitis
Case fatality rate
High
High
Low
Complications
Paralytic, hemorrhagic
Hemorrhagic
Obstructive
Predominance
In epidemic areas
Epidemic areas
Endemic areas
Spread
Rapid
Rapidly than mitis
Less rapid
Colony on TBA
‘Daisy head” colony ‘Frog’s egg colony ‘Poached egg’ colony Hemolysis
Variable
Nonhemolytic
Usually hemolytic
Based on colony morphology on the tellurite medium & other properties, McLeod classified diphtheria bacilli into three types:
Biotypes of Diphtheria bacilli
* Laboratory Diagnosis
* Laboratory DiagnosisVirulence tests - Test for toxigenicityInvivo tests – animal inoculation (guinea pigs)Subcutaneous testIntracutaneous testInvitro testsElek’s gel precipitation testTissue culture test
* Laboratory Diagnosis Virulence tests - Invivo tests
Bacterial growth from Loeffler’s serum slope is emulsified in 2-4 ml broth.Two guinea pigs (GP A and GP B) Subcutaneous test – 0.1 ml of emulsion is injected SC into each guinea pig GP A - has diphtheria antitoxin (500 units injected 18 to 24 hours before) GP B - Doesn't have antitoxinIn vivo Detection of Diphtheria Exotoxin
* Laboratory Diagnosis Virulence tests - Invitro testsElek's gel precipitation test filter paper saturated with antitoxin (1000units/ ml) is placed on agar plate with 20% horse serum bacterial culture streaked at right angles to filter paper
Detection of toxin: Elek’s Test Principle: It is toxin/antitoxin reaction Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin Procedure: A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar The organism is streaked at right angels to the filter paper Incubate the plate at 37C for 24 hrs
Resuls: After 48 hrs incubation, the antitoxin diffusing from filter paper strip and the toxigenic strains produce exotoxin, which diffuses and resulted in lines four precipitation lines radiating from intersection of the strip and the growth of organism
Filter paper saturated with diphtheria antitoxin
Inoculated M.O.
Positive Elek’s Test Lines of precipitations
* Laboratory Diagnosis Virulence tests - Invitro tests
Tissue culture test - incorporation of bacteria into agar overlay of eukaryotic cell culture monolayers. Result: toxin diffuses into cells and kills them*
Toxin - Antitoxin Neutralization test
* A small amount (0.1 ml) of diluted (1/50 MLD) diphtheria toxin is injected intradermally into the arm of the person. Minimum lethal dose (MLD, also LDmin) is the least amount of drug that can produce death in a given animal species under controlled conditions If a person does not have enough antibodies to fight it off, the skin around the injection will become red and swollen, indicating a positive result. This swelling disappears after a few days. If the person has immunity, then little or no swelling and redness will occur, indicating a negative result.
*
Results can be interpreted as: Positive: when the test results in a red necrotic area of 5-10 mm diameter Pseudo-positive: when there is only a red colored inflammation and it disappears rapidly Negative reaction: No Wheel and erythema
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