Corynebacteria

Gram positive rods Spore-forming AEROBIC ANAEROBIC Non spore-forming 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 segments Frequently show club shaped swellings – corynebacteria (coryne = club) C. diphtheriae : most important member of this genus, causes diphtheria Diphtheroids : 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 forming Non capsulated Non--hemolytic on sheep blood agar Facultative anaerobic C. diphtheriae is fastidious while diphtheriods are non-fastidious Catalase positive Oxidase negative

ØLipid-rich cell wall contains meso-diaminopimelic acid, arabino-galactan polymers, and short-chain mycolic acids ØLysogenic bacteriophage encodes for potent exotoxin in virulent strains

Species of Corynebacteria Corynebacterium diphtheriae Other Significant Corynebacterium species C. xerosis C. pseudodiphtheriticum C. pseudotuberculosis C. jeikeium, (skin) C. ulcerans Normal flora of RT, urethra, vagina, Skin

Introduction – C. diphtheriae Diphtheros – leather (tough, leathery pseudomembrane) Also known as Klebs–Loeffler bacillus Causes Diphtheria

Important features of C. diphtheriae Slender Gram positive bacilli Pleomorphic, non motile, non sporing Chinese 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 granules Metachromatic Granules: made up of polymetaphosphate Bluish purple color with Loeffler’s Methylene blue Special stains

Virulence factor Exotoxin – Diphtheria toxin: Protein in nature very powerful toxin Responsible for all pathogenic effects of the bacilli Produced by all the virulent strains Two 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 NAD

Mechanism of Action of Diphtheria Toxin: Mechanism of Action of Diphtheria Toxin: Inhibition of Protein Synthesis

Molecular Structure of Diphtheria Toxin Catalytic 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 phages Can be toxoided by - Prolonged storage Incubation at 37°C for 4 - 6 weeks Treatment with 0.2 – 0.4 % formalin or Acid pH. Antibodies to fragment B - protective

Epidemiology Habitat – nose, throat, nasopharynx & skin of carriers and patients Spread by respiratory droplets, usually by convalescent or asymptomatic carriers Nasal carriers harbour the bacilli for longer time than pharyngeal carriers Local infection of throat - toxemia Incubation period of diphtheria – 3 to 4 days In tropics, cutaneous infection is more common than respiratory infection

Diphtheria Site of infection Faucial (palatine tonsil) – commonest type Laryngeal Nasal Otitic Conjunctival Genital – vulval, vaginal, prepucial Cutaneous – 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 adenitis Septic – ulceration, cellulitis, & gangrene around the pseudomembrane Hemorrhagic – 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 passage Acute circulatory failure Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, palatine & ciliary, spontaneous recovery Sepsis – pneumonia & otitis media

Diphtheria Cased by C. diphtheriae Mycocarditis, neuroitis, Acute, Toxin mediated Recovery or complication & death (if more toxin absorbed) Childhood disease affect upper respiratory tract Diphtheria Respiratory obstruction due to extensive membrane formation Transmitted by droplet infection 2-6 days I.P. Sore throat, Pharyngitis 2-3 days, Bluish white adherent pseudo membrane

Laboratory Diagnosis Specimen – swab from the lesions Microscopy Gram stain: Gram +ve bacilli, chinese letter pattern Immunofluorescence Albert’s stain for metachromatic granules

Morphology 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 egg Blood agar Loeffler’s serum slope – rapid growth, 6 to 8 hrs Tellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies Hoyle’s media modifications of TBA McLeod’s media

Growth of diphtheria bacilli Blood agar Tellurite blood agar Loeffler’s serum slope

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.

Biotypes of Diphtheria bacilli Based on colony morphology on the tellurite medium & other properties, McLeod classified diphtheria bacilli into three types: Features 1. Gravis 2. Intermedius 3. Mitis Case fatality rate 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

Laboratory Diagnosis Biochemical reactions Ferments sugar with acid formation but not Gas ferments: glucose, galactose, maltose and dextrin, but not ferment sucrose. Resistant to light, desiccation and freezing Sterilization: sensitive to heat (destroyed in 10mins at 58°C or 1min in 100°C), chemical disinfectants

Laboratory Diagnosis Virulence tests - Test for toxigenicity Invivo tests – animal inoculation (guinea pigs) Subcutaneous test Intracutaneous test Invitro tests Elek’s gel precipitation test Tissue 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 antitoxin

In vivo Detection of Diphtheria Exotoxin

Laboratory Diagnosis Virulence tests - Invitro tests Elek'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

Filter paper saturated with diphtheria antitoxin 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 Lines of precipitations Inoculated M.O. Positive Elek’s Test

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 In Vivo (Schick test): Schick test - used to determine whether or not a person is susceptible to diphtheria (Corynebacterium diphtheriae). Named after its inventor, Béla Schick (1877-1967) It is a simple procedure. 40

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. 41

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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Treatment specific treatment must not be delayed if clinical picture suggests of diphtheria rapid suppression of toxin-producing bacteria with antimicrobial drugs (penicillin or erythromycin) early administration of antitoxin: 20,000 to 1,00,000 units for serious cases, half the dose being given IV 44

Prophylaxis Active Immunization (Vaccination) Formol toxoid (fluid toxoid) incubation of toxin with 0.3% formalin at pH 7.4 - 7.6 at 37°C for 3 to 4 weeks fluid toxoid is purified and standardized in flocculating units (Lf doses) Adsorbed toxoid (more immunogenic than fluid toxoid) purified toxoid adsorbed onto insoluble aluminium phosphate or aluminium hydroxide given IM (DTP or TD) 45

Prophylaxis Adsorbed Toxoid DPT - triple vaccine given to children; contains diphtheria toxoid, Tetanus toxoid and pertussis vaccine DaT - contains absorbed tetanus and ten-fold smaller dose of diphtheria toxoid. (smaller dose used to diminish likelihood of adverse reactions) Schedule i) Primary immunization - infants and children - 3 doses, 4-6 weeks interval - 4th dose after a year - booster at school entry ii) Booster immunization - adults -Td toxoids used (travelling adults may need more) SHICK test - to test susceptibility to vaccine, not done now-a-days

Prophylaxis Passive immunization ADS (Antidiphtheritic serum, antitoxin) - made from horse serum - 500 to1000 units subcutaneously Combined immunization First dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation

CONTROL isolate patients treat with antibiotics actively complete vaccination schedule should be used with booster every 5 years

Other Corynebacteria C. ulcerans – diphtheria like lesions in guinea pigs & cows, may get transmitted to humans by cow’s milk Diphtheroids – Normal commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva Stain uniformly Few or no metachromatic granules Arranged in parallel rows (palisades) Nontoxigenic