Corynebacterium

Second Faculty of Medicine, Charles University in Prague

MUDr. D. Lžičařová

2019

Coryneforms (diphtheroids)

morphological term, includes several taxonomically

diverse genera of gram-positive, non spore-forming, non

acid-fast, non-branching, pleomorphic rods

Genera: Actinomyces, Arachnia, Arcanobacterium, Arthrobacter,

Bacterionema, Bifidobacterium, Brevibacterium, Cellulomonas,

Corynebacterium, Erysipelothrix, Eubacterium, Jensenia, Kurthia,

Listeria, Mycobacterium, Nocardia, Oerskovia, Propionibacterium,

Rhodococcus, and Rothia.

Corynebacterium

Cell wall – meso-diaminopimelic acid and arabinogalactan

Short-chain mycolic acids (22 – 36 carbon atoms)

46 to 74 mol% G+C.

Microscopy - g+ (or uneven staining) straight or slightly curved

rods, snapping division – angular and palisade arrangements, club-

shaped rods occur

Metachromatic granules – Albert stain (high energy phosphate

reserves)

Catalase positive, all medically relevant species non-motile

Facultative anaerobic or aerobic, fermentative or oxidative

metabolism

Metachromatic granules – Albert stain, C. diphtheriae

www.textbookofbacteriology.net

Potentially toxinogenic Corynebacterium species

Corynebacterium diphtheriae – strictly human pathogen

Corynebacterium ulcerans - zoonotic

Corynebacterium pseudotuberculosis – animals, zoonotic

Diphtheria toxin – exotoxin

Tox gene encoding diphtheria toxin is bacteriophage-borne (β-phage)

Synthesized and released into the extracellular medium as a single

polypeptide

Irreversibly denatured by heat and acid

Receptor-binding domain – binds to diphtheria toxin receptor on

susceptible cells, fragment B

Transmembrane domain – inserts into the endosomal membrane,

catalytic domain transported into the cytosol – fragment B

Catalytic domain – stops protein synthesis by inactivating

elongation factor 2 – ADP ribosylation (fragment A)

Diphtheria toxin

Toxin gene expression regulation – C. diphtheriae, not the phage

Chromosome-encoded regulatory element Diphtheria toxin

repressor (DtxR)

DtxR – Fe2+ - activated protein (expression regulated by the bacterial

host)

sequence-specific DNA-binding activity for the diphtheria

toxin operator

Fe depletion enhances diphtheria toxin production

C. diphtheriae strains differ greatly in the yield of toxin – if tox+ phage

from weakly toxigenic strain introduced to other C. diphtheriae strain,

normal yields of toxin may occur

When toxin produced at a maximal rate, no evidence of other phage

proteins expression occurs

Diphtheria toxin – maximum production in the stationary phase of

bacterial growth

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Diphtheria – pathogenesis

Colonization of mucosal membrane of upper respiratory tract –

seldom invades the bloodstream

Toxin spreads through the mucosa via diffusion, cell death occurs in

superficial mucosal layers.

Pseudomembranes (coagulum of necrotic cells, fibrin and blood

elements) may spread futher into te aitways and cause their obturation

Toxin may further spread into the bloodstream, affects most

susceptible cells

Myocard

Peripheral nerves (periaxonal cells)

Epithelium of renal tubules

Clinical diphtheria

Incubation period 2-4 days

Pharyngeal – catarrhal – fibrinous – pseudomembranous inflammatory reaction

Cervical lymphadenopathy, low fever

Benign phase, prompt reaction to antibiotics administration

Laryngeal (croup) – life threatening particularly in infants

In the initial stage reminds viral laryngitis

Spreading of pseudomembranes to lower respiratory tract follows

Serious prognosis, tracheostomy not successful in all cases

Cutaneous diphtheria – ulcers with rolled edges, painless, grey pseudomembranes

Skin, (conjunctiva, ear, genitals)

Toxin-mediated impairment rare

Nasal – pseudomembranes, purulent secretion

High contagiosity

Clinical diphtheria

Malignant diphtheria – pseudomembranes further evolve, spread,

necrotic areas occur

Toxin absorbs and spreads into the bloodstream – high fever,

bilateral cervical lymphadenopathy and soft tissue edema

Peripheral neuropathy (acute – soft palate palsy, late – polyneuritis)

Myocard impairment (dysrythmia, collapses, heart dilatation and

failure),

Renal tubular necrosis

Most impairments due to diphtheria toxin are reversible, dysrythmia

may remain

Corynebacterium ulcerans

Zoonotic species, diphtheria-like disease

Pets, raw dairy products

No interhuman transmission occurs

Non-toxigenic strains – granulomes, pulmonary nodules in

immunocompetent hosts

Corynebacterium pseudotuberculosis

Human infections rare (professional exposure) – lymphadenitis,

abscesses,

farm animals (caseous lymphadenitis) – significant economic losses

in sheep and goat worldwide

Diphtheria – laboratory diagnosis

Culture (swabs recommended – throat, other)

Suspection of diphtheria must be mentioned in the request form

Special procedures must be applied for successful isolation and identification

of potentially toxinogenic corynebacteria

Culture media

Blood agar – routine processing of throat swab, all white colonies must be

isolated

Tellurite/cystin agar media (Clauberg, Tinsdale) – black colonies must be

isolated

Liquid media should be used – inoculation on blood and tellurite agar

follows

Identification – bichemical (API-Coryne, pyrazinamidase, urease, nitrate

reduction, sugar fermentation tests – Hiss serum, Hottinger broth)

MALDI-TOF – correct identification of potentially toxinogenic corynebacteria

Diphtheria – laboratory diagnosis

C. diphtheriae biovars

gravis, mitis, intermedius, belfanti – biochemically distinguishable,

all may produce toxin

Toxigenicity testing

PCR tox gene detection – preliminary test, excludes tox gene

negative strains

Tox gene positivity does not mean toxigenicity, not all positive

strains really produce the toxin

Toxin production must be confirmed by phenotypic test

Diphtheria – laboratory diagnosis

Toxigenicity testing

Gold standard – Elek test

Immunoprecipitation in agar medium

Metabolic-inhibition colorimetric test on tissue cultures

Inhibition of mammal cell growth by diphtheria toxin (monkey kidney

cells – vero, other)

Antibody levels testing possible with the same test – not for

diphtheria diagnosis!

Protection verification, antibody response testing (haematology).

Diphtheria – treatment

Antibiotics

Penicillin – drug of choice

Macrolides for decolonization

Lincosamides, ciprofloxacin, rifampicin - alternatives

Antitoxin (horse anti-diphtheria toxin serum) – i.m. or i.v. for toxin

inactivation

Skin test before administration (risk of anaphylaxis)

Non-toxigenic C. diphtheriae strains

Colonization of skin and mucosa not common in the vaccinated

population, colonization prevented by vaccine

Individuals with underlying conditions prone to colonization/infection:

Intravenous drug abuse

Alcoholism

Homelessness

Poor living conditions

Diabetes mellitus

Immunodeficiency

Non-toxigenic strains may invade the bloodstream, virulence factors not

known

Chronic cutaneous infections, wound infections occur most frequently

Bacteremia, sepsis, endocarditis, septic arthritis, osteomyelitis, brain

abscess..

Diphtheria – epidemiology

Populations with high vaccine coverage

Sporadic cases, very rare

Usually imported from countries where diphtheria is endemic

Clinical course may be very mild, atypical in vaccinated individuals

Low carriage rates

Endemic diphtheria countries

Low vaccine coverage, up to 5 % of healthy carriers

Transmission – respiratory droplets, direct contact, contaminated

surfaces

source – infected or colonized person

www.cdc.gov

Diphtheria – vaccination

Diphtheriae anatoxinum adsorbed to aluminium hydroxide

Vaccine protects from the effect of diphtheria toxin, not the

bacterium itself

Good herd immunity provided

Protective levels

Common population min. 0,01 IU/ml

Healthcere workers and other persons at risk min. 0,1 IU/ml

Booster administration every 10 years recommended

Thank you for your attention