Introduction

• Klebs--1883 discovered• Loefflers--1884 cultured• Also known as KLB • Emil von Behring- 1890

produced antitoxin• Awarded nobel prize

Emil Von Behring

Morphology

• Gram positive bacilli. 3-6 μ x 0.5-0.8 μ.• V or K or L shape.• Chinese letter pattern, angular ['æŋɡjələr] arran

gement

Chinese-letter morphology in Gram stain

Morphology

• Metachromatic 因温变色的 granules. volutin ['vɒljətɪn] 迂回体 granules, polymetaphosphate energy storage depots ['diːpoʊ]

methylene blue staining

Morphology

• Alberts stain – green and bluish black• Nonmotile noncapsulated, nonsporing• pleomorphic

Cultivation

• Loefflers serum slope– creamy white colonies in 6-8 hrs

• Potassium tellurite ['teljʊˌraɪt]medium—black colonies

• Blood agar

ClassificationClassification

3 morphological  types of 3 morphological  types of C. diphtheriaeC. diphtheriae are  found on are  found on

tellurite containing media: tellurite containing media:

△ △ MitisMitis ['m ['maiaitɪs]– black colonies with a gray tɪs]– black colonies with a gray

 periphery periphery

△ △ GravisGravis – large,  gray  colonies – large,  gray  colonies

△ △ IntermediusIntermedius – small, dull gray  to black. – small, dull gray  to black.

All produce an immunologically identical toxinAll produce an immunologically identical toxin

GravisGravisMitisMitis IntermediusIntermedius

Biochemical reactions

• Hiss [hɪs]serum

• Ferments glucose,maltose ['mɔːltoʊz] 麦芽糖 with acid only

• Lactose, sucrose ['suːkroʊs], mannitol not fermented

• Urease ['jʊriːˌeɪs] 尿素酶 negative

• Starch –only gravis type, not intermedius or mitis

Pathogenicity

• Produces exotoxin

• Lysogenic conversion with beta phage

• Toxin – heat labile protein

• A and B fraction

• Toxicity- disease

• Antigenicity- immunity

• Toxoid – toxin without toxicity

Pathogenesis

For For C. diphtheriasC. diphtherias to cause diphtheria an exotoxin must to cause diphtheria an exotoxin must

be  produced.be  produced.

△△ Is a heat-labile polypeptide produced during Is a heat-labile polypeptide produced during

lysogeny [laɪ'sɒdʒəniː]lysogeny [laɪ'sɒdʒəniː] of  a of  a phage that carries the phage that carries the

"tox” gene."tox” gene.

△△ Alkaline pH of  7.8- 8.0, aerobic conditions, and a Alkaline pH of  7.8- 8.0, aerobic conditions, and a

low environmental iron level are essential for toxin low environmental iron level are essential for toxin

production (occurs late in the growth of the organism).production (occurs late in the growth of the organism).

△△ The  toxin  inhibits protein  synthesis  by  ADP-The  toxin  inhibits protein  synthesis  by  ADP-

ribosylating elongation factor 2[EF-2]. ribosylating elongation factor 2[EF-2].

Pathogenesis

△ Trypsin  cleaves  the toxin into 2 fragments, A and B, that

are linked together by a disulfide [daɪ'sʌlfaɪd] bridge.

- Fragment A contains  the toxic activity(catalytic domain)

- Fragment B (transmembrane and receptor binding

domains [doʊ'meɪn] )

■ Receptor: heparin-binding epidermal growth factor

- rich on cardiac ['kɑːrdiæk]cells and nerve cells

△ One molecule of toxin can inhibit  90%  of the  protein

synthesis in a cell.

Pathogenesis

△ Toxin diffuses throughout body via blood

- Cardiac, neurologic complications

- Heart/respiratory damage, paralysis

△ Systemic effects  include heart failure, paralysis and

adrenal hypofunction leading to an Addison’s like disease.

C. diphtheriaC. diphtheria toxin toxin

※ ※ Toxin enters through receptor mediated endocytosisToxin enters through receptor mediated endocytosis

※ ※ Acidification of endocytic vesicle allows A to dissociate from BAcidification of endocytic vesicle allows A to dissociate from B

※ ※ A enters cycoplasmA enters cycoplasm

Biotypes

1)gravis(13 types)-most serious diseaseColonies: large, irregular, gray

2)mitis(40 types)-mild illnesscolonies: small, round, convex, black

3)intermedius(4 types)-intermediate severityColonies: small, flat and gray

Diphtheria

• Children, fatal if not treated in time• Exclusively human disease• Droplet infection- fomites• Fever, cervical lymphadenopathy, pseudomembr

ane• Myocarditis arrhythmia [ə'rɪθmɪə] 心律不齐 fatal• Polyneuropathy, palatine ['pælətaɪn] 上颚的 para

lysis• Rare in adults.

Pseudomembrane formation

Thick membrane lining throat that interferes with eating, drinking and breathingContents: WBC, RBC, bacteria, disintegrating epithelial cellsCause: fibrinous exudate from local necrosis by exotoxin (removal of PF may cause bleeding)Localization: faucial ['fɔːʃəl] 咽喉的 , laryngeal[lə'rɪndʒiəl]喉头的 , nasal, otitic, conjunctival, genital, cutaneous

[haɪ'pɑːksiə] 氧过少 [lɪmˌfædɪ'naɪtɪs] 淋巴腺炎

Faucial:

Signs: sore throat, fevercomplications: prostration [prɑː'streɪʃn] 疲劳 , dyspnea [dɪsp'niːə]

呼吸困难 , asphyxia [æs'fɪksiə] 窒息 , arhythmia['ɑːhiːðmjə] 心律不齐 ,

difficulties of speech, vision, swallowing, movement

Septic: Ulcerated['ʌlsəreɪtɪd]:

Clinical classification

i) Malignant (hypertoxic) diphtheria

Signs: severe toxemia and adenitis[ˌædə'naɪtɪs] 腺炎 , lymph glands

swelling in the neck Complications: death-circulatory failure, paralytic

sequelae [sɪ'kwiːliː]

ii) Septic diphtheria:Signs: ulceration with pseudomembrane formation and cellulites (gangrene and Amputation)

iii) Hemorrhagic diphtheriaSigns: local and general bleeding from edge of psudomembrane, conjunctival, epistaxis [ˌepɪ'stæksɪs] 鼻出血 and purpura ['pɜːpjʊrə] 紫癜

Complications

1)Asphyxia [æs'fɪksiə] 窒息 -obstruction of resp tract2)Acute circulatory failure3)kidney failure 4)paralysis-soft palate, eye muscles, extremities (3rd-4th week)5)septic sequelae-pnemonia, otitis media

 

Laboratory diagnosis

• Sample collection: Throat swab or swab from membrane

• Microscopy: Gram stain and Alberts stain

• Culture: Loefflers and PT

• Biochemicals

• Virulence test in vivo and in vitro

Virulence test

In vivoGuinea pig– subcutaneous

intradermal

Test and control animals to be used

In vitro

Eleks gel precipitation test

Toxigenicity test (virulence test)

i) Animal inocculation-bacteria culture emulsified[ɪ'mʌlsɪfaɪ] 使乳化 in water and 0.8 ml injected into 2 guinea pigsGP A-has dipht antitoxin (injected 2 hours before)GP B-Doesn't have antitoxin

Result: Guinea pig B dies.

ii) Elek's gel precipitation test-filter paper saturated with antitoxin is placed on agar plate with 20% horse serum-bacterial culture streaked [striːk] 条痕 at right angles to filter paper

iii) tissue culture test-incorporation of bacteria into agar overlay of eukaryotic cell culture monolayers.Result: toxin diffuses into cells and kills them

iv) PCR assays-test presence of specific bacteriophage gene (tox)

Elek's gel precipitation test

Schick test

• A small amount (0.1 ml) of diluted (1/50) diphtheria toxin is injected intradermally into the arm of the person. 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. If the person has an immunity, then little or no swelling and redness will occur, indicating a negative result.

EPIDEMIOLOGYEPIDEMIOLOGY

• Formally important pediatric diseaseFormally important pediatric disease• Developed countries - EradicatedDeveloped countries - Eradicated• Developing countries serious problemDeveloping countries serious problem• Rare in 1Rare in 1stst year – maternal antibodies year – maternal antibodies• Peak 1-5 years, 5-10 years- decreases there afterPeak 1-5 years, 5-10 years- decreases there after• Immunity – sub clinical infectionsImmunity – sub clinical infections• Carriers outnumber cases – throat & nasalCarriers outnumber cases – throat & nasal• Rarely spread through milkRarely spread through milk

Prevention• Active immunity- DPT dosage (diphtheria, pertussis (who

oping cough), and tetanus)– Antitoxin level – 0.01/ ml protectiveAntitoxin level – 0.01/ ml protective

• Passive immunity- ADS– When susceptibles are exposedWhen susceptibles are exposed– 500 – 1000 units sc of Anti Diphtheritic [ˌdɪfθə'rɪtɪ500 – 1000 units sc of Anti Diphtheritic [ˌdɪfθə'rɪtɪ]]Serum ( ADSerum ( AD

S)S)

• Combined immunisation– First dose of adsorbed toxoid on one armFirst dose of adsorbed toxoid on one arm– ADS on another armADS on another arm– To be continued with full course of active immunizationTo be continued with full course of active immunization

Preventiona) DTP (DPT)- • Formal toxoid - Incubation of Toxin at pH 7.4 -7.6 for 3 – 4 Formal toxoid - Incubation of Toxin at pH 7.4 -7.6 for 3 – 4

weeks at 37 °C weeks at 37 °C • Adsorbed toxoid – purified toxoid adsorbed on to Adsorbed toxoid – purified toxoid adsorbed on to

aluminum phosphate or hydroxidealuminum phosphate or hydroxide [haɪ'drɑːksaɪd] 氢氧化物• triple vaccine given to children. trivalent preparation • Diphtheria toxoid, Tetanus toxoid and Pertussis vaccine

Td- contains absorbed tetanus and ten-fold smaller dose of diphtheria toxoid.

b) Schedule

i) primary immunization –

- infants and children - 3 doses, 4-6 weeks - 4th dose after a year - booster at school entry

ii) Booster immunization

- adults - Td toxoids used (traveling adults may need more)

Shick test-to test sensitivity (allergic reaction)

Treatment

• Antitoxin – 20,000 to 1 lakh [læk]Antitoxin – 20,000 to 1 lakh [læk] 十万十万unitsunits

• Antibiotic therapy – PenicillinAntibiotic therapy – Penicillin

• Carriers - ErythromycinCarriers - Erythromycin

For therapy ofFor therapy of

AbDT

tumor

tumors !!

ProspectProspect

Patch in the throat

• Diphtheria

• Streptococcal tonsillitis

• Oral thrush– Candida albicans

• Spirochete- Borrelia vincenti

Other Corynebacteria

1. C. ulcerans:

– Diphtheria like lesions

– Spread thru milk

– Antitoxin protective, erythromycin

2. C.minutissimum- – Erythrasma [erɪ'θræzmə] 红癣

3. C. tenuis:- Trichomycosis axillaris

4. C.pseudotuberculosis: – Veterinary importance

5. C.parvum:– Immunomodulator

6. A. haemolyticum:– pharyngitis, skin ulcers

7. C.jakieum:– Skin inf/ Rx(medical prescriptions)- Vancomycin.

Diphtheroids

• Morphologically resembling diphtheria but do not cause any disease.

• Present as commensals in throat, skin

• Do not contain metachromatic granules.

• Do not produce any toxin ie virulence test is negative.

• C.hofmani, C.xerosis, C.pseudodiphtheriticum

Exercises

Elek's gel precipitation test

Schick test

1.Briefly describe the mechanisms of diphtheria exotoxindiphtheria exotoxin??