medical Microbiology lecture power point ppt corynebacterium diphtheriae

2. Introduction
• Klebs--1883 discovered
• Loefflers--1884 cultured
• Also known as KLB
• Emil von Behring- 1890
produced antitoxin
• Awarded nobel prize
Emil Von Behring

3. Morphology
• Gram positive bacilli. 3-6 μ x 0.5-0.8 μ.
• V or K or L shape.
• Chinese letter pattern, angular ['æŋ jələr]ɡ
arrangement
Chinese-letter morphology in Gram stain

4. Morphology
• Metachromatic 因温变色的 granules. volutin
['v ljət n]ɒ ɪ 迂回体 granules, polymetaphosphate
energy storage depots ['di po ]ː ʊ
methylene blue staining

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

6. Cultivation
• Loefflers serum slope– creamy white
colonies in 6-8 hrs
• Potassium tellurite ['telj ra t]medium—ʊˌ ɪ
black colonies
• Blood agar

8. ClassificationClassification
3 morphological types of3 morphological types of C. diphtheriaeC. diphtheriae are found onare found on
tellurite containing media:tellurite containing media:
△△ MitisMitis ['m['maiait s]– black colonies with a grayɪt s]– black colonies with a grayɪ
peripheryperiphery
△△ 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

9. Biochemical reactions
• Hiss [h s]serumɪ
• Ferments glucose,maltose ['m lto z]ɔː ʊ 麦芽
糖 with acid only
• Lactose, sucrose ['su kro s], mannitol notː ʊ
fermented
• Urease ['j ri e s]ʊ ːˌ ɪ 尿素酶 negative
• Starch –only gravis type, not intermedius
or mitis

10. Pathogenicity
• Produces exotoxin
• Lysogenic conversion with beta phage
• Toxin – heat labile protein
• A and B fraction
• Toxicity- disease
• Antigenicity- immunity
• Toxoid – toxin without toxicity

11. Pathogenesis
ForFor C. diphtheriasC. diphtherias to cause diphtheria an exotoxin mustto cause diphtheria an exotoxin must
be produced.be produced.
△△ Is a heat-labile polypeptide produced duringIs a heat-labile polypeptide produced during
lysogeny [la 's d əni ]ɪ ɒ ʒ ːlysogeny [la 's d əni ]ɪ ɒ ʒ ː of aof a ββ phage that carries thephage that carries the
"tox” gene."tox” gene.
△△ Alkaline pH of 7.8- 8.0, aerobic conditions, and aAlkaline pH of 7.8- 8.0, aerobic conditions, and a
low environmental iron level are essential for toxinlow 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].

12. Pathogenesis
△ Trypsin cleaves the toxin into 2 fragments, A and B, that
are linked together by a disulfide [da 's lfa d]ɪ ʌ ɪ bridge.
- Fragment A contains the toxic activity(catalytic domain)
- Fragment B (transmembrane and receptor binding
domains [do 'me n]ʊ ɪ )
■ Receptor: heparin-binding epidermal growth factor
- rich on cardiac ['k rdiæk]cells and nerve cellsɑː
△ One molecule of toxin can inhibit 90% of the protein
synthesis in a cell.

13. 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.

14. C. diphtheriaC. diphtheria toxintoxin
※※ 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

17. Biotypes
1)gravis(13 types)-most serious disease
Colonies: large, irregular, gray
2)mitis(40 types)-mild illness
colonies: small, round, convex, black
3)intermedius(4 types)-intermediate
severity
Colonies: small, flat and gray

18. Diphtheria
• Children, fatal if not treated in time
• Exclusively human disease
• Droplet infection- fomites
• Fever, cervical lymphadenopathy,
pseudomembrane
• Myocarditis arrhythmia [ə'r θm ə]ɪ ɪ 心律不齐 fatal
• Polyneuropathy, palatine ['pæləta n]ɪ 上颚的
paralysis
• Rare in adults.

19. Pseudomembrane formation
Thick membrane lining throat that interferes with eating,
drinking and breathing
Contents: WBC, RBC, bacteria, disintegrating epithelial
cells
Cause: fibrinous exudate from local necrosis by exotoxin
(removal of PF may cause bleeding)
Localization: faucial ['f əl]ɔːʃ 咽喉的 , laryngeal[lə'r nd iəl]ɪ ʒ
喉头的 , nasal, otitic, conjunctival, genital, cutaneous

20. [ha 'p ksiə]ɪ ɑː 氧过少 [l m fæd 'na t s]ɪ ˌ ɪ ɪ ɪ 淋巴腺炎

21. Faucial:
Signs: sore throat, fever
complications: prostration [pr 'stre n]ɑː ɪʃ 疲劳 , dyspnea [d sp'ni ə]ɪ ː
呼吸困难 , asphyxia [æs'f ksiə]ɪ 窒息 , arhythmia[' hi ðmjə]ɑː ː 心律不齐 ,
difficulties of speech, vision, swallowing, movement
Septic: Ulcerated[' lsəre t d]:ʌ ɪ ɪ

22. Clinical classification
i) Malignant (hypertoxic) diphtheria
Signs: severe toxemia and adenitis[ ædə'na t s]ˌ ɪ ɪ 腺炎 , lymph glands
swelling in the neck
Complications: death-circulatory failure, paralytic
sequelae [s 'kwi li ]ɪ ː ː
ii) Septic diphtheria:
Signs: ulceration with pseudomembrane formation and cellulites
(gangrene and Amputation)
iii) Hemorrhagic diphtheria
Signs: local and general bleeding from edge of psudomembrane,
conjunctival, epistaxis [ ep 'stæks s]ˌ ɪ ɪ 鼻出血 and purpura ['p pj rə]ɜː ʊ 紫
癜

23. Complications
1)Asphyxia [æs'f ksiə]ɪ 窒息 -obstruction of
resp tract
2)Acute circulatory failure
3)kidney failure
4)paralysis-soft palate, eye muscles,
extremities (3rd-4th week)
5)septic sequelae-pnemonia, otitis media

24. 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

25. Virulence test
In vivo
Guinea pig– subcutaneous
intradermal
Test and control animals to be used
In vitro
Eleks gel precipitation test

26. Toxigenicity test (virulence test)
i) Animal inocculation
-bacteria culture emulsified[ 'm ls fa ]ɪ ʌ ɪ ɪ 使乳化 in water and 0.8 ml injected into 2
guinea pigs
GP 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 [stri k]ː 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)

27. Elek's gel precipitation test

28. 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.

29. 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 antibodiesyear – 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

30. Prevention
• Active immunity- DPT dosage (diphtheria, pertussis
(whooping 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 [ d fθə'r tˌ ɪ ɪ ɪ500 – 1000 units sc of Anti Diphtheritic [ d fθə'r tˌ ɪ ɪ ɪ]]SerumSerum
( ADS)( ADS)
• 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

31. Prevention
a)DTP (DPT)-
• Formal toxoid - Incubation of Toxin at pH 7.4 -7.6 for 3 – 4Formal toxoid - Incubation of Toxin at pH 7.4 -7.6 for 3 – 4
weeks at 37 °Cweeks at 37 °C
• Adsorbed toxoid – purified toxoid adsorbed on toAdsorbed toxoid – purified toxoid adsorbed on to
aluminum phosphate or hydroxidealuminum phosphate or hydroxide [ha 'dr ksa d]ɪ ɑː ɪ 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.

32. 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)

33. 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

34. For therapy ofFor therapy of
Ab
DT
tumor
tumors !
!
ProspectProspect

35. Patch in the throat
• Diphtheria
• Streptococcal tonsillitis
• Oral thrush– Candida albicans
• Spirochete- Borrelia vincenti

36. Other Corynebacteria
1. C. ulcerans:
– Diphtheria like lesions
– Spread thru milk
– Antitoxin protective, erythromycin
2. C.minutissimum-
– Erythrasma [er 'θræzmə]ɪ 红癣
3. C. tenuis:
- Trichomycosis axillaris

37. 4. C.pseudotuberculosis:
– Veterinary importance
5. C.parvum:
– Immunomodulator
6. A. haemolyticum:
– pharyngitis, skin ulcers
7. C.jakieum:
– Skin inf/ Rx(medical prescriptions)- Vancomycin.

38. 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

39. Exercises
Elek's gel precipitation test
Schick test
1.Briefly describe the mechanisms of
diphtheria exotoxindiphtheria exotoxin??