1. Non forming spore Gram positive bacilli
Lecture 4

2. Corynebacterium spp
Gram positive bacilli, with characteristic morphology (club shaped and beaded)
Non motile
Non spore forming
Non capsulated
Facultative anaerobic
Breakdown glucose by oxidative and fermentative i.e. O+/F+
C. diphtheriae is fastidious while diphtheriods are non-fastidious
Catalase positive
Oxidase negative

3. Species of Corynebacteria
It caused diphtheria
C. diphtheriae is the only pathogenic members of this genus
Normal flora of RT, urethra, vagina, Skin

4. Gram Positive Bacilli Gram positive rods Spore forming
Non spore forming
Aerobic
Anaerobic
Corynebacterium
Bacillus spp
Clostridium spp

5. The non-spore-forming gram-positive bacilli are a diverse group of bacteria. Many members of the genus corynebacterium and their anaerobic equivalents, propionibacterium species, are members of the normal flora of skin and mucous membranes of humans.

6. Corynebacterium diphtheriae is the most important member of the group, as it can produce a powerful exotoxin that causes diphtheria in humans. Listeria monocytogenes
and Erysipelothrix rhusiopathiae are primarily found in animals and occasionally cause severe disease in humans

7. Based upon Gram stain morphology, formation of spores, and catalase reaction
 Spore-forming, catalase positive (aerobic): Bacillus sp.
 Regularly-shaped, non-spore forming
 Catalase positive: Listeria monocytogenes
 Catalase negative: Erysipelothrix rhusiopathiae, Lactobacillus sp., Gardnerella vaginalis
 Irregularly-shaped, non-spore forming, catalase positive: Corynebacterium sp. (“diphtheroids”)
 Branching: Nocardia sp., Actinomyces sp., & Streptomyces sp.

8. Corynebacterium species “coryneform bacteria” have a high guanosine plus cytosine content and include the genera corynebacterium, arcanobacterium, brevibacterium, mycobacterium, and others.

9. Actinomyces and propionibacterium
are classified as anaerobes, but some isolates grow well aerobically (aerotolerant) and must be differentiated from the aerobic coryneform bacteria.

10. species—than they are to the coryneform bacteria.
Other nonspore- forming gram-positive bacilli have more regular shapes and a lower guanosine plus cytosine content. The genera include listeria and erysipelothrix; these bacteria are more closely related to the anaerobic lactobacillus species, which sometimes grow well in air, to the sporeforming bacillus and clostridium species—and to the gram-positive cocci of the staphylococcus and streptococcus
species—than they are to the coryneform bacteria.

11. There is no unifying method for identification of the gram-positive bacilli. Few laboratories are equipped to measure guanosine plus cytosine content. Growth only under anaerobic conditions implies that the isolate is an anaerobe, but many isolates of lactobacillus, actinomyces, and propionibacterium species and others are aerotolerant.

12. CORYNEBACTERIUM DIPHTHERIAE Morphology & Identification
Corynebacteria are 0.5–1 μm in diameter and several micrometers long. Characteristically, they possess irregular swellings at one end that give them the “clubshaped” appearance.
Irregularly distrib uted within the rod (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules)
that give the rod a beaded appearance.

14. On blood agar, the C diphtheriae colonies are small, granular, and gray, with irregular edges, and may have small zones of hemolysis. On agar containing potassium tellurite, the colonies are brown to black with a brownblack halo because the tellurite is reduced intracellularly (staphylococci and streptococci can also produce black colonies).

15. C diphtheriae and other corynebacteria grow aerobically on most ordinary laboratory media. Propionibacterium is an anaerobe. On Loeffler’s serum medium, corynebacteria grow much more readily than other respiratory organisms, and the morphology of organisms is typical in smears.

16. Pathogenesis The principal human pathogen of the group is C diphtheriae. In nature, C diphtheriae occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers. It is spread by droplets or by contact to susceptible individuals; the bacilli then grow on mucous membranes or in skin abrasions, and those that are toxigenic start producing toxin.

17. All toxigenic C diphtheriae are capable of elaborating the same disease-producing exotoxin. In vitro production of this toxin depends largely on the concentration of iron. Toxin production is optimal at 0.14 μg of iron per milliliter of medium but is virtually suppressed at 0.5 μg/mL. Other factors influencing the yield of toxin in vitro are osmotic pressure, amino acid concentration, pH, and availability of suitable carbon and nitrogen sources. The factors that control toxin production in vivo are not well understood.

18. Diphtheria toxin is a heat-labile polypeptide that can be lethal in a dose of 0.1 μg/kg.

19. Pathology
Diphtheria toxin is absorbed into the mucous membranes
and causes destruction of epithelium and a
superficial inflammatory response. The necrotic epithelium .

20. becomes embedded in exuding fibrin and red and
white cells, so that a grayish “pseudomembrane” is
formed—commonly over the tonsils, pharynx, or larynx.
Any attempt to remove the pseudomembrane
exposes and tears the capillaries and thus results in
bleeding. The regional lymph nodes in the neck enlarge,
and there may be marked edema of the entire neck. The
diphtheria bacilli within the membrane continue to
produce toxin actively. This is absorbed and results in
distant toxic damage, particularly parenchymatous
degeneration, fatty infiltration, and necrosis in heart
muscle, liver, kidneys, and adrenals, sometimes accompanied
by gross hemorrhage. The toxin also produces
nerve damage, resulting often in paralysis of the soft
palate, eye muscles, or extremities

21. Diagnostic Laboratory Tests Swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered. Smears stained with alkaline methylene blue or Gram’s stain show beaded rods in typical arrangement.

22. Laboratory diagnosis of case
Specimen: A throat swap
Culture: The swap is inoculated on Loeffler's serum medium and/or on blood tellurite agar aerobically at 37C for 24.
On Loeffler's serum medium:
Corynebacteria grow much more readily than other respiratory pathogens
Deep blue or red metachromatic granules (accumulated inorganic polyphosphates) by methylene blue stain
Loefflers serum

23. The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges

24. Cultural characteristics
On blood tellurite agar
It is selective medium for isolation of C. diphtheriae (Potassium tellurite)

26. 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.

27. Biochemical Reaction
All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

28. 2- Carbohydrate Fermentation Test:
Principle:
Each species of corynebacteria has its specific carbohydrate fermentation pattern
C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only

29. Procedure:
Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism
Incubate the tubes at 37oC for 24 hrs.
Glucose
Maltose
Sucrose

30. 2- Carbohydrate Fermentation Test:
Principle:
Each species of corynebacteria has its specific carbohydrate fermentation pattern
C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only
Procedure:
Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism
Glucose
Maltose
Sucrose
Incubate the tubes at 37oC for 24 hrs.

31. Results:
Sugar fermentation can be indicated by change of color of the medium from red to yellow due to formation of acid which decrease the pH
Glucose
Maltose
Sucrose
Glucose
Maltose
Sucrose
+ve
+ve
+ve
+ve
+ve
- ve
C. diphtheriae
C. xerosis

32. 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

33. 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

34. 1-4 positve 2-3 negative

35. Resistance & Immunity Since diphtheria is principally the result of the action of the toxin formed by the organism rather than invasion

36. by the organism, resistance to the disease depends largely on the availability of specific neutralizing antitoxin in the bloodstream and tissues. It is generally true that diphtheria occurs only in persons who possess no antitoxin (or less than 0.01 Lf unit/mL). Assessment of immunity to diphtheria toxin for individual patients can best be made by review of documented diphtheria toxoid immunizations and primary or booster immunization if needed

37. Treatment The treatment of diphtheria
rests largely on rapid suppression of toxin-producing bacteria by antimicrobial drugs and the early administration of specific antitoxin against the toxin formed by the organisms at their site of entry and multiplication.

38. Antimicrobial drugs (penicillin, erythromycin) inhibit the growth of diphtheria bacilli. Although these drugs have virtually no effect on the disease process, they arrest toxin production. They also help to eliminate coexistent streptococci and C diphtheriae from the respiratory tracts of patients or carriers.

39. Anaerobic Corynebacteria
Propionibacterium species) reside in normal skin. Propionibacterium acnes,
is aerotolerant and grows aerobically. It participates in the pathogenesis of acne
Arcanobacterium haemolyticum produces betahemolysis on blood agar. It is occasionally associated with pharyngitis

40. LISTERIA MONOCYTOGENES
There are several species in the genus listeria. Of these, L monocytogenes is important as a cause of a wide spectrum
of disease in animals and humans.

41. Culture & Growth Characteristics Listeria grows on media such as Mueller-Hinton agar. Identification is enhanced if the primary cultures are done on agar containing sheep blood, because the characteristic small zone of hemolysis can be observed around and under colonies. Isolation can be enhanced if the tissue is kept at 4 °C for some days before inoculation bacteriologic media.

42. produces acid but not gas in a variety of carbohydrates.
The organism is a facultative anaerobe and is catalase-positive and motile. Listeria
produces acid but not gas in a variety of carbohydrates.
The motility at room temperature and hemolysin production are primary findings that help differentiate listeria from coryneform bacteria.

43. ACTINOMYCETES
Actinomycetes are a large, diverse group of gram-positive bacilli with a tendency to form chains or filaments.
They are related to the corynebacteria and mycobacteria as well as the stepomycete

44. The most famous species are
Actinomyces israelii
Actinomyces naeslundii

45. Diagnostic Laboratory Tests:
Pus from draining sinuses, sputum, or specimens of tissue are examined for the presence of sulfur granules.
The granules are hard, lobulated, and composed of tissue and bacterial filaments, which are club-shaped at the
periphery .Specimens are cultured in
thioglycolate broth and on brain-heart infusion blood

47. NOCARDIA
Causing Nocardiosis wich is caused by infection withNocardia asteroides
complex or, less frequently, Nocardia brasiliensis or Nocardia otitidiscaviarum, and only rarely by other species of nocardia. The Nocardia asteroides complex
includes Nocardia abscessus,
Nocardia farcinia, Nocardia nova,
and others

49. THANK YOU