1. Corynebacterium
Mycobacterium
Mary Joyce Saborrido-Teoxon, RMT, MD
Dept. of Microbiology and Parasitology

2. GENUS: CORYNEBACTERIUM
• Gram-positive, pleomorphic rods
• Nonspore-forming, nonmotile, non-
encapsulated
• Aerobic

3. Corynebacterium diphtheriae
• Distinguishing Characteristics:
– Kleb Loeffler’s Bacillus
– Club-shaped Gram-positive rods arranged in
V , L, X, Y shapes
– Granules (Babes Ernst) produced on
Loeffler’s coagulated serum medium stain
metachromatically

4. Corynebacterium diphtheriae
• Transmission
– Bacterium or phage via respiratory droplets from oropharynx
of infected person
• Pathogenesis
– Organism not invasive; colonizes epithelium of oropharynx
or skin in cutaneous diphtheria.
– Diphtheria toxin (A-B component) – inhibits protein
synthesis by adding ADP-ribose to EF-2.
– Effect on oropharynx:
– Dirty gray pseudomembrane (made up of dead cells and
fibrin exudates bacterial pigment)
– Extension into larynx/trachea → obstruction
– Effect of systemic circulation → heart & nerve damage.

5. Diphtheria

6. Corynebacterium diphtheriae
• LABORATORY DIAGNOSIS
• 1. DME (G/S, LAMB)
• 2. CULTURE
– Loeffler’s serum agar slant
– Pai coagulated egg
– Tinsdale (black  dark brown halos)
– Tellurite blood agar
– Cystine tellurite blood agar (black 
gray)

7. Corynebacterium diphtheriae
• LABORATORY DIAGNOSIS
• 3. Catalase test (+)
• 4. Urease test (-)
• 5. Toxigenicity test
– Elek test (in vitro)
– Animal inoculation test (in vivo)

8. Corynebacterium diphtheriae
• Treatment
– Erythromycin and antitoxin
• Prevention
– Toxoid vaccine (formaldehyde-modified toxin
is still immunogenic but with reduced toxicity),
part of DtaP, DTP, or Td

9. Corynebacterium minutissimum
• Agent of ERYTHRASMA
• “coral red fluorescence” on Wood’s light
– Presence of porphyrin

10. Diphtheroid
• C. pseudodiphthericum
• Hoffman’s Bacillus
• Causes diphtheria like disease

11. GENUS: MYCOBACTERIUM
• Acid fast rods with waxy cell wall
• Obligate aerobe
• Non-sporeforming, Non-encapsulated
• Slow-growers (except: M. fortuitum,
M. chelonei)
• Granules (Much)

12. GENUS: MYCOBACTERIUM
Three Groups:
• M. tuberculosis complex- cause TB
– M. tuberculosis – pulmunonary tuberculosis
– M. bovis – intestinal tuberculosis
– M. africanum – pulmonary tuberculosis (
Africa)
• MOTT
• M. leprae

13. Mycobacterium tuberculosis
• Distinguishing Characteristics
– Koch Bacillus
– Acid fast
– Aerobic, require CO2
– slow growing
– Produces niacin
– Produces a heat-sensitive catalase:
• Catalase negative at 68°C (standard catalase test)
– (other mycobacterial catalase are heat
insensitive)
• Catalase active at body temperature

14. Mycobacterium tuberculosis
• Reservoir
– Human lungs
• Transmission
– Respiratory droplets and droplet
• Predisposing Factor
– For active disease is poverty, HIV infections, or
any CMI system immunosuppression.

15. Mycobacterium tuberculosis
• Pathogenesis
– Facultative Intracellular Organism
– Sulfatides (sulfolipids in cell envelope)
• Inhibit the phagosome-lysosomal fusion allowing
intracellurlar survival. (If fusion occurs, waxy nature of cell
envelope reduces killing effect.)
– Cord factor (trehalose di-myoclate)
» Causes serpentine growth in vitro
» Inhibits leukocyte migration; disrupts mitochondrial
respiration and oxidative phosphorylation
• Tuberculin (surface protein) along with mycolic acid →
delayed hypersensitivity and CMI
– Granulomas and caseation mediated by cell-
mediated immunity (CMI)
– No exotoxins nor endotoxin; damage done by
immune system

16. Mycobacterium tuberculosis
Disease
• Tuberculosis
• Causative agents: Mycobacterium tuberculosis , M.
bovis, and M. africanum
• Complex disease: pulmonary, urinary tract, and organ or
military (disseminated)
• Primary infection: organisms replicate in naïve
macrophages, killing macrophages until CMI is set up.
• Most people heal without disease; some organisms
walled off in the Ghon complex remain viable unless
treated.
• Post primary (reactivational TB) erosion of granulomas
into airways (high oxygen) later in life under conditions of
reduced T-cell immunity leads to mycobacterial
replication and disease symptoms

17. SPECIMEN PROCESSING:
Specimen
Sterile Nonsterile

18. SPECIMEN PROCESSING:
NONSTERILE
LIQUEFICATION
DECONTAMINATION
NEUTRALIZATION
CENTRIFUGATION

19. 1.) Liquefy
• NALC
• Dithiothreitol (sputolysin)
• Enhance by mixing with a vortex type of
mixer in a closed container, stand 15 mins

20. 2.) Decontaminate
• NaOH
• Zephiran-trisodium
• 6% Oxalic acid (g-, Pseudomonas,
Proteus)

21. 3.) Neutralize
• Buffer
• H2O

22. Mycobacterium tuberculosis
• LABORATORY DIAGNOSIS
• 1. Gram stain – to qualify specimen
• 2. Acid Fast Stain
– Fuchsin stain
– Fluorochrome

23. Acid Fast Reporting
0 No AFB seen
1-2 / 300 fields Doubtful; request
another specimen
1-9/ 100 fields +1
1-9/ 10 fields +2
1-9/ field +3
>9 +4

24. Mycobacterium tuberculosis
• 3. Culture
A. Agar Base Media:
1. Duboi’s Oleic Acid Albumin medium
2. Mitchison’s medium
3. Middlebrook 7H10 – 7H11 – AST
B. Egg-Base Media: malachite green
1. Petragnani medium
2. Lowenstein-Jensen medium
3. American Thoracic Society medium
4. Dorset Egg medium
C. Liquid Media: Bactec 12B, Septi-Chek AFB,
Middlebrook 7H9

25. M. tuberculosis on Lowenstein-Jensen(LJ) agar.
Coagulated eggs, glycerol, potato flour, and salts,
Malachite green.

26. Young colonies of M. tuberculosis on(10 days)
Middlebrook 7H11 agar viewed microscopically.
Beginning of cording characteristic of M.tb

27. M. tuberculosis exhibiting cauliflower
colonies

28. M. Tuberculosis on Middlebrook 7H11 agar. Cream-
colored, dry, and wrinkled colonies. Contains casein
hydrolysates that improve recovery of INH resistant
strains of M.tb and shorten incubation time for M.
avium complex

29. Biochemical Tests
1. NIACIN TEST
 principle: NIACIN + NIACIN RIBONUCLEOTIDE +
ANILINE DYE + CYANOGEN BROMIDE
 M. tuberculosis = positive (yellow)
 M. bovis = negative

30. Biochemical Tests
2. Catalase test:
-medium: TWEEN 80
-reagent: 30 % H2O2
-all Mycobacteria (+)
types:
a. Semi-quantitative test
- column of bubbles
b. Heat stable catalase test
- 68 oC – denature enzyme
-M. tb. = negative
(+) M. kansasii

31. Biochemical Tests
3. Nitrate reduction test:
 nitroreductase
 detected by:
a. HCL
b. sulfanilamide
c. alpha napthyl amine
 (+) result = pink color
(+) M.tb
(-) M.avium

32. Biochemical Tests
4. ARYLSULFATASE TEST:
– Detects rapid growers
– Principle:
– Tripotasium Arylsulfatase Free
Phenolphthalein Phenolphthalein
Disulfide/sulfate (END PRODUCT)
– RESULT: (+) Red/ Pink
– Strongly (+)  M. fortuitum-chelonei
– (-)  M-avium

33. Biochemical Tests
5. TWEEN 80 HOH test:
Principle:
Tween 80 hydrolysis of tween 80
(polyoxyethelene (oleic acid +
Sorbitan polyoxyethylated
Monooleate) sorbitol)
(+) red = M. kansasii
(-) no red = M. avium

34. Biochemical Tests
6. Tellurite reduction test:
Px; Telurite --- black metallic tellurium
 used to ID M. avium (+) ; M. kansasii (-)

35. Biochemical Tests
7. TCH Susceptibility test
(+) susceptible = M. bovis
(-) resistant = M. tb
TCH  Thiophene-2-carboxylic acid hydrazide

36. Automated test for Mycobacterium
1. Bactec 460 Middlebrook 7H12 (RIA based)
Principle : 14C palmitic acid + orgs= 14 CO2
Result (+) : more than 10 growth index
2. Mycobacteria Growth Indicator Tube
(MGIT)
– Fluorometric based
3. Bactec 12B + NAP
– P-nitro acetylamino beta
hydroxypropiophenone (NAP)
AST = disk elution using S-I-R-E disks

37. • Diagnosis
– PPD skin test (Mantoux):
– >5 mm in HIV+ or anyone with recent TB
exposure; AIDS patients have reduced ability
to mount skin test.
– >10 mm in high-risk population: IV drug
abusers, people living in poverty, or
immigrants from high TB area.
– >15 mm in low-risk population
– Positive skin test indicates only exposure
but not necessarily active disease.

38. • Treatment
– Multiple drugs critical to treat infection
– Standard observed short-term therapy for
uncomplicated pulmonary TB (rate where acquired
<4%):
• First 2 months: isoniazid + rifampin + pyrazinamide
• Next 4 months: isoniazid and rifampin
– Ethambutol or streptomycin added for possible drug-
resistant cases until susceptibility tests are back (if
area acquired has >4% DRM TB

39. • Prevention
– Isoniazid taken for 6-9 months can prevent
TB in persons with infection but not clinical
symptoms.
– Bacille-Calmette-Guerin (BCG) vaccine
contains live, attenuated organisms may
prevent disseminated disease. Not commonly
used in the U.S.
– UV lights or HEPA filters used to treat
potentially contaminated air

40. Mycobateria Other Than
Tuberculosis (MOTTS)
• (MOTTS) = Non-tuberculous Mycobacteria
= atypical Mycobacteria
• Non-contagious!
• Found in surface waters, soil, cigarettes;
most common in southeastern U.S.

41. Table I. Runyon Grouping of
MOTTS
Runyun Runyon Group Dark Light Growth
Group # Name
I Photochromogen - + Slow
(+) Cream/buff
Orange/yellow in 10-21
days
II Scotochromogen + + Slow
(+) Orange/ Yellow 10-
21 days
III Non- - - Slow
photochromogen Cream buff in 10-21
days
IV Rapid growers Fast < 7days

42. Table I. Runyon Grouping of MOTTS
RUNYON’S Genus & specie
CLASSIFICATION
Photochromogen M. kansasii
M. marinum
M. asiaticum
M. simiae
Scotochromogen M. scrofulaceum (scrofula)
M. szulgai
M. gordonae (tap H2O bacillus)
Non- M. avium or
Photochromogen M. intracellulare (battey bacillus)
M. Ulcerans (Buruli)
M. xenopi ( hot ,cold H2o taps)
M. triviale
M.haemophilum
M. malmoense

43. Table I. Runyon Grouping of MOTTS
RUNYON’S CLASSIFICATION Genus & specie
Rapid growers M. fortuitum
M. chelonei
M. phlei
M. smegmatis

44. Mycobateria Other Than
Tuberculosis (MOTTS)
• Disease
– Pulmonary/Gastrointestinal/Disseminated
– Patients: AIDS (prophylaxis <75 CD4+ cells/mm3), cancer,
chronic lung disease
– M. avium-intracellulare, M. kansasii.
– Mycobacterial lymphadenitis
– Usually solitary cervical lymph nodes (surgically removed) in
kids.
• M. scrofulaceum.
– Soft-Tissue Infections
• M. marinum: cutaneous granolomas in tropical fish
enthusiast (fist tank granuloma) or scuba divers from
abrasions on coral

45. Mycobacterium leprae
• Distinguishing Characteristics
– Acid fast rods (seen in punch biopsy)
– Cigarette-packet/picket-fence
– Can hydrolyze 3,4-dihydroxy-phenylalanine (DOPA)
– Obligate intracellular parasite (cannot be cultured in vitro)
– Optimal growth at less than body temperature
• Reservoir
– Human mucosa, skin, and nerves are the only significant
reservoir.
– Some infected armadillon in Texas and Lousiana
• Transmission
– Nasal discharge from untreated lepromatous leprosy patients

46. Mycobacterium leprae
• Pathogenesis
– Obligate intracellular parasite
– Cooler parts of body e.g., skin, mucous membranes,
and peripheral nerves
• Disease
– Leprosy (Hansen’s)
A continuum of disease, which usually start out with an
indeterminate stage called “borderline “

47. Mycobacterium leprae
Tuberculoid Lepromatous
Cell-mediated immune Strong CMI Weak CMI
system
Lepromin skin test Lepromin test + Lepromin test -
Number of organisms Low B High (foam cells totally filled)
in tissue o
Damage form Immune response r Large number of intracellular
(CMI killing infected d organisms
cells) e Nerve damage from overgrowth
Granuloma formation r of bacteria in cells
→ nerve l Loss of sensation → burns and
enlargement/damage i trauma
Loss of sensation → n
burns and trauma e
Number of lesions and Fewer lesions: Numerous lesions becoming
other syndromes macular; nerve nodular; loss of eyebrows;
enlargement, destruction of nasal septum
paresthesia Paresthesia
Leonine facies

48. Mycobacterium leprae
• Laboratory Diagnosis
– Punch biopsy or nasal scrapings; acid fast stain
– Lepromin skin test is positive in the tuberculoid but
not in the lepromatous form.
– No cultures
• Treatment
– Multiple-drug therapy with dapsone and rifampin,
with clofazimineadded for lepromatous
• Prevention
– Dapsone for close family contacts