Corynebacterium (1)
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Corynebacterium
Mycobacterium
Mary Joyce Saborrido-Teoxon, RMT, MD
Dept. of Microbiology and Parasitology
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GENUS: CORYNEBACTERIUM
• Gram-positive, pleomorphic rods
• Nonspore-forming, nonmotile, non-
encapsulated
• Aerobic
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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
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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.
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Diphtheria
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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)
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Corynebacterium diphtheriae
• LABORATORY DIAGNOSIS
• 3. Catalase test (+)
• 4. Urease test (-)
• 5. Toxigenicity test
– Elek test (in vitro)
– Animal inoculation test (in vivo)
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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
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Corynebacterium minutissimum
• Agent of ERYTHRASMA
• “coral red fluorescence” on Wood’s light
– Presence of porphyrin
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Diphtheroid
• C. pseudodiphthericum
• Hoffman’s Bacillus
• Causes diphtheria like disease
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GENUS: MYCOBACTERIUM
• Acid fast rods with waxy cell wall
• Obligate aerobe
• Non-sporeforming, Non-encapsulated
• Slow-growers (except: M. fortuitum,
M. chelonei)
• Granules (Much)
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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
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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
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Mycobacterium tuberculosis
• Reservoir
– Human lungs
• Transmission
– Respiratory droplets and droplet
• Predisposing Factor
– For active disease is poverty, HIV infections, or
any CMI system immunosuppression.
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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
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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
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SPECIMEN PROCESSING:
Specimen
Sterile Nonsterile
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SPECIMEN PROCESSING: NONSTERILE
LIQUEFICATION
DECONTAMINATION
NEUTRALIZATION
CENTRIFUGATION
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1.) Liquefy
• NALC
• Dithiothreitol (sputolysin)
• Enhance by mixing with a vortex type of
mixer in a closed container, stand 15 mins
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2.) Decontaminate
• NaOH
• Zephiran-trisodium
• 6% Oxalic acid (g-, Pseudomonas,
Proteus)
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3.) Neutralize
• Buffer
• H2O
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Mycobacterium tuberculosis
• LABORATORY DIAGNOSIS
• 1. Gram stain – to qualify specimen
• 2. Acid Fast Stain
– Fuchsin stain
– Fluorochrome
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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
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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
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M. tuberculosis on Lowenstein-Jensen(LJ) agar.
Coagulated eggs, glycerol, potato flour, and salts,
Malachite green.
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Young colonies of M. tuberculosis on(10 days)
Middlebrook 7H11 agar viewed microscopically.
Beginning of cording characteristic of M.tb
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M. tuberculosis exhibiting cauliflower colonies
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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
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Biochemical Tests 1. NIACIN TEST
principle: NIACIN + NIACIN RIBONUCLEOTIDE +
ANILINE DYE + CYANOGEN BROMIDE
M. tuberculosis = positive (yellow)
M. bovis = negative
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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
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Biochemical Tests
3. Nitrate reduction test:
nitroreductase
detected by:
a. HCL
b. sulfanilamide
c. alpha napthyl amine
(+) result = pink color
(+) M.tb
(-) M.avium
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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
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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
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Biochemical Tests
6. Tellurite reduction test:
Px; Telurite --- black metallic tellurium
used to ID M. avium (+) ; M. kansasii (-)
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Biochemical Tests
7. TCH Susceptibility test
(+) susceptible = M. bovis
(-) resistant = M. tb
TCH Thiophene-2-carboxylic acid hydrazide
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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
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• 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.
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• 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
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• 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
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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.
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Table I. Runyon Grouping of
MOTTS Runyun
Group #
Runyon Group
Name
Dark Light Growth
I Photochromogen - + Slow
(+) Cream/buff
Orange/yellow in 10-21
days
II Scotochromogen + + Slow
(+) Orange/ Yellow 10-
21 days
III Non-
photochromogen
- - Slow
Cream buff in 10-21
days
IV Rapid growers Fast < 7days
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Table I. Runyon Grouping of MOTTS RUNYON’S
CLASSIFICATION
Genus & specie
Photochromogen M. kansasii
M. marinum
M. asiaticum
M. simiae
Scotochromogen M. scrofulaceum (scrofula)
M. szulgai
M. gordonae (tap H2O bacillus)
Non-
Photochromogen
M. avium or
M. intracellulare (battey bacillus)
M. Ulcerans (Buruli)
M. xenopi ( hot ,cold H2o taps)
M. triviale
M.haemophilum
M. malmoense
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Table I. Runyon Grouping of MOTTS
RUNYON’S CLASSIFICATION Genus & specie
Rapid growers M. fortuitum
M. chelonei
M. phlei
M. smegmatis
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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
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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
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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 “
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Mycobacterium leprae Tuberculoid
B
o
r
d
e
r
l
i
n
e
Lepromatous
Cell-mediated immune
system
Strong CMI Weak CMI
Lepromin skin test Lepromin test + Lepromin test -
Number of organisms
in tissue
Low High (foam cells totally filled)
Damage form Immune response
(CMI killing infected
cells)
Granuloma formation
→ nerve
enlargement/damage
Loss of sensation →
burns and trauma
Large number of intracellular
organisms
Nerve damage from overgrowth
of bacteria in cells
Loss of sensation → burns and
trauma
Number of lesions and
other syndromes
Fewer lesions:
macular; nerve
enlargement,
paresthesia
Numerous lesions becoming
nodular; loss of eyebrows;
destruction of nasal septum
Paresthesia
Leonine facies
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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