Corynebacterium (1)

Corynebacterium

Mycobacterium

Mary Joyce Saborrido-Teoxon, RMT, MD

Dept. of Microbiology and Parasitology

GENUS: CORYNEBACTERIUM

• Gram-positive, pleomorphic rods

• Nonspore-forming, nonmotile, non-

encapsulated

• Aerobic

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

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.

Diphtheria

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)

Corynebacterium diphtheriae

• LABORATORY DIAGNOSIS

• 3. Catalase test (+)

• 4. Urease test (-)

• 5. Toxigenicity test

– Elek test (in vitro)

– Animal inoculation test (in vivo)

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

Corynebacterium minutissimum

• Agent of ERYTHRASMA

• “coral red fluorescence” on Wood’s light

– Presence of porphyrin

Diphtheroid

• C. pseudodiphthericum

• Hoffman’s Bacillus

• Causes diphtheria like disease

GENUS: MYCOBACTERIUM

• Acid fast rods with waxy cell wall

• Obligate aerobe

• Non-sporeforming, Non-encapsulated

• Slow-growers (except: M. fortuitum,

M. chelonei)

• Granules (Much)

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

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

Mycobacterium tuberculosis

• Reservoir

– Human lungs

• Transmission

– Respiratory droplets and droplet

• Predisposing Factor

– For active disease is poverty, HIV infections, or

any CMI system immunosuppression.

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

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

SPECIMEN PROCESSING:

Specimen

Sterile Nonsterile

SPECIMEN PROCESSING: NONSTERILE

LIQUEFICATION

DECONTAMINATION

NEUTRALIZATION

CENTRIFUGATION

1.) Liquefy

• NALC

• Dithiothreitol (sputolysin)

• Enhance by mixing with a vortex type of

mixer in a closed container, stand 15 mins

2.) Decontaminate

• NaOH

• Zephiran-trisodium

• 6% Oxalic acid (g-, Pseudomonas,

Proteus)

3.) Neutralize

• Buffer

• H2O

Mycobacterium tuberculosis

• LABORATORY DIAGNOSIS

• 1. Gram stain – to qualify specimen

• 2. Acid Fast Stain

– Fuchsin stain

– Fluorochrome

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

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

M. tuberculosis on Lowenstein-Jensen(LJ) agar.

Coagulated eggs, glycerol, potato flour, and salts,

Malachite green.

Young colonies of M. tuberculosis on(10 days)

Middlebrook 7H11 agar viewed microscopically.

Beginning of cording characteristic of M.tb

M. tuberculosis exhibiting cauliflower colonies

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

Biochemical Tests 1. NIACIN TEST

principle: NIACIN + NIACIN RIBONUCLEOTIDE +

ANILINE DYE + CYANOGEN BROMIDE

M. tuberculosis = positive (yellow)

M. bovis = negative

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

Biochemical Tests

3. Nitrate reduction test:

nitroreductase

detected by:

a. HCL

b. sulfanilamide

c. alpha napthyl amine

(+) result = pink color

(+) M.tb

(-) M.avium

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

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

Biochemical Tests

6. Tellurite reduction test:

Px; Telurite --- black metallic tellurium

used to ID M. avium (+) ; M. kansasii (-)

Biochemical Tests

7. TCH Susceptibility test

(+) susceptible = M. bovis

(-) resistant = M. tb

TCH Thiophene-2-carboxylic acid hydrazide

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

• 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.

• 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

• 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

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.

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

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

Table I. Runyon Grouping of MOTTS

RUNYON’S CLASSIFICATION Genus & specie

Rapid growers M. fortuitum

M. chelonei

M. phlei

M. smegmatis

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

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

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 “

Mycobacterium leprae Tuberculoid

B

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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

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