(M) Lesson 8.1: Mycobacteria (Part 1) Flashcards
- Slender, slightly curved or straight rods
- Non-motile, strict aerobes
Mycobacterium
All Myconacterium are non-sporeforming except?
Mycobacterium marinum
All Mycobacterium are slow growers except?
- Mycobacterium fortuitum
- Mycobacterium chelonae
- Mycobacterium phlei
- Mycobacterium stegmatis
60% of the cell wall contains what?
Clue: Two answers
- High lipid content
- Lipid containing structures (Mycolic Acid and Cord Factor Wax D)
Mycobacterium is also referred to as?
Acid Fast Bacilli
- Characteristic or main feature of Mycobacteria
- Pertains to the ability of the bacterial cell wall to withstand strong acid decolorizer due to the presence of long chain waxy mycolic acid
Acid Fastness
Mycobacterium contains ____ instead of N-acetylmuramic acid
N-glucolyl muramic acid
T or F: There is low lipid content in Mycobacterium - which is why they are able to create a hydrophobic permeability barrier.
F (very high lipid content)
The reason as to why Mycobacteria grow more slowly (the organisms tend to clump together and the nutrients are not easily allowed into the cells)
Hydrophobicity
Classify if MTC or NTM
- M. tuberculosis
- M. bovis
- Mbovis BCG
- M. africanum
- M. caprae
- M. microti
- M. canetti
- M. mungi
- M. oygis
- M. pinnipedii
Mycobacterium tuberculosis complex (MTC)
Classify if MTC or NTM
Slow-growing pathogenic
* M. avian complex
Rapid-growing opportunistic pathogenic
* M.aviancomplex
* M. fortuitum group
* M.chelonae
* M.abssessus subs. Abscessus
Non-cultivable NTM
* M. leprae
Non-tuberculosis mycobacteria (NTM)
- Complex: Two or more species that are difficult to distinguish and have little medical importance
- Can cause tuberculosis
- Non-pigmented colonies
- Slow grower
Mycobacterium tuberculosis complex
How many days does it require for Mycobacterium tuberculosis complex to grow?
More than 7 days (2 to 60 days)
Identify the organism based on habitat and mode of transmission.
- Habitat: Patient with cavitary (lung) disease (primary reservoir)
- Mode of Transmission: P2P, ABT (single bacilli may cause infection, only 15 to 20% develop diseases years later)
M. tuberculosis
ABT - Airborn Transmission
P2P - Person to Person
Identify the organism based on habitat and mode of transmission.
- Habitat: Humans and animals
- Mode of Transmission: Ingestion of contaminated milk, ABT
M. bovis
Identify the organism based on habitat and mode of transmission.
- Habitat: Commercial vaccines
- Mode of Transmission: Via vaccination (rare)
M. bovis BCG
BCG - Bacillus Calmette-Guerin
Identify the organism based on habitat and mode of transmission.
- Habitat: Humans
- Mode of Transmission: ABT
M. africanum
Identify the organism based on habitat and mode of transmission.
- Habitat: Animals, Humans (rarely)
- Mode of Transmission: ABT
Clue: Three answers
- M. caprae
- M. microti
- M. pinnipedii
Hi idol, study the epidemiology of TB and TB-HIV !!
Go na please mwa
Ghon’s complex
* Focus of inflammation
* Pulmonary lesion caused by tuberculosis
Primary (Initial) Pulmonary Tuberculosis
- Also called reactivation
Cavity (Secondary) Pulmonary Tuberculosis
Cavity (Secondary) Pulmonary Tuberculosis
Hard tubercle in lung-cellular aggregate
Granuloma
Cavity (Secondary) Pulmonary Tuberculosis
Cheese-like dead tissue to macrophage enzymes
Caseous necrosis
- In patients infected with primary active TB, the disease may be spread through lymphatic system
- Progress to form cavities in lungs and other organs
- Rarely do granulomas become calcified and remain asymptomatic for years
Miliary (Disseminated) Pulmonary Tuberculosis
Common name of Mycobacterium tuberculosis
Koch bacillus
Who discovered the Mycobacterium tuberculosis?
Robert Koch (1882)
- Obligate aerobe
- Require CO2 for growth (5 to 10%)
- Virulence factor
Mycobacterium tuberculosis
Characteristics of Mycobacterium tuberculosis
Sticky response of the sputum
Cord Factor and Sulfatides
Disease
- The TB bacilli are inside the body but they cannot spread the disease to others
- However, if the patient do not get the treatment needed it may develop to Active TB
Latent TB infection
Disease
- It can spread the disease from one person to another
- Can cause death if not treated immediately
- The bacilli grows in the body and makes the person feel sick and have symptoms
Active TB Disease
Disease
What are the possible symptoms for TB?
- Productive cough
- Low grade fever
- Myalgia
- Chills
- Sweating
Disease
- Happens when the immune of an individual becomes weak because the body cannot control the bacteria from growing because the immune system is suppressed
- Can also mimic other diseases
Reactivation Tuberculosis
Disease
What are the diseases that TB can mimic?
- Influenza
- Acute Bronchitis
- Pneumonia
- Neoplasm
- Fungal Infections
The primary means of identifying individuals affected by mycobacterium
Tuberculin Skin Test
- Purified Protein Derivative (PPD)
- 2-4 Days: Delayed Hypersensitivity Reaction (Erythema)
Tuberculin Skin Test
- A culture extract of mycobacterium tuberculosis is injected intramuscularly or intracutaneously
- After 48-72 hours, the individual will show delayed hypersensitivity reaction to PPD and it is characterized by erythema or redness and also in duration or firmness as a result of the influx of the immune cells
- Not 100% specific or sensitive
Tuberculin Skin Test
- Blood incubated with Mtb Ag
- Measure released cytokines using enzyme-linked immunospot
T-Spot TB Test
- Measure T-cells activated by MTb Ag
- Should be interpreted in correlation with the patient’s signs and symptoms
T-Spot TB Test
- Enzyme-Linked Immunoassay (ELISA)
- Detects interferon-γ released from lymphocytes
Quantiferon-TB Gold
- Detects the interference gamma that is release from the lymphocytes
- Another important feature is that the result of the assay are not affected by the BCG vaccination
Quantiferon-TB Gold
What the different types of specimen for Mycobacterium tuberculosis?
- Sputum (Expectorated and Induced)
- Blood
- Urine
- CSF
- Pleural, Pericardial, and Peritoneal Fluid
The most common specimen for Mycobacterium tuberculosis?
Sputum
Sputum specimen must be collected over the course of?
Three (3) consecutive days
Types of Sputum
- Deep cough
Expectorated Sputum
Types of Sputum
- Through inhalation of NaCl using nebulizer
- Obtained through bronchoscopy
Induced Sputum
Sputum secretions are obtained by?
Bronchoscopy
Gram stain quality specimen is classified with?
Bartletts Classification
Saliva or sputum?
Greater than 10 EC with 25 Polymorphonuclear (PMN)
Saliva (rejected)
Saliva or sputum?
Less than 10 EC with less than 25 Polymorphonuclear (PMN)
Sputum (accepted)
CSF (Specimen for M. tuberculosis)
- Pellicle of web-like clot (positive in TB) indicates what?
Tubercular meningitis
T or F: There is increased ADA in Mycobacterium tuberculosis.
T
Produced by lymphocytes which is a useful marker for the diagnosis of tuberculous pleurisy
Adenosine Deaminase
- Pleural effusion secondary to infection of mycobacteria
Tuberculous pleurisy
- Presumptive for MTb
- Microscopic examination of specially stained smears to detect acid fast organism
AFB Smear Microscopy (DSSM)
What are the two different types of smears?
- Direct Smear
- Concentrated Smear
Types of Smears
- Prepare directly from a patient specimen prior to processing
Direct Smear
Types of Smear
- Prepared from a processed specimen after centrifugation to concentrate the material
Concentrated Smear
Identify the method.
- Carbol Fuchsin with heat
- Acid Alcohol (3% HCl in 95% ethanol
- Methylene Blue
Ziehl-Neelsen’s Method (Hot Method)
What is the mordant for Ziehl’s Neelsen’s Method?
Heat
Ziehl-Neelsen’s Method
Blue bacilli against blue background is reported as?
Non-Acid Fast
Ziehl-Neelsen’s Method
Red bacilli against blue background is reported as?
Acid Fast Bacilli
Identify the method.
- Carbol Fuchsin (no heat)
- Acid-alcohol 0.5% HCl in 95% alcohol
- Malachite Green
Kinyoun’s Method (Cold Method)
What is the mordant chemical of Kinyoun’s Method?
Tergitol
Kinyoun’s Method
Red bacilli against green background
Acid Fast Bacilli
Kinyoun’s Method
Green bacilli against green background
Non-Acid Fast
Identify the method.
- Carbol Fuchsin (with heat)
- Pappenheim’s differentiating stain
- Rosalic acid with Methylene Blue in glycerine and absolute alcohol
Pappenheim’s Method
Pappenheim’s Method
Red or pink bacilli is reported as?
M. tuberculosis
Pappenheim’s Method
Colorless bacilli is reported as?
M. stegmatis
Identify the method.
- Alcoholic Carbol Fuchsin
- 1% nitric acid in 95% alcohol
Baumgarten’s Method
Baumgarten’s Method
Blue bacilli is reported as?
M. tuberculosis
Baumgarten’s Method
Red bacillis is reported as?
M. leprae
Identify the method.
- Alcoholic Carbol Fuchsin
- Acid Alcohol (3% HCl in 95% ethanol)
- Gabbet’s Methylene Blue Solution
Gabbet Method
Gabbet Method
Red bacilli against blue background is reported as?
Acid Fast Bacilli
Gabbet Method
Blue bacilli against blue background is reported as?
Non-Acid Fast
T or F: A positive smear will provide a conclusive and vital clue for the clinician in the diagnosis of Tuberculosis.
F (presumptive not conclusive)
T or F: The acid fast stain can help in monitoring the course of treatment.
T
- The carbol fuchsin binds to the mycolic acid in the cell wall of the mycobacteria
- The fuchsin and the phenol are driven into the cell wall of the microorganism
Ziehl-Neelsen Method
- The tergitol forces the fuchsin into the cell wall
Kinyoun Technique
This acts as a counterstain for either hot or cold procedure.
Methylene Blue
- Its differentiating stain contains Rosalic acid, methylene blue, glycerin, absolute alcohol
- Differentiates M. stegmatis from M. tuberculosis
Pappenheim’s Method
- Differentiates M. leprae from M. tuberculosis
Baumgarten’s Method
- Contains methylene blue, sulfuric acid, absolute alcohol, and distilled water
Gabbet’s Methylene Blue Solution
Gabbet’s Methylene Blue Solution
Acts as the primary stain
Alcoholic carbol fuchsin
Gabbet’s Methylene Blue Solution
Acts as the decolorizer
Acid alcohol (3% HCl, 95% ethanol)
Gabbet’s Methylene Blue Solution
T or F: The interpretation is same as ZN method.
T
Gold standard method for TB diagnosis
TB Culture
Used for species identification, drug susceptibility testing, and genotyping
Culture
Two major categories of culture media
- Solid (Egg and Agar based)
- Liquid (Broth media)
Solid Media Comparison
- Selective: Malachite green
- Rate of Growth: Slower
- Area of Inoculum: Usually less but if present involves the entire surface
- Visual Examination: Difficult due to media opacity
- CO2: Not required
- DST: Not performed in LJ
- Shelf Life: Longer (6 to 12 months)
Egg-based Media
Solid Media Comparison
- Selective: Added antibacterial and antifungal
- Rate of Growth: Faster
- Area of Inoculum: More frequent, but can still isolate colonies
- Visual Examination: Media is clear, allows easier colony quantification and morphology confirmation
- CO2: Required
- DST: Preferred due to larger surface area and faster rate of growth
- Shelf Life: Shorter (1 to 2 months)
Agar-based Media
Solid Agar Based
- For Mycobacteria isolation
- Middlebrook 7H10
- Middlebrook 7H11
Solid Agar Based
- For Mycobacteria isolation from heavily contaminated cultures
Mitchison’s selective
Solid Egg Based
- For isolation and cultivation of Mycobacteria
- Wallenstein
- Lowenstein-Jensen
- L-J pyruvic acid
Solid Egg Based
- Contains higher concentration of malachite green
Petragani Medium
Liquid Based
- Automated System
- Bactec MGIT
- VERSATREK
- BacT/ALERT 3D
TB colonies resemble what?
Cauliflower-like colonies
Why do we need the methods of decontamination of bacteria?
- Most specimen are contaminated with normal flora and can overgrow the slow-growing Mycobacteria
- To isolate the Mycobacterium
T or F: Specimen like CSF must be sterile.
F (sterile already since they are collected aseptically)
Involves splitting the disulfide bonds in mucin that may trap bacteria
Digestion of sputum samples
Identify the reagent.
- Digestant and decontaminating agent
- 2-4% NaOH
- Benzalkonium chloride (Zephiran)
Identify the reagent.
- Liquefying agent
N-acetyl-L-cysteine (NALC)
Identify the reagent.
- Decontaminates specimen contained with P. aeruginosa
5 to 6% Oxalic Acid
Biochemical Tests
- Reagent strips impregnated with cyanogen bromide are inoculated with the organism
- Principle: Niacin + Niacin ribonucleotide + aniline dye + cyanogen bromide
- For M. tuberculosis identification
Niacin Test
Niacin Test
Positive result?
Yellow (accumulation of free niacin - lack of the enzyme)
Niacin Test
These species produce a positive result for Niacin Test.
- M. tuberculosis
- M. simiae
- M. marinum
- M. chelonae
Niacin Test
Negative results?
No color change
Niacin Test
This specie is the only one that produces a negative result.
M. bovis
Biochemical Tests
T or F: Most mycobacterium species are catalase positive.
T
Catalase Test at 68C or Heat-Stable Catalase
Positive results?
45 mm gas bubble height
Catalase Test at 68C or Heat-Stable Catalase
Negative results?
No bubbles
Catalase Test at 68C or Heat-Stable Catalase
The only specie that produces a positive result.
M. kansasii
Catalase Test at 68C or Heat-Stable Catalase
Species that produce negative results.
All members of the MTB complex react negatively.
Biochemical Test
- Mycobacteria possess the enzyme nitroreductase that converts nitrates to nitrites
- Principle: HCl + sulfanilamide + N-naphthylethylene diamine
Nitrate Reduction Test
Nitrate Reduction Test
Positive results?
Pink or red
Nitrate Reduction Test
Species that produce a positive result.
- M. kansasii
- M. szulgai
- M. fortuitum
- M. tuberculosis
Nitrate Reduction Test
Negative results?
No color change
Nitrate Reduction Test
Species that produce a negative result.
- M. avium-intracellulare complex
- M. bovis
- M. marinum
- M. simae
- M. gastri
Biochemical Tests
- Principle: Tween 80 converted to oleic acid by Tween 80 lipase
- Useful for identification of M. kansasii as quick as 6 hours
- Differentiates M. gordonae and M. scrofulaceum
Tween 80 Hydrolysis
Tween 80 Hydrolysis
Positive results?
Red color
Tween 80 Hydrolysis
Species that produce a positive result.
- M. kansasii
- M. gordonae
Tween 80 Hydrolysis
Negative results?
No red/amber
Tween 80 Hydrolysis
Species that produce a negative result.
- M. avium
- M. scrofulaceum
Biochemical Tests
- Principle: Certain mycobacteria can reduce tellurite salts to metallic tellurium forming a black precipitate
Tellurite Reduction Test
Telleurite Reduction Test
Positive results?
Smooth fine black precipitate
Tellurite Reduction Test
Specie that produces a positive result.
M. avium
Tellurite Reduction Test
Negative results?
Gray clumps
Tellurite Reduction Test
Specie that produces a negative result.
M. kansasii
Biochemical Tests
- For rapid growers
- Principle: Aryl sulfatase split sulfate group from tripotassium phenolphthalein disulfide/sulfate, producing free phenolphthalein
- For identification of M. fortuitum-chelonae complex that gives (+) result after 3 days
Aryl Sulfatase Test
Aryl Sufatase test
Positive results?
Pink or red color
Aryl Sulfatase Test
Specie that produces a positive result.
M. fortuitum-chelonae complex
Biochemical Tests
- A susceptibility test
- Casein hydrolysis serves as a growth stimulant for drug-resistant strains of M. tuberculosis
Thiphene-2-Carboxylic Acid Hydralize (TCH) Test
TCH Test
Components of the TCH Agar
- Inorganic salt essential (for mycobacterial growth)
- Glycerol(source of carbon and energy)
TCH Test
This serves as a growth stimulant for drug-resistant strains of M. tuberculosis.
Casein hydrolysis
TCH Test
Susceptible to TCH Test
M. bovis
TCH Test
Resistant to TCH Test
M. tuberculosis
Biochemical Tests
- Only rapid growers can convert 20% ferric ammonium citrate to an iron oxide
Iron Uptake
Iron Uptake
Positive results?
Rusty brown colonies
Iron Uptake
Species that produce a positive result.
M. phlei, M. smegmatis (rapid growers)
Iron Uptake
Negative results?
No color formation
Iron Uptake
Specie that produces a negative result.
M. chelonae (also a rapid grower)
Biochemical Tests
Converts pyrazinamide to pyrazinoic acid and ammonia in 4 days
Pyrazinamidase
Pyrazinamidase
Positive result?
Red pigment
Pyrazinamidase
Species that produces positive results.
- M. tuberculosis
- M. marinum
Pyrazinamidase
Negative results?
No color formation
Pyrazinamidase
Species that produces a negative result.
- M. bovis
- M. kansasii
Biochemical Tests
- The detection of urease activity
Urease
Urease
Specie that produce positive results.
M. scrofulaceum
Urease
Specie that produces negative results.
M. gordonae
Biochemical Tests
T or F: Most bacteria cannot grow in high salt concentration, 5% NaCl in egg-based media, inhibiting the growth of most media
T
Biochemical Tests
- For identification of M. triviale, the only slow grower that gives (+)
Sodium Chloride Tolerance
Sodium Chloride Tolerance
Species that gives positive results (growth)?
- M. flavescens
- M. trivale
- Mycobacterium rapid growers
