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

(M) Lesson 8.1: Mycobacteria (Part 1) Flashcards by Kchianna Lauren Coronel

Slender, slightly curved or straight rods
Non-motile, strict aerobes

Mycobacterium

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All Myconacterium are non-sporeforming except?

Mycobacterium marinum

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All Mycobacterium are slow growers except?

Mycobacterium fortuitum
Mycobacterium chelonae
Mycobacterium phlei
Mycobacterium stegmatis

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60% of the cell wall contains what?

Clue: Two answers

High lipid content
Lipid containing structures (Mycolic Acid and Cord Factor Wax D)

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Mycobacterium is also referred to as?

Acid Fast Bacilli

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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

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Mycobacterium contains ____ instead of N-acetylmuramic acid

N-glucolyl muramic acid

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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)

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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

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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)

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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)

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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

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How many days does it require for Mycobacterium tuberculosis complex to grow?

More than 7 days (2 to 60 days)

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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

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Identify the organism based on habitat and mode of transmission.

Habitat: Humans and animals
Mode of Transmission: Ingestion of contaminated milk, ABT

M. bovis

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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

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Identify the organism based on habitat and mode of transmission.

Habitat: Humans
Mode of Transmission: ABT

M. africanum

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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

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Hi idol, study the epidemiology of TB and TB-HIV !!

Go na please mwa

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Ghon’s complex
* Focus of inflammation
* Pulmonary lesion caused by tuberculosis

Primary (Initial) Pulmonary Tuberculosis

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Also called reactivation

Cavity (Secondary) Pulmonary Tuberculosis

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Cavity (Secondary) Pulmonary Tuberculosis

Hard tubercle in lung-cellular aggregate

Granuloma

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Cavity (Secondary) Pulmonary Tuberculosis

Cheese-like dead tissue to macrophage enzymes

Caseous necrosis

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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

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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.

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.

* 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.

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.

# 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

# 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