Week 5 - Mycobacteria

General Characteristics of Mycobacterium

Non-motile, slender, slightly curved, rod-shaped bacilli; non-spore forming
Cell wall has a high lipid content (mycolic acid)
Resist staining with Grams stain
Require complex media
Strictly aerobic
Slow-growers

M. tuberculosis lab safety

Bioterror level 3 - must use N-95 respirators, gloves, disposable gowns
Skin testing performed regularly
Lab separate from other areas of lab
Nonrecirculating bentilation system
Negative air pressure
Biological Safety Cabinet certified annually
Appropriate disinfectants and UV light

Acid Fast Staining used for Mycobacterium

Ziehl-Neelsen
Kinyoun modification
Auromine rhodamine

Ziehl-Neelsen

Uses heat to force stain into bacilli
Carbol fusion/methylene blue
Organisms stain red

Kinyoun modification

Does not require heat
Carbol fusion and malachite green

Auromine rhodamine

Fluorescent stain

Acceptable Respiratory Specimens

Expectorated sputum
Normal-saline-nebulized, induced sputum
Transtracheal aspirate
Bronchoalveolar lavage
Laryngeal swab

Acceptable Body fluid Specimens

Pleural fluid
Pericardial fluid
Joint aspirate
Gastric aspirate
Peritoneal fluid
Cerebrospinal fluid
Stool
Urine
Pus

Acceptable Body tissue Specimens

Blood
Bone marrow biopsy/aspirate
Solid organ
Lymph node
Bone
Skin

How to process uncontaminated samples like Blood and CSF

Centrifuge, stain and culture sediment

How to process contaminated samples like respiratory and stool

Digestion and Decontamination

Sodium hydroxide (NaOH) 2%, 3%, 4%

Serves as decontaminating agent and digestant

N-acetyl-L-cysteine

Serves as a liquefying or mucolytic agent

Benzalkonium chloride (Zephiran) with trisodium phosphate (Z-TSP)

Z-TSP Liquefies sputum rapidly but requires long exposure to decontaminating agent
Zephiran is bacteriostatic to tubercle bacilli

Oxalic acid 5%

Used to decontaminate specimens contaminated with Pseudomonas aeruginosa

Ways to Identify and Differentiate Mycobacterium

Acid-fast stain
Rate of growth
Colony morphology
Pigmentation
Nutritional requirements
Optimum incubation temperature
Biochemical tests
Culture

Lowenstein-Jensen, Petragnani

Broth and solid media
Egg-based medium
Contain malachite green to inhibit contaminants
Egg neutralizes toxic materials

Middlebrook 7H10 or 7H11

Non-selective agar
Clear medium for easier visualization

Selective agar

Increases recovery from contaminated samples

Other Media that can be used to identify Mycobacterium

Septi-check AFB system
Mycobacteria growth indicator tube broth
Bactec TB
Bactec 9000 MB
ESPII
MB/BacT

Photochromogens

Produce pigment when exposed to light

Scotochromogens

Produce pigment in light and dark

Non-chromogens

Nonpigmented
Tan or buff colored colonies

Slow Growing Nonchromogens

M. tuberculosis

M. avium

M. bovis

M. ulcerans

M. terrae complex

M. celatum

M. gastri

M. malmoense

Rapid Growing Nonchromogens

M. chelonae

M. fortuitum group

Slow Growing Scotochromogens

M. gordonae

M. szulgai

M. scrofulaceum

M. xenopi

Rapid Growing Scotochromogens

M. phlei

M. smegmatis group

Slow Growing Photochromogens

M. kansasii

M. marinum

M. simiae

M. asciaticum

Biochemical Tests for Mycobacterium

Niacin
Nitrate reduction
Catalase
Tween 80 hydrolysis
Tellurite reduction
Arylsulfatase test
Probes

Direct Susceptibility testing method

AFB demonstrated in direct smear

Indirect Susceptibility testing method

Subculture of primary culture

Niacin

Tests for production of niacin
Liquid from culture added to reagent
Positive = yellow
A non-chromogenic and slow growing organism that is niacin positive is indicative of a putative M. tuberculosis isolate

Nitrate reduction

Detects nitroreductase
Reduces nitrate to nitrite
Positive = red • (M. tuberculosis, M. kansasii, M. fortuitum, M. szulgai)

Catalase

Drop method (H2O2 dropped on colonies. INH resistant Mtb usually negative)
Semi-quantitative (Add Tween80-H2O2 reagent to tube culture, Measure height of bubbles)
Heat stable (Add H2O2 after heating organisms)

Tween 80 hydrolysis

Tests for lipase production
Hydrolysis of Tween 80
Distinguishes photochromogens, scotochromogens, and non-chromogens
Pathogenic strains usually negative
Other strains are commonly positive

Tellurite reduction

Tests ability to reduce tellurite
Tests performed over 3 days
MAC and all rapid growers are positive

Arylsulfatase test

Tests ability to split phenolphthalein
3-day test
Identifies M. fortuitum and M. chelonae

What Mycobacterium is nonchromogenic and nonphotoreactive?

M. tuberculosis

Mycobacterium leprae

Causes Chronic Infectious Disease - Leprosy or Hansen's Disease

Two major forms of Leprosy

Tuberculoid Leprosy
Lepromatous Leprosy

Tuberculoid Leprosy

Usually self-limiting
Single skin lesions
Nerve Involvement
Patches of anesthesia from inflammatory response
Organisms rarely found in tissues
Nerve Damage

Lepromatous Leprosy

Progressive and malignant
Extensive skin involvement
Organisms disseminate to all organ
Hypopigmentation of skin
Nodular skin lesions
Nose deformities
Nerve involvement not severe

Habitat of Mycobacterium leprae (Leprosy)

Humans are major source
Armadillos

Transmission of Mycobacterium leprae (Leprosy)

Unknown
Possibly skin or nasal secretion contact
Requires susceptible host
Children more susceptible
Males infected more than females

Control of Mycobacterium leprae (Leprosy)

Treat infected individuals
Incubation period 3 - 10 years
No longer use isolation

Treatment of Mycobacterium leprae (Leprosy)

Sulfones
May need to be medicated for life

Diagnosis of Mycobacterium leprae (Leprosy)

Tissue and skin scrapings
Can NOT be grown on artificial medium
Diagnosis based on clinical symptoms and positive acid-fast smears of scrapings
Skin scrapings incubated 12 weeks

Organisms found in the M. tuberculosis complex

M. tuberculosis

M. africanum

M. bovis

M. microti

M. cannetti

Primary Tuberculosis

Organisms enter lungs and infect macrophages
Can be directly cleared by host immunity
Granuloma formation
Caseous necrosis

Reactivation Tuberculosis

Organism begins replicating after a period of dormancy

Extrapulmonary TB

Less common than pulmonary TB
More common in HIV co-infected patients

Miliary TB

Seeding of any organ outside of pulmonary tract through hematogenous spread
Any organ can be infected

Skeletal TB

Pott's Disease

Risk Factors for TB

Individuals residing with infected patient
HIV/AIDS Patients
Poverty-stricken, homeless
IV Drug users
Nursing home residents
Age
Malnourishment
Health Care Professionals employed in "high risk" areas
Prisoners

Virulence Factors of TB

Cord factor
Mycolic acid in cell wall
Sulfatides

Transmission of TB

Droplet aerosols
May be acquired through ingestion

Control and Prevention of TB

BCG Vaccine (Bacille de Calmette et Guerin)
Identifying and treating infected individuals
PPD skin testing
X-ray

Acceptable samples used to diagnose TB

Sputum samples (Fresh specimens, Deep cough, 5-10 ml on three consecutive days)
Other pulmonary samples (Induced sputum, Bronchoalveolar lavage)
Extra-pulmonary samples (Blood, CSF, Tissue)

Microscopic morphology of Mycobacterium tuberculosis

Rod shaped
Filamentous
Categorized as a gram-positive organism

Treatment for TB

9 months of isoniazid and rifampin
Once per day for one month and then 2x per week
Regimens may include 2 to 8 week initial course of streptomycin or ethambutol
Pyrazinamide may be added if needed
Other second-line drugs include kanamycin, amikacin, capreomycin and fluorquinolones

Culture Conditions for Mycobacterium tuberculosis

Raised colonies with a dry, rough appearance
Nonpigmented (buff colored)
Optimum growth at 35º - 37ºC
Grows best in 5% - 10% CO2
35 - 37 degrees
Examined at 5 - 7 days and then weekly for 8 weeks

Multi-drug resistant Tuberculosis (MDR-TB)

14.9% of TB cases are resistant to at least one anti-mycobacterial agent
Usually acquired via spontaneous mutations
Risk factors: Previous treatment for TB, Living in endemic area, Close contact with MDR-TB infected patient

Mycobacterium bovis

Causes TB in cattle, dogs, cats, swine, parrots
In humans, closely resembles Mtb infection
Rare in U.S.
Member of Mtb complex
Grows slowly
Nonpigmented

Diagnostic Features of Mycobacterium bovis

Niacin negative
Do not reduce nitrate
Do not grow in presence of T2H

Mycobacterium Other Than Tuberculosis (MOTT)

Slow Growing Speices
Found in soil and water
Opportunistic pathogens
Usual presentation is pulmonary disease resembling TB
Some species associated with cutaneous infections
Not transmissible from person to person

Mycobacterium avium Complex

M. avium and M. intacellulare
Pulmonary disease similar to TB
Cough, fatigue, weight loss, low-grade fever, night sweats
Disseminated disease in immunocompromised patients
The most common systemic bacterial infection in AIDS patients
Difficult to manage

Laboratory Diagnosis of M. avium and M. intacellulare

Slow growth
Usually produce smooth colonies
Nonpigmented but will become yellow with age
Optimal growth at 37ºC
Coccobacilli appearance
Inactive in most physiologic tests
Growth on media containing thiophene-2-carboxylic acid hydrazide T2H
Produce heat-stable catalase

Mycobacterium avium subsp. paratuberculosis

Causative agent of Johne disease in cattle, sheep and goats

Some studies suggest a link to Crohn’s disease in humans

Difficult to cultivate due to VERY slow growth (3 – 4 months)

Mycobactin supplemented medium needed for primary isolation

Mycobacterium kansasii

Second to MAC for NTM lung disease
Most cases in US reported from Texas, Louisiana, Florida, Illinois, Missouri, and California
Natural source of human infection unclear
Upper-lobe chronic pulmonary disease
Rare extrapulmonary infection

Laboratory Diagnosis of Mycobacterium kansasii

Slow growing
Long rods with distinct cross-banding
Optimal growth at 37ºC
Smooth to rough colonies with wavy edges and dark centers
Photochromogenic
Strongly catalase positive
Ability to hydrolyze tween 80 in 3 days
Strong nitrate reduction and PZA production

M. marinum

Slow growing
Fish pathogen (Fresh and salt water)
Cutaneous granulomas (swimming pool granuloma)
Ulcers (from Swimming pools and aquariums)

M. scrofulaceum

Slow grower
Incubation 25 - 37 degrees
Scotochromogenic
Urease and catalase positive
Tween 80 hydrolysis and nitrate reduction negative
Cervical lymphadenitis in children

Cervical lymphadenitis in children

Caused by M. scrofulaceum

One or more enlarged lymph nodes

Mandible and high in neck

Little or no pain

M. ulcerans

Causes Cutaneous ulcers, Bairnsdale (Australia) ulcer, and Buruli (Africa) ulcer

M. ulcerans Habitat, Transmission, and Treatment

Habitat: Soil, plants, water
Transmission: Contact with environmental source
Treatment: Highly resistant to drugs, Surgical removal of lesions

Other Slow Growing MOTTS

M. asiaticum

M. celatum

M. gastri

M. genavense

M. gordonae

M. simiae

M. szulgai

M. terrae complex

M. xenopi

M. haemophilum

M. malmoense

MOTT - RAPID GROWERS

Growth within 2 – 5 days

Environmental organisms

M. chelonae, M. abscessus, M. fortuitum group, M. phlei, M. smegmatis complex

M. fortuitum and M. chelonae

Rapid growers (Growth in 3 - 5 days)
Local abscesses at site of infection
Trauma or surgical wounds
Corneal infections
Endocarditis