CLBACT1 LEC FINALS QUIZ 2 – MYCOBACTERIA

Week 10: Mycobacteria

Mycobacteria

General Characteristics

 Mycobacteria are slender, slightly curved or straight, rod-shaped organisms 0.2 to 0.6 μm × 1 to 10
μm in size; this bacteria has the tendency to clump

 They are nonmotile and do not form spores.

 The cell wall has extremely high lipid content; thus, mycobacterial cells resist staining with
commonly used basic aniline dyes, such as those used in the Gram stain, at room temperature

 Mycobacteria take up dye with increased staining time or application of heat but resist
decolorization with acid-ethanol

o This characteristic is referred to as acid fastness— hence, the term AFB (acid fast bacilli)—
and is a basic characteristic in distinguishing mycobacteria from most other genera

 Mycobacteria are also strictly aerobic, but increased carbon dioxide (CO2) will enhance the growth
of some species; the pathogenic mycobacteria grow more solely than most other bacteria pathogenic
for humans
 Produces Much’s granules

 Rapid growing species: 2-3 days at 20-4- C at simple media;

Species associated with diseases: 2-3 weeks of incubation at complex media
 Ph requirement: 6.5 - 6.8
 Generation time: > 12 hours
 Culture: Egg-based agar; colonies are smooth, soft or rough and friable growth
2 GROUPS

1. MTC (Mycobacterium tuberculosis complex)

2. NTM (Non-tuberculous mycobacteria)

Pathogenic Species

Mycobacterium tuberculosis (Koch bacillus)

 It has the longest replication time among the mycobacteria species; replication time is between 20-
22 hours

 TB is one of the oldest documented communicable diseases

 Virulence factor: Cord factor due to high lipid content in the cell wall they appear as rods that stick
together and develop long ropes
 Culture:
a. Rough colonies exhibit “cording”
b. Slow growing colonies, exhibit buff color; raised and dry, and have a “cauliflower-like
appearance”

Biochemical Tests for Mycobacterium tuberculosis:

 ( + ) Niacin Test
 Reduction of nitrate to nitrite
 ( + ) Catalase Test – destroyed after heating
 ( - ) T2H – thiophene-2-carboxylic acid hydrazide
 From this test species can be distinguished from Mycobacterium bovis by the inhibition of M. bovis
by the T2H and by the pyradinamidase activity
PATHOGENIC LIFE CYCLE

-way of transmission of M. tuberculosis

Mycobacterium tuberculosis infection initiates when fine aerosol particles containing the bacteria
coughed up by an individual with active disease are deposited in the lungs of a new host. The bacteria recruit
macrophages to the surface of the lungs which becomes infected, and they serve to transport the bacteria
across the lung epithelium to deeper tissues. A new round of macrophage recruitment to the original
macrophage is initiated forming the granuloma (an organized aggregate of differentiated macrophages and
other immune cells). The granuloma in its earliest stages expands infection by allowing the spread of bacteria
to the newly arriving macrophages. As adaptive immunity develops, the granuloma can restrict bacterial
growth, however under many circumstances, the infected granuloma, macrophages can undergo necrosis
forming a necrotic core that supports bacterial growth and transmission to the next host.

*inserted video about the Mantoux Test

Primary Tuberculosis

 TB is usually a disease of the respiratory tract.

 After exposure to M. tuberculosis, whether a person develops TB is determined by his or her

cellular immune response, amount of exposure and virulence of the strain.

 In many exposed individuals, the immune system does not eliminate the bacteria. The pathologic
features of TB are the result of a hypersensitivity reaction to mycobacterial antigen .

Reactivation Tuberculosis

 The risk of reactivation TB is about 3.3% during the first year after a positive PPD skin test and a
total of 5% to 15% thereafter in the person‘s lifetime.

 Progression from infection to active disease varies with age and the intensity and duration of
exposure
Extrapulmonary Tuberculosis

 Cases of extrapulmonary disease are a common presentation in individuals with human

immunodeficiency virus infection, although it is most often in association with pulmonary disease.

Identification of Mycobacterium tuberculosis

 Colonies are typically raised with a dry, rough appearance. The colonies are nonpigmented and
classically described as being buff-colored.

 Biochemically, It is characteristically positive for niacin accumulation, reduction of nitrate to nitrite,

and production of catalase

Mycobacterium bovis

 Mycobacterium bovis produces TB primarily in cattle but also in other ruminants, as well as in dogs, cats,
swine, parrots, and humans.

 Closely resembles that one that is caused by Mycobacterium tuberculosis and is also treated
similarly.
 Its attenuated strain (strain that is weakened) is used for the vaccination (BACILLE-CALMETTE-
GUERIN or BCG) of newborns against TB (tuberculosis)
 It is acquired through the ingestion of contaminated milk or carcasses of infected animals
 Culture: slow growing, small, granular, rounded, and non-pigmented

 Biochemical tests:
Niacin Test ( - ); do not reduce nitrate; and do not grow in the presence of T2H

Transmission of Mycobacterium bovis

It is transmitted by inhalation of aerosol or by the direct inoculation. Main source is the cattle or the cow.
Transmission by ingestion of contaminated milk from infected cows did occur prior to routine pasteurization.
Aerosols are also transmitted to different animals.

Clinical syndromes associated with M. bovis: soft tissue infection

Mycobacterium canettii

 Smooth strain of M. tuberculosis

 First human isolate is from a cervical lymph node
 Grows rapidly than M. tuberculosis; it only takes 6 days on solid media for colonies to grow
 It is also isolated from an AIDS patient with mesenteric tuberculosis
 Biochemical ( + ), reduces nitrate to nitrite

Clinical infections

Tuberculosis:

 disease of the respiratory tract; bacteria is deposited in the lung alveoli or in the cells of the alveoli
 Chronic granulomatous infection (phagocytes bursts that leads to infection)
 Mode of transmission: Inhalation of infectious droplets by cough, sneezing and talking
 Signs and symptoms: low grade fever, night sweats, anorexia, and weight loss

-in chronic stage of tuberculosis there is a loss of lung volume and calcification

Granuloma:

 Pott’s disease aka Tuberculosis spondylitis this is a rare infection of the spine; typically caused by an
extra spinal infection; this disease is a combination osteomyelitis and arthritis which involve multiple
vertebrae

Military Tuberculosis

 Also known as the seeding of organism in different organs

 This occurs when a large number of bacteria travel through the blood stream and spread throughout
the body
Nontuberculous Mycobacteria – slow growers species

- Also known as anonymous, atypical, unclassified and MOTT (mycobacteria other than tuberculosis)
- Present everywhere in the environment
- Causes chronic pulmonary disease
- Not transmissible from person to person

Slow Growing Species

 Mycobacterium avium Complex: Mycobacterium avium and Mycobacterium intracellulare

o These organisms are common environmental saprophytes and have been recovered from
soil, water, house dust, and other environmental sources.

o Disease in poultry and swine

o Most common cause of pulmonary infections in humans similar to M. tuberculosis
o It is self-limiting
o Pathogen in immunocompetent and immunocompromised persons
o Reservoir: natural water also (seen in soil, water and house dust)
o Portal entry: Respiratory tract and GIT (gastro intestinal tract)
o Microscopy: pleomorphic, short, coccobacilli without beading; positive with PAS
(periodic acid shift) staining

 Mycobacterium avium subsp. Paratuberculosis

o Agent of Johne’s disease

o It is the causative agent of Johne disease, an intestinal infection occurring as a chronic

diarrhea in cattle, sheep, goats, and other ruminants

 Mycobacterium kansasii

o Not normally contagious from person to person

o Microscopy: long rods with distinct crossbanding

o Resemble Shepherd’s crook

o Culture:
a. MB (Middle Brook) 7H10 – colonies are smooth tp rough with dark centers and
waxy edges
b. Photochromogenic colonies exhibit a dark red crystal color

o It second to MAC as the cause of Nontuberculous Mycobacteria

o The most common manifestation is chronic pulmonary disease involving the upper lobes,
usually with evidence of cavitation and scarring.
 Mycobacterium genavense

o It is the cause of disseminated infections in patients with AIDS

 Mycobacterium haemophilum

o It occurs primarily in patients who are immunocompromised.

o Submandibular lymphadenitis, subcutaneous nodules, painful swellings, ulcers progressing

to abscesses, and draining fistulas are often the clinical manifestations

 Mycobacterium malmoense

o M. malmoense appears as a short coccobacillus without cross bands on acidfast–stained

smears

 Mycobacterium marinum

o Caused disease in fish and is isolated from aquariums

o Cause swimming pool granuloma in humans; this swimming pool granuloma is a
tender red subcutaneous modules in traumatized skin that made contact with
inadequately chlorinated fresh water or salt water
o Culture: rough and wrinkled yellow colonies (photochromogen)

o Prefers low temperature; they grow between 28-32 C

o Cutaneous infections in humans occur when traumatized skin comes into contact with salt
water or inadequately chlorinated fresh water containing the organism

 Mycobacterium scrofulaceum

o Causes cervical lymphadenitis in children.

 Mycobacterium simiae

o Original strains were isolated from the lymph nodes of monkeys

 Mycobacterium szulgal

o The most common manifestation is pulmonary disease similar to TB

 Mycobacterium ulcerans

o Third most common mycobacterium species after M. tuberculosis and M. leprae

o Causative agent of Buruli ulcer
o Culture: nonpigmented smooth to rough colonies
o 6-12 weeks incubation; grow best between 30-33 C

o It is a rare cause of mycobacteriosis, also referred to as Buruli ulcer

 Mycobacterium gordonae ( tap water bacillus)

o A contaminant in the preparation of bacteriologic smears
o This is present in tap water
o Culture: colonies are smooth, and yellowish-orange colored (scotochromogen)

 Mycobacterium xenopi

o Isolated from an African tode

o Recovered from hot and cold water taps – specially those water storage tanks
o Cause pulmonary infections in adults
o Colonies: cultures can either be non-photochromogenic or scotochromogenic; small
colonies with dense center and filamentous adge, young colonies: stickline
projections “Bird’s nest appearance”

o It has been recovered from hot and cold water taps and birds
  Mycobacterium terrae complex - normal saprophytes; rarely cause infections to humans
o M. triviale – rough and dry colonies
o M. terrae – smooth colonies
o M. nonchromogenicum – smooth to rough and white to buff colonies
o Microscopy: short to medium coccobacilli

Non-tuberculous rapid bacteria – RAPID growers

o Potential pathogens to humans; they are pathogenic to humans

o They are saprophytic meaning they are seen in soil, water supplies and marine and terrestrial life
forms
o They can grow in routine bacteriologic media and mycobacterial media
o Microscopy: weakly gram positive bacilli that resemble diphtheroids
o Colonies appear on solid media in 7 days or less
o common isolates: M. fortuitum, M. chelonae and M. abscessus

Laboratory Diagnosis:

Specimen:

 Sputum
o Early morning (for 3 consecutive days)
o 5-10 microliters expectorated sample
o Microscopy prior to culture: <10 EC and >25 pus cells
o This procedure is done in a class 2 or class 3 biological safety cabinet
o This are processed should have a negative pair pressure
o Pooled sputum are not acceptable because it might cause contaminations
o If only one sputum is tested positive for the 3 consecutive days collected, yield at
least <10 EC and >25 pus cells in each specimen
o If only one sputum is tested positive another specimen should be submitted for
culture
 Gastric Lavage
o Preferred over the bronchul alveolar lavage for the detection of Mycobacteria in
children
o Use to recover bacteria that has been swallowed
o 3 specimen after 3 days
o 20-25 mL gastric secretion
o Before collection there should be an overnight fasting
 Urine
o First morning and midstream catch ( 3 consecutive days)
o 15 ml of urine
 Bronchoscopy specimen
o BAL and bronchial washing
o Specimen of choice for NTM
Culture media – solid

o Incubation: 35 C in the drak with 5-10% CO2

o 30-32 C: M. marinum and M. ulcerans
o 42 C: M. xenopi
o Tube media: incubated slanted with loose cap ( for evaporation of excess water and entry of C02)
o growth must be examined weekly
o Malachite green: inhibitory agent

1. Egg based media

-fresh whole eggs, potato flour, glycerol, milk, potato, and malachite green
-egg yolk is the source of lipid in the media; promotes the growth of mycobacteria
-short life of egg based media: 1 year
a. Lowenstein Jensen Agar
- protein rich medium
-most commonly used for mycobacteria
b. Petragani medium
-inhibitory agent: 0.052 g/dL malachite green
-used for the recovery of Mycobacteria from heavily contaminated specimen
c. Wallenstein medium
-use for detection of mycobacterium avium complex

2. Serum Agar-based medium (transparent medium)

-vitamins, salt, cofactors, glycerol, malachite green, agar, oleic acid, bovine albumin, glucose and
beef catalase
a. Middlebrook 7H10-11
b. Mitchison’s medium 7H11

Culture media – Liquid / broth

BACTEC 12B and BACTEC 13BA

-improves isolation rate of mycobacteria and reduce recovery time

-reading is about 4 days after inoculation

-growth enhancer: Polyoxythylene stearate

-grow indicator: release of C02

-can also be used for antimicrobial susceptibility testing for Mycobacterium tuberculosis

Staining

General rule

o 2 of the first 3 sample must be positive to confirm diagnosis

o 5,000-10,000 organisms/mL – needed for positive result
o AFB staining method
-Ziehl-Neelsen
-Kinyoun method
Reporting results of Acid-fast bacilli smears

Fuchsin Stain – 1000 magnification

Flurochrome stain – 450 magnification

 Mycobacterium chelonae–Mycobacterium abscessus Group

o The three most important rapidly growing mycobacteria causing human infections are M.
abscessus subsp. Abscessus, M. chelonae, and M. fortuitum.

o M. chelonae is the species of rapidly growing mycobacteria most likely isolated from
disseminated cutaneous infections in immunocompromised patients.

o Both M. fortuitum and M. chelonae have been associated with a variety of infections of the
skin, lungs, bone, central nervous system, and prosthetic heart valves.

 Mycobacterium fortuitum Group

o The M. fortuitum group contained the species M. fortuitum, M. peregrinum.

o The M. fortuitum group is the most common rapidly growing mycobacteria associated with
localized cutaneous infections.

 Mycobacterium smegmatis Group

o The M. smegmatis group contains two species, M. smegmatis and M. goodie

o It has been implicated in rare cases of pulmonary, skin, soft tissue, and bone infections.
NON-CULTIVATABLE NTM

Mycobacterium leprae (Hansen’s bacillus)

-cannot be cultivated in vitro

-invades peripheral nerve and skin cells

-mostly found on SCHWANN Cells

-microscopy: cigar pocket or pocket fence appearance

-culture: only grows on biological living tissue media, they survive only in vivo , so we need an animal, foot
pads of mouse is used

-optimal growth: 30 C

-skin test: fernandez and mitzuda reaction

Clinical Infection

Hansen’s disease /Leprosy

-lepros means scaly, scabby or rough

-transmission:

-person to person through inhalation of nasal secretion

-contact with infected skin

-breast feeding

-vertical transfer

2 forms of leprosy

1. Tuberculoid / localized leprosy

-benign form, nonprogressive disease
-asscociated with delayed type hypersensitivity
-s/s: skin lesion, damaged nerves
-positive for Lepromin test
2. Lepromatous / Disseminated leprosy
-malignant form and slowly progressive
-s/s: loss or deformities of facial features and fingers, toes and anatomical structure
-negative for Lepromin test

*inserted video for performing Lepromin test

Laboratory Test for M. leprae

Specimen: nasal/mucosal smear and skin nips

Serologic testing: ELISA

Definitive test: inoculation of specimen to the mouse footpods

Specimen: biopsy material

Positive result: Hansen’s disease

 Mycobacterium leprae is the causative agent of Hansen disease, an infection of the skin, mucous
membranes, and peripheral nerves

 Isolation and Identification of the Mycobacteria

o Direct Detection Methods

 Microscopy is considered a reasonably sensitive and rapid procedure for the

presumptive identification of Mycobacterium spp. in clinical specimens.
 Acid-Fast Stains- Visualization of acid-fast bacilli in sputum or other clinical
material should be considered only presumptive evidence of tuberculosis
 Culture