Module 9: Mycobacteria -once called “consumption”

General Characteristics -Has a Gram-positive cell wall, but they are not
stained during Gram’s staining , thus called
 Slender, slightly curved or straight rod-
Gram-ghost
shaped organisms
 Non-motile -cannot penetrate the cell wall due to high lipid
 Non spore-forming composition
 Non-encapsulated
-acid-fast because of mycolic acid
 Strictly aerobic; catalase (+)
 They produce Much Granules (inclusion -has tapered ends and may exhibit “cording”
bodies)
-( intertwining of bacilli in serpentine
 Various species found in the soil and
cords especially in Tween-80 medium).
Water
-causes tuberculosis and Pott’s disease
Cell Wall
-Pott’s disease is TB of Bone and Muscles , cause
 extremely high lipid content
deformity of the bone affecting the spine
-mycolic acid (acid responsible for its
 Cell wall contains a waxy lipid called
acid fast staining characteristics)
mycolic acid.
-assists in resisting harsh environments  The unusual cell wall results in a
number of unique characteristics
-assisting in penetrating host immune system
-Slow Growth (8 weeks /2
 Consequences of high lipid content months)
- Staining requires longer time or -Protection from lysis once the
application of heat bacteria are phagocytized
- Once stained, resist decolorization -capacity for intracellular
with acid-alcohol (acid-fast) growth
- Long generation time -resistance to Gram-staining,
detergents, many antimicrobial
THREE MAJOR GROUPS drugs, and desiccation
- Mycobacterium tuberculosis complex

- Non-tuberculous mycobacteria (NTM) TUBERCULOSIS

-Mycobacterium leprae  Respiratory disease cause by
Mycobacterium tuberculosis
 Virulent strains of M. tuberculosis
THE MYCOBACTERIUM TUBERCULOSIS contain the cell wall component cord
COMPLEX factor, that is necessary to cause
M. Photochromo Scotochromo Nonphotochrom Rapid disease.
tubercul gens gens ogens Growers
osis  3 types of tuberculosis:
complex
M.
tubercul
M. kanasii M.scrofulace
um
M.
aviumcomplex
M.
fortuitu -Primary TB- Results from the initial
osis
M. bovis M. marinum M. szulgai M. xenopi
m
M.
infection with M. tuberculosis
chelonae
M.
africanu
M. simiae M. gordonae M. mamoense M.absces
sus
-Secondary TB – Reestablishment of an
m
M. M.
active infection after a period of
microti
M.cane
paratuberculosis dormancy
tti
-Disseminated TB- Results when the
infection spreads throughout the body
Mycobacterium tuberculosis

-Primarily a pathogen of the respiratory tract

(“TB”)

-One of the oldest communicable diseases

 PRIMARY TUBERCULOSIS -primary agent of intestinal TB in man through
ingestion of contaminated milk
-spread by coughing, sneezing, or
talking -source of BCG vaccine (Bacille Calmette-Guerin)

- inhaled into alveoli, where the

organisms are phagocytized
MYCOBACTERIA OTHER THAN TUBERCLE
-if the organism does not cause BACILLUS (MOTT)
immediate infection, the organism can
be “walled of” in a granuloma
ATYPICAL MYCOBACTERIA
-granulomas can break down in future
and the organism can cause infection -also known as MOTT (Mycobacteria other than
later (secondary TB) Tuberculosis) or NTM (Non-Tubercle Bacillus)

-most found in soil and water

METHODS -Chronic pulmonary disease resembling TB, skin
infections, chronic lymphadenitis
 PPS test (Purified Protein Derivative
Test) / unreliable / skin test -opportunistic pathogen in patients with liver
- Detects patient’s cell-mediated disease, immunocompromised, percutaneous
immune response to bacterial trauma
antigens (macrophage can react
other than MTB)
- Positive result Bump in skin RUNYOUN’S CLASSIFICATION OF MOTT

 Interferon-Gamma Release Assays  Classification of Mycobacterium

-Blood Test -Photoreactivity (ability to produce pigment)

-Measure person’s immune reactivity to  Group 1 (Photochromogens)- produce

specific mycobacterial antigens (test antibodies) carotene pigment upon exposure to
light
Advantages  Group 2 (Scotochromogens) – produce
-Single patient visit carotene pigment in light or dark
 Group 3 (Nonphotochromogens)- no
-No booster phenomenon pigment ; these colonies are buff color
 Group 4 (Rapid Growers)-
-Less reader bias in interpretation
nonpathogenic group which grow in less
Disadvantage/ Limitation than 7 days
-Sample must be processed within 8-16 hours
Photochromogens Scotochromogens Non-pathochromogens Rapid growers

-Limited data on certain populations

-M. kansasii -M. gordonae -M. avium Complex -Mycobacterium
-M. marinum -M. scrofulaceum (MAC) fortiutumchelonei
-M. simiae -M. flavescens -M. gastri complex
-M. asiaticum -M. terrae complex -M. smegmatis
-M. xenopi -M. phlei
Mycobacterium bovis (Zoonotic) / Part of MTB -M. ulcerans
complex

-Primarily in cattle, dog, cats, swine , parrots

and humans

-small, granular , rounded white colonies with

irregular margins

-slow grower, non-pigmented

-Similar to M. tuberculosis which causes

tuberculosis in cattles
Group 1 -Photochromogen - grows on approximately 7 days at 37
degC as yellow to orange colonies
1. Mycobacterium kanasasii (Yellow
- (+) tween 80 and heat stable catalase
Bacillus)
4. Mycobacterium xenopi
-grow best at 37 degree Celsius and growth is
evident in 14-28 days, grow slowly at 24 degree - slow grower and produce small colonies
Celsius but not at 42 degree Celsius - optimal growth at 42degC; (+) at 37 but
not as 25 degC.
-Reactions to identify M. kansassi :
- on Cornmeal agar produces branching
-Tween 80 hydrolysis (+) in 3 days colonies “Bird’s Nest”
- (+) catalase, (+) in 2 weeks in Arylsulfatase
-Nitrate reduction (+) Test
-Rapid Catalase Activity and - causes pulmonary infection in patients
Pyrazinamidase Negative with pre-existing lung pathogens.

-causes chronic pulmonary disease 5. Mycobacterium thermoresistible

2. Mycobacterium marinum 6. Mycobacterium flavescens

-grows optimally at 30-32 degree Celsius; - a normal flora

growth at 24 degree Celsius and 32 degree - produces smooth yellow colonies and
Celsius occurs in 2 weeks grows moderately at 37 deg C

-grows poorly or fails to grow at 35-37 degree

Celsius

- (positive) to Niacine test , tween 80 , urease

and Pyrazinamidase
GROUP III- NON-PHOTOCHROMOGENS
-causes “swimming pool granuloma”
1. Mycobacterium malmoense
3. Mycobacterium simiae - gray to white smooth raise colonies
4.Mycobacterium asiaticum in 2-3 weeks at 37 Deg C
- growth is slow at 22-24 degC & may
require 1-2 weeks to gw=row
GROUP II-SCOTOCHROMOGENS - a rare agent of pulmonary disease
- (+) 80 & heat stable catalase
1. Mycobacterium scrofulaceum 2. Mycobacterium haemophilum
causes cervical lymphadenitis - grows at 28-32 degC in 2-4 weeks
produces smooth, buttery, yellow to but requires 4-8 weeks to grow at
orange colonies in 4-6 weeks 25 degc or 25 degc
pigment production evident in both the - unable to grow at 37 DegC
absence and presence of light - requires HEMIN for growth
(+) heat stable catalase - biochemically inert
- agent of subcutaneous lesions,
2. Mycobacterium szulgai
ulcers, abscesses in
- classified both as photochromogen (25 immunosuppresses patient
deg C) or scotochromogen (35-37 degC) 3. Mycobacterium gastri
- (+) nitrate reduction; slow (+) for tween 4. Mycobacterium avium-intracellular
80 fails to grow on 5% NaCl complex (MAI)
- may cause pulmonary & cutaneous - M. avium aka battery bacillus
disease - slow growers that produce buff-
colored colonies
3. Mycobacterium gordonae (Tap water
- biochemically inert
Bacillus)
- (+) heat stable catalase &
- usually, non-pathogenic pyrazinamide test
- -previously known as M. aquae - both are stained positive for PAS
 5. Mycobacterium terrae-triviale complex - transmission: direct contact or through
- rare agents of arthritis and breaks in the skin
osteomyelitis 2 different forms of disease
- grows slowly at 25 &37 degC
*Tuberculoid leprosy: nonprogressive
- M. terrae aka Raddish bacillus
disease that is characterized by loss of
sensation in regions of the skin
GROUP IV-RAPID GROWERS
1. Mycobacterium fortuitum *Lepromatous leprosy: produces gradual
- found in water, soil & dust tissue destruction that results in the loos of
- grows as smooth, waxy, heaped facial features, digits, and other body
colonies in 2-4 days structures
- hyphae are present in young colonies in
1-2 days LAB SAFETY
- stains poorly with fluorochrome stain Safety considerations
2. Mycobacterium chelonei
- colonies re round, smooth, colorless, & do - mycobacteriology workers are three times
not produce hyphae more likely to seroconvert (develop positive
skin test)
 both are Arylsulfatase & can grow on Mc - adequate safety equipment
Conkey - safe laboratory produces training
 *M. fortuitum (+) Nitrate reduction & - information on hazards
iron uptake, while M. chelonei is - preparations for unexpected accidents
negative on these tests - staff must be monitored regularly by
medical personnel
3. Mycobacterium smegmatis - -PPD/Mantoux test
- now known as mycolicibacterium PROPER VENTILATION
smegmatis
- saprophytic mycobacterium - separate from other parts of lab
- has fast doubling time an requires only BSL- - non-recirculating ventilation systems
1 - negative air pressure
- causes skin disease in some cases and - air flows from clean areas to less clean
isolated in SMEGMA areas
- 6-12 room air changes/hour
- biological safety cabinet
MYCOBACTERIUM LEPRAE - use of proper disinfectant
- causes leprosy or Hansen’s disease bactericidal for mycobacteria
- infection of the skin, mucous membranes also called tuberculocidal
and peripheral nerves - other precautions
- most cases are from warm climates disposables
- bacteria infect the coolers areas of the body protective clothing, face masks
(ears, nose, eyebrows, finger, toes) SPECIMEN COLLECTION
- exhibit cigar pack, picket fence or palisade
arrangements - variety of clinical specimens, including
- cannot grow in artificial media, thus they respiratory, urine, feces, blood, CSF, tissue
are grown in armadillos and footpad and and aspirations
earlobe of mice. - should be collected before antibiotic
therapy and process ASAP
LEPROSY - swabs are discouraged due to decreases
- caused by Mycobacterium leprae recovery
- bacteria have never been in cell-free
culture
- cases of leprosy are becoming relatively
rare
 2. CONCENTRATION
- after decontamination and
digestion, the specimen is
centrifuged in a closed, vented
centrifuge for 15 minutes @
3000g to concentrate the
organisms
3. ACID FAST STAINS
- after centrifugation, the button at
the bottom of the tube is used to
make a smear and to inoculate media
Ziehl-Neelsen
SPECIMEN PROCESSING
Kinyoun
1. Digestion & decontamination of specimens Auramine or auramine-rhodamine
- because Mycobacterium grow so slowly fluorochrome stain
and are often collected from non-sterile
CULTURE AND ISOLATION
body sites, they are easily overgrown by
other bacteria - strictly aerobic
- specimens from non-sterile sites, - 5-10% CO2
therefore, must be “decontaminated” - 35-37oC
- sputum or other viscous specimens also - slow growers; cultures held for 6 weeks
must be “digested” before calling negative
- specimens from sterile sites (CSF, etc.) do
3 types of media:
not need contamination
EGG-BASED with malachite green=
Lowenstein-Jensen (LJ)
PURPOSES:
AGAR-BASED= Middlebrook 7H10 and
- to liquefy the sample to clear 7H11 agar
proteinaceous material LIQUID MEDIA= Middlebrook 7H9 broth
- agents kill non-mycobacterial organism

IDENTIFICATION OF MYCOBACTERIA
DECONTAMINATION
 traditional characteristics used to identify
- specimen from non-sterile site is mixed mycobacterium
with an agent that will kill non-  rate of growth
mycobacterium bacteria  colony morphology
- common decontamination agents  pigment production
- NaOh is most common  nutritional requirements
- Benzalkonium chloride (Zephiran)  optimum incubation temperature
- oxalic acid (used with Ps. aeruginosa)  biochemical test results
- after decontamination, the agent must
be neutralized so that it will not
FIRST STEP IS TO CONFIRM ORGANISM AS ACID
eventually kill the mycobacterium
FAST

 growth rate
DIGESTION  rapid growers- colonies in <7 days
- liquefying mucus enable the  slow growers- colonies in >7 days
mycobacterium to contact and use  temperature- range can vary from 20C-
the nutrients in the agar medium 42C
- common digestion agents  photoreactivity
- N-acetyl-cysteine- most common  biochemical identification
- Trisodium phosphate (Z-TSP)- used  niacin accumulation
with Zephiran  nitrate reduction
  catalase
 iron uptake
 arylsulfatase
 pyrainamidase
 telluride reduction
 urease
 hydrolysis of tween 80

IDENTIFICATION of Mycobacterium
tuberculosis

 slow grower
 colonies are thin. flat spreading and friable
with a rough appearance
 may exhibit characteristic “cording”
formation
 grows best at 35 to 37C colonies are not
photoreactive

ANTIBIOTIC SENSITIVITY TESTING FOR

MYCOBACTERIUM

- mycobacterium is fairly resistant and

only a few organisms left can cause
reinfection
- development of drug-resistance

common antibiotics (usually two or more are

given)

o isoniazid
o rifampin
o ethambutol
o streptomycin
o pyrazinamide

“STRIPE”/ PRIEST”