MYCOBACTERIOLOGY
Transmittanc
e and
NonPersonal
chromogens
safety
Vaccine and
Treatments
Photochromo
gens
Leprosy
Scotochromo
gens
Staining
method
Rapid
Growers
Isolation
technique
Key
Identification
Specimen
Collection
TRANSMITTANCE AND PERSONAL SAFETY

Transmitted via:



Airborne droplets (e.g. coughing )
In a confined space (e.g. jail, at home, school, work etc)
General safety:


Lab safety:


Adequate ventilation; wear masks; early detection and treatment
Use biosafety level 3 cabinets with directional airflow; aerosol free centrifuges;
protective coverings, UV light; periodic TB testing, etc.
Symptoms:

Fatigue, weight loss, cough, fever, night sweats. X-ray illustrates cavitary disease of solitary
nodules

M. tuberculosis




Rough, buff, about 21 days
Key reactions: Niacin and Nitrate
Confirmation: Nucleic acid probes or HPLC
(high performance liquid chromatography).
Mainly causes pulmonary illness but can be
anywhere.

M. bovis



M. avium-intracelluare (M.A.C)




Rough or smooth, slow
Can be other Runyoun groups
Key reactions: lack of niacin and nitrate,
growth in NAP and TCH. Semi-quant cat and
tellurite are positive
Pulmonary, disseminated
NON-CHROMOGENS

Generally smooth, buff colonies with
irregular margins seen after 21 days of
incubation at 37ºC.
Key reactions: Urease positive, sensitive
to TCH
Causes pulmonary illness mainly in
livestock but can be transmitted from
cattle to humans.

M. xenopi







Recovered from water, hot and cold taps
Can be scotochromogenic
Small, slow growing colonies with dense centers
and filamentous edges
Optimum growth at 42 degrees Celsius
Key reactions: Niacin negative, nitrate negative,
Catalase positive, arylsulfatase positive, PZA
positive
Slow progressive pulmonary infection
M. haemophilum




Rough to smooth buff colonies
Optimum growth at 28-32oC
Requires heme/hemoglobin for growth
Cause lymphadenitis, abscesses, draining
fistulas (but mostly in immunocompromised
patients)
NON-CHROMOGENS

M. ulcerans




Slow growth, buff smooth and rough
colonies
Optimum growth 30-33oC , 6-12 weeks
incubation
Key reactions: Biochemically inert
Bairnsdale ulcer, cutaneous lesions in
tropical regions following soil disturbances.

M. kansasii

Yellow carotene pigment when exposed to light

Rough colonies with wavy edges and dark centers ,
slow growth at 37 degrees C

M. marinuum





Key reaction: Niacin negative, strongly positive for
nitrate and catalase, PZA and Tween
80 positive


Chronic pulmonary disease

M. simiae

Smooth, slow growing colonies, 10-21 days at
37 degrees C

Key reactions : Niacin positive (can easily be
mistake n for M. tuberculosis), Nitrate negative ,
catalase positive

Pulmonary disease
PHOTOCHROMOGENS

Optimum growth at 28oC-32oC
Buff colonies seen in the dark, yellow when
exposed to light
Slow growing, smooth to rough colonies.
Key reactions: Niacin and nitrate negative,
catalase negative, PZA
positive
Cutaneous infections after trauma in water
M. asiaticum

Dysgenic and smooth
Growth after 15 to 21 days at 37oC
Key reactions: Niacin negative, Nitrate
negative, Catalase positive,
Tween 80 positive

Rarely causes human infection



M. gordonae






Smooth yellow-orange colonies , 10-14 days
Optimum temperature: 22-37oC
Key reactions: Nitrate negative, catalase
positive, Tween 80 positive, urea positive
Rarely causes infection
Contaminant from tap water (referred to as “tapwater bacillus”)
M. szulgai





Pigment production is temperature dependent
Photochromogen at 22oC
Smooth and rough colonies
Key reactions: slow hydrolysis of Tween 80,
nitrate positive, inability to grow in the presence
of 5% NaCl
Pulmonary disease
SCOTOCHROMOGENS

M. scrofulaceum




4-6 weeks growth at 25-37 degrees
Celsius
Smooth light yellow to deep orange
colonies with dense centers
Key reactions: Nitrate negative, catalase
positive, Tween 80 negative, urea positive
Associated with cervical lymphadenitis in
children

M. fortuitum





Growth in 3-5 days of incubation at 37oC
Buff rough or smooth colonies
Key reactions: Positive 3-day arylsulfatase test,
nitrate positive, NaCl positive, iron positive
Skin and soft tissue , “whirlpool footbath”
infections





M. chelonae





Related to M. abscessus
Most commonly isolated rapid grower
Rough or smooth buff colonies
Growth within 3-5 days of incubation at 37oC
Key reactions: Positive 3-day arylsulfatase,
nitrate negative, iron negative, NaCl negative
M. smegmatis complex



Disseminated nodular disease in
immunocompromised patients


RAPID GROWERS
3-5 days growth; optimum temperature 30oC
Coarsely wrinkled with deep yellow to orange pigment
Key reactions: negative arylsulfatase, iron positive, NaCl
negative
Rare cause of pulmonary and soft tissue infections
M. thermoresistable


Pulmonary, skin, soft tissue and bone infections
M. phlei


Buff, rough, wrinkled or coarsely folded growth after 2-4
days
Key reactions: negative arylsulfatase, iron positive,
nitrate positive, positive growth in NaCl
Rare
Grows at 52oC
M. abscessus


Subspecies of M. chelonae
Chronic lung disease, otitis media following
tympanostomy, disseminated cutaneous infections
Pigment
Scotochromogen
Photochromogen
Non-chromogen
Strong
Nitrate
Positive
Nitrate
negative
M. kansaii
Cat (+)
M. marinum
Niacin
negative
Niacin
positive
M.
Tuberculosis
TCH (+)/
PZA (+)
Tween 80
negative
M. gordonae
M.
scrofulaceum
Urease (-)
Urease (+)
Nitrate (+) ,
TCH (+) cat (-)
NAP (-)
M. Avium
complex
Tellurite and
cat (+)

TCH (-)/
PZA (-)
Tween 80
posistive
M. bovis
Rapid Growers : M. chelonae - Arylsulfatase (+); 5% NaCl, iron uptake and Nitrate (-)
M. fortuitum - Arylsulfatase (+) ; 5% NaCl, iron uptake and nitrate (+)
SPECIMEN COLLECTION

Sterile wide mouth cup with tightly fitted lid

Respiratory:



First morning deep cough sputum, bronchial wash or brushing, BAL
or bronchial lavage
Cutanaeous lesions
Body Fluids

Pleural, ascitic pericardial, gastric, CSF, urine, joint aspirate, feces,
etc.
SPECIMEN COLLECTION

Culture media
 Egg

based media (Lowenstein Jensen)-
classical TB medium. Contains malachite green which suppresses the growth of gram
positive bacteria
 Agar

based media (Middlebrook 7H10) –
contains casein hydrolysate (helps isolate isoniazid resistant stains), biotin and catalase
(helps revive damaged bacilli); and albumin (binds toxins)
ISOLATION TECHNIQUE: DIGESTION, DECONTAMINATION AND CONCENTRATION

1. Digestion:


2.Decontamination


N-acetyl-L-cysteine (NALC) disrupts disulfide bonds of mucus,
releasing organism
4% sodium hydroxide (NaOH) equally mixed with NALC destroys
bacterial species (i.e. normal flora) other than mycobacterium
Concentration
Neutralization: Phosphate buffer of low specific gravity is added to
mixture, and centrifuged at a high rate to concentrate the organism
 Albumin dilutes the mixture and heal the damaged cells

STAINING METHOD

Gram stains poorly due to high lipid content
(mycolic acid) in cell wall

Fluorescence or Carbolfuschin technique (i.e.
Ziel Neelson or Kinyoun stain)
DETECTION METHODS

Mantoux screening (tuberculin skin test)


Read 48 hours later
Positive skin test due to:
 1) exposure to the antigen



2) Recovery from past infection(delayed type (IV)
hypersensitivity)
Reading the test:

Healthy individuals – Induration (raised hardened area) of
15mm or greater is positive result

Immunocompromised individuals–Induration greater than or
equal to 5 mm is a positive result
Quantiferon-TB gold
Use whole blood
Latent or active TB will stimulate interferon-gamma release from leukocytes
Measured with ELISA
LEPROSY (HANSEN’S BACILLUS)

Spread through direct contact or by inhalation via aerosols

Slow growth (very difficult to culture in routine lab)

Acid fast positive

Two forms:

Tuberculoid form – non-contagious hypo-pigmented skin macules with
underlying nerve involvement causing loss of feeling

Lepromatous form - highly contagious. Cell mediated immunity absent
causing an increase development of the organism
VACCINES AND TREATMENT

B.C.G NOT used as a vaccine for TB due to:




Interference with skin test
U.S. is a low risk population
Varies in its ability to trigger immunity
Dapsone + Rifampin + Clofazimine
multidrug therapy which
effectively treats both TB and leprosy
(must be used in combination to prevent development of resistant organisms)