1. Mycobacteriology
Update 2018
Margie Morgan, PhD

2. Mycobacteria
 Acid Fast Bacilli (AFB)
 Cell walls contain complex mycolic acids and free
lipids, many properties of the mycobacteria due to
their presence
 Once stained, the rods resist de-colorization with acid
alcohol (HCl)
 AFB stain vs. Partial Acid Fast (PAF) stain
 AFB – HCl used to decolorize – identify Mycobacteria
 PAF – H2SO4 used to decolorize – identify Nocardia
 Gram stain – Gram positive, weekly stained and
appear beaded
 Aerobic bacilli, no spores,
rarely branch

3. Identification of the Mycobacteria
 For decades, identification algorithm started with the
determination of pigment in the light and dark; followed
by biochemical reactions, growth rate, and optimum
temperature. Obsolete
 With expanding taxonomy, biochemical reactions were
unable to separate and identify most of the newly
recognized species, replaced with High Performance
Liquid Chromatography (HPLC) methods. Obsolete
 Current methods:
 Genetic probes/Amplification
 MALDI-TOF Mass Spectrometry to analyze proteins
 Sequencing 16 sRNA for precise genetic sequence
information

4. Mycobacteria Taxonomy >170 species
 TB complex:
 Mycobacterium tuberculosis
 M. bovis and the Bacillus Calmette-Guerin strain (BCG
– live attenuated strain of M. bovis)
 M. africanum
 And some very rare species:
 Mycobacterium microti
 Mycobacterium canetti
 Mycobacterium caprae
 Mycobacterium pinnipedii
 Mycobacterium suricattae
 Mycobacterium mungi
 Mycobacteria other than TB – “MOTT”

5. Mycobacteria that cause disease?
 Mycobacterium tuberculosis – the major pathogen of this genus
The others: MOTT
 M. avium-intracullare complex
 M. genavense
 M. haemophilum
 M. kansasii
 M. malmoense
 M. marinum
 M. simiae
 M. szulgai
 M. ulcerans
 M. xenopi
 M. fortuitum group
 M. abscessus
 M. chelonae
 M. mucogenicum

6. Mycobacteria that rarely if ever cause
disease! If so, in immune compromised!
 Mycobacterium gordonae
 M. gastri
 M. celatum
 M. scrofulaceum
 M. terrae complex
 M. smegmatis

7. Algorithm for identification of MOTT
- historically speaking
 Runyon System
 Classify species not in the TB complex (MOTT)
 Based on pigment production when exposed to light & growth rate
 Runyon separates MOTT into four groups using the light
test that promotes pigment production
 Photochromogen = Pigment produced with light
 Scotochromogen = Pigment in both light and dark
 Non-photochromogen = No pigment produced
 Rapid Grower = Growth rate (<= 7 days)

8. Light Test – Determine does it produce a yellow
pigment after being exposed to 8 hours of light?
bulb.
Group I Photochromogen
Turns yellow after light exposure
Group III
Non-photochromogen – No
pigment produced after light exposure
Group II Scotochromogen
Always has yellow pigment

9. Runyon Classification System Results
 Group I - Photochromogen – turns yellow when
exposed to light, no color in the dark
 M. kansasii
 M. simiae
 M. szulgai when incubated at 25˚C***
 M. marinum
 Group II - Scotochromogen – yellow pigment in
dark or exposure to light
 M. gordonae
 M. scrofulaceum
 M. szulgai when incubated at 37°C***

10. Runyon Classification cont’d
 Group III – Non-photochromogen – No pigment
produced in the light or dark
 M. avium-intracellulare
 M. haemophilum
 M. xenopi
 Group IV – Rapid growers – grow in 7 days or less
 M. fortuitum group
 M. abscessus
 M. chelonae
 M. mucogenicum
 M. smegmatis

11. Safety in the
AFB Laboratory
 Level 3 biosafety precautions required in AFB laboratories that
process, identify and perform susceptibility testing
 Level 2 Hepa filter approved biosafety cabinet with return air vented
to outside of the laboratory
 Safety cabinets must be certified at least yearly for safe use
 Negative air flow, anteroom, contiguous autoclave
 95 respirator masks or PAPR (powered air purifying respiratory
mask), gloves, disposable gowns must be worn
 Plastic cups with protected lids for centrifugation of specimens
Biosafety Cabinet
PAPR

12. Specimen collection
 Sputum – 3 early morning collection, or
1 early morning, 2 at least 8 hours apart
 Bronchial lavage fluid
 Tissues or Lesions
 CSF or sterile body fluids
 Urine – 3 to 5 early morning collection
 Stool – for M. avium-intracellulare complex
 Gastric – children, must neutralize pH (7) of
specimen after collection for AFB to survive
 Blood – for detection of disseminated disease
 Automated systems – AFB blood culture
bottles manufactured for AFB isolation

13. Specimen Processing
Start to Finish!
 5 ml of specimen pipetted into conical tube
 Decontaminate and Liquify specimen for 15 minutes
 Add 5 ml of 4% NaOH (Increases the pH to 9)
 plus N-acetyl-L-Cysteine (breaks up the mucus)
 Fill tube with phosphate buffer to neutralize pH (7.0)
 Centrifuge for 30 minutes – safety cups
 3000 X g to pellet the specimen
 Pour off the supernatant
 Prepare slides from pellet for AFB staining
 Dilute the pellet with small amount of sterile saline
for culture
 Incubate cultures @ 37˚C, 5-10% CO2 for 6–8 wks

14. Why Decontaminate Specimens?
 For the slow growing mycobacteria to be cultured
 Must eliminate competing bacteria/yeast that grow 24
x faster – killed by pH change of the 4% NaOH
 Must also release the AFB from mucus plugs in the
sputum specimens - L-acetyl-cysteine action

15. Specimen Decontamination/Digestion
Most often used :
 4% NaOH / N-acetyl-L-cysteine
 Used in Special circumstances:
 Oxalic acid for sputum collected from patients with
cystic fibrosis -eliminate mucoid strains of
Pseudomonas aeruginosa
 Oxalic acid should not be used routinely
 Will decrease isolation of AFB in culture
 These solutions kill bacteria and can also kill
AFB if left on specimen > 15 minutes

16. Specimen Centrifugation
 Centrifugation at 3000 X g (fast) with safety cups
 Speed of centrifugation is important - AFB are lipid
laden and they will float if not rapidly centrifuged –
must pellet to bottom of tube so they are not
decanted with supernatant
 Can determine the sensitivity of the AFB stain
 Pour off supernatant into waste
 Use pellet for stains/culture

17. Manual Plating/ Culture Media
 Middlebrook – Synthetic media
 Clear solid agar and liquid media
 Synthetic = chemical ingredients added for optimal growth
 Used for culture and susceptibility testing
 Autoclave for sterility
 Lowenstein-Jensen – Egg based
 Green from addition of malachite green
 Hens egg, glycerol, and potato flour
 Sterilize by inspissation – drying
 Cultures incubated at 37˚C , 5-10% C0₂ for 6-8 weeks

18. Automated Detection of AFB
Bactec MGIT 960, Bacti-Alert and VersaTREK
Liquid Middlebrook 7H9 tubed media for growth
Bactec and Bacti-Alert have same detection method
 As AFB grow in the tubes, the AFB respire CO₂ and the O₂ is decreased.
The lower level of O₂ leads to fluorescence of the tube indicator and indicates
growth in the tube.
Incubation at 37˚C for 6 weeks
Fluoresce
 NAP test – Identification for TB complex
NAP = (p-nitro-α-acetylamino-B-hydroxypropiophenone)
Automated test run only on the MGIT 960 instrument
TB complex does not grow in the tube containing NAP
MOTT species grow in NAP
MGIT 960
BACTEC
Bacti-Alert
VersaTrek

19. Acid Fast Staining for Mycobacteria -
only concentrated specimen should be stained
 Carbol Fuchsin based stains
 Carbol Fuchsin is a red colored stain
 Potassium permanganate is the background blue
counterstain
 Two methods:
 Ziehl-Neelsen (ZN) – uses heat to drive stain into lipid
laden AFB
 Kinyoun – uses increased % of phenol to drive stain
into AFB
 Read numerous microscopic fields (5 min)using light
microscope/100x oil objective

20. Fluorochrome based stain
Auramine Rhodamine –
 Fluorescent stain, organisms stain fluorescent
yellow with black background,
 Read on 25X or 40X for 2 min, viewing numerous
fields using a fluorescence microscope
 Based on nonspecific fluorochrome that binds to
mycolic acids
Considered more sensitive than ZN or Kinyoun
stains for concentrated specimen patient slide
examination

21. Acid Fast staining of the Mycobacteria
Mycobacterium avium complex
Organisms are routinely shorter than TB
(Kinyoun stain) No cording
M. Tuberculosis - Organisms are
long rods and can appear as if they
are sticking together [cord factor]
In broth
cultures -
ropes of TB
will form due to
cord factor

22. Direct Detection of TB from Respiratory
Specimens by Molecular Amplification
 FDA cleared to detect TB complex organisms in smear
positive and smear negative sputum specimens:
 Cepheid Xpert-TB RIF (rtPCR) – detects TB complex and Rifampin
resistance (rpoB gene)
 Amplify a 16S rRNA gene sequence of TB complex
 Sensitivity of assays @ 90% AFB smear (+) specimens and
75% (+) for AFB smear (-) specimens
 Test of diagnosis not cure
 Residual rRNA and DNA can be present up to 6m after a
positive diagnostic test
 Must confirm all specimens with AFB culture and sensitivity
 Currently, no FDA cleared assays for MOTT species

23. PPD (Purified Protein Derivative)
Mantoux test/TB skin test
 Detects past or current TB exposure
 False positive reaction in those with BCG
immunization
 @25% false negative reactions – usually due to low
T cell reactivity or problems with administration
 Measures delayed hypersensitivity (T cell response)
to TB complex antigens
 Measure (mm) area of induration at injection site
 >=15mm positive rxn (check for history of BCG)
 >=10mm positive in immune suppressed or just exposed

24. Cell Mitogen assays – Interferon
Gamma Release Assays
 QuantiFERON-TB-Gold (QFT) and T-spot
assays
 Whole blood tests to screen for cell mediated
immunity to TB specific antigens
 Quantitative measurement of gamma interferon due to
the CD4 T cell response to TB specific antigens
 Specificity for TB is helpful in screening patients
immunized with BCG
 Detects latent and active infection – does not
replace culture
 Sensitivity similar to PPD/ improved specificity

25. Mycobacterium tuberculosis
 Optimal Temp 37˚ C, Grows in 12 –25 days
 Buff colored, dry cauliflower-like colony
 Manual tests for identification – old school
 Niacin accumulation Positive
 Niacin produced from growth of TB on egg
containing medium (LJ medium)
 Nitrate reduction Positive
 Is it really M. tuberculosis or could it be M. bovis?
 Both in TB complex and diseases are similar
 M. bovis = nitrate reduction Negative
 M. bovis does not grow in Thiophene-2-carboxylic hydrazide (T2H),
however TB does grow on this substrate
 Current ID uses Molecular probe, MALDI-TOF, & Sequencing

26. Mycobacterium
tuberculosis
Demonstrates Cord factor –
due to high lipid content in cell
wall, rods stick together and
develop long ropes – unique
to M. tuberculosis
Long AFB – stick together

27. Tuberculosis
 Classic Disease
 Slowly progressive pulmonary infection cough, weight loss, and
low grade fever
 Lesion with calcified foci of infection and an associated lymph node known as
Ghon’s complex
 Dissemination occurs most often in AIDS, elderly, children and with
some medications (Remicade- infliximab)
 Secondary tuberculosis: mostly in adults as reactivation of
previous infection (or reinfection), Granulomatous inflammation
much more florid and widespread. Upper lung lobes are most
affected, and cavitation can occur.
 TB is spread by respiratory droplets
 All patients with a positive concentrated smear require
respiratory isolation precautions
 All patients with high level of suspicion require precautions

28. Pathology of M. tuberculosis
Lung with pulmonary TB has apical cavitations with
numerous areas of caseous necrosis and massive tissue
consolidation

29. TB in HIV/AIDS patients
 Worldwide -TB is the most common opportunistic infection
affecting HIV/AIDS patients
 AIDS patients have higher likelihood to be multi-drug
resistant (resistant to at least INH and Rifampin)
 With progressive decline of cell mediated immunity (low
CD4 count) – greater risk of extra pulmonary dissemination
 Granulomas with/without caseation can occur
Miliary TB

30. M. tuberculosis outside lung
 Scrofula -Unilateral lymphadenitis (cervical lymph node)
most often seen in immune compromised (50%)
 Fine needle aspiration for diagnostic specimen
 M. tuberculosis most common in adults
 M. avium complex and other MOTT (2-10%)
most common in children
 Pott’s disease - TB infection of the spine
 Secondary to an extra-spinal source of infection
 Manifests as a combination of osteomyelitis and arthritis that
usually involves more than 1 vertebra.

31. Susceptibility testing of TB
 Two methods for testing
 (1) Agar dilution (indirect proportion method)
 antibiotics embedded in solid agar and TB inoculated onto media
 (2) Bactec liquid 7H9 medium containing antibiotic solutions
 Tested on automated MGIT 960 system
 Primary TB drug panel / 5 drugs
 Isoniazid Ethambutol
 Pyrazinamide Streptomycin
 Rifampin
 If patient culture positive after four
months/retest/possible resistant strain

32. Mycobacterium bovis
 Produces disease in cattle and other animals
 Spread to humans by raw milk ingestion
 Disease in humans similar to that caused by TB
 Bladder infections in patients treated with BCG [Bacille Calmette-
Guerin] when used as an immune adjuvant to treat bladder cancer
 BCG is an attenuated strain of M. bovis
 It can become “active” and infect the bladder
 M. bovis will be isolated from urine specimens
 Is it M bovis or is it M tb?
M.bovis M. tb
Nitrate Negative Positive
Growth in T2H* No growth Growth
Pyrazinamidase Negative Positive
*(Thiophene-2-carboxylic hydrazide)

33. Mycobacterium ulcerans
 Bairnsdale or Buruli ulcer boil that progresses into a
painless skin ulcer
 Can progress into avascular coagulation necrosis
 African continent and tropical environments
 Optimum growth temp 30˚ C
 All skin lesions should be
cultured at both 30˚ and 37˚C
 Slow growing requiring 3- 4 weeks

34. Mycobacterium kansasii
 Culture 37* C, 10-20 days
 Photochromogen
 Niacin accumulation test = negative
 Nitrate reduction = positive
 Tween 80 positive / tests for presence of lipase enzyme
 68*C catalase positive
 AFB are larger than TB - rectangular and beaded
Shepherd’s crook shape
 Clinical disease mimics pulmonary TB but usually does
not disseminate from lung –
 Predisposition diseased lung (COPD)
 Immune suppressed, pneumoconiosis, alcohol abuse, HIV
 Granulomatous inflammation in lung

35. Mycobacterium marinum
 Optimum temp for growth is 30˚ C
 Grows poorly or not at all at 37°C
 Grows in 5-14 days
 Photochromogen
 M. marinum found in fresh and salt water
 Infection associated with trauma occurring in water:
 swimming pools (swimming pool granuloma)
 cleaning fish tanks
 ocean (surfing)

36. Mycobacterium marinum
 Disease: Tender, red or blue/red subcutaneous nodules
develop following trauma
 Biopsy (skin) specimens for culture and histopathology
 Lesions can spread up arm and along lymphatics,
 Clinically appears similar to infections with Sporothrix,
Nocardia and rapid growing Mycobacteria species.

37. Mycobacterium szulgai
 Grows at 37 ˚C in 12 - 25 days
 Scotochromogen at 37˚C / Photochromogen at 25˚C
 Only AFB that has a different light test result based on
temperature of incubation
 Rare cause of pulmonary
disease in adults
Symptoms similar to TB 25˚ C - Photochromogen
37˚ C - Scotochromogen

38. Mycobacterium xenopi
 Optimum temp 42˚C,
 Capable of growing in hot water systems
 Grows in 14 - 28 days in culture
 Scotochromogen
 Egg nest type colony on agar plate
 Rare cause of pulmonary disease
 Clinically disease is like TB
 Occurs in patients with preexisting lung disease and
HIV/AIDS

39. M. avium-intracellulare complex
 M avium & M intracellulare most common but complex
includes eight species of environmental and animal related AFB
 Biochemically and genetically very difficult to distinguish
 Growth at 37 ˚C / 7 – 21 days
 Non-photochromogen
 Smooth colony
 Inert in biochemical reactions
 Identify using
 GenProbe (AccuProbe) molecular DNA probe
 MALDI-TOF
 Genetic 16s rRNA Sequencing

40. M. avium-intracellulare complex
 Opportunistic infection
High organism load in intestine, liver, spleen and bone marrow
Can be recovered from blood cultures
Involvement of GI tract causes diarrhea
 Positive AFB stool smears
 Tissue Biopsy with inflammation
AFB small and not beaded
 Do not have cord factor
Pathology - Necrotizing rather than
granulomatous inflammation

41. M avium-complex in lymph node tissue
(Kinyoun stain)
Granulomatous inflammation
lung tissue (H&E stain)
Bowel -Lamina propria expanded from
predominately Lymphohistocytic infiltration

42. M. avium complex clinical correlation
 HIV/AIDS
 Disseminated infection in end stage AIDS disease
 Nonspecific low grade fever, weakness, weight loss,
picture of fever of unknown origin
 Abdominal pain and/or diarrhea with malabsorption
 Normal host
 In adults mostly pulmonary disease, much like TB
 marked % of cases – elderly adults with lung
damage (COPD)
 M. chimaera
 Contaminated Heater–cooler units using tap water identified as a
source of surgical site infections. Units caused an airborne
transmission of M. chimaera over an open surgical field

43.  Low virulence, found in nature
 Cause: Infections in soft tissue, venous catheter sites
and shunts @ 20 species – 5 species most common
 M. fortuitum – skin and surgical wound infections
 M. chelonae- skin infections in immune suppressed
 M. abscessus - chronic lung infection and skin infection in
immune suppressed
 M. smegmatis
 M. mucogenicum
 Grows in <=7 days = rapid grower
 Biochemical reactions:
 All species are Arylsulfatase test positive
 M. fortuitum: Nitrate Positive and Iron Uptake positive
 M. chelonae and M. abscessus: Nitrate Negative
 MALDI-TOF and 16 S RNA Sequencing for identification
Rapid Growers

44. Miscellaneous
 M. gordonae –
 Rare! Cause of Infection
 Scotochromogen
 Major laboratory water contaminant in cultures
because it is commonly contaminating water
systems
 Use sterile/distilled water in AFB culture
processing to prevent contamination

45. Miscellaneous pathogenic species
 Mycobacterium haemophilum
 Requires addition of hemoglobin or hemin to
culture media for growth
 Will not grow on LJ or in automated systems without the
addition of hemin supplements
 Disease: Painful subcutaneous nodules and
ulcers, primarily in AIDS patients or immune
suppressed
 Lymphadenitis in children

46. Mycobacterium leprae
 Leprosy – Hansen’s disease
 Begins with anesthetic skin lesions,
peripheral neuropathy with nerve thickening, numbness in earlobes
or nose, loss of eye brows, completely curable with early
diagnosis and therapy
 Two types of Leprosy
 Lapromatous – can lead to severe disfiguring lesions, large
numbers of AFB in lesions / +/- co-infection with Strongyloides
 Tuberculoid -less severe/fewer lesions, lower numbers of AFB
in lesions
 Will not grow on laboratory AFB media
 PCR on tissue for definitive diagnosis
 Armadillo is the natural reservoir
 Found in tropical Africa and Asia

47. Tuberculoid leprosy
Lapromatous leprosy
Skin biopsy - AFB seen in nerve fiber
AFB in “cigar packets”