This document discusses mycobacteriology and the identification and testing of mycobacteria. It covers the following key points: 1. Mycobacteria are acid-fast bacilli that can be identified using genetic probes, MALDI-TOF mass spectrometry, or 16S rRNA sequencing. There are over 170 mycobacterial species separated into the TB complex and non-tuberculous mycobacteria. 2. Identification of non-tuberculous mycobacteria was historically based on pigment production and growth rate, categorized using the Runyon system. Current best methods are genetic or proteomic. 3. Specimen processing involves decontamination to eliminate competing bacteria, centrifug

1. Mycobacteriology
Update 2019
Margie Morgan, PhD, D(ABMM)

2. Mycobacteria
◼ Acid Fast Bacilli (AFB)
❑ Cell wall contains complex mycolic acids and free lipids
❑ Once stained, AFB resist de-colorization with acid alcohol (HCl) hence
the term acid fast
❑ Difference of AFB stain vs. modified or partial acid fast (PAF) stain
◼ AFB stain uses HCl is to decolorize Mycobacteria (+) Nocardia (-)
◼ PAF stain uses H2SO4 to decolorize Mycobacteria (+) Nocardia (+)
◼ Gram stain = poorly stained / appear beaded Gram positive
◼ Aerobic, no spores, rarely branch
Gram
stain

3. Identification of the Mycobacteria
◼ For decades, identification algorithm started with the ability to form
pigment in the light or dark; followed by biochemical reactions,
growth rate, and optimum temperature for growth. Obsolete
◼ With expanding taxonomy, biochemical reactions were unable to
separate and identify most of the newly recognized species, so
replaced with High Performance Liquid Chromatography (HPLC)
methods. Now also obsolete
◼ Current best methods for identification:
❑ Genetic probes (DNA/RNA hybridization)
❑ MALDI-TOF Mass Spectrometry to analyze cellular proteins
❑ Sequencing 16 sRNA for precise genetic sequence information

4. Mycobacteria Taxonomy currently >170 species
2 Taxonomic groups
◼ Group 1 - TB complex
❑ Mycobacterium tuberculosis
❑ M. bovis Bacillus Calmette-Guerin (BCG) strain
◼ Attenuated strain of M. bovis used for vaccination
❑ M. africanum
❑ Rare species of mycobacteria
◼ Mycobacterium microti
◼ Mycobacterium canetti
◼ Mycobacterium caprae
◼ Mycobacterium pinnipedii
◼ Mycobacterium suricattae
◼ Mycobacterium mungi
◼ Group 2 - Mycobacteria other than TB – “MOTT” also known
as Non-Tuberculous Mycobacteria

5. MOTT/ non tuberculous mycobacteria/ cause disease
(1) Slowly growing non tuberculosis
mycobacteria
◼ M. avium-intracullare complex
◼ M. genavense
◼ M. haemophilum
◼ M. kansasii (3) Mycobacterium leprae
◼ M. malmoense
◼ M. marinum (4) Mycobacterium tuberculosis complex/
◼ M. simiae including M. bovis
◼ M. szulgai
◼ M. ulcerans
◼ M. xenopi
◼ M. smegmatis
(2) Rapid growing mycobacteria
◼ M. fortuitum group
◼ M. abscessus
◼ M. chelonae
◼ M. mucogenicum

6. Mycobacteria that rarely if ever cause disease!
If so, only in the immunocompromised!
Slowly growing nontuberculous
mycobacteria
◼ Mycobacterium gordonae
◼ M. gastri
◼ M. celatum
◼ M. scrofulaceum
◼ M. terrae complex

7. Algorithm for identification of MOTT
- historically speaking
◼ Based on the “Runyon System”
❑ System to classify species not in the TB complex
❑ Based on pigment production when exposed to incandescent light bulb
& growth rate
◼ Four Runyon groups - separated by pigment production when exposed to
light and growth rate
❑ Photochromogen = Pigment produced only when exposed to light
❑ Scotochromogen = Pigment produced in both light and dark
❑ Non-photochromogen = No pigment produced in either light or dark
❑ Rapid Grower = Growth rate (<= 7 days)

8. The Light Test – Determine if mycobacteria can produce a yellow
pigment after being exposed to 8 hours of light bulb
Group I Photochromogen
Turns yellow only after light exposure Group III
Non-photochromogen
No pigment produced in dark
or after light exposure
Group II Scotochromogen
Yellow pigment present in dark or
with light exposure

9. Runyon Classification System Classification
◼ Group I - Photochromogen – turns yellow when exposed
to light, no color in the dark. Growth in 2 -3 weeks
❑ M. kansasii
❑ M. simiae
❑ M. szulgai when incubated at 25˚C*
❑ M. marinum
◼ Group II - Scotochromogen – yellow pigment in dark or
exposure to light. Growth 2 – 3 weeks
❑ M. gordonae
❑ M. scrofulaceum
❑ M. xenopi (most strains)
❑ M. szulgai when incubated at 37°C*

10. Runyon Classification continued
◼ Group III – Non-photochromogen – No pigment produced in
the light or dark, growth in 2-3 weeks
❑ M. avium-intracellulare complex
❑ M. haemophilum
❑ M. xenopi (some strains)
◼ Group IV – Rapid growers, grow in 7 days or less incubation
❑ M. fortuitum group
❑ M. abscessus
❑ M. chelonae
❑ M. mucogenicum
❑ M. smegmatis

11. AFB Laboratory
Safety
◼ Level 3 biosafety precautions required in AFB laboratories that process, identify and
perform susceptibility testing on mycobacteria species
❑ Level 2 Hepa filter biosafety cabinet with return air vented to outside and must be
certified per regulation to insure proper operations at least yearly
◼ Room under negative air flow, anteroom for safety gear, and contiguous autoclave
◼ Must wear a 95 respirator mask (yearly mask fit required) or PAPR (powered air purifying
respiratory mask) when working with organisms even when inside the biosafety cabinet
◼ Gloves and disposable surgical gowns for culture work
◼ Plastic cups with lids to contain specimen for centrifugation and prevent exposure
Biosafety Cabinet
PAPR
Vented to outside

12. Specimen collection
◼ Sputum – 3 early morning collections, or
1 early morning, plus 2 collected at least 8 hours apart
◼ Bronchial lavage fluid
◼ Tissues or Lesions
◼ CSF or sterile body fluids
◼ Urine – 3 to 5 early morning collections
◼ Stool – Primarily for M. avium-intracellulare complex
◼ Gastric – usually children unable to produce sputum,
must neutralize gastric to pH 7.0 after
collection in order for AFB to survive
◼ Blood – in suspected disseminated disease
❑ Automated systems – AFB blood culture bottles
manufactured specifically for AFB

13. AFB Specimen Processing
Start to Finish!
◼ 5 ml of specimen pipetted into conical tube
◼ Decontaminate and liquify sputum specimen for 15 minutes with:
❑ 5 ml of 4% NaOH (increases the pH to 9) – kill contaminating bacteria
❑ plus N-acetyl-L-Cysteine (breaks up the mucus) – liquify mucus plugs
◼ After 15 min - fill tube with phosphate buffer to neutralize to pH (7.0)
◼ Centrifuge for 30 minutes – using safety cups
❑ 3000 X g to pellet the specimen (speed is important – AFB are buoyant)
◼ Pour off the supernatant
◼ Prepare concentrated slide from pellet for AFB staining
◼ Dilute the pellet with small amount of sterile saline for culture
◼ Incubate culture media @ 37˚C, 5-10% CO2 for 6–8 weeks

14. Why Decontaminate Specimens?
Slow growing mycobacteria will be overgrown by bacteria in the
specimen unless they are killed in the decontamination step:
❑ Must eliminate competing bacteria/yeast that grow 24 x faster
– these organisms are killed by the increased pH with the
addition of 4% NaOH
❑ Must also release the AFB from mucus plugs in the sputum
specimen - this is accomplished with the addition of the L-
acetyl-cysteine

15. Specimen Decontamination/Digestion of potentially
contaminated specimen types (respiratory)
◼ Routine processing:
❑ 4% NaOH with N-acetyl-L-cysteine
◼ Processing: special circumstances/ cystic fibrosis
❑ Process sputum with oxalic acid to eliminate mucoid
strains of Pseudomonas aeruginosa
◼ Oxalic acid should not be used routinely for processing
◼ Will decrease yield 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
◼ Pellet used for stains/culture

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

18. Automated Detection of AFB
BD BACTEC MGIT 960 most used automated instrument for AFB
❑ Middlebrook 12B liquid media
❑ BACTEC detection method
◼ As AFB grow in the 12B media, the AFB respire CO₂ and the amount of O₂ is
decreased over time. The lower level of O₂ causes fluorescence of the tube indicator
and indicates growth in the tube.
❑ Incubation at 37˚C for 6 weeks
Fluoresce
◼ NAP test /Identification of TB complex organisms
using MGIT 960 instrument
NAP = chemical (p-nitro-α-acetylamino-B-hydroxypropiophenone)
TB complex does not grow in the tube containing NAP
Non-tuberculous species grow in NAP media
MGIT 960

19. Acid Fast Stains for Mycobacteria
◼ Carbol Fuchsin based stains
❑ Carbol Fuchsin is red colored stain
❑ Potassium permanganate is the background blue counterstain
◼ Two methods:
❑ Ziehl-Neelsen (ZN) – heat used to drive stain into lipid laden AFB
❑ Kinyoun – High % of phenol in stain drives stain into mycobacteria
◼ Read numerous microscopic fields for 5 minutes, using light microscopy,
100x oil objective

20. Fluorochrome based stain
◼ Auramine Rhodamine stain
❑ Fluorescent stain
❑ Rods stain fluorescent yellow with black background
❑ Read on 25X or 40X for 2 min, viewing numerous fields using
a fluorescence microscope
❑ Based on nonspecific fluoro-chrome that binds to the mycolic
acids in the mycobacteria
◼ Considered more sensitive than ZN or Kinyoun stains for
concentrated patient specimen slide examination

21. Acid Fast Mycobacteria morphology
Mycobacterium avium complex
Organisms are routinely shorter than TB
(Kinyoun stain) without cording
M. tuberculosis - Organisms are long
rods and can appear as if they are sticking
together [cord factor]
In broth
cultures -
ropes of AFB
will form due to
cord factor

22. Direct Detection of TB from Respiratory Specimens
using Molecular Amplification
◼ FDA cleared molecular assay to detect TB complex organisms in
concentrate AFB smear positive and negative sputum specimens:
❑ Cepheid Xpert-TB RIF Assay (rtPCR)
❑ Detects TB complex organism gene sequence and rifampin resistance gene (rpoB)
◼ Sensitivity of assays
❑ 90% for AFB concentrated smear (+) specimens
❑ 75% for AFB concentrated smear (-) specimens
◼ Test of diagnosis, not cure
❑ Residual rRNA and DNA can be present for up to 6 months after initial
diagnosis and start of therapy
◼ Must confirm all tests with AFB culture and sensitivity (gold standard)
◼ Currently, no FDA cleared assay for non-tuberculous mycobacteria spp.

23. PPD (Purified Protein Derivative)
Mantoux test/TB skin test
◼ Detects latent or current TB exposure
◼ False positive reactions can occur in patients immunized with BCG
◼ @ 25% false negative reactions – usually due to low T cell counts or T cell
reactivity, such as occurs with high dose steroids. Problems with PPD
administration and reading can also lead to false negative reactions
◼ Measures delayed hypersensitivity (T cell response) to TB complex
antigens (not specific for TB)
❑ Measure (mm) area of induration at injection site
◼ >=15mm positive (check for history of BCG)
◼ >=10mm positive in immune suppressed or just exposed to TB and part of an
outbreak investigation

24. Cell Mitogen assays –
Interferon Gamma Release Assays (IGRA)s
◼ QuantiFERON-TB-Gold Plus (QFTP)
❑ CD4 and CD8 T cells in patient whole blood samples are stimulated
with TB specific antigens.
❑ If patient has active or latent exposure to TB, the stimulated T cells
will produce gamma interferon
❑ Automated EIA assay measures amount of gamma interferon
produced
❑ Quantitative endpoint indicates a positive or negative reaction
❑ Indeterminate reactions can occur, if T cells are absent or inactive
due to medications
◼ No false positive reactions from immunization with BCG
◼ Sensitivity >=80%, does NOT replace culture for disease diagnosis
◼ Sensitivity similar to PPD/ but has improved specificity

25. Mycobacterium tuberculosis
◼ Optimal Temp 37˚ C, Growth in 12 –25 days
◼ Buff colored, dry cauliflower-like colony
◼ Manual tests for identification – old school
❑ Niacin accumulation test positive
◼ Niacin produced from growth of TB on egg containing medium (LJ)
❑ Nitrate reduction test positive
◼ Current identification methods:
❑ Molecular DNA/RNA hybridization probe for MTB complex
❑ MALDI-TOF mass spectrometry for M. tuberculosis
❑ 16 sRNA sequencing for M. tuberculosis

26. Mycobacterium tuberculosis
Cord factor – Due to high lipid content
in cell wall, rods stick together and
develop long ropes when grown in broth
media – unique to M. tuberculosis
Long AFB / stick together

27. Susceptibility testing of TB
◼ Two methods for testing
❑ (1) Agar dilution (indirect proportion method)
◼ Antibiotics embedded in solid agar and TB inoculated onto media quadrants
❑ (2) Liquid 7H9 medium containing anti-TB antibiotic solutions
◼ Tested on automated Bactec MGIT 960 system
◼ Primary TB drug panel / 5 drugs
❑ Isoniazid Ethambutol
❑ Pyrazinamide Streptomycin
❑ Rifampin
◼ If resistant to primary drugs, second line drugs tested
◼ If patient remains culture positive after four months on treatment/
retest the TB susceptibility / possible drug resistant strain

28. Tuberculosis
◼ Classic disease is slowly progressive pulmonary infection cough,
weight loss, and low grade fever, presentation can vary depending
on degree of immunosuppression
◼ Ghon’s complex: Lesion with calcified foci of infection and an associated lymph
node
◼ Milliary TB, wide spread dissemination via the blood occurs most often in AIDS,
elderly, children, immunosuppression and some medications (Remicade-
infliximab)
◼ Secondary tuberculosis: mostly in adults as reactivation of previous infection
❑ Granulomatous inflammation much more florid and widespread than in primary disease.
❑ Upper lung lobes are most affected, and cavitation can occur.
◼ TB is spread by respiratory droplets
◼ All patients with high level of suspicion require respiratory isolation precautions

29. Pathology of Mycobacterium tuberculosis
The hallmark of Mycobacterium tuberculosis infected tissue is
necrotizing granulomatous inflammation, composed of
epithelioid histiocytes surrounding a central necrotic zone, and
can be accompanied by a variable number of multinucleated
giant cells and lymphocytes. Non-necrotizing granulomas can
be present as well.

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

31. M. tuberculosis outside the lung
◼ Scrofula –Unilateral lymphadenitis (cervical lymph node)
most often seen in immunocompromised adult or children/
caseous necrosis often present
❑ Fine needle aspiration for diagnostic specimen
◼ M. tuberculosis most common in adults
◼ M. avium complex and other MOTT (2-10%) in children
◼ Pott’s disease - TB infection of the spine
❑ Secondary to an extra-spinal source of infection (1* pulmonary)
❑ Manifests as a combination of osteomyelitis and arthritis
that usually involves more than 1 vertebra.

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
◼ Can cause bladder infection in patients treated with BCG [Bacille Calmette-Guerin] /
used as an immune adjuvant to treat bladder cancer
❑ BCG is an attenuated strain of M. bovis, it can actibate and infect the bladder
❑ M. bovis isolated in culture from urine specimens
◼ Need to differentiate M. bovis from M. tuberculosis in such circumstances
M.bovis M. tb
Nitrate Negative Positive
Growth in T2H* No growth Growth
Pyrazinamidase enzyme Negative Positive
Pyrazinamide susceptibility Resistant Susceptible
*(Thiophene-2-carboxylic hydrazide)

33. Mycobacterium ulcerans
◼ Cause of Buruli ulcer
◼ Localized painless skin nodule evolves rapidly to ulceration and can
become disabling, due to production of mycolactones (cell cytotoxin)
◼ Endemic in the African continent and other tropical areas with limited
medical care, skin trauma with exposure to contaminated water, peak age
group 5-15 years of age
◼ Optimum growth temp 30˚ C
❑ Does not grow well or at all at 37*C
❑ **Skin lesions suspect for AFB 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 the lipase enzyme
❑ 68*C catalase = positive
◼ AFB are larger than TB - rectangular and very beaded with Shepherd’s
crook shape
◼ Clinical disease mimics pulmonary TB but usually does not disseminate
from lung – acquire disease from tap water
❑ Predisposition for diseased lung (COPD)
❑ Immune suppressed, pneumoconiosis, alcohol abuse, and HIV
❑ Produces granulomatous inflammation in lung

35. Mycobacterium marinum
◼ Photochromogen
◼ Optimum temp for growth is 30˚ C
❑ Grows poorly or not at all at 37°C
❑ Grows in 5-14 days
◼ Normal habitat is fresh and salt water
❑ Infection associated with trauma occurring in water, such as
◼ Swimming pools (swimming pool granuloma)
◼ Cleaning fish tanks
◼ Ocean (surfing)

36. Mycobacterium marinum
◼ Disease: Tender, red or blue/red subcutaneous nodules develop at
the site of trauma
◼ Biopsy (skin) specimens for culture and histopathology
◼ Lesions classically spread up arm along lymphatics,
❑ Clinically appears similar to diseases Sporotrichosis, Nocardiosis,
and infection with rapid growing Mycobacteria species

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

38. Mycobacterium xenopi
◼ Scotochromogen
◼ Optimum growth temperature 42˚C,
❑ Capable of growing in hot water systems
◼ Growth in 14 - 28 days
◼ Egg nest type colony on agar plate
◼ Rare cause of pulmonary disease
❑ Clinical disease is like TB
❑ Occurs in patients with preexisting lung disease and HIV/AIDS

39. M. avium complex
◼ M avium & M intracellulare most common species in complex, complex
includes eight species of environmental and animal related AFB
❑ Non-photochromogen
❑ All species biochemically and genetically very similar
❑ Growth at 37 ˚C / 7 – 21 days
❑ Smooth / creamy colony
❑ Short rods, not beaded
❑ Inert in biochemical reactions
❑ Identify using
◼ Molecular DNA/RNA hybridization probe
◼ MALDI-TOF mass spectometry
◼ 16s rRNA sequencing

40. M. avium complex clinical correlation
◼ Disease in the somewhat normal host
❑ Adults mostly chronic pulmonary disease, Fibro-cavitary or nodular
bronchietatic disease presentation
❑ Elderly adults with prior lung damage (COPD) produces chronic
cough
❑ Children – chronic granulomatous inflammation with lymph node
involvement / scrofula
◼ M. chimaera (species within the M. avium complex)
❑ Environmental / nosocomial pathogen
❑ Recently in the news as a contaminate in heater–cooler units used in cardiac
surgery/ aerosolized water from instrument culture positive for M. chimaera
❑ The airborne transmission of M. chimaera over open surgical field caused deep
tissue infection post surgery

41. M. avium complex (MAC)
◼ HIV/AIDS – common opportunistic infection in AIDS
❑ Nonspecific low grade fever, weakness, weight loss, picture of
fever of unknown origin
◼ Diagnosis:Isolation of MAC from an AFB blood
culture or bone marrow culture
❑ Abdominal pain and/or diarrhea with malabsorption
◼ Positive AFB smears from stool specimens
◼ In tissue:
❑ Pathology: necrotizing rather than granulomatous
❑ High organism load in infected tissue
❑ AFB small (short) rods and not beaded
◼ Does not have cord factor

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

43. ◼ Grows in <=7 days, more rapid than all other mycobacteria species
◼ Low virulence organisms found as environmental contaminates
◼ 20 species – 3 species most common
◼ M. fortuitum skin and surgical wound infections, catheter sites
◼ M. chelonae skin infections in immune suppressed, catheter sites
◼ M. abscessus chronic lung infection and skin infection in immune suppressed
❑ Biochemical reactions:
◼ All species Arylsulfatase positive
◼ M. fortuitum: Nitrate reduction positive and iron uptake positive
◼ M. chelonae and M. abscessus: Nitrate Negative
❑ MALDI-TOF and 16 sRNA sequencing for identification
Rapid Grower Mycobacteria species

44. Miscellaneous
◼ M. gordonae –
❑ Scotochromogen
❑ Rarely if ever cause infection
❑ Commonly found in tap water
❑ Find it’s way into AFB cultures as a laboratory contaminant
❑ Use sterile/distilled water in AFB culture processing to prevent
culture contamination

45. Miscellaneous pathogenic species
◼ Mycobacterium haemophilum
❑ Requires addition of hemoglobin or hemin to culture media for
growth
◼ Will not grow on LJ media or in automated systems (Middlebrook 12B
media) without the addition of hemin supplements
❑ Disease:
◼ Painful subcutaneous nodules and ulcers, primarily in AIDS patients or
immunosuppressed
◼ Lymphadenitis in children

46. Mycobacterium leprae
◼ Leprosy – Hansen’s disease
◼ Infection begins with anesthetic skin lesions
❑ Peripheral neuropathy with nerve thickening, numbness in earlobes or nose, loss
of eye brows,
❑ Curable with early diagnosis and appropriate therapy (dapsone with rifampin and
clofazimine
◼ Two types of leprosy found in Africa and Asia
❑ Lapromatous – Severe disfiguring lesions, large numbers of AFB in lesions
❑ Tuberculoid - Less severe and fewer lesions, lower numbers of AFB in lesions
◼ Will not grow on any laboratory media
◼ PCR performed from tissue for definitive diagnosis
◼ Armadillo is a natural reservoir

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