Mycobacteriology 2016 Microbes with Dr. Morgan

1. Mycobacteriology
Review
Margie Morgan, PhD, D(ABMM)

2. PPD (Purified Protein Derivative)
aka the TB skin test
 Determined exposure to TB for decades
 Detects past or current TB exposure
 X-reacts with BCG immunization
 @ 25% false negative reactions
 Measures delayed hypersensitivity response to TB Ags r
 T cells react to TB related antigens---
 Lymphokine is released ----
 Forms area of induration at the injection site
 Measure induration in mm at 48 hr
 >=15 mm positive rxn or check for BCG immunization in past
 >=10 mm positive in immune suppressed or just exposed

3. Cell Mitogen assays for TB screening-IGRAs
(Interferon Gamma Release Assays)
 QuantiFERON-TB Gold (QFT) and T-Spot
 Whole blood tests to screen for cell mediated immunity
to TB complex antigens
 Able to detect both latent TB and active infection
 Specific for TB complex - Useful for screening BCG
immunized population where PPD falsely positive
 Useful if patient cannot return to have PPD reaction
interpreted
 Tests quantitate the immune response to TB following
the stimulation of the patient’s T cells by TB specific
antigens

4. Mycobacteria –Acid Fast Bacilli (AFB)
 Related to the genera Nocardia, Corynebacterium,
and Rhodococcus
 What does the term “Acid Fast” refer to?
 Once stained the rods resist decolorization
with acid alcohol (HCl)
 Very beaded and faded on Gram stain
 Gram stain is NOT a good stain to detect AFB
 Note: Differentiate AFB staining of the mycobacteria from partial
acid fast (PAF) staining used for the Nocardia species.
 AFB acid fast stains use HCl to decolorize
 PAF acid fast stains use H2SO4 to decolorize
 This is a more gentle process and Nocardia will be PAF + but will
stain negative in the “true” AFB stains meant for the mycobacteria.
Gram
AFB

5. Mycobacteria –
General Characteristics
 Aerobic, no spores, slightly curved
or straight rods, rarely branch, vary in length
depending on species
 Hardy in the environment for months
 Most species grow on simple substrates
 Mycobacteria include obligate pathogens,
opportunists and saprophytic species
 High amount of complex mycolic acids and free
lipids in cell wall which give many properties to this
genus including AFB staining properties

6. Identification of the Mycobacteria
 For decades the identification algorithm started with
the determination of pigment in the light and dark
followed by biochemical reactions
 With expanding taxonomy, biochemical reactions
were not able to separate and identify most of the
newly recognized species so we evolved into HPLC
methods. HPLC is now obsolete.
 Current methods for identification:
 Genetic probes – RNA/DNA hybridization probes
 MALDI-TOF Mass Spectrometry to analyze proteins
 Sequencing 16 sRNA for genetic sequence information

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

8. Mycobacteria that cause disease?
 Mycobacterium tuberculosis – the major pathogen of this genus
The others:
 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

9. 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

10. Algorithm for identification of MOTT
- historically speaking
 The Runyon System is used to classify those species
not in the TB complex (MOTT)
 Divided into four groups:
 Photochromogen = Pigment when exposed to light in
Light Test
 Scotochromogen =Pigment in both light and dark in
the Light Test
 Non-photochromogen = No pigment produced in light
or dark in the Light Test
 Rapid Grower = Growth rate (<= 7 days)

11. Light Test – does it produce a yellow pigment
after being exposed to light ?
Group I Photochromogen
Turn yellow after light exposure
Group III
Non-photochromogen –
No pigment produced
Group II Scotochromogen
Always has yellow pigment

12. 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***

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

14. Specimen Processing
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 for dressing and washing hands
 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

15. Diagnosis: Specimen collection
 Sputum – 3 early morning collection
or 3 specimens at least 8 hours apart
 Bronchial lavage fluid
 Tissues or Lesions
 CSF or sterile body fluids
 Urine – 3 to 5 early morning collection
 Stool – M. avium-intraculluare 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

16. 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
 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

17. Why do you Decontaminate Specimens?
 Mycobacteria are more resistant to killing by acids and
alkaline solutions than most bacteria due to the high
amount of complex lipids in the cell wall – this is used to
rid the specimen of contaminating bacteria and yeast
 For the slow growing mycobacteria to be cultured
 Must eliminate competing bacteria that grow 24 x faster
 Must also release the AFB from mucus plugs in the sputum
specimens
 Accomplished by exposing the sputum to alkaline/acid solutions
and mucolytic reagents such as 4% NaOH and L-acetyl-L
cysteine

18. Specimen Decontamination/Digestion
Most often used :
 4% NaOH – for decontamination
 N-acetyl-L-cysteine – liquid faction of mucus
 Expose specimen to solution for 15 minutes
 Used in Special circumstances:
 Oxalic acid can be used for sputum collected from
patients with cystic fibrosis to kill mucoid
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

19. Specimen Centrifugation
 Centrifugation at 3000 X g (fast)
 Speed of centrifugation is important - AFB are lipid
laden and they will float if not spun fast enough –
must pellet to bottom of tube so AFB are not poured
off into waste
 Helps to determine the sensitivity of the AFB stain
 Pour off supernatant into waste
 Use pellet for stains/culture

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

21. Automated Detection of AFB
Bactec MGIT 960, Bacti-Alert and VersaTREK
Use 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
 BACTEC MGIT 960 NAP test – Identification for TB
NAP = (p-nitro-α-acetylamino-B-hydroxypropiophenone)
Automated test on NGIT 960 for TB identification
TB does not grow in the NAP containing tube
Other Mycobacteria species grow in NAP
MGIT 960
BACTEC
Bacti-Alert
VersaTrek

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

23. Fluorochrome based stain
Auramine Rhodamine –
 fluorescent stain, organisms stain fluorescent
yellowish with black background,
 Read on 25X or 40X for 2 min, viewing numerous
fields using a fluorescence microscope
 Nonspecific Fluorochromes that bind to mycolic
acids
Considered more sensitive than ZN or Kinyoun
stains for patient slide examination

24. Acid Fast staining of the Mycobacteria
Mycobacterium avium complex
Organisms are routinely shorter than TB
(Kinyoun stain)
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

25. Direct Detection of TB from Respiratory
Specimens by Amplification
 Two tests are FDA cleared to detect TB complex organisms
in smear positive and smear negative sputum specimens:
 Hologics Amplified MTD Test - Transcription Mediated Amplification
(TMA)
 Cepheid Xpert-TB RIF (rtPCR) – detects TB complex and Rifampin
resistance (rpoB gene)
 Amplify a 16S rRNA gene sequence of TB
 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
diagnosis
 Must confirm with AFB culture and sensitivity

26. Mycobacterium tuberculosis
 Optimal Temp 37˚ C, Grows in 12 –25 days
 Buff colored, dry cauliflower-like colony
 Manual tests used for identification
 Niacin accumulation Positive – Niacin
produced from growth of TB on egg containing
medium (LJ medium)
 Nitrate reduction Positive– Reduces nitrate to nitrite
 Is it reallly 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
 Currently ****Molecular probe, MALDI-TOF, & Sequencing

27. 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

28. Tuberculosis
 Classic presentation
 Slowly progressive pulmonary infection cough, weight loss, and
low grade fever
 Dissemination occurs most often in AIDS, elderly, children and
with some medications
 Initial focus: lesion with calcified focus of infection and an
associated lymph node known as Ghon’s complex
 Secondary tuberculosis: seen mostly in adults as a 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/ suspect for transmission
 All patients with high level of suspicion require precautions

29. TB in HIV/AIDS patients
 Worldwide -TB is the most common opportunistic infection
affecting HIV/AIDS patients
 AIDS patients have high likelihood to be muti-drug resistant
TB (MDRTB)
 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 specimen
 M. tuberculosis (adults)
 M. avium complex and other MOTT (2-10%)
most common in children
 Pott’s disease - TB infection of the spine
 Secondary to an extraspinal 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 plated onto media
 (2) Bactec liquid 7H9 medium with antibiotic solutions
 Tested on the automated MGIT 960 system
 Primary TB drug panel / 5 drugs
 Isoniazid * Ethambutol*
 Pyrazinamide* Streptomycin
 Rifampin *
 PCR Methods– Hybridization probes used in combination with
RT PCR assay to quantify target DNA. Used for rapid determination
of MDRTB (multi drug resistance)

32. Mycobacterium bovis
 M. bovis produces disease in cattle and other animals
 Spread to humans by raw milk ingestion
 Disease in humans similar to that caused by TB
 M. bovis can be the cause of 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 TB – or is it M Bovis?
 M. bovis = nitrate negative, M. TB = nitrate positive
 M. bovis does not grow in Thiophene-2-carboxylic hydrazide
 TB grows in the T2H compound
 M. bovis does not produce Pyrazinamidase (PYRZ) enzyme
 TB produces PYRZ enzyme

33. Mycobacterium ulcerans
 Infection begins as boil and develops a painless skin
ulcer known as Bairnsdale or Buruli ulcer
 Can progress into avascular coagulation necrosis
 Found primarily in the African continent and tropical
environments
 Optimum growth temp 30˚ C
 All skin lesions should be
cultured at both 30˚ and 37˚C
 Slow growing 3- 4 weeks
 Negative – niacin accumulation
and nitrate test

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
 Acid fast stain: cells are long, rectangular and beaded,
larger than TB/ Shepherd’s crook
 Clinical disease in adult mimics pulmonary TB but does
not disseminate from lung – predisposition diseased lung
 Causes granulomatous inflammation in lung

35. Mycobacterium marinum
 Optimum temp for growth is 30˚ C
 Growth in 5-14 days
 Photochromogen
 M. marinum found in both fresh and salt water sources
 Associated with trauma in Swimming pools, fish tanks,
water cooling towers
 Disease known as “Swimming pool granuloma”

36. Mycobacterium marinum Disease
 Tender, red or blue/red subcutaneous nodules following
trauma to skin
 Biopsy (skin) specimens must be cultured at 30*C
 Lesions can continue to extend up arm and spread 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
 Niacin accumulation = negative
 Nitrate test = positive
 Unusual 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
 Could be considered “contaminate”
 Clinically disease is like TB
 Occurs in patients with preexisting lung
lung disease
 Can be seen in HIV/AIDS patients

39. M. avium-intracellulare complex
 M avium and M intracellulare – two species
 Biochemically and genetically very difficult to distinguish
 Opportunistic infection in HIV/AIDS
 High organism load in AFB stains of in
intestine, liver, spleen and bone marrow
 Isolated from blood cultures
 Involvement of GI tract can cause diarrhea
 Positive AFB smears in stool
 Tissue Biopsy with inflammation
 Organisms mostly short rods
 Shorter than TB organisms
 Do not have cord factor
 Pathology - Necrotizing rather than
granulomatous inflammation

40. M avium-complex in lymph node tissue –
Kinyoun AFB stain
M. avium complex
Kinyoun AFB stain variable in
size – absence of cording
Granulomatous inflammation
In lung tissue – H&E stain
Bowel -Lamina propria expanded from
predominately Lymphohistocytic infiltration

41. M. avium-intracellulare
 Laboratory –
 Growth at 37 ˚C / 7 – 21 days
 Non-photochromogen
 Smooth colony
 Inert in biochemicals
 Identify using
 GenProbe (AccuProbe) molecular identification
 MALDI-TOF
 Genetic 16s rRNA Sequencing

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 pulmonary disease, much like TB,
 marked % of cases – elderly adults with lung
damage (history of smoking)
 In children – lymphadenitis
 M. avium ssp. paratuberculosis –
 ? Association with Crohn’s disease
 Inconclusive evidence for causation

43. Rapid growing Mycobacteria species
 Laboratory
 Growth at 37˚C in <=7 days
 Positive Arylsulfatase test
 @ 20 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
 Biochemical reactions:
 M. fortuitum Nitrate Positive and Iron Uptake positive
 M. chelonae and M. abscessus Nitrate Negative
 MALDI-TOF and Sequencing for identification

44. Miscellaneous
 M. gordonae –
 Rare! Cause of Infection
 Major laboratory water contaminant in cultures
”tap water bacillus” – present in water systems
 Use sterile water in culture processing to prevent
contamination
 Scotochromogen

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

46. Mycobacterium leprae
 Leprosy – also known as Hansen’s disease
 Begins with anesthetic skin lesions and
peripheral neuropathy with nerve thickening
 Presenting presentation numbness in earlobes or nose
 Lapromatous leprosy – severe disfiguring lesions,
large numbers of AFB in lesions / associataed with
co-infection with Strongyloides stercoralis
 Tuberculoid leprosy -less severe/fewer lesions, lower
numbers of AFB in lesions
 Will not grow on laboratory AFB media
 Armadillo is the natural reservoir
 PCR on tissue for definitive diagnosis

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