Mycobacteria

(Nontuberculous Mycobacteria and M. leprae )

Martin E. Evans, MD
Division of Infectious Diseases
Department of Internal Medicine
February 15, 2013
Objectives
Understand basic concepts of mycobacterial taxonomy, biology, and host immune response
Be familiar with nontuberculous mycobacteria (NTM) and the diseases they cause
Be able to describe the transmission, pathophysiology, and clinical syndromes of leprosy
Mycobacteria Microbiology/Taxonomy
Taxonomic Tree: genus mycobacterium, species: M. tuberculosis complex, NTM, m. leprae
M. tuberculosis
complex

m. leprae

M. tuberculosis
M. bovis (BCG
strain used as
vaccine)
M. africanum
M. microti

NTM:
Nontuberculous
Mycobacteria
Slow growers
Rapid growers

Nontuberculous Mycobacteria

Slow growers (NTM)

Photochromogens
o M. kansasii
o M. marinum
Scotochromogens
o M. scrofulaceum (Kings Evil)
o (Also M. gordonae)
Nonchromogens
o M. avium complex
o (Also M. terrae, M. xenopi, m. ulcerans)

Scotochromogenic bacteria develop

pigment in the dark or in the light.
Runyon Group II nontuberculous
mycobacteria are examples but the term
could apply to many other organisms.

Rapid growers (NTM)

1. M. fortuitim (this one is a common pathogen)
2. M. chelonae
3. M. abscessus
Mycobacterial differences
M. tuberculosis and M. leprae
Pathogenic for healthy humans
Major reservoir is humans (exception that M. bovis can be in cows milk)
Transmitted from person to person
Nontuberculous mycobacteria (NTM)
Usually only pathogenic for unhealthy humans (high dose steroids, COPD, AIDS, etc)
Exist in free-living state in the environment
Not transmitted from person to person (NTM is NOT transmitted via droplet nuclei)
Mycobacterial Cell Wall (see bacterial architecture lecture)
Lipoarabinomannan (LAM)
LAM is a lipid polysaccharide virulence factor

Lipoarabinomannan, also called LAM, is a glycolipid, and a virulence factor

associated with Mycobacterium tuberculosis, the bacteria responsible for
tuberculosis.
Its primaryby
function
is to inactivate
macrophages and scavenge
Inhibits processing of mycobacterial
peptides/proteins
antigen-presenting
cells
Induces tumor-necrosis factor oxidative radicals.
Mycolic Acids
The inactivation of macrophages allows for the dissemination of mycobacteria
unique to mycobacteria
probably responsible for acid-fastness to other parts of the body. The destruction of oxidative radicals allows for the
survival of the bacteria, as oxidative free radicals are an important mechanism
by which our bodies try to rid ourselves of infection.

Red Snapper appearance (acid fast

stain)

Carbol Fuchsin - Acid-Alcohol Counterstain

Methylene Blue
Zhiel-Neelson Stain (Acid Fast Stain)
Patient sample taken, often sputum
Red carbol-fuchsin stain, decolorize w/ acid-alcohol, methylene blue counterstain
You get red staining bacteria against a blue counterstaining background
Can also stain with Auramine-Rhodamine (fluoresce)
Pathophysiology

Transmission of M. tuberculosis
M. tb spread via airborne particles called droplet nuclei (5um)

Expelled when person with infectious TB coughs, sneezes, shouts, or sings

Transmission occurs when droplet nuclei inhaled and reach the alveoli of the lungs, via nasal passages,
respiratory tract, and bronchi
Phase I: Infection or Not?
Hardiness of the bacterium
o How well survived transmission to alveoli through droplet nuclei
o Intrinsic virulence

Inhibitory activity of alveolar macrophage

o Genetics
o Prior, non-specific stimulation
Primarily CR3
Phase II: Early proliferation and spread (days to weeks)
Only 1-3 bacilli typically reach the terminal alveolus in a droplet nucleus
Paradoxin order to survive, the tubercule bacillus must promote its uptake by macrophages
They are opsonized and then phagocytosed by macrophages via CR1, CR3, and CR4 and also
mannose receptors (binds LAM)
Once inside the macrophage
AM = Alveolar Macrophages
The bacilli replicate
Macrophages elaborate cytokines and chemokines that attract additional AMs, peripheral
blood ,monocytes, dendritic cells, lympocytes, PMNs
1. TB endocytosis via mannose receptors binding LAM, and complement receptors
2. Phagosome-lysosome fusion blocked, bacteria not killed
3. AM releases TNF- which stimulates the AM itself
4. AM releases IL-12 which attracts and stimulates CD4 and CD8 / T lymphocytes
5. AM releases chemokines which attract cells from the circulation
Proliferation
During this phase, the macrophages are not activated and the bacteria multiply.
Fatally damaged macrophages disgorge their burden of bacilli.
These bacilli are engulfed by incompetent phagocytic cells that have migrated to the site.
Creating additional breeding capacity for the bacillus
TB bacilli replicate every 12-16h 540,000,000 bacilli in 20 days!!
Spread
Mycobacteria are transported via centripetal lymphatics to hilar lymph nodes.
and bacteria spread hematogenously
From terminal alveolus mediastinal lymphatics thoracic duct left subclavian vein

 By direct invasion into alveolar capillaries

Via transportation by macrophages or PMNs that have left the bloodstream, traversed the
inflammatory site, engulfed bacteria, and returned to the bloodstream
Seeding most common to apices of lungs, kidneys, bone growth plates, vertebrae
Phase III: Evolution of Cell Mediated Immunity
1. Mycobacterial proteins broken down in phagolysosome
2. Ag remaining in the phagosome is presented via CD1a or MHC-II pathway to CD4 cells
3. Mycobacterial Ag that has escaped into the cytosol is presented via MHC-I pathway to CD8 cells
4. Activated T-cells release IFN- which further activates the macrophage
5. IFN-
a. MHC-II expression for improved Ag presentation
CD1a presents lipid and glycolipid antigens
b. LRG-47 formation of phagolysosome
c. expression of inducible nitric oxide synthase (iNOS) which produces nitric oxide resulting in
bacterial killing
Option 1 mild DTH (Delayed Type Hypersensitivity/Type IV Hypersensitivity->Cell-Mediated)
Bacterial proliferation is halted and the bacillary population falls substantially
The primary lesion and metastatic foci involute leaving minimal residual
The tuberculin skin test (TST)/PPD Test and INF- release assay (IGRA) become positive
Bacterial killing.
Option 2 aggressive DTH (Delayed Type Hypersensitivity)
Bacilli not killed
Chronic antigen stimulation and release of cytokines (IFN-, TNF-, and IL-2)
inflammation
Macrophages & CD4 T cells maintain granulomas
Granuloma formation
Caseating necrosis (destruction & liquefaction of tissues)
Increased bacterial numbers

Nontuberculous Mycobacteria

M. kansasii, M. marinum, M. scrofulaceum, M. avium complex

Clinical Syndromes of Nontuberculous Mycobacteria

Progressive pulmonary disease resembling TB

M. avium complex (MAC) or M. kansasi
Disseminated disease in immunosuppressed
MAC
Skin and soft-tissue infection
Rapidly growing Mycobacteria (M. chelonae, abscessus, fortuitum)
Cervical lymphadenitis (usually in children)
MAC (M. Avium Complex)
M. scrofulaceum (Kings Evil)
M. malmoense (mainly in Europe)
Swimming pool granuloma
M. marinum

2 Clinical Presentations of Pulmonary MAC Disease

Older male with COPD

Smoker
Cough, weight loss
Apical infiltrates that look like TB
Lady Windermere Syndrome
Thin elderly female, pectus excavatum
Often non-smoker, bronchiectasis (bronchi dont taper distally, so difficult to cough
secretions)
Lower lobe disease

Disseminated MAC Disease

Patients with AIDS, hematological malignancies, etc.

Bacteremia with fever, fatigue, weight loss.
Multiple organ involvement:
-bone marrow (anemia, leukopenia)
-liver/spleen (organomegaly)
-GI tract (diarrhea, abdominal pain)
-pulmonary (cough, dyspnea, diffuse infiltrates)
Diagnose with blood culture

Rapidly Growing Atypical Mycobacteria

M. fortuitum, chelonei, abscessus
Infection by direct inoculation, often skin/soft tissue sites
Clinical setting: surgical wounds, central vascular catheters, peritoneal dialysis catheters, IM injection
sites
M. scrofulaceum (The Kings Evil)
Found in soil
Cause of cervical lymphadenitis in children (along with MAC)
Treat by removing the lymph node
M. marinum
Found in fresh and salt water
Swimming pools
Aquaria
Grows at 30 degrees but not 37 degrees
Distal skin ulceration with sporotrichoid lymphadenopathy progression of raised bumps that
travel up lymph nodes

Leprosy = Hansens Disease

Historical Perspective
Clinical description known since writing was possible
Image of physical mutilation
Social exclusion coined the term Leper
Cause discovered by Norwegian physician, G.A. Hansen in 1873
Named Hansens disease to decrease social stigma
Leprosy Distribution
246,007 cases reported globally in 2008
90% of cases came from 11 counties led by India and Brazil
About 150 new cases in US each year (1/3 acquired locally)
Probable Zoonotic Leprosy in the Southern United States
A unique M. leprae genotype (3I-2-v1) was found in 28 of 33 wild armadillos and 25 of 39 U.S. leprosy
patients who resided in areas where the armadillos lived

 This genotype has not been reported elsewhere in the world

Conclusion: Armadillos are a large natural reservoir for M. leprae and leprosy may be a zoonosis in
this region.
Mycobacterium leprae
Slow growinggeneration time 12.5 days
Grows best at 27C to 33C, which correlates with its affecting cooler areas of the body (skin, nerve
segments close to the skin, mucous membranes of the upper respiratory tract)

Johnston Lecture (Biofilms): Mycobacterium leprae/ leprosy cannot grow at 37oC; requires lower
T so it forms lesions on skin, esp. extremities (nose, fingers). Bacteria not found in warmer areas
of body (core).

Never grown in culture, but grows in tissues of the armadillo which has a core body temperature of
34C
Transmission of M. leprae
Definitive route never identified
May be aerosols from respiratory tract since nasal secretions of patients with lepromatous leprosy
contain large
numbers of bacilli
Direct contact with ulcerated skin lesions may be important
Pathophysiology
Inhaled bacteria taken up by alveolar macrophages or dendritic (antigen-presenting) cells in the nasal
mucosa or skin
Disseminates hematogenously, but replicates only in relatively cool parts of the body. Preferred cells
are macrophages and Schwann cells.
Incubation period: 3 months to 40 years (average 2 to 7 years)
Subsequent cytokine/chemokine production directs adaptive CMI into a Th1 or Th2 response to M.
leprae.
Cytokine Gene Expression in Leprosy
T-cell type 1 (Th1) like response
Predominance of IL-2, lymphotoxin, and INF- which promotes host resistance
(Kaplan) Tuberculoid T cells are Th1and T cells respond to IL-12
T-cell type 2 (Th2) like response
Predominance of IL-4, IL-5, IL-10, and much less IFN leading to little host resistance
(Kaplan) Lepromatous T cells are Th2, and T cells do not respond to IL-12
M. leprae Clinical Classification reflects progression of disease based on host immune response
Tuberculoid (TT)
Borderline Tuberculoid (BT)
Mid Borderline (BB)
Borderline Lepromatous (BL)
Lepromatous (LL)

The Hansens Disease Spectrum

TT > BT
>
BB
>
Tuberculoid
Th1
o High resistance to M.
leprae
o Few bacilli
(paucibacillary
form)
o IL-1, IL-2, IL-12, IL18, TNF-,
and IFN- produced
o Low antibody
Granulomatous
lesions
Nerve Injury in Leprosy
Immune Mediated (tuberculoid)

BL
>
LL
Lepromatous
Th2
o Low resistance to M.
leprae
o Many bacilli
(multibacillary
form)
o IL-4, IL-5, IL-10
produced
o High antibody
(ineffective)
Foam cell lesions

 Marked increase in pro-inflammatory cytokines (TNF-, IL-1, and interferon-) within and
around the nerve
Immune mediated injury can occur before diagnosis, and during and after treatment
Direct Invasion (lepromatous)
Bacillus interacts with specific protein (lamina 2) found in basal lamina of the Schwann cell
and binds to it
Bacteria binding to 2 chain of Laminin produces Schwann cell tropism (Kaplan)
Binding mediates invasion followed by rapid demyelination independent of cellular immunity.
Genetic Factors Affecting Host Response
95% of the worlds population is immune to leprosy
Some persons cannot mount a response to lepromin, a bacterial extract
Occurrence of leprosy more common in identical twins
Susceptibility linked to polymorphisms in the NRAMP1 gene (a transmembrane protein to pump Fe++
out of lysosome)
HLA type important
HLA-DR3 tuberculoid
HLA-DQ1 or HLA-MT1 lepromatous
(Kaplan)Mutations in promoter region of PARK2 is a risk factor for leprosy
PARK2 codes for a ligase in the ubiquitin-proteosome pathway
regulates antigen processing within M and may influence antigen presentation
(Kaplan) NOD2 mutations are risk factor for LL
NOD2 responds to muramyl dipeptide, and helps limit spread of M. leprae
Distribution of clinical forms
In India and Africa, 90% of patients are tuberculoid
In Southeast Asia, 50% are lepromatous and 50% are tuberculoid
In Mexico, 90% are lepromatous
Tuberculoid Leprosy
Anesthetic lesions: cannot feel pinprick/touch within the lesion. Hypopigmented lesion w/ pigmented
border.
Can involve great auricular nerve
Burns due to anesthetic infection (no sensation thru nerves)
Claw hand as result of ulnar nerve involvement
Lepromatous Leprosy
Loss of eyebrows
Involves cool areas (face, nose, earlobe, extremities, can include testicular involvement)
Clinical Features of Leprosy
Nerve involvement
Sensory or motor loss
Fingers/toesresorption of tips
Ulnar/median nerve = claw hand
Peroneal nerve = foot drop
Posterior tibial nerve = claw foot/ulcer
Loss of eyebrows/lashes
Perforated nasal septum ("leonine appearance")
Hoarseness due to laryngeal involvement
Diagnosis of Leprosy
Kaplan: Fernandez reaction is Delayed Type Hypersensitivity-bases skin test for Leprosy
Lepromin injected under skin
History of travel to or residence in an endemic area (ingestion of armadillos?)
Anesthetic skin or mucous membrane lesions
Thickened peripheral nerves
Detection of AFB in slit-skin smears or skin biopsies that include sub-epidermal tissue confirms the
diagnosis
Fite stain of tissue biopsy red snapper appearance with stain
Leprosy: Infection Prevention and Control 2012 AD
Tuberculoid patients are generally not contagious because of the small numbers of bacteria

 When a patient with either tuberculoid or lepromatous leprosy is placed on medication, most of
bacteria are killed within a few days.
Within two weeks of starting the treatment, there is no risk of transmission.
It is not necessary to isolate a patient at anytime.
It is not transmitted through sexual contact or pregnancy.

M. tuberculosis

Transmission mainly human to human,

animal reservoircows milk
Transmission via inhalation of infectious
nuclei
Latent TB infection identified by TST and
treated
Target site: lung
Can be cultured on artificial media. Does
not require animal inoculation

M. leprae

Transmission mainly human to human,

animal reservoirarmadillos
Route of transmission unknown, but may
be inhalation of infected respiratory
secretions
Patient identified by clinical disease only
Target site: skin/nerve
Cannot be cultured on artificial media.
Need animal inoculation armadillo or
mouse foot pad

Summary
Global control of leprosy has been successful with only 3 countries currently reporting annual
rates of >1 new case/10,000 persons at risk.
The sine qua non of leprosy is its ability to infect peripheral nerves.
The organism still eludes culture in artificial media.
New knowledge of leprosy has delineated the impact of leprosy on the immune responses by the host
but much remains to be understood.
Effective treatment is available and is most successful when begun early in the course of the illness

Exam #1 (Murphy)
Caseous necrosis - Tissue grossly appears "cheesy". Microscopically, necrotic focus of fragmented
cells and granular cellular debris surrounded by border of granulomatous inflammation. (See
"Chronic Inflammation" lecture.) Characteristic of tuberculosis (TB).
Granulomatous Inflammation - A distinctive pattern of chronic inflammation characterized by focal
aggregates of granulomas within the tissue. At its most basic, a granuloma is a focal aggregate
of macrophages. There are 2 types of granulomas, which differ in their pathogenesis.
A. Immune granuloma.
1. Forms under 2 conditions:
a. Macrophages phagocytose a substance that they are unable to degrade. Usually a
microorganism, such as Mycobacterium tuberculosis or Treponema pallidum (syphilis).
b. The indigestible substance is immunogenic, in that it induces a cell-mediated immune
response by T-lymphocytes. This results in transformation of the macrophages into
epithelioid cells (see below).
2. Morphologically, the immune granuloma consists of:
a. Epithelioid cells - i.e. macrophages that are transformed to resemble epithelial cells.
b. Langhans-type giant cells - Huge cells formed by the fusion of many epithelioid
macrophages. Voluminous cytoplasm with up to 50 macrophage nuclei within. Nuclei are
arranged around the periphery of the cell.
c. Other cell types - Surrounding the mixture of epithelioid and giant cells is a collar of
lymphocytes, plasma cells and fibroblasts, which may produce collagen.
d. Necrosis - Depending on the specific disease, the granuloma may have a necrotic center
(caseous, or cheesy, necrosis). Classic feature of TB granulomas, as well as some fungi.
3. Diseases that can cause immune granulomas:
Disease
Tuberculosis
Leprosy (tuberculoid type)
Histoplasmosis
Coccidioidomycosis
Q fever
Brucellosis
Syphilis
Sarcoidosis
Crohns disease

Antigen
Mycobacterium tuberculosis
Mycobacterium leprae
Histoplasma capsulatum
Coccidioides immitis
Coxiella burnetii (rickettsial organism)
Brucella species
Treponema pallidum
Unknown
Unknown

Note: Common name of TB granuloma is the tubercle. Syphilis granuloma is called the
gumma (rubbery).
Exam #4 (Stevenson)

Antibiotics against Tuberculosis:

NOTE: Stevenson is discussing antibiotics that target Acid Fast Cell Wall
Synthesis
Cycloserine : blocks Alanine Racemase & D-Alanine Ligase (targets are not in notes)

Isoniazid (INH)
Inhibits mycolic acid synthesis
Highly specific for Mycobacterium tuberculosis
Bactericidal
1st line drug in combination therapy of tuberculosis infections

Ethambutol (EMB)
Inhibits synthesis of arabinogalactan and lipoarabinomannan
Bacteriostatic within macrophages
1st line drug in combination therapy of tuberculosis infections
Pyrazinamide (PZA)
Appears to inhibit lipid metabolism
Bactericidal
Active in acidic environment
Activated by bacterial enzyme
1st line drug in combination therapy of tuberculosis

Exam #4 (Various)
Problems with Anti-TB drugs due to:
1.Very slow growth (antibiotics target metabolically active/dividing cells)
2.The thick, waxy outer covering of mycobacterium excludes many drugs
3.Mycobacterium Tb is an obligate intracellular parasite-drugs have to get into eukaryotic
cells, too.
Exam #4 (Straley)

Mycobacterium Tuberculosis -> Aerobe: Use respiration.

Require O2; cannot ferment.
Catalase +/Peroxidase +/SOD +

Tetrahydrofolate: critical cofactor in the

synthesis of thymidylate, nucleotides,
and amino acids.

Two enzymes in bacterial

Tetrahydrofolate (AKA
TH4/FH4/FAH4) synthesis:
1.Dihydropterate synthetase
(Sulfonamides, p-Aminosalicylic
acid)
2.Dihydrofolate reductase (DHFR)
(Trimethoprim, and the chemo drug
methotrexate)

p-Aminosalicylic acid resembles p-ABA. Although it isn't a sulfonamide, it has a similar

function. This drug is a competitive inhibitor of dihydropteroate synthetase
dihydropteroate synthetase inhibits the conversion of p-ABA to dihydrofolate (FAH2)
Bacteria can't take up FAH2, so this enzyme is essential
This is bacteriostatic

Aminoglycosides inhibit peptide chain elongation

Dead initiation complex, miscoding
Effective vs. aerobic and facultative bacteria, incl. intracellular pathogens such as M. tuberculosis
Streptomycin, gentamicin, neomycin, amikacin, kanamycin, capreomycin,
Penetrate eukaryotic cell membranes well
Rifamycins inhibit prokaryotic RNA polymerase

Block transcription by binding to RNAP (there's only one RNA

polymerase in bacteria).

 Protein synthesis also ceases as secondary effect of inhibition of

transcription.
Rifampin is a front-line antitubercularChemo
drug.drugs that inhibit HUMAN
Topoisomerase II:
Fluoroquinolones inhibit Topoisomerase II.

1.Doxorubicin (makes your pee red)

2.Etoposide

First Line Tb Drugs

Second Line Tb Drugs

Pyrazinamide

p-Aminosalicylic acid

Isoniazid (INH)

Ethionamide

= isonicotinyl hydrazine

Cycloserine

Ethambutol

Fluoroquinolones

Rifamycins

Aminoglycosides

The drugs in red target Acid Fast Cell Wall Synthesis

Kaplan (Exam #4)

-Lepromin-acute induration, 24-72 hr (Fernandez reaction: Skin Test for Leprosy that
uses Delayed Type Hypersensitivity to test for M. Leprae)
M. Tb infects macrophages
M. leprae infects macrophages and Schwann cells; Bacteria binding to 2 chain of laminin
appears responsible for tropism for Schwann cells

Mutations in promoter region of PARK2 is a risk factors for leprosy. PARK2 codes for a
ligase in the ubiquitin-proteosome pathway which regulates ag processing within M and
may influence ag presentation.

Immune Response to M. Leprae:

Mutations in NOD2 are a risk factor for LL (Lepromatous Leprosy)

Activation of M by TLR2/1 (triacylated lipopeptide) but not NOD2 (MDP) triggers M
differentiation whereas activation of M VIA NOD2 preferentially triggers DC
differentiation.
Both live M.leprae and NOD2 but not TLR2/1 induced IL-32 which was required for NOD2mediated induction of M differentiation into DCs and was sufficient to induce DC
differentiation.
Skin lesions and PB monocytes from patients with leprosy demonstrated that the NOD2induced pathway correlated with self-limited versus the disseminated form of the disease.

The critical role of TNF- in resistance to M.tuberculosis in humans is demonstrated by the

increased risk of reactivation in rheumatoid arthritis patients treated with anti- TNF- mAb (ex:
using the anti-TNF drug Etanercept -> Norris Immunosuppressant lecture)

Immune Response to Tuberculosis

1.Lipoproteins activate Ms and DCs via TLR1/TLR2
Results in IL-12 & NOS2 expression (iNOS)
2.One of the major ways IFN- and TNF- kill mycobacteria is by causing Nitric Oxide (NO) to be made
Ms iNOS makes NO!
TNF-also helps with granuloma formation
3.IFN- induces LRG-47
Tuberculosis is good at blocking the phagosome from killing it
LRG-47: GTPase that is too powerful for tuberculosis; phagosome kills tuberculosis
4.Th1 response is critical for controlling tuberculosis
IL-12 drives Th1 response!
Mutations in IL-12p40 or IL-12 receptor genes will mean less IFN- and increase
Susceptibility to mycobacteria in general (M. bovis, M. avium, etc.)
5.TNF-(TNF) is critical for controlling tuberculosis
Enbrel (Etanercept) blocks TNF and increases reactivation of tuberculosis
6.IFN- is critical for control tuberculosis. Block it or the receptor and youll have problems with
tuberculosis (or any intracellular bacteria)
7. Activated T cells see lipoarabinomannan (LAM) and glycolipids via CD1 presentation
Produces IFN- or kill infected host cells
8.MHC class II presentation activates CD4+ T cells
Tuberculosis lives in vacuole in M
This is where MHC class II get the antigens they present
9.CD8+ CTLs secrete IFN- and lyse infected Ms
MCH Class I presentation is also important
M. tuberculosis Immune evasion
1.Impaired MHC class II presentation
2.Anti-inflammatory mediator Lipoxin A4
Arachidonic acid metabolite
Inhibits neutrophil chemotaxis & endothelium adherence
3.Regulatory T cell (Treg) induction
4.Downregulate bacterial antigens
5.Resistance to the Mactivating effects of IFN-