M. tuberculosis

Report
Microbiology 2011
5/11/2011
Yu Chun-Keung
Chapter 28
Mycobacterium
Chapter 27
Nocardia
Actinomyces
Tuberculosis
Tubercular decay has been
found in the spines of Egyptian
mummies. Pictured: Egyptian
mummy in the British Museum
[Source: Wikipedia]
One new case / 36 min
One death / 6 hours
Genus Mycobacterium (分枝桿菌屬)
Nonmotile, non-spore-forming aerobic bacilli
Ubiquitous presence, 130 species; 7 species cause most
infections
Pathogenic Mycobacteria
M. tuberculosis (結核分枝桿菌) : airborne
M. leprae (痲瘋分枝桿菌) : close contact
M. avium complex : water/soil
M. kansasii : water/soil
M. fortuitum : water/soil
M. chelonae : water/soil
M. abscessus : water/soil
Cell Wall of Mycobacteria
G(+) bacteria
Inner cytoplasmic membrane
Anchor : proteins, phosphotidylinositol mannosides,
lipoarabinomannan (LAM)
Thick peptidoglycan layer
Attach arabinogalactan (a branched polysaccharide) + mycolic acid
(70-90 carbon)
No outer membrane
Cell Wall of Mycobacteria
Rich in lipids: responsible for many
characteristic properties (acid-fastness, resistant
to disinfectants and antibiotics, antigenicity, slow
growth, clumping), 60% of dry weight.
Acid-fastness: mycolic acid -resistant to common
laboratory stain. Once stained, cannot be
decolorized with acid solutions
Polypeptides: transport proteins and porins;
15% dry weight; PPD (purified protein derivatives)
- induce cell-mediated immunity.
Preliminary classification of mycobacteria by
growth properties and colonial morphology
1. M. tuberculosis complex
slow-growing, no pigmentation
2. Non-tuberculous mycobacteria (NTM)
Runyon group I
slow-growing, yellow pigment (+) in light
Runyon group II
slow-growing, yellow pigment (+) in dark
Runyon group III
slow-growing, nonpigmented
Runyon group IV
rapidly growing
A pigmented and / or a rapidly growing mycobacterium
 M. tuberculosis
1. Mycobacterium tuberculosis complex
Tuberculosis

M. tuberculosis
(airborne/droplet)

M. bovis
(unpasturized
products)
milk
M. tuberculosis - pathogenesis
Inhalation of aerosolized infectious particles to alveoli
Intracellular pathogen, infect inactivated alveolar macrophages,
lifelong infections
Prevent phagosome-lysosome fusion by blocking early
endosomal autoantigen 1 (EEA1)
Acquire nutrients from other intracellular vesicle
Resist killing by nitric oxide and superoxide anions
Induce protective immunity: macrophages release IL-12 and
TNFα, and recruit T cells and NK cells; induce TH1 cell; secrete
IFN-γ; activate macrophages; increase intracellular killing
Infected cells spread locally (lymph nodes) or hematogenous
spread (bone marrow, spleen, kidneys, CNS), and attract
macrophages and lymphocytes; formation of granuloma
Caseous necrosis
(乾酪壞死)
Granuloma (肉牙腫)
Inner mass : alveolar macrophages, epithelioid cells, Langhans
giant cells (fused epithelioid cells)
Dense wall : CD4, CD8, NK, T cells, macrophages
Caseous necrosis (乾酪壞死)
Signs and symptoms
Most infections are restricted to the lungs
Insidious at onset
Non-specific : malaise, weight loss, cough,
night sweats, sputum (purulent, bloody =
lung tissue destruction, cavity)
Primary focus is present in middle or lower
lung fields.
Hematogenous spread : disseminated
(miliary粟粒狀) tuberculosis
Clinical disease
In most patients replication ceases within
3-6 wks after CMI is activated. PPD (+), x-ray (-)
~5% infected patients will progress to
active disease within 2 years. PPD (+), x-ray (+)
Other 5-10%, Latent PPD (+), x-ray (-), will
develop disease sometime later in life. PPD
(+), x-ray (+)
Progression to disease is related to
infectious dose and the patient’s immune
competence.
Pathogenesis
No toxins and enzymes
Histological signs (i.e., granuloma) are host
immune responses to infection (DTH
response)
Immunopathology results in tissue necrosis
(cytokine toxicity, complement activation,
ischemia, etc.)
Pathogenesis
Small antigenic burden + protective immunity:
activated macrophages can penetrate small
granulomas (< 3 mm) and kill all bacteria with
minimal tissue damage.
But if many bacilli are present, cellular immune
response (over-reactive, impaired) results in
formation of large, necrotic or caseous
granulomas encapsulated with fibrin, which
protect bacteria from macrophage killing (latent),
thus may be reactivated years later when
patients’ immunologic responsiveness wanes.
Epidemiology
Humans - natural reservoir
Non-human primates and guinea pigs experimental infection
Person-to-person contact

Inhalation: small particles containing one to
three tubercle bacilli can reach alveolar
spaces and establish infection.
 Patients with tubercle bacilli in sputum infect 10-
15 people/year.

Ingestion of unpasteurized milk products (M.
bovis)
Epidemiology
One third of the world’s
population is infected.
9 million new cases and
2 million deaths annually.
Endemic area:
Southeast Asia, subSaharan Africa, Eastern
Europe
In USA, immigrants,
homeless persons, drug
and alcohol abusers,
prisoners, HIV patients
World TB incidence. Cases per
100,000; Red = >300, orange =
200–300; yellow = 100–200;
green 50–100 and grey <50.
Data from WHO, 2006.
Clinical Diagnosis of M. tuberculosis
In most patients, the only
evidence of infection :
• Radiographic evidence of
calcification in lungs
• Lifelong positive tuberculin
skin test to PPD
[Source: Wikipedia]
• Lab detection of mycobacteria :
microscopy or culture.
Immunodiagnosis
(for M. tuberculosis)
Tuberculin skin test
[Source: Wikipedia]
• 5 unit PPD, intradermal; diameter of the
area of induration after 48h
• Patient’s response to exposure to M.
tuberculosis (a DTH response)
• Differentiate between infected and noninfected people.
Reactivity to PPD
10 mm (positive): 3-4 wks after infection;
lifelong
 acute disease or protective immunity
= has been infected & develop immune
response
False negative: anergy (eg. HIV infection),
overwhelming TB
False positive: BCG vaccination, isoniazid
treatment
In vitro IFN-γ release assays
Patient’s whole blood (contain sensitized
T cells)
Stimulate with M. tuberculosis-specific
antigens (i.e., ESAT-6, CFP-10)
Monitor IFN-γ production
More sensitive and specific alternative to
PPD skin test
Microscopy
The most rapid method to confirm mycobacterial disease acid-fast stain
Carbolfuchsin strain :
Ziehl-Neelsen method or
Kinyoun method
Auramine-rhodamine strain :
Truant-fluorochrome method
Specificity > 95% (mycolic acid)
Sensitivity relate to (1) respiratory
specimens and (2) specimens with high
bacterial count
Positive acid-fast stain = high infectivity
Cannot identify mycobacterial species

Sputum specimens with acid-fast stain
“positive” : must differentiate between true
infection with M. tuberculosis and transient
colonization of other non-pathogenic spp.
Culture
Specimens:
Pulmonary infection – abundant organisms in
respiratory secretions
Extrapulmonary infection – need repeated
samplings
Most isolates grow slowly; thus obscured by
rapidly growing bacteria
Specimen (sputum) decontaminated with 2%
NaOH.
Egg-based medium: Löwenstein-Jensen
Agar-based medium: Middle-brook
Broth culture of special formula : reduce
culture time of most mycobacteria (from 34 wks to 10-14 days)
Microscopic observation drug susceptibility
assay (MODS):




24-well plate with broth and antimycobacterial
drug
Add specimen
Mycobacteria growth is detected by an
inverted microscope
Susceptibility test done simultaneously
2. Mycobacterium leprae
Leprosy (癩病) (Hansen’s disease): a
chronic infection that affects skin and
peripheral nerves (Schwann cells and
macrophages)
Since 1985, Global prevalence drops
dramatically
Endemic in few countries
in Asia, Africa and Latin
American
Endemic in armadillos
Nine-banded Armadillo (犰)
Leprosy
Long incubation period > 20 years
Several clinical manifestations - depend on
patient’s immune reaction to the bacilli
Tuberculoid leprosy (類結核型痲瘋病)
Lepromatous leprosy (痲瘋結節型痲瘋病)
Tuberculoid
leprosy
Hypopigmented
skin macula
Peripheral nerve
damage with
complete
sensory loss
Lepromatous
leprosy
Many erythematous
macula, papules or
nodules
Extensive disfiguring
skin lesions (nasal
cartilage, ears)
Tuberculoid leprosy
• Paucibacillary Hansen disease
• Strong cell-mediated immunity
• Granuloma in tissues with few bacilli; low
infectivity
Lepromatous leprosy
• Multibacillary Hansen disease
• Strong humoral immunity
• Abundance bacilli in skin and peripheral
nerves; infectious
Transmission
Person-to-person contact
Inhalation of infectious aerosols
or skin contact with respiratory
secretions and wound exudates
Diagnosis of M. lepare
Base on clinical disease
M. leprae cannot grow in cell-free cultures
Histopathology - presence of acid-fast
bacteria in lesions
Skin test to lepromin

Lepromin: derived from inactivated M. leprae

Confirmative test for tuberculoid leprosy

No use for lepromatous leprosy (anergy)
3. M. avium complex (MAC)
Common environmental isolate in soil, water, plants.
Developed after ingestion of contaminated food or
water, no person-to-person spread
In immunocompetent patients:
M. avium subsp. intracellulare
Recover from clinical specimens – mostly transient
colonization (= infection,  disease)



Three forms:
Middle-age or older men with a history of smoking and
underlying pulmonary disease;
Elderly, female nonsmokers (pneumonitis, bronchiectasis)
Solitary pulmonary nodule
In HIV-infected patients:
• M. avium subsp. hominissuis
• The most common
mycobacterial disease in
USA, typically disseminated
• All organs are involved,
especially in terminal stages
with CD4 T counts < 10
cells/mm3
Tissue from a patient with AIDS who is infected
with Mycbacterium avium complex
4. Slow-growing mycobacteria (3-8 weeks)
Present in soil and water
Opportunistic pathogens for immunocompromised patients
Thus, isolation in specimens of immunocompetent patients mostly represents a
transient colonization
No person-to-person spread (except
M. bovis)
Slow-growing mycobacteria
M. bovis
M. kansasii
Pulmonary tuberculosis
M. scrofulaceum
Lymphatic tissue infection
M. ulcerans
M. marinum
M. haemophilum
Cutaneous infection
New species and spectrum
of diseases are expending.
5. Rapidly growing mycobacteria (< 7 days)
M. fortuitum, M. chelonae, M. abscessus.
Relatively low virulence, susceptible to
“conventional” antibacterial antibiotics
Infections establish in deep subQ tissues
after introduced by trauma or iatrogenic
infections.
No person-to-person spread
Incidence increases as invasive procedures
increases.
Preliminary classification of mycobacteria by
growth properties and colonial morphology
1. M. tuberculosis complex
slow-growing, no pigmentation
2. Nontuberculous mycobacteria (NTM)
Runyon group I
slow-growing, yellow pigment (+) in light
Runyon group II
slow-growing, yellow pigment (+) in dark
Runyon group III
slow-growing, nonpigmented
Runyon group IV
rapidly growing
A pigmented and / or a rapidly growing mycobacterium
 M. tuberculosis
Preliminary identification
Colonial morphological (pigmentation) and
growth properties for preliminary
identification is important.
Only M. tuberculosis are transmitted from
person to person. Thus only these
patients are needed to be isolated and
close contacts given prophylactic
antibiotics.
Guide empirical antimicrobial therapy
Definitive identification
Biochemical tests : production of nicain and
reduction of nitrate (need > 3 weeks)
Chromatographic analysis of cell wall lipids
Species-specific nucleic acid probes for
popular species
For species without specific probe:
amplification of species-specific 16S
ribosomal RNA gene or SecA gene
followed by sequence analysis
Treatment (complex and controversial)
Slow-growing mycobacteria:
resistant to most common antibiotics, need
multiple antimycobacterial agents for
extended period (6-9 m)
Isoniazid (INH) + rifampin + pyrazinamide +
ethambutol for 2 months;
then isoniazid + rifampin for 4 to 6 months
Treatment
Rapidly growing mycobacteria: resistant to
commonly used antimycobacterial agents;
susceptible to antibiotics - amikacin,
imipenem, clarithromycin
M. avium complex
Clarithromycin + ethambutol + rifabutin
M. leprae
tuberculoid form: dapsone + rifampin for
6 m; lepromatous form: dapsone +
rifampi + clofazimine for 12 m
Effective of Treatment
Right drug combination
Sufficient treatment time
Compliance
Otherwise drug resistant strains may develop
Multidrug-resistant M. tuberculosis (MDR-TB) :
isoniazid and rifampin
Extensively drug-resistant (XDR)-TB :
drugs for MDR-TB + second line drugs
(kanamycin, amikacin, capreomycin);
untreatable
Vaccination for M. tuberculosis
Attenuated M. bovis (BCG strain) (France
1921)
Effective in young people; less effective in
adult
Cannot be used for immunocompromised
patients (i.e., HIV patients)
Low skin test reactivity develops after
vaccination – false postive

IFN-γ release assays are not affected by BCG.
Control
Elimination is highly unlikely
Active surveillance + prophylactic
intervention + therapeutic
intervention + case monitoring
Directly Observed Treatment, Short
Course (DOTS) 短期直接觀察療法
送藥到口
服藥入口
吃了再走
Chapter 27
Nocardia
Gram (+), aerobic rod; form beaded
filaments in tissues and cultures
(similar to fungal hyphae).
Contain mycolic acid (50-62 carbon),
thus stain weakly acid-fast (use a
weak decolorizing solution of
hydrochloric acid).
Epidemiology
Ubiquitous presence, widespread in soil
and in nature, > 100 species.
Infections are exogenous.
Cause disease in immuno-suppressed
patients (AIDS, leukemia, transplant
recipients)
Transmission: unknown, no person-to
person spread
Pathogenesis
Pathogenic strains
Resist phagocytic killing
Secrete catalase and superoxide
dismutase
Replicate in macrophages
Cord factor (trehalose-6,6’-dimycolate):
prevent phagosome-lysosome fusion
Clinical disease - nocardiosis
Lung and skin are primary sites of
infections.
Necrosis and abscess formation,
sinus tract
Highly predilection for hematogenous
spread to CNS or skin
Clinical diseases
Bronchopulmonary infections:
immunocompromised patient + pneumonia
with cavitation + CNS or skin involvement
Brain abscess : develop in 1/3
of patients, single or multiple
Cutaneous infection:
Primary (following trauma) or
secondary (from a pulmonary site)
Mycetoma,
lymphocutaneous infections,
skin infections with abscess and cellulitis
Laboratory diagnosis
Specimen: sputum, infected tissues
Microscopic examination:
Partially acid-fast, filamentous
Culture:
Medium for Legionella (BCYE agar)
Aerial hyphae = hyphae protrude
upward from the surface of a colony
T/P/C
Antibiotic – amikacin + cephalosporin +
sulfonamides for > 6 wks
Surgical debridement
Localized infections – good prognosis
Disseminated disease in
immunocompromised patients – poor
prognosis
Actinomyces
Greek words for “ray
fungus”
Form filamentous
hyphae in specimens
or culture
G(+), anaerobic bacilli
Contain no mycolic
acid, non-acid-fast
Pathogenesis
normal flora of the mouth, alimentary tract
and vagina (but not skin)
low virulence potential, invade and cause
disease when tissue is injured (trauma,
surgery, or infection)
unlike Nocardia, actinomyces are
pathogenic to normal hosts, all infection
are derived endogenously, no person-toperson spread.
Actinomycosis
Chronic granulomatous lesions
Suppurative, abscess formation with
sinus tracts
Discharge (wound exudates) contain
sulfur granules (pigmented microcolonies
of organisms + calcium phosphate)
Actinomycosis
Cervicofacial (lumpy jaw)
Poor oral hygiene, invasive dental
procedure, oral trauma
Patient with cervicofacial
actinomycosis. Note the
draining sinus tract.
Actinomycosis
Thoracic (lung): History of aspiration
Abdominal: GI surgery, bowel trauma
Pelvic: Intrauterine device user
CNS: Solitary brain abscess,
hematogenous spread
Laboratory diagnosis
Confirmation is difficult
(normal population on mucosal surface)
Sulfur granules for Gram’s stain
Anaerobic culture: grow slowly, >2 wks.
Colonies resemble top of a molar
Treatment
Surgical debridement of
involved tissues
Prolonged administration of
antibiotics – penicillin,
erythromycin, clindamycin
Nocardia
Actinomyces
Mycolic acid
50-60 carbon
No
Acid-fast stain
weak
Negative
Present
Ubiquitous
Normal flora
Infection mode
Exogenous
Endogenous
Host
Immunosuppressed
Colony
Aerial hyphae
Immunocompetent
Resemble top
of a molar

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