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Transcript of 20 Mycobacteria
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Mycobacteria
MICROBIOLOGY LECTURE SERIES
LUZ GREGORIA LAZO-VELASCO, MD
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Mycobacteria Rod-shaped, aerobic bacteria
do NOT form spores
acid-fast bacilli
do not stain readily but once stained, resist
decolorization by acid or alcohol
Mycobacterium tuberculosis tuberculosis
Mycobacterium leprae leprosy
Mycobacterium avium- intracellulare (M. avium complex,or MAC) /other nontuberculous mycobacteria (NTM) -
infect patients w/ AIDS; opportunistic pathogens in other
immunocompromised persons; occasionally cause disease
in patients with normal immune system
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MycobacteriaSPECIES RESERVOIR COMMON CLINICAL MANIFESTATION;COMMENT
SPECIES ALWAYS CONSIDERED PATHOGENS
M. tuberculosis Humans Pulmonary and disseminated TB
M. leprae Humans Leprosy
M. bovis Humans, cattle Tuberculosis-like diseaseClosely related to M. tuberculosis
SPECIES POTENTIALLY PATHOGENIC IN HUMANS
MODERATELY COMMON CAUSES OF DISEASE
M. avium
complex
Soil, water, birds,
fowl, swine, cattle,envt.
Disseminated, pulmonary; very common in AIDS
pts.; occurs in other immunosuppressed pts.Uncommon in pts w/ normal immune system
M. kansasii Water, cattle Pulmonary, other sites
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MORPHOLOGY AND IDENTIFICATION
A. TYPICAL ORGANISM
characterized by acid-fastness (95% ethyl alcohol
containing 3% hydrochloric acid/acid-alcohol quicklydecolorizes all bacteria EXCEPT mycobacteria)
acid fastness depends on integrity of waxy envelope
Yellow-orange fluorescence with fluorochrome stain
(auramine, rhodamine)
Mycobacterium tuberculosis
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Acid-Fast Stains
(Ziehl-Neelsen) Principle
The lipid capsule of the acid fast bacteria contains mycolic acid(long chain fatty acid) which is responsible for its waxycharacteristic that resists the penetration of an aqueous based
solution (i.e. Crystal violet). The lipid capsule takes up thecarbolfuchsin and resists decolorization with an acid alcoholrinse.
Primary Stain: Carbolfuchsin
lipid-soluble and contains phenol, which helps the stain penetrate
the cell wall; assisted by the addition of heat Smear is rinsed with a very strong decolorizer, which strips the
stain from all non-acid-fast cells but does not permeate thecell wall of acid-fast organisms
The decolorized non-acid-fast cells then take up the
counterstain.
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Counter Stain: Methylene Blue
Malachite Green
Acid-Fast Stains
(Ziehl-Neelsen)
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MORPHOLOGY AND IDENTIFICATION
B. CULTURE
media for primary culture:
nonselective mediumselective medium- antibiotics to prevent the overgrowth
of contaminating bacteria and fungi
1. Semisynthetic Agar media
2. Inspissated Egg Media3. Broth Media
Mycobacterium tuberculosis
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MORPHOLOGY AND IDENTIFICATION
B. CULTURE
1. SEMISYNTHETIC AGAR MEDIA
Middlebrook 7H10 and 7H11 contain:
- defined salts - cofactors - albumin - glycerol
- vitamins - oleic acid - catalase
7H11 medium (+) casein hydrolysateused for observing colony morphology, susceptibility
testing, as selective media (add antibiotics)
Mycobacterium tuberculosis
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MORPHOLOGY AND IDENTIFICATION
B. CULTURE
2. INSPISSATED EGG MEDIA
Lwenstein-Jensen contain:- defined salts - complex organic substances
- glycerol (fresh eggs or egg yolks, potato flour)
- MALACHITE GREEN (inhibit other bacteria)
small inocula in specimens will grow on these media in 3-6
wks
serve as selective media (antibiotics added)
Mycobacterium tuberculosis
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MORPHOLOGY AND IDENTIFICATION
B. CULTURE
3. BROTH MEDIAMiddlebrook 7H9 and 7H12
- support proliferation of small inocula
- ordinarily, mycobacteria grow in clumps or masses
(hydrophobic character of cell surface)- growth is more rapid than on complex media
Mycobacterium tuberculosis
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Culture of M. tuberculosis on egg nutrient substrate:
after four weeks of incubation- rough, yellowish, cauliflowerlike colonies
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MORPHOLOGY AND IDENTIFICATION
C. GROWTH CHARACTERISTICS
obligate aerobes
derive energy from oxidation of many simple carboncompounds
increased CO2 tension enhances growth
biochemical activities are not characteristic
growth rate is much slower than that of most bacteria
doubling time: 18hrs
Mycobacterium tuberculosis
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MORPHOLOGY AND IDENTIFICATION
D. REACTION TO PHYSICAL AND CHEMICAL AGENTS
more resistant to chemical agents than other bacteria
(hydrophobic nature of cell surface and their clumped
growth)
dyes (malachite green) and antibacterial agents (penicillin)
can be incorporated into media without inhibiting the
growth of tubercle bacilli
acids and alkalies permit the survival of some exposedtubercle bacilli; used to help eliminate contaminating
organisms and for concentration of clinical specimens
resistant to drying and survive long periods in dried sputum
Mycobacterium tuberculosis
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MORPHOLOGY AND IDENTIFICATION
E. PATHOGENICITY OF MYCOBACTERIA
Humans and guinea pigs highly susceptible to M. tuberculosis
infection; fowls and cattle are resistant
M. tuberculosis and M. bovis are equally pathogenic to
humans
route of infection (resp. vs. intestinal) determines the pattern
of lesions
Some atypical mycobacteria (nontuberculous)
M. kansasiiproduce human disease indistiguishable from
tuberculosis
others cause only surface lesions or act as opportunists
Mycobacterium tuberculosis
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Constituents of Tubercle bacilli
1. Lipids- largely bound to proteins & polysaccharides
- mycolic acids (long-chain FA C78-C90)
- waxes
- phosphatides
Muramyl dipeptide complexed with mycolic acids -
granuloma formation
Phospholipids caseous necrosis
Lipids responsible for acid-fastness
Mycobacterium tuberculosis
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Constituents of Tubercle bacilli
1. Lipids
Virulent strains of tubercle bacilli form microscopic
serpentine cords (acid-fast bacilli are arranged in parallel
chains) related to virulence
Cord factor (trehalose-6,6 dimycolate) extracted
from virulent bacilli; inhibits migration of leukocytes, causes
chronic granulomas, serve as immunologic adjuvant
Mycobacterium tuberculosis
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Constituents of Tubercle bacilli
2. Proteins elicit the tuberculin reaction; elicit formation
of a variety of antibodies
3. Polysaccharides role in pathogenesis of disease
uncertain; induce immediate type of hypersensitivity;
serve as antigens in reactions with sera of infected
persons
Mycobacterium tuberculosis
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Pathogenesis
Mycobacteria emitted in droplets
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PathologyProduction and development of lesions and healing or
progression determined by:
1. number of mycobacteria in inoculum and their
subsequent multiplication
2. type of host (resistance and hypersensitivity)
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Pathology
PRINCIPAL LESIONS
Exudative type
Acute inflammatory reaction, with edema fluid, PMN
leukocytes, and monocytes around tubercle bacilliSeen in lung tissue resembling bacterial pneumonia
May: heal by resolution
lead to massive necrosis of tissue
develop to second (productive) type of lesions
tuberculin test positive
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Pathology
PRINCIPAL LESIONS
Productive type
Chronic granuloma with 3 zones:
1. CENTRAL AREA of large, multinucleated giant cellscontaining tubercle bacilli
2. MID ZONE of pale epithelioid cells
3. PERIPHERAL ZONE of fibroblasts, lymphocytes and
monocytes
Later, peripheral fibrous tissue develops and central area
undergoes caseation necrosis
Caseous tubercle may break into a bronchus, empty itself and
form a cavity
Heal by fibrosis or calcification
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Pathology
SPREAD OF ORGANISMS IN THE HOST
- direct extension through lymphatic channels and
bloodstream
- via bronchi and GI tract
- in first infection, tubercle bacilli always spread from the
initial site via lymphatics to regional lymph node
- bacilli may spread farther and reach the bloodstream
distributes bacilli to all organs (miliary distribution)
- bloodstream can be invaded also by erosion of a vein or
caseating tubercle or lymph node
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PathologyINTRACELLULAR SITE OF GROWTH- Once mycobacteria establish themselves in tissue, they reside
principally intracellularly in:
monocytes giant cells
reticuloendothelial cells- INTRACELLULAR LOCATION makes chemotherapy difficult and
favors microbial persistence
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Primary Infection of Tuberculosis
when a host has first contact with tubercle bacilli, the
following features are observed:
1. acute exudative lesion develops and rapidly spreads to
lymphatics and regional lymph nodes
2. lymph node undergoes massive caseation, usually calcifies(Ghon lesion)
3. tuberculin test becomes positive
Primary infection type: past (childhood) now in adults who
have remained free from infection (tuberculin-negative) inearly life
Involves any part of the lung , most often the base
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Reactivation Type of Tuberculosis
- usually caused by tubercle bacilli that survived in the primarylesion
- characterized by:
1. chronic tissue lesion
2. tubercle formation
3. caseation
4. fibrosis
- Regional LN slightly involved, do not caseate
- almost always begins at the APEX of lung, where oxygen tension
is highest- differences between primary infection and reinfection/
reactivation is attributed to:
1. resistance
2. hypersensitivity induced by first infection
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Immunity and
Hypersensitivity
During the first infection with tubercle bacilli, certain
resistance is acquired and there is increased capacity to
localize tubercle bacilli, retard their multiplication, limit
their spread, reduce lymphatic dissemination
(cellular immunity)
In the course of primary infection, host also acquires
HYPERSENSITIVITY to tubercle bacilli (positive tuberculin
reaction)
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Tuberculin test
A. MATERIAL
Old tuberculin (concentrated filtrate of broth in
which tubercle bacilli have grown for 6 weeks)
PPD - obtained by chemical fractionation of old
tuberculin
B. DOSE OF TUBERCULIN
5 TU , 1 TU (extreme hypersensitivity)
0.1mL injected intracutaneously
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Tuberculin test
C. REACTIONS TO TUBERCULIN
examined for presence of induration no later than 72 hrs
POSITIVE: induration of > 10mm in diameter
>5mm in pts at highest risk of developingactive disease (HIV-infected, + exposure)
> 15mm in persons with low risk
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Tuberculin test
C. REACTIONS TO TUBERCULIN- tuberculin test becomes positive w/in 4-6
weeks after infection
- may be negative in the presence of tuberculousinfection when anergy develops due to:
overwhelming tuberculosis
measles
Hodgkins disease
Sarcoidosis
AIDS or immunosuppression
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Tuberculin test
C. REACTIONS TO TUBERCULIN
Positive tuberculin test indicates that and
individual has been infected in the past
it does NOT imply that an active disease or
immunity to disease is present
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Tuberculin test
D. INTERFERON-GAMMA RELEASE ASSAYS FORDETECTION OF TUBERCULOSIS
- improve diagnostic accuracy
- whole-blood gamma-interferon release assays (IGRAs)
- based on the hosts immune responses to specificM tuberculosis antigens ESAT-6 (early secretory antigenictarget-6) & CFP-10 (culture filtrate protein-10) which areabsent from NTM mycobacteria & BCG
- detects interferon-gamma released by sensitized CD4 T cells in
response to these antigens- Commercial assays (US): QFT-GIT (Quantiferon-Gold In-Tube
Test), T-SPOT-TB
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Clinical Findings
can involve every organ system, its clinical manifestations
are protean
fatigue, weakness, weight loss, fever, night sweats
pulmonary involvement (chronic cough and spitting of
blood) is associated with far-advanced lesions
meningitis/ urinary tract involvement can occur in the
absence of other signs of TB
bloodstream dissemination leads to miliary TB with
lesions in many organs and a high mortality rate
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Diagnostic Laboratory Tests
positive tuberclin test does NOT prove the presence of
active disease due to tubercle bacilli
Isolation of tubercle bacilli provides proof
Specimens:
fresh sputum gastric washings
urine blood
joint fluid pleural fluid
biopsy material CSFother suspected material
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Diagnostic Laboratory Tests
SMEARS
- Sputum, exudates or other material is examined for acid fast
bacilli by (Ziehl-Neelsen) staining
- Stains of gastric washings and urine generally are NOTrecommended because saprophytic bacteria may be present
and yield a positive stain
- Fluorescence microscopy (auramine-rhodamine stain) ismore sensitive than acid fast stain; preferred method for
clinical material
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Diagnostic Laboratory Tests Culture, Identification and Susceptibility testing
- selective broth culture - most sensitive method and provides
results more rapidly (Lowenstein-Jensen, Middlebrook
7H10/H11 biplate with antibiotics)
- incubated at 35-37
o
C in 5-10% CO2 for up to 8weeks- Blood for culture of mycobacteria (MAC) should be
anticoagulated and processed by 1 of 2 methods:
1. commercially available lysis centrifugation system
2. inoculation into commercially available broth mediaspecifically designed for blood cultures
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Diagnostic Laboratory Tests
Culture, Identification and Susceptibility testing Growth rates separates the rapid growers (growth in < 7
days) from other mycobacteria
Photochromogens produce pigments in light but not
in darkness Scotochromogens develop pigment when growing
in the dark
Nonchromogen (nonphotochromogen) are nonpigmented
or have light tan or buff-colored colonies
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CLASSIFICATION ORGANISM
TUBERCULOSIS COMPLEX - M. tuberculosis
- M. africanum- M. bovis
PHOTOCHROMOGENS - M. asiaticum
- M. kansasii
- M. marinum
- M. simiae
SCOTOCHROMOGENS - M. flavescens
- M. gordonae- M. scrofulaceum
- M. szulgai
TRADITIONAL RUNYON CLASSIFICATION OF MYCOBACTERIA
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NONCHROMOGENS -M. avium complex - M. celatum
- M. haemophilum - M. gastri
- M. genavense - M. malmoense- M nonchromogenicum
- M. shimoidei - M. terrae
- M. trivale - M. ulcerans
- M. xenopiRAPID GROWERS - M. abscessus
- M. fortuitum group
- M. chelonae group
- M. immunogenum- M. mucogenicum
- M. phlei
- M. smegmatis
- M. vaccae
TRADITIONAL RUNYON CLASSIFICATION OF MYCOBACTERIA
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Diagnostic Laboratory Tests
Culture, Identification and Susceptibility testing Molecular probe methods faster than conventional
methods
High Performance Liquid Chromatography (HPLC)
- based on development of profiles of mycolic acids whichvary from one species to another
MALDI-TOF-MS (Matrix-assisted laser desorption ionization-
time of flight mass spectrometry) not useful
Susceptibility Testing - adjunct in selecting drugs for effectivetherapy
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Diagnostic Laboratory Tests
DNA detection
Polymerase chain reaction - rapid and direct detection of
M. tuberculosis in clinical specimens
- overall sensitivity: 55-90%; specificity: 99%- highest sensitivity when applied to specimens that have
smear positive for acid-fast bacilli
DNA fingerprinting - done using standardized protocol based
on restriction fragment length polymorphism- DNA fragments are generated by restriction
endonuclease digestion and separated by electrophoresis
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Treatment
4-drug regimen: INH, RFM, PZA, EMB
Multidrug-resistant M tuberculosis
resistant to both INH, RMP
treated according to susceptibility tests Extensively drug-resistant (XDR)
resistant to both INH, RMP, any fluoroquinolone,
and at least one of the three injectable 2nd
line drugs (amikacin, capreomycin,kanamycin)
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Epidemiology
most frequent source of infection is the human who excretes,particularly from the respiratory tract, large numbers of
tubercle bacilli
Close contact (in the family)
Massive exposure (in medical personnel)
Development of clinical disease influenced by:
- age (high risk in infancy and in the elderly)
- undernutrition
- immunologic status
- coexisting disease (silicosis, diabetes)
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Prevention and Control
Prompt and effective treatment of patients with active
tuberculosis and careful follow-up of their contacts with
tuberculin tests, x-rays, and appropriate treatment are
the mainstays of public health tuberculosis control
Drug treatment of asymptomatic tuberculin-positive
persons in the age groups most prone to develop
complications (children) and in tuberculin-positive
persons who must receive immunosuppressive drugs
greatly reduces reactivation of infection
P ti d C t l
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Prevention and Control Individual host resistance: nonspecific factors may reduce
host resistance, thus favouring the conversion ofasymptomatic infection into a disease
- factors: starvation, gastrectomy, and suppression of cellularimmunity by drugs (corticosteroid) or infection, HIV infectionis a major risk factor for tuberculosis
Immunization: various living avirulent tubercle bacilli,particularly BCG (bacillus-calmette-guerin, an attenuatedbovine organism), have been used to induced certain amountof resistance in those heavily exposed to infection.
The eradication of tuberculosis in cattle and thepasteurization of milk have greatly reduced M bovis infection