20 Mycobacteria

7/26/2019 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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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



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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)



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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)



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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



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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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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



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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



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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



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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



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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



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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



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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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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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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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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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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

20 Mycobacteria

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