Chapter 28 Mycobacterium

Chapter 27 Nocardia

Actinomyces

Microbiology 20115/11/2011Yu Chun-Keung

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 milk products)

M. tuberculosis - pathogenesisInhalation 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 symptomsMost 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 diseaseIn 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.

EpidemiologyHumans - 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)

EpidemiologyOne third of the world’s population is infected.

9 million new cases and 2 million deaths annually.

Endemic area: Southeast Asia, sub-Saharan 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.

In most patients, the only evidence of infection :

• Radiographic evidence of calcification in lungs

• Lifelong positive tuberculin skin test to PPD

• Lab detection of mycobacteria : microscopy or culture.

Clinical Diagnosis of M. tuberculosis

[Source: Wikipedia]

Immunodiagnosis (for M. tuberculosis)

Tuberculin skin test

•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 non-infected people.

[Source: Wikipedia]

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

MicroscopyThe most rapid method to confirm mycobacterial disease - acid-fast stain

Auramine-rhodamine strain : Truant-fluorochrome method

Carbolfuchsin strain : Ziehl-Neelsen method or Kinyoun 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.

CultureSpecimens: 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 3-4 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

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

2. Mycobacterium leprae

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. intracellulareRecover 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 immuno-compromised patients

Thus, isolation in specimens of immuno-competent patients mostly represents a transient colonization

No person-to-person spread (except M. bovis)

Slow-growing mycobacteria

M. bovis Pulmonary tuberculosis M. kansasii

M. scrofulaceum Lymphatic tissue infection

M. ulcerans Cutaneous infection M. marinum M. haemophilumNew 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 identificationColonial 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 identificationBiochemical 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 TreatmentRight 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/CAntibiotic – 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-to-person 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

NocardiaActinomyce

s

Mycolic acid 50-60 carbon No

Acid-fast stain

weak Negative

Present Ubiquitous Normal flora

Infection mode

Exogenous Endogenous

HostImmuno-

suppressed Immuno-

competent

ColonyAerial

hyphae

Resemble top of a molar