Mycobacteria

MICRO

MYCOBACTERIUM MYCOBACTERIA

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70 species of mycobacteria 2 major pathogens o M. tuberculosis o M. leprae MOTTS

ORDER: Actinomycetales FAMILY : Mycobacteriaceae GENUS: Mycobacterium

Mycobacteriaceae

GENUS

Mycobacterium

Actinomycetacae

Nocaria

SPECIES

Mycobacterium tuberculosis complex M. tuberculosis M. africanum M. bovis M. microti

Streptomycetacea ee

Streptomycea

NTMM

Actinomyces

Di na tlga mabasa (sori)

S. proteus May isa pa di ko na din mabasa

All Mycobacteria Are; ö Acid fast ö Aerobic ö Contain mycolic acids ö Genomes have 59-66% GC content

RUNYON CLASSIFICATION SCHEME Medical Significant Mycobacteria Representati Growt Pigment ve Species h

Slow

Pigmented

+

Runyon Group I

M. kansasii M. marinum

Slow

Photochro -mogen (1)

-

Runyon Group II

M. gordonae M. scofulaceum M. intracellulare M. avium M. xenopi

Slow

Scotochromogen (2)

-

Slow

Nonpigmente d

-

Runyon M. fortuitum Rapid Group M. chelonei (3) IV (1) pigment formed in the presence of light only (2) pigment formed in the presence or absence of light (3) growth in 1 week

Actinomycetales

FAMILY

M. tuberculosis M. bovis

Runyon Group III

The Taxonomic Tree For Selected Mycobacteria And Related Species ORDER

TB Comple x

THE   

GLOBAL EMERGENCY 8-12 M new infections/yr 2-3M people die from TB/yr Emergency of MDR(Multi-drug resistance) M.tb

CELL WALL  Unique : waxy, hydrophobic and high lipid content 60% of dry weight : o Mycolic acids – long chain, branches fatty acids  Form pseudo outer membrane responsible for unusual staining  Responsible for hydrophobicity  (+) adjuvant properties, responsible for dev’t of DTH  All mycobacterial pathogens are intracellular pathogens o The walls help organism to survive w/in the macrophages TUBERCULOSIS  Chronic granulomatous disease caused by M. tuberculosis Ancient disease  Prehistoric human bones o tuberculous damage  Mummies (+) AFB  Drawing from ancient Egyptian times: o Tuberculous deformities

Niaci n

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MICRO

MYCOBACTERIUM

MYCOBACTERIUM TUBERCULOSIS BACTERIA

PATHOGENESIS

TB INFECTION

Mycobacterium tuberculosis Complex  M. tuberculosis  M. africanum  M. bovis  M. microti  Obligate pathogen Man : principal host

Antigenic Structure  Cell wall structures; o Polysaccharides, proteins, peptides cytoplasmic proteins  Seibert proteins A and B elicit skin reactions more potent than PPD

Site of Infection  Inhalation of droplet nuclei  Aerosols, contain 1-3 org  Suspended in the air indefinitely  Inhaled by person being infected  Taken up by alveolar macrophages  1 org enough to establish infection  Tb is spread from person to person through the air. The dots in the air represent droplet nuclei containing tubercle bacilli

Morphology  Slender, straight or slightly curved, non-motilr nonsporogenous, nonencapsulated bacilli  0.2-0.5 x 1-4μ  Gm(+) ZIEHL-NEELSEN STAIN  Red rods in blue background AFB  Attributable to high lipid content Physiology  Cultural characteristics o Slow growing (divides q1824 hrs) o Lowenstein – Jensen or BACTEC

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Small, dry, scaly colonies with corrugated surfaces Obligate aerobes

Virulence factors  NO toxins  Pattern of culture o Virulent: serpentine cords w/ bacilli in parallel arrangement o Avirulent : random brush – heap pattern  Cord factor – trehalose dimycolate  Catalase  Tuberculoproteins Pathogenesis Airborne  deposit in alveolar space of lungs  engulfed by macrophages Portion : resist intracellular destruction  persist  multiply and kill the macrophages stimulate and inflammatory focus  Mature into a granulomatous lesion (tubercle)  caseous necrosis  erosion of tubercle into an adjacent airway  cavitation  release of massive

Common Sites of TB  Brain  Larynx  Bone  Kidney  Lymph node  Pleura  Lung  Spine

Infects macrophages

TB disease 1. 1° TB or primary complex diseases

2. 3.

4.

2° TB or post primary TB or reactivation TB seen after a prolonged period of LTBI relapse after an inerim period of being cured M. tb survivors of previous tx grow again. Effective tx does not completely eliminate M. tb pop’n Re-infection TB • Successful infection by another Tb strain occurs despite previously acquired immunity

TB Inactive Disease  (+) residual organ damage  Tb org no longer actively multiplying  Viable dormant org persist and contained by the granulomatous rxn Occurs: spontaneously (30%) As treatment outcome (95%) Completely treated non-R cases

Lesions 1. Exudative : lungs – primary lesion 2. Ghon Complex: diagnosis from X ray Parenchymal exudative lesion + draining lymph nodes 3. Granulomatous tubercle: Heal by fibrosis and calcification 

TB DISEASE

Progression of TB People who are exposed to TB may or may not develop TB infection. People with TB infection may or may not develop TB disease. The risk of developing TB disease is highest in the first two years after infection. TB Infection vs. TB Disease

MORBIDITY AND MORTALITY Clinical Symptoms: • due to overprodu ction of TNF • severe weight loss • night sweats • chronic cough (> 2 weeks duration) • hemoptysi s

LABORATORY DIAGNOSIS based an demo of M. tb in a clinical specimen sputum, pleural biopsy, brochoalveolar washings, fibreoptic bronchoscopy, biopsy specimen of lung, CSF, gastric washing, biopsy from other anatomical site Interpretation of AFB Stain

TB DEATHS Mortalities dependent on • site and type of disease • timeliness of diagnosis • appropriat eness of interventi on 30-40% of sputum (+) untreated die within 1 yr 50-70% die within 5-7 yrs 1.5 M people die annually

alveolar

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TREATMENT, CHEMOPROPHYLAXIS, AND PREVENTION Drug Therapy • DOTS ( Direct Observed Therapy, short -course) o cornerstone of Tb treatment  3 mos -sterilize lesion  3 anti-Tb agents  INH, RIF, PZA, (Ethambutol) • INH, PZA - need to be activated by mycobacterial enzymes (KatG and Pyrazinamidase) o Mutations in the activating enzymes can lead to R • Multi-Drug Resistance (MDR) - simultaneous R to INH and RIF (rifampicin) o R to the first line anti-Tb agents is due to mutations in the target proteins for these drugs o mutations in target proteins  INH : katG, inhA and inhB  RIF: B sub-unit of the DNA dependent RNA polymerase, rpaB  PZt.: pyrazinamidas e  Streptomycin: ribosomal

MICRO

MYCOBACTERIUM

Microbial Robustness 1. Resistant to effects of

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Drying – remains alive and virulent in dried sputum; stored x 12 yrs

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2.

Sunlight – destroyed in direct sunlight within o 2hrs in cultures o 20-30 hrs in sputum  If (-) sunlight o Sputum remains viable o 6-8 months in dried sputum o Weeks in putrefying sputum  Droplets adhering to dust are infectious x 1-1 ½ weeks Resistant effects of chemicals  Difficult to disinfect 5% solution of phenol  S: heat, pasteurization, UV light

Transmission  Inhalation of droplet nuclei  Aerosols, contain 1-3 org  Suspended in the air indefinitely  Inhaled by person

numbers of bacilli into the sputum

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Resistant host : tubercle  calcified Cheesy necrosis Early in infection : spread distally Indirectly thru lymphatics  hilar or mediastinal LN Directly into the circulation by erosion into pulmonary vessel

(1) Droplet nuclei containing tubercle bacilli are inhaled, enter the lungs, and travel to the alveoli (2) Tubercle bacilli in alveoli (3) Brain, lung, kidney, bone (figur eto.. di ko Makita) (4) Special immune cells form a hard shell (in this example, bacilli are in the lungs) (5) Hard shell breaks down and tubercle bacilli escape and multiply (in this example TB disease develops in the lungs)

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Extrapulmonary hematogenous dissemination ; seeding of other organs ( spleen, liver, kidneys ) eventually re-inoculation of the lungs  serve as origin of reactivation

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Phagocytosed by the macrophage Found in membrane – bound particles known as phagosomes Infected mphagosome does not mature, remains in a state similar to early endosome Phagosome does not acidify or fuse w/ lysosome Infected macrophage is unable to kill the organism

Exposure to M.Tb Major factors that determine risk: 1. Number of infectious cases in the community 2. Duration of their infectiousness 3. Number and nature of interactions between an infectious case and a S contact (susceptible) population density, family size, age of source of infection, gender 4. Climactic conditios Infection with M.Tb Risk factors 1. The infectious patient • (+) droplets • # bacilli in the sputum 2. Exogenous/environme ntal factors • The number and size of infectious droplet nuclei • Duration of exposure of the S person to the particle density • Air circulation

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mutations (s12 protein and 16S rRNA)

Number of bacilli seen Interpretation

Chemoprophylaxis Indication: • patients with (+) PPD test >10mm w/in 48-72 hrs • children exposed to risk of infection • patients with (+) PPD who undergo immune suppression

0 in at least 100 fields NO AFB Seen

1-9 +

10-299 ++

Prevention • live, attenuated M bovis strain (BCG) • Newer vaccines? • attenuated M. tb strains or subunit vaccines presented as naked DNA

300 and above +++

Microbiological Diagnosis microscopic investigation • Ziehl Neelsen • Auramine O stain (AFB) culture (2-8 weeks) • decont'n and conc'n of specimen • solid medium Lowenstein-Jensen • liquid medium (Middlebrook) Acid fast Characteristic: • Red rods

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Staining with

Zeihl-

MICRO

  

MYCOBACTERIUM

being infected Taken up by alveolar macrophages 1 org enough to establish infection Tb is spread from person to person through the air. The dots in the air represent droplet nuclei containing tubercle bacilli

and ventilation dilutes the conc of infectious droplet nuclei 3. Host factors immune competence

Neilson (carbol-fuchsin) stain due to high glycolipid content Laboratory • Sputum culture after concentration or digestion with KOH (destroys other undesired organisms) • Collect several specimens • Collect sputum early in the morning (increases bacterial concentration) • Acid fast staining (ZeihlNeilson or Kinyoun) • Fluorescent microscopy techniques • Culture of the organisms on specialized media (L-J and MIddlebrook agars) • PPD skin testing (DTH) Molecular diagnosis • demo of M. tb DNA or RNA in the specimen • RNA and mRNA - better indication of mycobacterial cell viability Tuberculin (Mantoux)

•

•

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Skin

Test

tuberculin partially purified extract of M. tuberculosis proteins (PPD) evokes a DTH response (basis)

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MICRO

MYCOBACTERIUM C lass T ype s D esc ripti on

0 No exp osu re to TB Not infe cte d No hx of exp osu re, neg ativ e rxn s to tub erc ulin skin test

1 E xpo sur e of TB No evi den ce of infe ctio n Hx

TB Infection TB Disease (in the lungs)

Guidelines For Interpretation of the Mantoux Test

Tubercle bacilli in the body

Diameter of Induration (in mm) Persons for Whom the Reaction is Considered Positive

Tuberculin skin test reaction usually positive

Chest x-ray usually normal Chest x-ray usually abnormal

Sputum smears and cultures negative. Sputum smears and cultures positive.

No symptoms Symptoms such as cough, fever, weight loss

Not infectious Often infectious before treatment

Not a case of TB A case of TB

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≥5 Immunosuppressed, a recent close contact with active TB, abnormal chest X-ray consistent with TB

≥ 10 Foreign-born (country with high TB prevalence), low income, injection drug users. residents of correctional facility or nursing home, > 70 yr, < 18 yr, healthcare workers, mycobacterial lab employee, medical condition associated with increased risk of TB (DM, prolonged corticosteroids, gastrectomy, chronic malabsorption, silicosis, ≥ 10 % below IBW)

≥ 15 All others

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MICRO

MYCOBACTERIUM

MYCOBACTERIUM LEPRAE BACTERIA

EPIDEMIOLOGY

PATHOGENESIS

Leprosy • Ancient disease • M. leprae - first pathogen to be recognized and demonstrated in human tissue ( Hansen, 1872) • Occurs predominantly in SEA, but also in Africa and the Americas o 1 M active cases worldwide in 1997 o 2 M ; irreversible deformities due to leprosy o incidence:  2: 1 males to females  1:1 Africa

Endemic Areas of the World WHO: Brazil, Madagascar, Mozambique, Tanzania, and Nepal have 90% of cases (1-2 million new cases annually worldwide)

CLINICAL POINTS • Macules, papules or nodular lesions • Areas of anesthesia • Thickened nerves • Superficial infection on cooler areas of the body (skin – nose, outer ears), testicles, superficial nerve endings • (+) Continuum of disease from Lepromatous Leprosy to Tuberculoid Leprosy • Immediate forms: o Borderline leprosy o Indeterminate Leprosy

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•

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Chronic, communicable disease caused by M. leprae Clinical spectrum of disease reflects: bacterial proliferation and accumulation at the site of infection immunological response peripheral neuritis Peripheral nerves, skin and mucous membranes are affected M. leprae only grows on the colder surfaces of the body Rarely an immediate cause of death

Incubation Period o a few weeks following exposure o several years (30 yrs) Transmission o NOT highly infectious o common among household contacts o inhalation of infectious agents o ? Insect bites, inoculation through broken (intact) skin o infectious: shed from nasal mucous membranes esp if (+) ulceration o can survive in nasal secretions x 36 hrs o humans: reservoir of infection o found: wild, ninebanded armadillos

CLINICAL FEATURES: DUE TO

• • •

Skin anesthesia results in burns or trauma → infected Resorption of bone leads to loss of features (nose; fingertips) Infiltration of the skin and nerves leads to thickening and folding of the skin

FEATURES OF LEPROSY • Localized areas of anesthesia due to nerve degeneration • Nasal secretions are most likely infectious material; low infectious ability, may take 3-4 yrs • Children are more susceptible than adults • Tx: 1-2 year combination therapy – Dapsone, Rifampin, Clofazimine

LEPREMATOUS LEPROSY • • • • •

• • •

Diffuse or nodular lesions (+) many AFB’s (multibacillary) Clumps of cells may occur intra- and extracellular masses in capsular material (globi) Sensory loss due to nerve damage Predominant humoral response, little CMI Multiple nodular lesions → Typical leonine (lionlike) facies After onset of tx, patients often develop erythema nodosum leprosum (ENL) – sign of restoration of CMI Painful nodules along extensor surfaces of tibia and ulna; neuritis and uveitis

DIAGNOSIS • AFB Stain of skin lesions or nasal scrapings • Foam cells – lipid laden macrophages contain many AFB in the skin • False (+) serologic test for SY • A person living in endemic area with the following cardinal signs: o Skin lesions + Definite sensory loss, with or without thickened nerves o (+) Skin smear (found in small proportion of cases) • Clinical Classification: o If lesion:  Single: Paucibacillary leprosy

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TUBERCULO ID LEPROSY

MICROBIOLO GY

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•

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•

Few well defined lesions Few AFB’s (paucibac illary) Predomin ant CMI Spontane ous regressio n occurs Cases in children

DIAGNOSIS • Few organism s seen • Typical granulom a – sufficient

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Resembles M. tb under the light microscop e; (+) pleomorph ic forms NEVER been cultured in the lab Cultured transiently in the mouse footpad Intracellul ar pathogen – escapes into the cytoplasm of the host cell Takes 20 – 30 days to divide WHO recommen ds two indices for quantificat ion of infection 1. BI

-

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Bacteri al index Measu re

WORLD HEALTH ORGANIZATION Case Definition Leprosy

of

1 or more of the following features: o Hypopigmented or reddish skin lesion(s) with definite loss of sensation o Involvement of the peripheral nerves (thickening with loss of sensation) o Skin smear (+) for AFB Eradication Programs Classified on the basis of clinical manifestations or on results of skin smears o Skin smears:  (-) : Paucibacillar y Leprosy  (+) : Multibacillar y Leprosy Diagnosis Lepromin skin test: o PPD based on proteins isolated from M. leprae o Used in epidemiologica l suveys to detect occurrence of subclinical

MICRO

MYCOBACTERIUM

Mycobacterium leprae • 1400 BC – Leprosy dates back to biblical times • 50-100 cases are diagnosed in the US annually now (increase in 1985 due to immigration from SE Asia • Morbidity is more important than mortality today: 1520 million cases worldwide • Clinical picture is 99% of the diagnosis

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main source for research Classification of Leprosy

2.

-

M. leprae Growth Characteristics • Does not grow well above 30°C. No systemic disease • Obligate intracellular parasite; won’t grow on artificial medium

•

Armadillo tissue is also used for culture

bacter ial load MI Morpho logical index Measu re bacter ial viabilit y

infection Treatment • Dapsone: Mainstay of treatment • Multidrug therapy (MDT) o Never treated with a single drug o Treatment for at least 2 years or until lesions free of organisms o Thalidomide: severe ENL reactions supportive ● Multibacillary leprosy o Rifampicin: 600 mg once a month o Dapsone: 100 mg daily o Clofazimine: 300 mg once a month and 50 mg OD • Paucibacillary leprosy o Rifampicin: 600 mg once a month o Dapsone: 100 mg daily o Duration: 6 months o Single skin lesion (ROM) single dose Rifampicin, Ofloxacin, Minocycline Prevention • Isolation of ALL lepromatous px

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MICRO

MYCOBACTERIUM • •

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Chemoprophylaxi s: Dapsone NO vaccine

MICRO

MYCOBACTERIUM RESISTANCE NO RESISTANCE

APPEARANCE OF DISEASE

Tuberculoid Lepromatous TUBERCULOID BORDERLINE

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Intact CMI Deficient CMI

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Non-progressive disease Progressive disease

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Macular skin lesions Nodular skin lesions

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Few bacilli present Abundant bacilli

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Severe asymmetric nerve involvement Symmetric nerve involvement

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Sudden onset Slow onset

•

Skin infiltrated with TH1 cells Ab levels are high

•

IL-2, IFN-γ & IL-2 IL-4 & IL-10

LEPROMATOUS

• • • • •

• • • • •

Non-progressing disease Macular skin lesions Few bacilli in lesions CD4+ helper T-cells IL-2, IFN-gamma & IL-12 promote healing Progressive disease Nodular skin lesions Abundant bacilli in lesions CD8+ suppressor Tcells IL-4 & IL-10 suppress healing

INDETERMINATE

INFECTION

•

•

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MICRO

MYCOBACTERIUM

MYCOBACTERIA OTHER THAN TUBERCULOSIS (MOTT) BACTERIA •

•

Environmental mycobacteria o Generally cause opportunistic infections o Person to person transfer does NOT take place o Generally R to drugs used for tx to Tb o Combination of 5 or 6 agents may have to be used Classified according to: rate of growth, whether they produce pigment o Group I  Photochromogens  Yellow to orange colony when exposed to light o Group II  Scotochromogens  Produce pigment in the dark o Group III  Nonchromogens  Little or no yellow orange pigment irrespective of (+) or (-) of light o Group IV  Grow rapidly (< 7 days)

GROUP I – PHOTOCHROMOGENS o Mycobacterium kansasii  Produce lung and systemic disease identical to Tb, frequently PPD (+)  Can infect old tuberculous lung lesions o Treatment  RIF, INH, Ethambutol  Surgical resection

GROUP II SCOTOCHROMOGENS o Mycobacterium scrofulaceum  Scrofula  Granulomatous adenitis enters through oropharynx  Infects draining lymph nodes o Habitat  Environmental water source  Saprophyte from human RT o Treatment  Surgical incision

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o

o o

GROUP III NONCHROMOGENS Mycobacterium aviumintracellulare complex (MAI or MAC)  M. avium; M. intracellulare  Most common bacterial cause of disease in AIDS  AIDS: CD4, 200/uL Habitat  Water and soil Treatment  For life  Highly R to antituberculous drugs  Initial: macrolide+ others: Rifabutin

GROUP IV – RAPIDLY GROWING o Mycobacterium fortuitum-chelonei complex  M. fortuitum; M. chelonei  Saprophytes found in soil and water  Rarely cause human disease  Skin and soft tissue infections (_____-ndi nnman xa makita, 1 word lng xa kc maliit lng un space. Senxa) of puncture wounds o Occur in:  Immunocompr omised px  Individuals with prosthetic hip joints and indwelling catheters o Treatment  R to anti-tb drugs  Multiple drugs + surgical excision  DOC: Amikacin & Doxycycline o Mycobacterium abscessus  Causes chronic lung infections  Infections of skin, bone, joints

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OTHER MOTT •

Mycobacterium ulcerans 3rd most common mycobacterial infection in humans o Causes Buruli ulcer o Occurs widely throughout west Africa o Treatment  Antibiotics unsuccessful  Surgical excision

o

MICRO

MYCOBACTERIUM Highly antibiotic R Mycobacterium smegamatis  Not associated with human disease  Part of N flora of smegma 

o

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MICRO

MYCOBACTERIUM Runyon’s classification of Atypical Mycobacteria

Group Growth Rate Pigment Formation in Typical Species

Light Dark

Slow M. kansasii M. marinum

Slow M. scrofulaceum

Slow M. avium M. intracellularae

Rapid M. fortulum M. fortulum-chelonei complex

I + -

II + +

III -

IV -

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MICRO

MYCOBACTERIUM

…Brother is called a “genius” but he paid the cost through “effort,” so Brother is like this now.

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