DEGEE O. GONZALES, R.M.T.

College of Medical Technology Velez College

 Aerobic,

slightly curved or straight rods Measures 0.2 to 0.4 x 2 to 10 Nonmotile Nonsporogenous, Nonencapsulated Most distinctive property ACID FASTNESS Some are saprophytes in soil, others are parasites 2 species causing 2 most dreaded diseases:

Tuberculosis and Leprosy

Some

species of environmental saprophytes cause human infections

 Most

distinctive structure of cell:

Cell wall contains N-glycolylmuramic acid in lieu of N-acetylmuramic acid and very high lipid content:
Contributes to acid fastness Difficult to stain with commonly used aniline dyes Unusual resistance to the effects of drying and harsh decontaminating agents

Hydrophobic cell surface makes these organisms grow more slowly (does not easily allows nutrients to enter the cell)

colonies visible in 2-60 days at optimum temperature

 Have

important characteristics in common with Corynebacterium and Nocardia called CNM group produce mycolic acid and similar guanine-plus-cytosine (G+C) content

All

 71

recognized or proposed species Divided into 2 MAJOR GROUPS based on fundamental differences in epidemiology and association with the disease:
Mycobacterium tuberculosis Complex Nontuberculous Mycobacteria (NTM)

 Mycobacterial

species that occur in humans

include:

M. M. M. M.

tuberculosis bovis bovis BCG africanum

All

species capable of causing TUBERCULOSIS Slow-growers Colonies are nonpigmented

ORGANISM M. tuberculosis

HABITAT

PRIMARY ROUTES OF TRANSMISSION

DISTRIBUTION Worldwide

Primary reservoir: inhalation of droplet nuclei patients with (infectious aerosols, 1-5, cavitary disease reach the lungs alveoli): Person to person contact Manipulation of lesions / clinical specimens in the lab Airborne transmission Humans Wide range of animals Humans Ingestion of contaminated milk from infection cows Airborne transmission Inhalation of droplet nuclei

M. bovis

Worldwide

M. Africanum

East and West Africa

 Slender,

straight or slightly curved rods with rounded/tapered ends

 Appear

beaded in tissue and sputum due to vacuoles and polyphosphate content

 May

exhibit cording/intertwining of bacilli in serpentine cords

 Acid

fast (due to long chain of mycolic acid and physical integrity of the cell)
LIPID BARRIER PRINCIPLE: increased hydrophobicity of the surface layers follows the complexing of dye with mycolic acid residues present in cell wall > prevents exit of carbol fuchsin trapped in the cell

True

branching in old cultures Cell Wall Structure:

Thick wall - 3 layers enclosing a 3 layered structure plasma membrane Wall is very complex with complex lipophilic macromolecules 60% of dry weight of cell wall is lipids which enable organisms to resist adverse environmental conditions

 Obligate

aerobe Grow on ordinary simple synthetic medium, but for 1o isolation from clinical material, requires a more complex medium Grow very slowly: colonies seen in 2-3 weeks of incubation at 37oC on egg media but no growth at 25oC and 45oC Forms PELLICLE on surface of Liquid medium after 14-15 hours Eugonic Catalase (+) Niacin (+) Nitrate Reduction Test (+)

Solid Medium: DRY, FRIABLE, ROUGH, WATERY, GRANULAR, AND BUFF COLORED WITH A FLAT, IRREGULAR MARGIN AND A CAULIFLOWER CENTER

 Highly

resistant to drying Cultures kept for 12 years at 37oC still viable and virulent When exposed to direct sunlight:

Organism from cultures killed in 2 hours Organisms in sputum killed in 20-30 hours

Bacilli

in dried sputum protected from sunlight: viable for 6-8 months More resistant to chemical agents Killed by pasteurization

 No

exotoxin or endotoxin Lipid-rich cell wall of mycobacteria:

Survival and growth inside monocytes and macrophages (inhibits phagolysosomal fusion) Resistant to many disinfectants

Mycolic

acids

Large (C60-C90), saturated, -alkyl, -hydroxyl fatty acids found in both waxes (esters of fatty acids with fatty alcohols) and glycolipids (lipid linked to carbohydrates).

 Cord

Factor

Contributes to the virulence of M. tb strains a mycoside of 6,6'-dimycolate of trehalose (dimer of 1,1 linked glucose nonreducing sugar) contributes to the hydrophobic character of the organism Toxic - causes profound disturbances of microsomal enzymes, mitochondria, and lipid metabolism in the liver responsible for neutral red reactivity associated with virulent strains

Sulfatides

 Lipoarabinomannan

CW glycolipid suppresses T cell proliferation may interfere with interferon-gamma secretion extracellular soluble compound that robs iron from ferritin (storage form of iron in the mammalian cell)

exochelin

mycobactins

cell associated and transport the iron through the mycobacterial cell wall

 Causative

agent of Bovine Tuberculosis Infection occurs in humans s by consuming raw or un-pasteurized milk from TB infected cows.

 Requires

longer period of incubation: 3-6 weeks at 35C Colony: TINY, TRANSLUSCENT, SMOOTH, PYRAMIDAL COLONIES

7H10: colonies resemble M. tuberculosis

 Dysgonic

Antigenic,

non-infectious Forms serpentine cords Niacin (-) Nitrate Reductase test (-) SUSCEPTIBLE to TCH (Thiopene-2-carboxylic acid Hydrazide)

 Tuberculous

cervical lymphadenitis
Formerly called SCROFULA tuberculosis of the cervical lymph nodes condition characterized by enlarged, inflamed, and tender lymph nodes of the neck (seen in certain infectious diseases of children) Also called cervical adenitis

 Mesenteric

Adenitis

condition clinically resembling acute appendicitis there is inflammation of the mesenteric lymph nodes receiving lymph from the intestine.

 Tuberculosis

of the Bones

and Joints

encountered in any age group No bone is immune from involvement by TB The most common location in childhood is spine and the most frequently involved joints are the weight-bearing joints such as hip, knee, shoulders, or elbow

 First

identified and reported as a separate sub-species of M. tuberculosis complex (MTBC) in 1968 Isolated from sputum samples of TB patients in Europe, the United States, and Africa Traditionally been identified by phenotypic criteria, occupying a position between M. bovis and M. tuberculosis according to biochemical characteristics Divided into M. africanum subtype I and M. africanum subtype II based on biochemical criteria

M. africanum subtype II has been identified as a major cause of human tuberculosis in Uganda

 Live

attenuated bacillus vaccine derived from the closely related virulent strain of M. bovis for the prevention of Tuberculosis Developed by Calmette and Gurin at the Pasteur Institute of Lille in 1908

original virulent strain isolated from an infected cattle was grown for 13 years on potato slices cooked in beef bile supplemented by glycerol First human vaccination with this attenuated strain, named Bacille Calmette-Gurin (BCG), was applied in 1921 in Paris

Introduced

into the WHO Expanded Programme on Immunization (EPI) in 1974

 Freeze-dried

BCG vaccine

international name and proper name of manufactured BCG vaccine as recommended by WHO intended for intradermal injection Freeze-dried BCG vaccine, Percutaneous name of BCG vaccine intended for percutaneous vaccination Immunize susceptible individuals versus Tuberculosis Boost the nonspecific cellular immune response of certain immunologically deficient patients

Uses:

 Includes

all other species that do not belong to M. tuberculosis Complex Initially considered as strictly saprophytes Ubiquitos a.k.a. as ATYPICAL Mycobacteria or MOTT Can produce severe and even fatal disease in humans through:

Inhalation of infectious aerosols / ingestion Iatrogenically acquired Nosocomially aquired

 RUNYONs

CLASSIFICATION

Based on phenotypic and characteristics of the various species: GROWTH RATE and COLONIAL PIGMENTATION

NONCULTIVABLE

NTM

Mycobacterium leprae

NTM

RECOVERED FROM HUMANS INTO FOUR MAJOR GROUPINGS

Based on the clinical disease the mycobacteria species caused

RUNYON GROUP NO. I

GROUP NAME

DESCRIPTION
Colonies that develop pigment following exposure to light after being grown in the dark and take more than 7 days to appear on solid media Colonies that develop pigment in the dark or light and take more than 7 days to appear on solid media

PHOTOCHROMOGENS

II

SCOTOCHROMOGENS

III
IV

Colonies that are nonpigmented regardless of NONPHTOCHROMOGENS whether grown in the dark or light and take more than 7 days to appear on solid media RAPID GROWERS Colonies that appear on solid media in less than 7 days

 synthesize

carotenoids (yellow to red pigment) in varying amounts Some are considered potentially pathogenic for humans, others are rarely associated with diseases. Divided into THREE groups:

Group I : Group II : Group III :

Photochromogens Scotochromogens Nonphotochromogens

ORGANISM

EPIDEMIOLOGY

PATHOGENICITY

TYPES OF INFECTION Chronic Pulmonary Diseases; Extrapulmonary Diseases (Cervical Lymphadenitis & Cutaneous Dss.) Pulmonary Disease

M. kansasii

Infection more common in white males. Natural reservoir is tap water Not commonly encountered

Potentially Pathogenic

M. asiaticum

Potentially Pathogenic

M. marinum

Natural reservoir is fresh water & saltwater as a result of contamination Potentially from infected fish & Pathogenic other marine life (entry by trauma or small breaks in skin) UNKNOWN UNKNOWN Potentially Pathogenic Potentially Pathogenic

Pulmonary Disease

M. intermedium M. movocastrense

Pulmonary Disease Pulmonary Disease

 Epidemiology

of the potentially pathogenic scotochromogens has not been definitevely described Organisms are rarely recovered in clinical laboratories

ORGANISM

EPIDEMIOLOGY

PATHOGENICITY Potentially Pathogenic

TYPES OF INFECTION Pulmonary Disease (middle-aged men), Cervical adenitis, Bursitis

M. szulgai

Water and soil

M. scrofulaceum M. interjectum

Raw milk, soil, water, Potentially dairy products Pathogenic

UNKNOWN Potentially Pathogenic Potentially Pathogenic Potentially Pathogenic NonPathogenic NonPathogenic NonPathogenic

Cervical adenitis in children

Chronic lymphadenitis (1case) Pulmonary disease (2 cases) Cervical lymphadenitis (1case) NA NA NA

M. heckeshornense UNKNOWN
M. tusciae M. gordonae M. cookii M. hiberniae UNKNOWN Tap water, soil Sphagnum, surface waters in NZ Sphagnum, soil in Ireland

M. kubicae

UNKNOWN

NonPathogenic

NA

 Nonpathogenic

to humans

M. terrae Complex: M. terrae, M. triviale, & M. chromogenicum M. gastri

Others

are potentially pathogenic and many are frequently encountered in the clinical laboratory Mycobacterium avium complex

Frequently isolated in the clinical laboratory Able to cause infection in the human host

ORGANISM M. avium complex

EPIDEMIOLOGY Environmental sources inc. natural waters

TYPES OF INFECTION Pulmonary infections in patients w/ preexisting pulmonary disease; Cervical lymphadenitis Disseminated disease in immunocompromised, HIVnegative patients,& patients w/ AIDS 1 Pulmonary infections in adults Less common: Extrapulmonary infections, & disseminated disease Indolent cutaneous and subcutaneous infections

M. xenopi

Water (especially hot water taps in hospitals); believed to be transmitted in aerosols Not isolated in the environment but infections occur in Tropical / temperate climates

M. ulcerans

ORGANISM M. malmoense

EPIDEMIOLOGY Majority of cases isolated from England, Wales, & Sweden Rarely isolated from patients with HIV Isolated only from humans & captured armadillos

TYPES OF INFECTION Chronic Pulmonary infections primarily in patients w/ preexisting pulmonary disease; Cervical lymphadenitis in children Less common infections of the skin or bursa

M. genavense

Isolated from pet birds and Disseminated disease in patients w/ dogs. Mode of acquisition is AIDS UNKNOWN

M. haemophilum

UNKNOWN

Disseminated disease Cutaneous infections in immunosuppressed adults Mild & limited skin infections in pre- or early adolescence Cervical lymphadenitis in children
Lymphadenitis in children Isolated from sputum, urine, and gastric aspirate

M. UNKNOWN heidelbergense

ORGANISM M. shimoidei

EPIDEMIOLOGY Not isolated from environmental sources Few case reports Widely spread geographically Not well delineated; rarely isolated

TYPES OF INFECTION Tuberculosis-like pulmonary infection Disseminated disease

M. simiae

Tuberculosis-like pulmonary infections

 Comprised

of : M. avium, M. intracellulare, M. paratuberculosis, M. lepraemurium, M. silvaticum Pathogenic to humans: M. avium, M. intracellulare, M. paratuberculosis M. avium & M. intracellulare:

Closely resemble to each other Distinction cannot be made by routine laboratory determination / clinical grounds Referred to as M. avium-intracellulare

 EPIDEMIOLOGY

and PATHOGENESIS

Ubiquitous in the environment Isolated from natural water, soil, dairy products, pigs, chickens, cats, and dogs Natural waters serve as the major reservoir for most human infections Infections acquired through inhalation or ingestion Pathogenesis of infection not clearly understood Most commonly isolated mycobacterial species in the United States:

High prevalence in individuals affected with HIV Approximately 90% of mycobacterial infections in patients with AIDS involve either MAC or Mtb

 CULTURAL

CHARACTERISTICS

Exhibit OPAQUE and TRANSLUCENT/TRANSPARENT colony morphology Transparent colonies are more virulent:

More drug resistant Isolated more frequently from blood of patients with AIDS Appearing more virulent in macrophage and animal models

 EPIDEMIOLOGY

and PATHOGENESIS

Causes Johnes Disease in cattle, sheep, and goats Isolated from the bowel mucosa of patients with Crohns Disease (chronic inflammatory bowel disease in humans)

CULTURAL

CHARACTERISTICS

Extremely fastidious Requires MYCOBACTIN (growth factor produced by other mycobacterial species, such as M. phlei) May take as long as 6-18 months for primary isolation

 GENERAL CHARACTERISTICS: Eight (8) taxonomic groups of potentially

pathogenic rapidly growing mycobcateria are currently recognized Most species are purely environmental saprophytes Can grow on routine bacteriologic media, and on media specific for cultivation of mycobacteria Colonies appear in 72 hours of incubation at 37oC Appear as weakly gram-positive rods resembling diptheroids in Gram Staining

 EPIDEMIOLOGY and PATHOGENESIS Ubiquitous in the environment & present worldwide Found in soil, marshes, rivers, municipal water

supplies, and in marine & terrestrial form. Mode of acquisition:

Community acquired from environmental sources Nosocomially acquired as a result of medical intervention Commensals of the skin

Chronic Pulmonary infections caused by rapidly growing NTM suggests a possible respiratory route for acquisition of organisms present in the environment No known 1o animal host but apparently exist in the soil No evidence of direct transmission of organisms from man to man

 EPIDEMIOLOGY Disease results

and PATHOGENESIS
from 2 coinciding events:

Colonization of a large number of mycobacteria Impairment of bodys defense mechanism

Account for approximately 90% of clinical disease occasional pathogens of humans, birds and animals share similar characteristics and seen in same type of infection do not cause disease

M. fortuitum, M. chelonae, and M. abscessus

M. fortuitum and M. chelonae

M. fortuitum-M. chelonei complex

M. smegmatis and M. phlei

ORGANISM
M. abscessus

COMMON TYPES OF INFECTION

Disseminated disease 1 in immuncocompromised patients, skin & soft issue infections, pulmonary infections, postoperative infections Postoperative infections in breast augmentation and median sternotomy; skin and soft tissue infections

M. fortuitum

M. chelonae
M. fortuitum third biovariant complex sorbitol (+/-)

Skin and soft tissue infections, postoperative infections, keratitis

Skin and soft tissue infections

M. peregrinum M. mucogenicum
M. smegmatis

Skin and soft tissue infections Posttraumatic wound infections, catheter-related sepsis
Skin or soft tissue infections

 Most

common manifestation in adults in USA Most common cause:

M. kansasii M. fortuitum, M. avium-intracellulare complex (MAC)

Usually

seen in children M. scrofulaceaum

 M.

marinum M. ulcerans

: USA : Africa and Southeast Pacific

M.

kansasii M. fortuitum M. avium-intracellulare Complex (MAC)

Causes Disseminated infections in patients with AIDS

NON-CULTIVABLE MYCOBACTERIUM

 An

ancient disease HANSENs Disease Chronic disease of the skin, mucous membranes, and nerve tissues A disease that continues to threaten the quality of life of more than 12 million people world wide. With increase in population and rapid means of transportation, there is increased contact between susceptible travelers and millions of patients who have leprosy.

 Cannot

be cultured in vitro experimentally only in animals

Not grown in tissue cultures of various types of human cells

cultivated in the armadillo and the footpads of mice

Grown

Difficult

to propagate and transmit to experimental animals. Slow growth in both animals and in patients An obligate intracellular parasite that multiplies very slowly within the mononuclear phagocytes, especially the histiocytes of the skin and Schwann cells of the nerves

 Acid fast can be removed distinguishes M. Structure

by extraction with pyridine leprae from other mycobacteria

resembles that of M. TB Phenolase in M. leprae

obtained from lepromatous nodules separates M. leprae from other mycobacteria and nocardias.

 Most

prevalent in Tropical countries Primary reservoir is infected humans Infectivity is very low Transmission of Leprosy (Person to Person):

Infection thru contact with patients with Lepromatous Leprosy who shed organisms in nasal secretions. Prolonged close contact

Major

portal of entry: respiratory tract Potential source of infection: insect bites and breast milk Cutaneous route thru excoriations no significant role

 Spectrum

of disease characterized by pronounced variations in clinical, histopathologic and immunologic findings

 TUBERCULOID

LEPROSY (TT)

Disease is localized to the skin and nerves No immune defect Few organisms in skin lesions

LEPROMATOUS

LEPROSY (LL)

Disseminated form Defect in Cell Mediated Immunity (CMI) Patient is anergic to M. leprae Bacterial growth is impeded Extensive skin lesions containing numerous bacilli

 Borderline

tuberculoid leprosy (BT) Borderline leprosy (BB) Borderline lepromatous leprosy (BL)

 Long

incubation period: 2-3 years (40 years) Earliest symptoms:

Asymptomatic slightly hypopigmented macule usually in trunk or distal portion of extremities

of patients has solitary lesion and heal spontaneously Disease progress from 1 form to form to another

 Immunologic

prognosis:

status of patient, determines the

TUBERCULOID LEPROSY (TT)

Benign, few skin lesions Bacilli multiply in the skin within macrophages Intermediate in position Bacilli multiply in peripheral nerves and begin to cause sensory impairment

BORDERLINE LEPROSY (BB)

LEPROMATOUS LEPROSY (LL)

most severe and extensive form of disease Leonine facies (deeply furrowed lumpy face with prominent superciliary arches) Trauma Secondary infection

 Hansen

in 1874 described the myriads of bacilli in the lesions of leprosy patients. Acid fast in modified mononuclear or epitheloid cells called lepra cells arranged like packets of cigars.

 Organisms

are found singly or in large masses called globi.

 Bacilli

are usually straight or curved and may stain uniformly or show granular beads.
Uniformly stained bacilli are healthy bacilli Beaded bacilli are probably non viable

 Footpads

of normal mice are injected with materials from leprosy patients In mouse, infection can be initiated with as low as 1-10 bacilli Footpad temperature is 30C

maintained by keeping room tempt at 20-30oC secret in the footpad success

Generation

time is 12 days

 Multiplication

continues for 150-180 days until number of bacilli reaches 1 X 106 bacteria. Multiplication stops because of CMI. Used for drug screening and vaccination experiments

 Nine-banded

armadillos is used Armadillos has disseminated leprosy Used to study immunologic factors that control development of disease Provide large numbers of M. leprae for vaccination study

 POLYMERASE

CHAIN REACTION (PCR)

Used to detect and identify M. leprae in infected tissues

Suspect

leprosy

Symptoms Type and distribution of lesion History of living in endemic area

 Demonstration

of AFB in smears / histopathologic examination of materials

Sources: skin lesion, nasal scraping, ear lobe, tissue secretions, lymph nodes, bone marrow, human milk (+) on stained smears: large numbers of AFB are seen packed in lepra cells (mononuclear epithelioid cells) in parallel bundles suggesting packets of cigors/globi Staining Methods Used:

Ziehl Neelsen Method Cold Kinyoun Technique Wade-Fite Technique used in paraffinized tissue and demonstration of AFB in tissue secretions

 LEPROMIN

TEST

Not diagnostically useful of value in determining the position of the patient on the immunologic spectrum Lepromin - suspension of heat killed M. leprae Detects for:

Early reaction Fernandez reaction Late reaction Mitsuda reaction Tuberculoid Leprosy: (+) for Early and Late Reactions Lepromatous Leprosy: (-) for Early and Late Reactions Lacks specificity Causes False (+): persons with Tuberculosis healthy children with BCG vaccine

Results:

Disadvantages: