Mycobacterium

Virginia P. Mesola, MD Cebu Institute of Medicine

Mycobacterium
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 Aerobic, slightly curved or straight rods

Mycobacterium
Most distinctive structure of cell multilayered structure with abundance of complex lipids some unique to mycobacteria Have important characteristics in common with Corynebacterium and Nocardia called CNM group All produce mycolic acid and similar guanine-plus-cytosine (G+C) content

Tuberculosis
Pulmonary tuberculosis most common
manifestation Primary tuberculosis primary complex Secondary tuberculosis

Morphology M. tuberculosis
Slender, straight or slightly curved rods
with rounded ends True branching in old cultures Acid fast, no spores, no capsule Appear beaded in tissue and sputum due to vacuoles and polyphosphate content Acid fastness due to mycolic acid and physical integrity of the cell

Morphology M. tuberculosis
Acid fastness explained by 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 Difficult to stain with gram stain -> failure of dye to penetrate the cell wall considered gram positive Gram stains of clinical materials invalid for identification of mycobacteria

M. Tuberculosis cell wall

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

Cultural characteristics M. tuberculosis

Obligate aerobe Grow on ordinary simple synthetic med.,

but for 1o isolation from clinical material requires a more complex medium Grow very slowly colonies seen in 11-12 days of incubation at 37oC Colonies small, dry, scaly (cauliflower) Liquid media pellicle on surface of med. Generation time 14-15 hours

Resistance M. tuberculosis
Highly resistant to drying Cultures kept for 12 yrs 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

Determinants of pathogenicity
No exotoxin or endotoxin No single structure, antigen or mechanism
can explain the virulence of the bacteria No simple in-vitro test based on colonial morphology & serologic differences distinguish virulent from avirulent strains. Possible only with virulence testing in animals

Determinants of pathogenicity
Has properties associated with virulent
strains -> progressive disease, none can account completely for virulence, each plays a role in pathogenesis of disease
Cord factor serpentine cords Sulfatides responsible for neutral red reactivity associated with virulent strains

Laboratory diagnosis
Many species of mycobacteria both
saprophytes and potential pathogens may be isolated from humans If the isolate is mycobacteria other than M. tuberculosis, the lab should be able to identify the species in by in vitro tests.

Collection of specimen
Collect before anti-TB drug therapy is
started Use sterile container Send specimen to the lab immediately A series of 3-5 single early morning samples of sputum of 5-10 ml is collected Other specimens pus, CSF, urine, gastric lavage. Fluids from inflammed serous cavities

Microscopic examination
Detection of AFB in stained smears
easiest and most rapid procedure for evaluating a clinical specimen Most symptomatic TB patients sputum is positive for AFB. Sputum exam has important role in TB control program Making smear > get caseous areas in sputum > spread thin > stain with AFS

Microscopic examination
Reading smear make 3 longitudinal
sweeps of the stained area parallel to the length of the slide

Microscopic examination
Reading recommended by American Lung
Association
Number of bacilli 0 1-2/slide Report No AFB seen report # found and request repeat specimen 3-9/slide rare or + 10 or more per slide few or ++ 1 or more per field Numerous or ++++

Digestion and Decontamination

Decontamination before culture destroy
contaminants which grow faster than the Mycobacteria Digestion since bacteria are usually trapped in cellular & organic debris, exudates must be liquified before culture N-acetyl cysteine for digestion and NaOH for decontamination are used

Culture
Some culture media can detect as few as 10

Egg-potato based media (Lowenstein Jensen) Agar based media (Middlebrook 7H-10)

bacteria/ml of digested concentrated material Two types of media:

Incubate inoculated media at 37oC in 5-10%

CO2 Test for M. tuberculosis positive niacin test Report a negative culture after 2 months

M. TB complex
M. tuberculosis M. bovis seen in countries where raw
milk is ingested. Almost completely eliminated with pasteurization of milk M. africanum isolated in Africa

Bacillus of Calmette and Guerin (BCG)

attenuated mutant of M. bovis

Mycobacteria associated with nontuberculous infection (MOTT)

Initially considered as strictly saprophytes Last 40 years recognized clinical

significance Also called atypical mycobacteria Can produce severe and even fatal disease in humans

Runyon classification 4 groups of mycobacteria

Group I photochromogens Group II scotochromogens Group III nonphotochromogens Group IV rapid growers visible growth
in less than 7 days Groups I,II,III slow growers requiring 7 days or more to yield visible colonies

Slowly growing Mycobacteria

Photochromgen
M. kansasii M. marinum

Scotochromogen
M. scrofulaceum

Non-photochromogen
M. avium-intracellulare

Rapidly growing mycobacteria

Most species are purely environmental

saprophytes M. fortuitum and M. chelonei occasional pathogens of humans, birds and animals M. fortuitum-M. chelonei complex share similar characteristics and seen in same type of infection Colonies appear in 72 hours of incubation at 37oC M. smegmatis and M. phlei do not cause disease

Laboratory diagnosis
Many species of mycobacteria both
saprophytes and potential pathogens may be isolated from humans If the isolate is mycobacteria other than M. tuberculosis, the lab should be able to identify the species in by in vitro tests.

Laboratory diagnosis
Diagnosis of specific organism crucial in

determining therapy ID by Runyon classification is inadequate Do bacteriologic ID of organism and repeated demonstration in patient secretions in the absence of other potential pathogens Tuberculin test is not helpful A + AF smear and a () tuberculin test may lead one to suspect the disease.

Identification
Grow well on Lowenstein-Jensen (LJ),
Middlebrook 7H10 and other media used for culturing M. TB Niacin test negative Only a few of these are known human pathogens

Epidemiology
Ubiquitous and found in all parts of the world Endemic in certain geographic areas No known 1o animal host but apparently exist in

the soil No evidence of direct transmission of organisms from man to man Disease usually occurs in man with previous lung damage Disease results from 2 coinciding events:
Colonization of a large # of mycobacteria Impairment of bodys defense mechanism

Disease is group according to organ involvement

Pulmonary disease
Most common manifestation in adults in USA Most common cause: M. kansasii; M. fortuitum, M. avium-intracellulare complex (MAC) M. scrofulaceum

Lymphadentis usually seen in children

Disease is group according to organ involvement

Cutaneous lesions (skin lesions)
USA M. marinum Africa and Southeast Pacific M. ulcerans

Disseminated infections
M. kansasii; M. fortuitum; MAC AIDS - disseminated infection usually caused by M. avium-intracellulare complex (MAC)

Mycobacterium leprae
Leprosy is an ancient disease. Hansen in 1874 described the myriads of
bacilli in the lesions of leprosy patients. Cannot be cultured in vitro. Difficult to propagate and transmit to experimental animals. Slow growth in both animals and in patients.

Mycobacterium leprae
Leprosy is 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. Leprosy is of global importance.

Mycobacterium leprae Morphology

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.

Mycobacterium leprae Morphology

Acid fastness can be removed by extraction with

pyridine distinguishes M. leprae from other mycobacteria Structure resembles that of M. TB Not grown in tissue cultures of various types of human cells Grown experimentally only in animals Phenolase in M. leprae has been obtained from lepromatous nodules separates M. leprae from other mycobacteria and nocardias.

M. leprae experimental disease in animals

Animal models extensively used for study of

the organisms and experimental leprosy Footpads of normal mice injected with materials from leprosy patients In mouse, infection can be initiated with as low as 1-10 bacilli Footpad temp. is 30oC maintained by keeping room tempt at 20-30oC, the secret in the footpad success Footpads of mice generation time is 12 days

M. leprae experimental disease in animals footpads of mice

Multiplication continues for 150-180 days -> until # of

bacilli reaches 1 X 106 bacteria. Multiplication stops because of CMI. Used for drug screening and vaccination experiments Nine-banded armadillos has been used Armadillos has disseminated leprosy Used to study immunologic factors that control development of disease. Provide large #s of M. leprae for vaccination study. Man is highly resistant to experimental infection.

M. Leprae - epidemiology
12 million persons have leprosy worldwide Man is only natural host. Infection thru contact with patients with LL who shed

organisms in nasal secretions. Major portal of entry respiratory tract Potential source of infection insect bites and breast milk Cutaneous route thru excoriations no significant role Genetic factors contributes to susceptibility and type of response to infection with M. leprae

Ridley and Jopling classification

Tuberculoid leprosy (TT) Borderline tuberculoid leprosy (BT) Borderline leprosy (BB) Borderline lepromatous leprosy (BL) Lepromatous leprosy (LL) Only TT and LL are stable, other forms are unstable Spectrum of disease characterized by pronounced variations in clinical, histopathologic and immunologic findings

Clinical manifestations
Long incubation period 2-3 yrs (40 yrs) 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

Clinical manifestations
Immunologic status of patient
determines the prognosis TT benign, few skin lesions BB intermediate in position LL most severe, extensive form of disease leonine facies, trauma and 2o infection

M. leprae nerve involvement

M. leprae ia an obligate intracellular parasite
that multiplies very slowly within the mononuclear phagocytes, especially the histiocytes of the skin and Schwann cells of the nerves. It has strong predilection for the nerves. TT nerve damage is non specific due to CMI LL nerves are infected with numerous bacteria in Schwann cells. LL nerve damage is less than in TT

M. leprae - immunity
Leprosy is a disease of low infectivity. Lepers are immunologically defective with
respect to M. leprae CMI and clinical forms:
TT strong DTH to lepromin -> LL loss of CMI

Antibodies to M. leprae - no protective role Antibodies to M. leprae cross react with

other mycobacteria

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 Early reaction Fernandez reaction Late reaction Mitsuda reaction TT - + early and late reactions LL (-) Lacks specificity + reaction persons with TB, healthy children with BCG

Laboratory diagnosis
Suspect leprosy from symptoms, type and distribution

of lesion, history of living in endemic area Demonstration of AFB in smears of skin lesion, nasal scrapings, early lobes, tissue secretions LL bacilli numerous; TT very difficult to impossible to detect Histopathologic response in biopsy material helpful in TT Wade Fite technique used in tissue secretions to demonstrate the AFB

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