Mycobacteria

Mycobacteria

1. Mycobacteria
• To know various species of the Mycobacteria
• To know the various diseases caused by the Mycobacteria
• To know the pathogenesis of Mycobacteria
• To know various tests for diagnosis of diseases caused by
Mycobacteria
• To know the treatment of diseases caused by Mycobacteria

2. Mycobacteria
Classification:
A: Tubercle bacilli
•Human: M. tuberculosis
•Bovine: M. bovis
•Murine : M. microti
•Avian: M. marinum
B: Lepra bacilli
•Human: M.leprae
•Rat: M.leprae marium
C: Mycobacteria causing skin ulcers:
M. ulcerans, M. balnei
D: Atypical Mycobacteria:
–Photochromogen
–Scotochromogen
–Nonphotochromogen
–Rapid growers
E: JOHNE’s bacillus:
M. paratuberculosis
F: Saprophytic Mycobacteria:
M. butyricum, M. pheli, M. stercoris, M. smegmatis

3. Mycobacteria are either:

a)Obligate pathogens:
M. tuberculosis
M. bovis
M. africanum
b) Opportunistic pathogen:
Atypical Mycobacteria or MOTT:
M. kansasii
M.avium
c) Saprophytic Mycobacteria:
M. smegmatis
M. pheli
M. stercoris
M. butyrijcum

4. Mycobacterium tuberculosis
•Fungus like bacteria: filamentous
forms resembling fungal
mycelium, acid fast, aerobic, non
motile, non capsulated & non
sporing
•Morphology: 2-3um x 0.4 um,
straight or slightly curved rods
with rounded ends, branching &
filamentous forms , beaded,
arranged singly or in small
clumps,

5. Cultural characteristic:
–an obligate aerobe,
–37
0
c, pH- 7,
–slow growth – take 2-8 weeks to grow
–generation time- 14-15 hrs,
–grows well on enriched media containing
serum, potato, blood & egg
–M. bovis is microaerophilic

6. 1)Solid media:
LJ, Dorset egg medium
•Containing blood: Tarshis medium
•Containing serum: Loeffler’s serum slope
•Containing potato: Pawolosky’s medium
2) Liquid medium:
Dubbo’s, Middle brook’s(7H-10 & H-12) , proskaur’s medium
Colony characters:
on solid media: dry, rough, raised wrinkled, irregular
•Liquid media:
growth occurs as bottom creeps up the sides, forms prominent
surface pellicle, extends along sides above the medium
•New technique: BACTEC system

7. Biochemical tests:
•Niacin test
•Neutral red test
•Aryl sulphtase test
•Nitrate reduction test
•Amidase test
•Catalase & peroxidase test
•Susceptibility to pyrazinamide
•Susceptibility to thiophen 2-carboxylic acid hydrazide
•Tween 80 hydrolysis

8. Resistance:
•viable in sputum for 20-30 hrs
•in droplet nuclei for 8-10 hrs
•in culture for 6-8 months
•resistant to disinfectant
•sensitive to formaldehyde & Glutaraldehyde
•killed by sunlight for 2-3 hrs exposure
•sensitive to ethanol

9. Pathogenesis:
Inhalation of M. tuberculosis

Reach lung
Ingested by alveolar macrophages

Multiplication in macrophages
Ghon’s focus in lower lobe

10. Hilar lymph node involvement
Primary complex
healing & calcification haematogeneous

Cause latent infection milliary TB
Reactivation or exogenous infection
Secondary tuberculosis
Usually pulmonary TB

11. •Tubercle formation:
avascular granuloma composed of central zone containing giant
cells with or without caseation & necrosis, surrounded by a zone of
epitheloid cells, with a peripheral zone of lymphocytes & fibroblast
•Pathogenicity due to biological activities of the bacteria
–Cell wall:
delayed type of hypersensitivity
–Lipid:
formation of macrophages, monocyte, epitheloid, giant cell
–Polysccaharide :
immediate type of hypersensitivity
–Phospitidase:
helps in tubercle formation by forming epitheloid cell & giant cell

12. •Tubercle lesion :
1) exudative
2) productive
•Infection depends on:
–dose of organism
–virulence
–mode of entry
–age
–resistance
–hypersensitivity of patient
•Antigens present in mycobacterium spp:
–Group specific polysaccharide antigen
–Type specific protein antigen : used in detection of antibodies
•Phage types of mycobacterium: A, B, C, type I

13. Various systems involved in TB:
•Pulmonary ,renal, tubercular meningitis, bone
marrow & joint , miliary, intestinal, skin
tuberculosis
•Clinical symptoms:
fever, cough with expectoration, haemoptysis,
weight loss, loss of appetite, signs of pleural
effusion/consolidation/cavity

14. LAB diagnosis:
•Specimens:
Sputum
Gastric lavage
CSF
•Microscopy:
Ziehl - Neelsen staining: grading (RNTCP)
3+ = > 10 AFB / Oil immersion field
2+ = 1 - 10 AFB / Oil immersion field
1+ = 10 - 99 AFB / 100 Oil immersion field
Exact = 1 – 9 AFB / 100 Oil immersion field

15. •Petroff’s method for sputum and oxalic acid for urine
•Culture on Lowenstein Jensen (LJ) medium
•ELISA, RIA, latex agglutination
•Nucleic acid detection: PCR,LCR,DNA probes
•BACTEC, VersaTrek, MGIT
•Animal inoculation in guinea pig
•Antibiotic Sensitivity test

16. Tuberculin test: Mantoux test
•Type IV hypersensitivity test
•Purified protein derivative (PPD) intradermal inoculation on forearm
•Site observed after 72 hrs for erythema & induration
•Interpretation:
–10 mm indicates positive
–5 mm negative,
–between 5-9 mm indicates doubtful
•Use: to diagnose active infection in infants

17. Treatment :
•Bactericidal : rifampicin, isoniazide, pyrizinamide, streptomycin
•Bacteriostatic: ethambutol, cycloserine, capreomycin, kanamycin, ciprofloxacin
•DOTS,RNTCP
Prophylaxis:
–BCG: live attenuated vaccine
–Strains used: M.bovis
–Route of inoculation: intradermal
–Age: at birth,shedule: single dose
•Immunity: 10- 12 yrs, protects individuals from complicated forms of tuberculosis
•Adverse effects : local, regional, systemic
•Contraindicated in AIDS, measles

18. Thought for dental professionals
38 yr Female with non painful swelling of the gingiva right upper side since 2
years
Gradually increasing in size with time
Experienced weight loss since 4/5 months
There was a cough and weakness since 15 days
Medical history reveals non systemic problem except cough and
expectoration since 15 days
Patient never visited dentist in her lifetime no dental trauma history or
surgery
Intra oral exmn showed gingival enlargement especially in upper and lower
anterior labial and upper posterior buccal areas
Gingiva was fiery red, irregular, papillary, pebbled and granular in
appearance
Lesion was slightly painful on touch with spontaneous bleeding on
provocation
No significant lymphadenopathy, swelling on lips, CBC was normal, HIV
negative, elevated ESR, CXR normal

20. •Gingival tuberculosis Published in J
Ind Soc Periodontol Aug 2009
•Conclusion:
–It can be occupational risk for dentists
–It is rare entity
–Consider for diff diagnosis
–Diff Diag :
•Gingival enlargement due to drugs
•Infections (bacerial, fungal and viral)
•Malignancy – leukemia
•Traumatic ulcer
•Squamous cell carcinoma
•A major concern for dentists so Infection prevention practices
meticulously

21. Atypical Mycobacteria
•Mycobacteria other than mammalian tubercle bacilli
•Occasionally cause human disease resembling TB
•Are opportunistic pathogens
•Also referred as Nontuberculous mycobacteria or MOTT
•Mycobacteria other than tubercle
•Classifed by Runyon (1959) based on pigment
production and rate of growth

22. Classification of atypical mycobacteria
•Photochromogens:
–pigments in sunlight,
–M. kansasii, M.simiae, M.marinum
•Scotochromogen :
–pigments in light as well as in dark,
–M. scrofulaceum, M.szulgai

23. Classification of atypical mycobacteria
•Nonchromogen:
–not produce pigment in light also,
M.avium,M.intracillularae,M.xenopi, M. ulcerans
•Rapid growers:
–grow within 7 days,
–M. fortuitum, M. chelonei

24. Atypical mycobacteria
•Opportunistic pathogen
•Some are rapid growers
•Some produce
pigments
•Niacin: negative
•Aryl sulphtase: positive
•Strong catalase positive
•Non pathogenic to
guinea pig
•R to antituberculous
drugs
•Some grow at 25
0
C
&some at 45
0
C
Typical
•Obligate pathogen
•Slow growers
•Non pigmented
•Niacin : positive
•Aryl sulphtase :
negative
•Weak catalase positive
•Pathogenic to guinea
pig
•S to antituberculous
drugs
•Growth does not occur
below 25
0
C & above
44
0
C

25. • Disease
•Pulmonary infection like
tuberculosis
•Lymphadenopathy usually
cervical
•Cutaneous ,subcutaneous
lesions
•a. chronic ulcers
•b. Abscess
•c. Swimming pool
granuloma
•d.Buruli ulcer
•e. Surgical wound infection
•Systemic disseminated
disease

•Usual atypical agents
•M.kansasii, M.avium, M.
intracellulare, M.simiae
•M.szulgae, M.xenopi,
M.chelonei, M. avium-
intracellulare,
M.scrofulaceum, M.kansasii
•M. marinum
•M.fortuitum, M.chelonei
•M.marinum
•M.ulcerans M. chelonei,
M.fortuitum
•M.avium, M.intracellularae

26. Laboratory diagnosis
•Microscopy
–Ziehl Neelsen staining
•Culture
–on Lowenstein Jensen (LJ)
–Incubated at 25
0
C, 37
0
C, 45
0
C,
–slants observed for pigment production

27. Identification
•Identification by different tests such as
–Nitrate reduction
–Aryl sulphtase
–Tween 80 hydrolysis
–growth at different temperatures
•Antibiotic susceptibility testing
•Treatment :
–prolonged treatment with INH,
–rifampicin,
–ethambutol, clofazimine,
–quinolones,
–macrolides

28. Mycobacterium leprae
•Leprosy is a old disease since vedic times
•Probably originated in the tropics and
spread to the rest of the world
•Lepra bacilli was first observed by Hansen
in 1868

29. Morphology
•L. leprae is a straight or slightly curved rod
•1-8 um x 0.2-0.5 um in size
•Polar bodies and other intracellular elements
present
•Clubbed forms, lateral buds or branching
•It is Gram positive readily stain than Tb bacilli
•Acid fast but less than M. tuberculosis
•5% H2SO4 is used as decolorizer
•Bacilli are seen singly and in groups intracellular or
lying free outside the cells

30. Cultivation:
•Not been able to cultivate in artificial media
•Can be cultivated in mice, nine banded armadillo
•Generation time 12-13 days
Resistance:
Remain viable in warm humid envt. For 9-16 days
For 46 days in moist soils
Survive exposure to direct sunlight for 2 hrs and UV rays for
30 min

31. Leprosy
•Is a chronic granulomatous disease primarily of skin,
peripheral nerves and nasal mucosa
•Classified as
–Lepromatous
–Tuberculoid
–Dimorphous
–Indeterminate
Type of disease is reflection of immune status of the host

32. Lepromatous:
•Lepromatous is seen in where the host immune response os
low
•Bacilli are seen in large numbers (multibacillary stage)
•Bacilli invade nose, mouth and upper respiratory tract and
are shed in large numbers in nasal and oral secretions
•More infective and prognosis is very poor
•Cell mediated immunity is deficient and lepromin test is
negative
•Humoral immunity is more

33. Tuberculoid:
Seen in patients with high degree of resistance
Skin lesions are few and sharply demarcated
Bacilli are scanty (paucibacillary)
Cell mediated immunity is adequate
Lepromin test is positive
Prognosis is good but humoral immune response is poor

34. Borderline or dimorphous:
Lesions possessing both tuberculoid and lepromatous
typesI
Indeterminate:
Is early unstable tissue reaction
Which is not characteristics of either
It may undergo healing spontaneously

35. Ridely and jopling classification
•1966
•Five groups
1.Tuberculoid (TT)
2.Borderline tuberculoid (BT)
3.Borderline (BT)
4.Borderline lepromatous (BL)
5.Lepromatous (LL)

36. epidemiology
•Exclusively human disease
•Source – patient
•Mode of transmission is still not clear
•Large no. of bacilli shed in nasal secretions
•Patients may discharge 8x10
8
bacilli in one nose blow
•Mode of entry
–Respiratory tract
–Skin
It is not highly communicable
Disease develops in 5% in spouses
Incubation period is 2-5 yrs
Worldwide in distribution
India – Orisa and Bihar (highest prevalence)

37. •Lepromin test:
–skin test for delayed hypersensitivity & used to study immunity in
leprosy patients, antigen is Mitsuda’s Ag or bacillary lepromin
•Procedure : 0.1ml injected intradermally on forearm
•Two reactions:
1. early or Fernandez reaction: is an acute inflammatory area of more than
10 mm in diameter appearing in 24-48 hrs & disappears in 3-4 days
2. Late or Mitsuda’s reaction: appears 3-4 weeks after injection in the form
of nodule,may ulcerate or subside in few weeks
•Mitsuda’s reaction is more meaningful is a manifestation of
CMI,induced by lepromin while Fernandez reaction indicates past
reaction
•Uses:
1. classify lesions of leprosy
2. to assess prognosis & response to treatment
3. to assess resistance of an individual to leprosy
4. to verify candidate lepra bacillus

38. Lab diagnosis:
•Specimens: skin clippings, nasal samples,skin
nerve biopsy, lymph node puncture
•Microscopy:
–ZN staining with 5% sulphuric acid
•Gradings: Ridely scale
•1+: 1-10 bacilli/ 100field
•2+: 1-10 bacilli/ 10 field
•3+: 1-10 bacilli/field
•4+: 10-100/ field
•5+: 100-1000/field
•6+: more than 1000, clumps/field

39. •Morphological & bacteriological index calculated
•Culture: footpads of mouse, granuloma developes in 1-6
months
•Serological: latex agglutination, ELISA
•Lepromin test not for diagnosis but to resitance of patient
•Treatment:
–for paucibacillary leprosy: rifampicin 600mg once in a month &
Dapsone (4’,4’- diaminodiphenyl sulphone,DDS)100mg daily for 6
months
–For multibacillary leprosy : rifampicin, 600mg once a month
Dapsone 100mg daily &clofazimine 50mg daily for 2 years

40. Department of Microbiology
Mr. Mukhit Kazi, Lecturer