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,
– 370
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 250
C
&some at 450
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 250
C & above
440
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 250
C, 370
C, 450
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 8x108
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