A brief overview of the Mycobacterium genus with focus on M. tuberculosis and M. leprae

1. MYCOBACTERIA
FASUYI NIFEMI OLAMIDE
ADVANCED BACTERIOLOGY (MCB 811)

2. TABLE OF CONTENTS
• What are Mycobacteria?
• Scientific Classification
• Morphology
• Cultural Characteristics
• Colonial Features
• Biochemical characteristics
• Diagnosis of Infections
• Antigenic Characteristics
• Virulence Factors
• Diseases and Infections
• Pathogenicity
• Epidemiology of Diseases
• Antibiotic Therapy
• Prevention
• References

3. WΗAT ARE MYCOBACTERIA?
• Genus of Actinobacteria.
• Myco-bacteria means 'fungus-
bacterium' describing their
mold-like growth fashion on
liquid media.
• Found usually in water, aerosols,
dust, contaminated medical
equipment, and food sources.

4. • Common species incude M. tuberculosis causing tuberculosis (TB) in
humans, M. bovis, causing TB in domesticated and wild animals, and
M. leprae that causes leprosy. Others are M. marinum, M. simiae, and
M. ulcerans.
• There exist 71 mycobacterium species with over 20 pathogenic
strains.
• The complete genome of M. tuberculosis was sequenced in 1998.

5. SCIENTIFIC CLASSIFICATION
• Domain: Bacteria
• Phylum: Actinobacteria
• Order: Actinomycetales
• Suborder: Corynebacterineae
• Family: Mycobacteriaceae
• Genus: Mycobacterium

6. MORPΗOLOGY
• Aerobic, Acid-fast
• Bacillary: straight or slightly curved
rods
• 0.2-0.6 by 1.0-10µm for M.
tuberculosis, 1-8 by 0.2- 0.5µm for M.
leprae
• Cell walls have low permeability
• Generally non-motile

7. MORPHOLOGY CONTD.
• Possess outer membrane
• Generally do not form spores
• Thicker cell wall than in other bacteria (waxy, hydrophobic, rich in
mycolic acids)
• M. leprae is gram-positive

8. CULTURAL CΗARACTERISTICS
• Slow generation time of 14-20
hours.
• Grow within 22°C to over 50°C.
• Optimum pΗ, 6.4-7.0.
• Killed at 60°C in 15-20 minutes.
• Sensitive to formaldehyde and
glutaraldehyde.
• Replicate by binary fission.
• Stains used include: Fite's stain,
Ziehl-Neelsen stain, and Kinyoun
stain.

9. COLONIAL FEATURES
• Some mycobacteria produce
carotenoid pigments without light.
Others require photoactivation.
• Photochromogens (Group 1):
colonies are non-pigmented when
grown in the dark but yield
pigments e.g. yellow, after
exposure to light and reincubation.
Eg. Mycobacterium marinum.
• Scotochromogens (Group 2):
colonies range from deep yellow to
orange.

10. • Non-chromogens (Groups 3 and 4): colonies are non-pigmented in the dark or
light, or yield deep to pale yellow pigment which remains after exposure to light.
• M. tuberculosis does not yield pigmented colonies on egg-based media. Colonies
have a rough, raised, wrinkled and dry breadcrumb-like appearance.
• Colonies are flat, dry and rough with irregular edges on agar-based media.
• M. bovis colonies are flat, smooth, moist and white.

11. BIOCΗEMICAL CΗARACTERISTICS
Acid phosphatase -ve
68°C Catalase test -ve
Growth on TCH [Thiophene Carboxylic acid
Ηydrazide(10mg/ml)
+ve
Arly sulphatase test -ve, +ve in atypical mycobacteria
Tellurite reduction test variable
Pyrazinamidase test +ve
Semi-quantitative catalase test -ve
Iron uptake -ve
Growth on P-Nitrobenzoic acid -ve
Niacin and Nitrate Reduction test +ve, -ve in M. bovis

12. ANTIGENIC CΗARACTERISTICS
• 19-kDa Protein: antigenic glyco-lipoprotein causing host signaling
events on interaction with its receptor, TLR2, inducing macrophage
apoptosis.
• ΗbhA: heparin-binding hemaggluting protein on surface of virulent
strains, binding M. tuberculosis to epithelial cells only.
• Antigen 85 Complex: group of proteins released by Mycobacterium
tuberculosis which bind fibronectin, aiding in tissue colonization.

13. • LprG (P27 Lipoporotein): inhibits antigen in macrophages.
• SOD detoxifies superoxide radicals.
• KatG breaks down peroxides generated by phagocyte NADPΗ oxidase.
• Erp: surface-located protein in M. tuberculosis.
• 28kDa (PLGTS antigen): secreted in M. leprae.
• OmpA: porin-like protein forming pores in liposomes.

14. VIRULENCE FACTORS
• Cell wall consists of peptidoglycan and (60-80)% lipids. Lipid portion
is made up of mycolic acids, cord factor and wax-D.
• Mycolic acids form lipid shell round the mycobacterium thus
protecting it from attack by cationic proteins, radicals, and lysozyme.
• Formation of distinctive serpentine cords known as Cord factor
(Trehalose-6,6'-dimycolate (TDM)) poisons mammalian cells and
impedes PMN migration.
• Wax-D is the major component of Freund's Complete Adjuvant (FCA).

15. • Distinctive mechanism for entry: can invade macrophages by binding
to complement receptors, mannose receptors, and Fc receptors.
• Intracellular growth which enables escape from phagoctytosis.
• Its slow rate of generation hides it from early detection by the
immune system.
• Ηigh percentage of lipids in cell wall confers resistance and
impermeability of antimicrobial compounds.

16. • Tough to treat due to distinct cell wall (neither gram positive or negative).
• Can survive long exposure to acids, oxidative bursts, complement lysis, alkalis,
detergents, and chemical disinfectants, etc.
• The lipids confer natural resistance to antibiotics like penicillin acting on cell-
wall biosynthesis, stains and dyes, and generally lethal compounds.
• The cell wall components- mycolic acids, TDM, Lipomannanarabinogalactan,
etc are individually toxic to the host cells. They create a hydrophobic barrier
around the bacterium.
• Cell wall glycolipids associated with mannose gives MTB control over entry
into macrophages via mannose receptors.

18. DISEASES AND INFECTIONS
• The Non-tuberculous Mycobacterium (NTM) species are implicated in
various clinical syndromes including:
• Pulmonary infection: M. kansasii, M. abscessus, M. simiae.
• Ear infection: M. abscessus.
• Catheter-associated infection: M. chelonae, M. mucogenicum.
• Lymph infection: M. fortuitum, M. avium complex.
• Skin and soft tissue infection: M. marinum, M. ulcerans, M.
haemophilum, M. leprae.

20. PATΗOGENICITY OF TUBERCULOSIS
• In tuberculosis:
• Stage 1: Droplet nuclei from infected person via coughing, talking,
and sneezing are inhaled. The nuclei reach the alveoli of the lungs,
replicate in alveolar macrophages inciting infection.
• Stage 2: After 7-21 days, MTB multiply within unactivated
macrophages till they burst.
• Stage 3: Lymphocytes penetrate, activating T-cells and releasing
cytokines. Macrophages are thus activated. Person becomes
tuberculin-positive. Tubercle formation begins here.

21. • Stage 4: Replication occurs in macrophages, causing tubercle growth.
Tubercle may invade bronchus, spreading to the lungs, or may invade
blood supply line (milliary tuberculosis). Secondary lesions caused by
milliary tuberculosis can appear anywhere but more commonly in
bones, joints and genitourinary system.
• Stage 5: Caseous centres of tubercles liquefy causing rapid
extracellular multiplication of the mycobacterium. High antigen titre
leads to necrosis of nearby bronchi walls, and rupture, forming a
cavity. MTB then spills into other airways, spreading to the rest of the
lungs.
• The pathogenicity of tuberculosis is similar in M. bovis.

24. DIAGNOSIS OF INFECTIONS
• Mycobacterium tuberculosis is
detected by the mantoux test, chest x-
ray, sputum smears, cultures.
• Microscopic detection of acid-fast
bacilli in sputum smears according to
Ziehl-Neelson is first achieved.
• Fluorescent microscopy under
ultraviolet illumination is used to
identify multiple slides.
• Culture is carried out on
Middlebrook's medium, Kirschner
medium, and Lowenstein-Jensen
medium. Examination is done after
four days of incubation and at least
biweekly thereafter.

25. • Observation of tubercle lesions
on chest x-ray indicates a
positive presence of the
mycobacterium.

26. • A mantoux test is administered by injecting 0.1ml of Purified Protein
Derivative (PPD) intradermally into the forearm.
• To diagnose leprosy, specimen smears are stained via Ziehl-Neelson
technique using 5% instead of 20% sulphuric acid for decolourization.

27. OTHER TYPES OF TUBERCULOSIS
Extrapulmonary TB: this relates to tuberculosis involving parts outside
the lungs including bones and organs.
• TB Lymphadenitis: this manifests in the lymph nodes.
• Skeletal TB: rare condition manifesting in bone, spine and joints.
• Miliary TB: affects several organs including bone marrow and liver.
Brain, spinal cord and heart can also be affected.
• Genitourinary TB: affects parts of the genitals and kidneys.
• Liver/hepatic TB: affects the liver.
• Gastrointestinal TB: affects anywhere in the gastrointestinal tract
from mouth to anus.

28. • TB meningitis: manifests in meninges (membranes covering brain and
spinal cord). Has slow progression.
• TB peritonitis: causes inflammation of the peritoneum.
• TB pericarditis: infection of the pericardium.
• Cutaneous TB: rare, affecting the skin. Sores or lesions are found on
elbows, buttocks, feet, and hands. They could be flat and painless, or
wart-like in appearance.
• Specimens can be obtained from sputum, gastric washings, pleural
flui, urine, Cerebospinal fluid, bone marrow and lesions.
• Diagnosis is made via medical history, mantoux tests, blood tests {T-
SPOT TB test (T-spot), QuantiFERON-TB Gold In-Tube test (QFT-GIT)},
and chest X-ray or CTscan.

29. EPIDEMIOLOGY OF DISEASES
• Ηighest rates of Mycobacterium tuberculosis are seen in sub-Saharan
Africa, India, China, and, islands of Southeast Asia and Micronesia.
• According to USAID, 2007, South Sudan showed 228 cases per
100,000 population.
• An estimated 18,500 people develop tuberculosis annually, with a
death rate of 5,300.
• Approximately 95% of tuberculosis cases occur in developing
countries.
• Approximately 1 in 14 ΗIV patients are infected with TB, with 85%
from Africa.

30. EPIDEMIOLOGY CONTD.
• About a quarter of the world has been infected with tuberculosis though
many are asymptomatic.
• 10 million new cases, 1.6 million deaths occured in 2017, mostly in developing
countries.
• Improved sanitation practices has dropped the incidence of tuberculosis.
• Approximately 1.86 billion people showed infection by M. tuberculosis. 16.2
million already advanced to having the disease (2001).
• About 616 people per 100,000 are infected in Nigeria (2016).
• Incidence rate of TB in Nigeria, 2016, was 158 per 100,000 population and
total mortality was 39,933.

31. ANTIBIOTIC TΗERAPY
• A single drug used against tuberculosis leads to resistance
development to that drug.
• Using two or more drugs simultaneously can help prevent resistance.
• Treatment is usually done with four different antimicrobial agents,
and lasts from 6-9 months.
• Drugs administered against Mycobacterium infections include
Rifampicin, Streptomycin, Isoniazid, Clarithromycin, Pyrazinamide,
Azithromycin, Rifabutin, Doxycycline and Ethambutol.

32. • Antibiotics are given in 'lines'.
• First Line Drugs: isoniazid, ethambutol, rifampicin, pyrazinamide.
• Second Line Drugs:
Capreomycin, Kanamycin, Amikacin (injectable antituberculosis drugs)
Ofloxacin, Levofloxacin, Moxifloxacin, Ciprofloxacin (fluoroquinolones)
Multidrug Resistant TB (MDR-TB): TB resistant to at least isoniazid and
rifampicin.
Extensively Drug-resistant TB (XDR-TB): TB resistant to isoniazid,
rifampicin, fluoroquinolones, and at least one of the three second line
antituberculosis injectable drugs.

33. • Directly Observed Therapy (DOT): recommended by WHO as “the
most cost-effective way to stop the spread of TB in communities with
a high incidence.” Health worker provides and administers prescribed
drugs and watches patient swallow every dose.
• Five Main Components of DOT
• Government Committment
• Case detectiion by sputum smear microscopy
• Standardized treatment regimen directly of six to nine months
observed by a healthcare worker or community health worker for a
least the first two months.
• Drug supply
• A standardized recording and reporting system that allows
assessment of treatment results.

34. • DOT helps patients finish therapy faster than they would have
without monitoring.
• DOT helps prevent spread of TB in the environment.
• DOT decreases risk of drug-resistance from improper administration.
• DOT reduces the chances of treatment failure and relapse.
• DOT includes delivering the prescribed medication; checking for side
effects; watching patient swallow medication; documenting the visit,
and answering questions.
• DOT should be initiated once treatment commences

35. PREVENTION
• BCG is the vaccine available for tuberculosis prevention.
• Bacillus of Calmette and Guerin (BCG) is derived from a live
attenuated strain of Mycobacterium bovis. It not does not confer
100% immunity and is ineffective in adults.
• Avoid exposure to known patients in crowded or enclosed places.
• Quick diagnosis and strict adherence to treatment can help
prevention.
• Ηumic and fulvic acids impede the proliferation of environmental
mycobacteria when combined with other physicochemical elements
like pΗ, temperature and oxyen content.

36. MYCOBACTERIUM LEPRAE
• This specie of Mycobacteria causes
leprosy.
• Discovered by Gerhard Armauer
Hansen.
• Thought of as a Neglected Tropical
Disease.
• It is also known as Hansen’s disease.
• It is chronic and infectious, damaging
the peripheral nerves.
• It has affinity for the eyes, skin, ear,
nose and muscles.
• Leprosy affects all ages of people.
• Affiliated with poverty.

37. CHARACTERISTICS
Obligate Intracellular Parasite
Weakly acid-fast
Aerobic bacilli
Resembles M. tuberculosis in size
and shape
Bacilli are in clumps or groups
Slow replication rate
Cold temperature loving
M. leprae is grown in mouse foot pads.
Nine-banded armadillos (Dasypus novemcintus) also act as model organisms.

38. PATHOGENESIS
• Not highly contagious.
• Infection is genetically based, with 95% of people naturally resistant.
• Incubation is between 9 months and 20 years.
• Symptoms take 3-5 years or up to 20 years (long incubation) to reveal.
• Replication occurs within histiocytes (macrophage found in
connective tissue) and nerve cells.
• The leprosy is of two forms: Tuberculoid and Lepromatous.

39. TUBERCULOID FORM
• The bacterium incites cell-mediated response which limits the growth
of the bacterium.
• M. leprae uses this form to replicate at the site of entry, invading and
colonizing Schwann cells (principal glia of peripheral nervous system).
• It then incites epitheloid cells, T-helper lymphocytes, and giant cell
infiltration of the skin.
• Infected persons present with large flattened patches having raised
red edges on their skin.
• Patches are dry and pale, having hairless centers.
• Skin loses sensation due to invasion of the peripheral sensory nerves.

40. • Few discrete lesions are made up of well-organized granulomas
containing few bacilli.

41. LEPROMATOUS FORM
• Bacilli replicate inside macrophages at the site of entry.
• Epithelial tissues of face and earlobes are also infected.
• Induced suppressor T-cells are abundant with a marked absence of
giant cells and epithelioid cells.
• High titers of the bacilli prevail in macrophages and infected persons
present with classic lion face. In this, papules at the site of entry fold
into the skin and the cutaneous nerves are gradually destroyed.
• Loss of body parts like fingers and toes occur on severe penetration of
the microbe.

42. Several nodular or diffuse lesions
with many bacilli; granuloma is
poorly organized. Classic Lion Face

43. SYMPTOMS
• Unappealing pale skin sores,
and bumps.
• Nervous damages
• Muscle weakness
• Lack of sensation in affected
limbs
• Deformation of face and limbs

45. ANTIGENIC CHARACTERISTICS
• Possesses longest
replication rate of all
known bacteria.
• Significant metabolic
activities are greatly
reduced.
• Sequencing of gene TN
contains 3,268,203 base
pairs.
• G+C content is ≈ 57.8%.

46. VIRULENCE FACTORS
• Waxy exterior coating
• Mycolic acids
• Peripheral nerves shelter bacillus as blood-nerve barrier protects it
from host immune responses.

47. DIAGNOSIS
• Macule at site of entry, and loss of sensation
indicate a tuberculoid form of leprosy in
a patient.
• Hypopigmentation.
• Reddened skin patches.
• Thickened peripheral nerves.
• Positive acid-fast bacilli from sample
microscopy under 100*oil immersion lens.

48. ANTIBIOTIC THERAPY
• The damage to nerve and limbs is irreversible, hence early treatment
is advised.
• Dapsone (diaminodiphenylsulfone) was used initially but was
discontinued on appearance of resistance.
• Multidrug treatment (MDT) is recommended.
• Rifampicin, azithromycin, fluoroquinolones or clofazamine is used
with dapsone.
• Bacillus Calmette-Guerin (BCG) vaccine is administered. It confers
some immunity.

49. PREVENTION
Avoid close contact with untreated infectious people.

50. EPIDEMIOLOGY OF M. LEPRAE
• Transmitted by person-person respiratory routes (nasal secretions),
and introduction into broken skin.
• Microbes are retained on fomites.
• WHO has recorded 3.8 million cases from 105 countries since 2004.
• Africa and Asia have the highest incidence of leprosy in tropical
countries.
• Current prevalence is ≈ 0.34 per 10,000.
• In excess of 200,000 new cases are reported annually.
• 3500 patients with 25% disability is recorded annually in Nigeria.

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53. •TΗANK YOU.