2. GENUS: MYCOBACTERIUM
Classification –
-Family Mycobacteriaceae
-1 genus of medical importence = Mycobacteria
-All are slow growing
-All are acid-fast and contain large amounts of
lipids in their cell walls
-Tubercle bacilli = M. tuberculosis, M. africanum,
and M. bovis
3. •Mycobacteria other than tubercle bacilli
(MOTT) or the atypicals. All other species.
•The Mycobacteria are divided into 4 groups
(Runyon groups) based on growth rate and
pigmentation:
1. Photochromagens:
- are non-pigmented when grown in the
dark.
- produce photoactivated pigments upon
exposure to light
e.g M. kansasii, M. marinum, M.
asiaticum, M. simiae
4. 2. Scotochromogens:
-Produce deep yellow to orange pigments when
grown in light or dark,
- The color deepens upon two weeks exposure to
light e.g M. gordonae, M. scrofulaceum, M. szulgai, M.
xenopi.
3. Nonphotochromogens:
-May produce pigment ranging from white to yellow,
-The pigment does not intensify upon exposure to
light. e.g. M. tuberculosis. M. avium, M.
intracellulare, M. terrae, M. ulcerans.
4. Rapid growers:
- organisms that form colonies within seven days.
eg. M.phlei, M. smegamtis, M. fortuitum, M. chelonei.
5. Morphology and cultural characteristics
Obligate aerobe, Gram-positive rods
Acid fast
Complex cell wall lipids
– include mycolic acids
– protects vs. phagolysosomal components
Peptidoglycan, glycolipids
– acid-fastness
NB: Always work under biosafety cabinet!
6. Heating required for stain penetration
due to the high lipid content of the cell wall
(mycolic acid and waxD).
Several acid fast stains that may be used:
1. Ziehl-Neelsen:
-uses heat to get the primary stain of
carbol fuchsin to penetrate the cell wall;
- acid alcohol destaining;
- methylene blue as the counterstain.
7. 2. Kinyon:
– Uses a higher content of phenol
(organic solvent) in the carbol fuchsin
primary stain to allow penetration of
the stain without the need to apply
heat.
-Acid alcohol for destaining and
-ethylene blue as the counterstain.
8. 3. Auramine-rhodamine fluorochrome (a
fluorescent stain):
-Requires a fluorescsnt microscope.
-Stain with auramine-rhodamine for 10
minutes (phenol in the solution allows for
penetration)
-Destain with acid alcohol
-Counterstain with acridine orange
-A positive result is a bright yellow
fluorescence.
10. Highly contaminated specimens with organic debris and
normal flora, should be digested and decontaminated with NaOH.
Most grow on simple media.
For primary isolation complex media should be used
– Use of a nonselective, a selective and possibly a liquid
media is recommended.
1. Nonselective
-May be egg or agar based.
- May include malachite green to suppress growth of contaminating
bacteria.
a. Lowenstein-Jensen
-egg based;
-Colonies grow in 18-24 days.
b. Middlebrook 7H10 and 7H11
– agar based;
- colonies grow in 12-14 days.
11. 2. Selective media:
– Consists of one of the nonselective media plus
added antimicrobial agents (malachite green,
cyclohexamide, and nalidixic acid are often used)
-The colonies of M. tuberculosis on the solid media are
rough, dry, granular, nonpigmented to buff colored
colonies.
3. Liquid media:
- Media usually contains tween 80 and albumin and
the organisms will grow faster than on solid media
NB: Most Mycobacteria grow best in 5-10% CO2 and at
35-370 C.
12. Identification
Rate of growth and growth in relation to temperature
Pigmentation and photoreactivity
Further biochemical testing includes:
1. Niacin reduction
-M. tb. Is nitrate reduction+ and – for catalase at
680 C
2. Tween hydrolysis,
3. Arylsulfatase production,
4. Tellurite reduction,
5. Salt tolerance, and
6. Pyrazinamidase production
14. Virulence factors.
Cord factor :
– A glycolipid, trehalose 6,6’ dimycolate responsible
for the serpentine growth (filaments or cords) of M. tb. in
which the bacilli grow in close parallel arrangement.
-Is toxic to leukocytes,
-antichemotactic,
-interfees with mitochondrial function in mice and
-plays a role in the development of granulomatous lesions
Iron capturing ability – required for survival inside
phagocytes
Sulfolipids prevent phgosome-lysosome fusion so that
the organisms are not exposed to lysosomal enzymes
(important in intracellular survival)
15. Pathogenecity
1. M. bovis:
• Hosts: cattle- natural host, swine,horses, dogs
and sheep!? Cats also susceptible and may
perpetuate bovine disease.
• In cattle- pulmonary d’se with involvement of
associated lymph nodes.
• Viscera and bone infections- occur in human
• Chickens- resistant.
• Rabbits, mice and guinea pigs more
susceptible.
16. 2. M. avium
Chicken most susceptible
Other birds-Yes
Not all infected chickens show gross lesions.
Water fowls – resistant
In swine- disease found in lymph nodes of the
head.
Cattle refractory but sensitized
Sporadic cases in horses, dogs and cats
Infection in human- little consequence.
17. 3. M. tuberculosis
• In human and primates
• Cattle sensitized by the human organism
• Swine- diseases in lymph nodes of the head
• Parrots – susceptible
• Dogs- can
• Cats – resistant
• Chicken-rare
• Guinea pigs and mice- very susceptible
• Rabitts- susceptible
18. CULTURE CHARACTERISTICS
On primary isolation:
– visible growth after up to 8 weeks
Colonies:
– Buff colour, dry bread crumb-like appearance
– Growth is eugonic (M. bovis = dysgonic)
Growth temperature:
– 35-37oC
Obligate aerobe
Heat-sensitive
Susceptible to alcohol, glutaraldehyde and
formaldehyde.
19. Differential characteristics of
tuberculle bacilli causing animal/human disease
____________________________________________________
Species Atmospheric
preference Nitratase TCH Pyrazinamide
---------------------------------------------------------------------------------------
M. tuberculosis Aerobic + S S
M. bovis Microaerophilic -- R R
_______________________________________
TCH = thiophen-2-carboxylic acid hydrazide
S= Sensitive, R= Resistant
20. THE DISEASE
Not highly contagious:
–transmission with prolonged contact between
susceptible and active case
–usually transmitted by airborne droplets, must
penetrate deep into respiratory tree
–infection can be via other routes: vingestion =>
infection through cervical or mesenteric LN
22. TUBERCULIN TEST
Tuberculin: a heat-concentrated filtrate of a
broth in which tubercle bacilli had been grown.
Injection of tuberculin into the skin >>
– Large, indurated reactions >>Post-Primary
Tuberculosis.
– No induration >> Protective immunity
Purified Protein Derivatives (PPD):
– Mantoux Method (Intracutaneous)
– Heaf Method (Spring-loaded gun)
– Tine Tests (Disposable single tests)
32. • TREATMENT
– Penicillin, Ciprofloxacin
• IMMUNIZATION
–Animals > Live spore vaccine (Sterne strain)
– Workers at Risk of Exposure >
Anthrax Vaccine Absorbed (AVA) >>
―Alum precipitated toxoid‖
33. II. Bacillus cereus
•Food Poisoning.
• Clinical Syndromes:
i. Severe Nausea &Vomiting.
ii. Abdominal Cramps & Diarrhoea.
34. PATHOGENICITY:
>> Due to an Enterotoxin.
• Also Causes Disease in Patients with Underlying
Disease.
TREATMENT:
>> Tetracycline, Erythromycin.
• iii. B. subtilis:
• iv. B. stearothermophilus.
36. Distinctive properties
•Large rods with rounded ends, occur singly in
short chains,or as long filaments
•Gram +ve bacilli
• Anaerobic (some facultative microaerophilic)
•Most are motile (except C. Perfrigens) and
nonencapsulated
• Spore Forming
-Spores: can be central, subterminal, or terminally
•Fermentative
•Catalse -ve
37. Groups of Clostridial spores
1. Subterminal spores
Gelatin not hydrolysed- group I e.g C. colinum
Gelatin hydrolysed- group II e.g C. sordellii, C.
botulinum, C.novyi, C .perfrigens, C hemolyticum,
C. chauvoei, C. septicum.
2. Terminal spores
Gelatin not hydrolyzed- group III (not associated
with animal diseases)
Gelatin hydrolized- group IV e.g C. tetani
38. Ink Stain of Sporulating Clostridiumspores
appear clear, vegetative cells dark
39. Distribution
•Clostridia are free-living saprophytes in soil
•Some spp are found in the GIT
•Only few spp (>60) cause disease.
Mode of infection
•Ingestion: Black leg (cattle); botulinum (food),
enterotoxemia, bacillary hemoglobinuria.
•Wounds: C. tetani, C chauvoei (sheep), C.
septicum and other gas gangreen organisms infect
wounds.
40. I. Clostridium perfringens
• Nonmotile
• Spores Not Produced in Ordinary Media.
• Aerotolerant Anaerobe.
• 5 Types: A - E
41. Clostridium perfrigens
• Synm: C. welchii.
• Disease: enterotoxemia
• Occurrence: C. perfrigens type A more
widespread, present in air, soil, dust, manure,
water of lakes, streams, and rivers.
– Has been isolated from vegetables, milk, cheese,
canned food, fresh meat, shellfish and mollusks.
42. FOOD POISONING:
• Cl. perfringens Type A >> Enterotoxin.
> Acute Abdominal Pain and Diarrhoea.
43. PATHOGENICITY & CLINICAL INFECTION
•α-Toxin: Acts on lecithin-containing lipoprotein
complexes in the cell membrane.
• Predisposing Factors:
i. Trauma with deep and lacerated or crush
wounds of muscle Etc.
ii. Require a reduced oxygen tension and
reduced oxidation reduction potential
for growth.
48. LABORATORY IDENTIFICATION
• In Chopped Meat - Glucose Medium:
•Colonies are 1-3 mm in diameter, round or
slightly irregular, slightly raised, granular, and
transparent or transluscent.
• On BA:
• On Egg Yolk Agar:
>> Precipitation (Opalescence).
• Milk Media: Stormy Formation.
• Nagler Reacrion:
49. Blood agar plate with Cl. Perfringens characteristic
double zone of hemolysis
50. Clostridium chauvoei
synonym: C. feseri
• Disease: blackleg
• Wide spread, found in intestine and in normal
tissues
• Toxins
– α toxin: hemolysin, necrotoxin
– ß toxin: deoxyribonuclease
– γ toxin: hyaluronidase
– ∆ toxin: hemolysin
51. Phathogenicity
• Ruminats: Blackleg (cattle 4 months to
2yr)- ingestion/endogenous, sheep and
goats- wounds
• lession dry, dark, with gas bubles, and a
rancid odor, there may be bacteremia.
• Immunity:
– Formalized whole-broth cultures- life long
– Recovery from disease—renders the animal
immune for life
52. Clostridium septicum
Disease: Malignant oedema
Occurnce:Ww, in soil and intestine
Toxins:
1. α toxin: lethal, lecithinase, necrotizing and
hemolytic
2. ß toxin: a deoxyribonuclease and leukocidal
3. gamma toxin:hyaluronidase
4. Delta toxin: a hemolyzing and necrotizing.
Pathogenicity: as for gangrene caused by C.
chauvoei
• affects horses, cattle, sheep, pigs.
53. Clostridium hemolyticum
Synm:C. novyi, type D
Disease: bacillary hemoglobinuria
Occurrence:Ww, especially where liver fluke
occur??
Subclinical infections may occur in some animals
(serves as carriers) sheding the organisms via the
intestinal tract.
Toxins: ß toxin, phosphplipase C, which is lethal,
necrotizing, and cause lysis of erythrocytes
Pathogenicity: infection limited o cattle, and
sheep.
54. LABORATORY DIAGNOSIS:
• Important: Diagnosis of Clostridium Myonecrosis
Should Be Rapid and Made on Clinical Grounds.
i. Direct Smear and Gram Stain of Material from
Deep Within the Wound.
ii. Culture:
Tissue Aspirates or Deep Swabs Taken from
Affected Muscle.
55. TREATMENT:
• Clostridium myonecrosis:
i. Surgical removal of all infected and necrotic
tissue.
ii. Antibiotic and Antitoxin therapy.
iii. Adminstration of hyperbaric oxygen.
56. Clostridia that may be associated with gas
gangrene:
• Cl. perfringens Type A
• Cl. Septicum
• Cl. novyi Type A
• Cl. Histolyticum
• Cl. Sordellii
59. VIRULENCE FACTORS:
• Tetanus Toxin (Tetanospasmin) > Neurotoxin.
i. An Intercellular Toxin Released by Cellular
Autolysis.
ii. Inhibits the Release of Inhibitory Transmitters.
iii. Toxoid.
•Hemolysin (tetanolysin or cytotoxin)
•Nonspasmogenic toxin
•Horses and human are more susceptible to
tetanus
60. CLINICAL INFECTION & PATHOGENESIS
• "Tetanus is Generalized in Nature".
• Spores germinate in dirty and neglected wounds
with some necrosis
•Toxin is elaborated after spore germination.
•Predisposing factors:
•Docking and castration wounds, umbilical
infections (tetanus neonatorum), parturition
(puperperal tetanus), and dehorning.
Immunity:
•Totally antitoxic
•Strains with different heat-stable and heat labile
antigens and 10 serotypes present based on
flagellar antigens.
61. LABORATORY DIAGNOSIS:
• > Diagnosis on clinical grounds.
TREATMENT:
• Antitoxin- applied prophylactically??
•Toxoid- widely used in horses.
•Debridement of wound and removal of any foreign
bodies.
•Pencillin >In large doses.
•Mild Tetanospasm: >> Barbiturates.
62. III. Clostridium botulinum
• > Botulism.
• > Gram +ve, spore forming bacilli.
• > Strict anaerobe.
•Gram stain of Cl. botulinum, characteristic long
rods
65. VIRULENCE FACTORS
• Botulinum Toxin >>> Neurotoxin.
– Serologically 8 types of Toxins >>A, B, C1, C2,
D, E, F & G.
> Affect the Cholinergic System > Blocks the
Release of Acetylcholine (at Points in Peripheral
Nervous System).
66. DISEASE IN HUMANS
1. Food – borne botulism:
Incubation period: 12-36 Hours to 8 days.
2. Infant botulism:
LABORATORY DIAGNOSIS
i. Diagnosis made clinically.
ii. Detection of organism or its toxin in the suspected
food
iii. Samples of stool or vomit
67. TREATMENT & PREVENTION
Important: Specific Treatment should begin as
quick as possible.
>Polyvalent Antitoxin >>> Immediately.
>Physiological support
>NEVER Use a swollen or defective can.
