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 intheir cell walls -Tubercle bacilli = M. tuberculosis, M. africanum, andM. bovis
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:
Photochromagens: - are non-pigmented when grown in the dark. - produce photoactivated pigments upon exposure to light e.gM. kansasii, M. marinum, M. asiaticum, M. simiae
2.Scotochromogens: -Produce deep yellow to orange pigments when grown in light or dark, - The color deepens upon two weeks exposure to light e.gM. 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.
Morphology and cultural characteristics
Obligate aerobe, Gram-positive rods
Complex cell wall lipids
– include mycolic acids – protects vs. phagolysosomal components
– acid-fastness NB: Always work under biosafety cabinet!
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 carbolfuchsin to penetrate the cell wall; - acid alcohol destaining; - methylene blue as the counterstain.
2. Kinyon: – Uses a higher content of phenol (organic solvent) in the carbolfuchsin primary stain to allow penetration of the stain without the need to apply heat.
Acid alcohol for destaining and
ethylene blue as the counterstain.
3. Auramine-rhodaminefluorochrome (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.
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. Nonselective -May be egg or agar based. - May include malachite green to suppress growth of contaminating bacteria. Lowenstein-Jensen -egg based; -Colonies grow in 18-24 days. b. Middlebrook 7H10 and 7H11 – agar based; - colonies grow in 12-14 days.
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.
Rate of growth and growth in relation to temperature
Pigmentation and photoreactivity
Further biochemical testing includes:
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
M. tuberculosis culture
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,
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)
Pathogenecity 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
Rabbits, mice and guinea pigs more susceptible.
2. M. avium
Chicken most susceptible
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.
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
Susceptible to alcohol, glutaraldehyde and formaldehyde.
Differential characteristics oftuberculle 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
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
I. Bacillus anthracis •Causative agent of Anthrax. Distinctive Properties • Large, Square - ended Rods, Arranged in Chains. • Non-Motile. • Spores: • Capsule: – Purple Stained >> McFadyan's Method (Polychrome Methylene Blue). • Colonies on BA: "Medusa Head Appearance"
PATHOGENESIS • Capsule > Invasiveness – D-glutamicacid • Exotoxin (Plasmid mediated) i. Protective Factor (Antigen). ii. Oedema Factor. iii. Lethal Factor. Blocks the AdenylCyclase Pathway > Increases vascular Permeability > Shock
LABORATORY DIAGNOSIS: • Specimens obtained from: a malignant pustule, sputum, blood. - Gram stain + fluorescent-antibody stain. - Motility - Capsule formation: Sodium bicarbonate +CO2 - String-of-pearls reaction: - Mouse test: - API >> Demonstration of Abs to the organism:
Bicarbonate agar and blood agar plate cultures of Bacillus anthracis
Negative encapsulation: Blood agar and bicarbonate agar plate cultures of Bacillus cereus
• TREATMENT – Penicillin, Ciprofloxacin • IMMUNIZATION –Animals > Live spore vaccine (Sterne strain) – Workers at Risk of Exposure > Anthrax Vaccine Absorbed (AVA) >> “Alum precipitated toxoid”
II. Bacillus cereus
• Clinical Syndromes: Severe Nausea &Vomiting. ii. Abdominal Cramps & Diarrhoea.
PATHOGENICITY: >> Due to an Enterotoxin. • Also Causes Disease in Patients with Underlying Disease. TREATMENT: >> Tetracycline, Erythromycin. • iii. B. subtilis: • iv. B. stearothermophilus.
2. GENUS: CLOSTRIDIUM
Large rods with rounded ends, occur singly in short chains,or as long filaments
• Anaerobic (some facultative microaerophilic)
Most are motile (except C. Perfrigens) and nonencapsulated
• Spore Forming
Spores: can be central, subterminal, or terminally
Groups of Clostridial spores Subterminal spores
Gelatin not hydrolysed- group I e.gC. colinum
Gelatin hydrolysed- group II e.gC. sordellii, C. botulinum, C.novyi, C .perfrigens, C hemolyticum, C. chauvoei, C. septicum.
Gelatin not hydrolyzed- group III (not associated with animal diseases)
Gelatinhydrolized- group IV e.gC. tetani
Ink Stain of SporulatingClostridiumspores appear clear, vegetative cells dark
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.
I. Clostridium perfringens • Nonmotile • Spores Not Produced in Ordinary Media. • Aerotolerant Anaerobe. • 5 Types: A - E
Clostridium perfrigens Synm: C. welchii. Disease:enterotoxemia Occurrence: C. perfrigenstype 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.
FOOD POISONING: • Cl. perfringens Type A >> Enterotoxin. > Acute Abdominal Pain and Diarrhoea.
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.
Gram stain of Clostridium perfringens
Exudate smear of Clostridium perfringens
Tissue smear of Clostridium perfringens
DISEASE: • Clostridialmyonecrosis. • Less severe wound infections. • Food poisoning.
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. • NaglerReacrion:
Blood agar plate with Cl. Perfringenscharacteristic double zone of hemolysis
Clostridium chauvoeisynonym: 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
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
Clostridium septicum Disease: Malignant oedema Occurnce:Ww, in soil and intestine Toxins: α toxin: lethal, lecithinase, necrotizing and hemolytic ß toxin: a deoxyribonuclease and leukocidal gamma toxin:hyaluronidase Delta toxin: a hemolyzing and necrotizing. Pathogenicity: as for gangrene caused by C. chauvoei
affects horses, cattle, sheep, pigs.
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.
LABORATORY DIAGNOSIS: • Important: Diagnosis of Clostridium MyonecrosisShould Be Rapid and Made on Clinical Grounds. Direct Smear and Gram Stain of Material from Deep Within the Wound. ii. Culture: Tissue Aspirates or Deep Swabs Taken from Affected Muscle.
TREATMENT: • Clostridium myonecrosis: Surgical removal of all infected and necrotic tissue. ii. Antibiotic and Antitoxin therapy. iii. Adminstration of hyperbaric oxygen.
Clostridia that may be associated with gas gangrene: • Cl. perfringens Type A • Cl. Septicum • Cl. novyi Type A • Cl. Histolyticum • Cl. Sordellii
II. Clostridium tetani
> Terminal Spores with drumstick appearance. • Obligate anaerobe.
Gram positive rods
VIRULENCE FACTORS: • Tetanus Toxin (Tetanospasmin) > Neurotoxin. An Intercellular Toxin Released by Cellular Autolysis. ii. Inhibits the Release of Inhibitory Transmitters. iii. Toxoid.
Hemolysin (tetanolysin or cytotoxin)
Horses and human are more susceptible to tetanus
CLINICAL INFECTION & PATHOGENESIS • "Tetanus is Generalized in Nature".
Spores germinate in dirty and neglected wounds with some necrosis
Toxin is elaborated after spore germination.
Docking and castration wounds, umbilical infections (tetanus neonatorum), parturition (puperperal tetanus), and dehorning.
Strains with different heat-stable and heat labile antigens and 10 serotypes present based on flagellar antigens.
Gram stain of Cl. botulinum, characteristic long rods
A photomicrograph of Clostridium botulinum type A
Blood Agar plate with C. botulinum
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).
DISEASE IN HUMANS 1. Food – borne botulism:
Incubation period: 12-36 Hours to 8 days.
2. Infant botulism: LABORATORY DIAGNOSIS Diagnosis made clinically. ii. Detection of organism or its toxin in the suspected food iii. Samples of stool or vomit
TREATMENT & PREVENTION Important: Specific Treatment should begin as quick as possible. >Polyvalent Antitoxin >>> Immediately. >Physiological support >NEVER Use a swollen or defective can.