GENUS: MYCOBACTERIUMClassification – -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
•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 andpigmentation: 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
2. Scotochromogens: -Produce deep yellow to orange pigments whengrown in light or dark,- The color deepens upon two weeks exposure tolight 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 tolight. 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 characteristicsObligate aerobe, Gram-positive rodsAcid fastComplex cell wall lipids – include mycolic acids – protects vs. phagolysosomal componentsPeptidoglycan, glycolipids – acid-fastnessNB: Always work under biosafety cabinet!
Heating required for stain penetrationdue 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.
2. Kinyon:– Uses a higher content of phenol(organic solvent) in the carbol fuchsinprimary stain to allow penetration ofthe stain without the need to applyheat.-Acid alcohol for destaining and-ethylene blue as the counterstain.
3. Auramine-rhodamine fluorochrome (afluorescent stain):-Requires a fluorescsnt microscope.-Stain with auramine-rhodamine for 10minutes (phenol in the solution allows forpenetration)-Destain with acid alcohol-Counterstain with acridine orange-A positive result is a bright yellowfluorescence.
Highly contaminated specimens with organic debris andnormal 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 liquidmedia 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.
2. Selective media:– Consists of one of the nonselective media plusadded antimicrobial agents (malachite green,cyclohexamide, and nalidixic acid are often used)-The colonies of M. tuberculosis on the solid media arerough, dry, granular, nonpigmented to buff coloredcolonies.3. Liquid media: - Media usually contains tween 80 and albumin andthe organisms will grow faster than on solid mediaNB: Most Mycobacteria grow best in 5-10% CO2 and at35-370 C.
IdentificationRate of growth and growth in relation to temperaturePigmentation and photoreactivityFurther 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
M. tuberculosis culture
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)
Pathogenecity1. 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.
2. M. aviumChicken most susceptibleOther birds-YesNot all infected chickens show gross lesions.Water fowls – resistantIn swine- disease found in lymph nodes of thehead.Cattle refractory but sensitizedSporadic cases in horses, dogs and catsInfection 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
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.
Differential characteristics of tuberculle bacilli causing animal/human disease____________________________________________________Species Atmospheric preference Nitratase TCH Pyrazinamide---------------------------------------------------------------------------------------M. tuberculosis Aerobic + S SM. bovis Microaerophilic -- R R_______________________________________TCH = thiophen-2-carboxylic acid hydrazideS= Sensitive, R= Resistant
THE DISEASENot 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
Virulence – Ability to Survive within Macrophages
TUBERCULIN TESTTuberculin: a heat-concentrated filtrate of abroth in which tubercle bacilli had been grown.Injection of tuberculin into the skin >> – Large, indurated reactions >>Post-Primary Tuberculosis. – No induration >> Protective immunityPurified Protein Derivatives (PPD): – Mantoux Method (Intracutaneous) – Heaf Method (Spring-loaded gun) – Tine Tests (Disposable single tests)
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 culturesof Bacillus anthracis
Negative encapsulation: Blood agar and bicarbonateagar 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•Food Poisoning.• Clinical Syndromes:i. Severe Nausea &Vomiting.ii. Abdominal Cramps & Diarrhoea.
PATHOGENICITY:>> Due to an Enterotoxin.• Also Causes Disease in Patients with UnderlyingDisease. TREATMENT:>> Tetracycline, Erythromycin.• iii. B. subtilis:• iv. B. stearothermophilus.
2. GENUS: CLOSTRIDIUM
Distinctive properties•Large rods with rounded ends, occur singly inshort chains,or as long filaments•Gram +ve bacilli• Anaerobic (some facultative microaerophilic)•Most are motile (except C. Perfrigens) andnonencapsulated• Spore Forming-Spores: can be central, subterminal, or terminally•Fermentative•Catalse -ve
Groups of Clostridial spores1. 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
Ink Stain of Sporulating Clostridiumsporesappear clear, vegetative cells dark
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 infectwounds.
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. 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.
FOOD POISONING:• Cl. perfringens Type A >> Enterotoxin. > Acute Abdominal Pain and Diarrhoea.
PATHOGENICITY & CLINICAL INFECTION•α-Toxin: Acts on lecithin-containing lipoproteincomplexes in the cell membrane.• Predisposing Factors:i. Trauma with deep and lacerated or crushwounds of muscle Etc.ii. Require a reduced oxygen tension andreduced oxidation reduction potentialfor growth.
Gram stain of Clostridium perfringens
Exudate smear of Clostridium perfringens
Tissue smear of Clostridium perfringens
DISEASE:• Clostridial myonecrosis.• Less severe wound infections.• Food poisoning.
LABORATORY IDENTIFICATION• In Chopped Meat - Glucose Medium:•Colonies are 1-3 mm in diameter, round orslightly irregular, slightly raised, granular, andtransparent or transluscent.• On BA:• On Egg Yolk Agar:>> Precipitation (Opalescence).• Milk Media: Stormy Formation.• Nagler Reacrion:
Blood agar plate with Cl. Perfringens characteristicdouble zone of hemolysis
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
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 septicumDisease: Malignant oedemaOccurnce:Ww, in soil and intestineToxins:1. α toxin: lethal, lecithinase, necrotizing and hemolytic2. ß toxin: a deoxyribonuclease and leukocidal3. gamma toxin:hyaluronidase4. Delta toxin: a hemolyzing and necrotizing.Pathogenicity: as for gangrene caused by C. chauvoei• affects horses, cattle, sheep, pigs.
Clostridium hemolyticumSynm:C. novyi, type DDisease: bacillary hemoglobinuriaOccurrence:Ww, especially where liver flukeoccur??Subclinical infections may occur in some animals(serves as carriers) sheding the organisms via theintestinal tract.Toxins: ß toxin, phosphplipase C, which is lethal,necrotizing, and cause lysis of erythrocytesPathogenicity: infection limited o cattle, andsheep.
LABORATORY DIAGNOSIS:• Important: Diagnosis of Clostridium MyonecrosisShould 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 fromAffected Muscle.
TREATMENT:• Clostridium myonecrosis:i. Surgical removal of all infected and necrotic tissue.ii. Antibiotic and Antitoxin therapy.iii. Adminstration of hyperbaric oxygen.
Clostridia that may be associated with gasgangrene:• Cl. perfringens Type A• Cl. Septicum• Cl. novyi Type A• Cl. Histolyticum• Cl. Sordellii
II. Clostridium tetaniTetanus. > Terminal Spores with drumstick appearance.• Obligate anaerobe.•Gram positive rods
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 totetanus
CLINICAL INFECTION & PATHOGENESIS• "Tetanus is Generalized in Nature".• Spores germinate in dirty and neglected woundswith 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.
LABORATORY DIAGNOSIS:• > Diagnosis on clinical grounds.TREATMENT:• Antitoxin- applied prophylactically??•Toxoid- widely used in horses.•Debridement of wound and removal of any foreignbodies.•Pencillin >In large doses.•Mild Tetanospasm: >> Barbiturates.
III. Clostridium botulinum• > Botulism.• > Gram +ve, spore forming bacilli.• > Strict anaerobe.•Gram stain of Cl. botulinum, characteristic longrods
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 theRelease of Acetylcholine (at Points in PeripheralNervous System).
DISEASE IN HUMANS1. Food – borne botulism:Incubation period: 12-36 Hours to 8 days.2. Infant botulism:LABORATORY DIAGNOSISi. Diagnosis made clinically.ii. Detection of organism or its toxin in the suspectedfoodiii. Samples of stool or vomit
TREATMENT & PREVENTIONImportant: Specific Treatment should begin asquick as possible.>Polyvalent Antitoxin >>> Immediately.>Physiological support>NEVER Use a swollen or defective can.