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Selected gram positives bls 206


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Selected gram positives bls 206

  2. 2. 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
  3. 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 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
  4. 4. 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 M.phlei, M. smegamtis, M. fortuitum, M. chelonei.
  5. 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-fastnessNB: Always work under biosafety cabinet!
  6. 6. 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.
  7. 7. 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.
  8. 8. 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.
  9. 9. Acid-fast bacilli
  10. 10. 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.
  11. 11. 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.
  12. 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
  13. 13. M. tuberculosis culture
  14. 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. 15. 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.
  16. 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 thehead.Cattle refractory but sensitizedSporadic cases in horses, dogs and catsInfection in human- little consequence.
  17. 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. 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. 19. 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
  20. 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
  21. 21. Virulence – Ability to Survive within Macrophages
  22. 22. TUBERCULIN TESTTuberculin: 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 immunityPurified Protein Derivatives (PPD): – Mantoux Method (Intracutaneous) – Heaf Method (Spring-loaded gun) – Tine Tests (Disposable single tests)
  23. 23. LABORATOY DIAGNOSIS2. Microscopy: – Ziehl-Neelsen Stain – Fluorescent dyes3. Culture: – Decontamination: – Lowenstein Jensen medium4. Nucleic Acid Methods:
  25. 25. 1. GENUS: BACILLUS•Gram +ve bacilli• Aerobic• Spore-Forming
  26. 26. I. Bacillus anthracis•Causative agent of Anthrax.Distinctive Properties• Large, Square - ended Rods, Arranged in Chains.• Non-Motile.• Spores:• Capsule:– Purple Stained >> McFadyans Method(Polychrome Methylene Blue).• Colonies on BA: "Medusa Head Appearance"
  27. 27. Bacillus anthracis
  28. 28. PATHOGENESIS• Capsule > Invasiveness– D-glutamic acid• Exotoxin (Plasmid mediated)i. Protective Factor (Antigen).ii. Oedema Factor.iii. Lethal Factor.Blocks the Adenyl Cyclase Pathway >Increases vascular Permeability > Shock
  29. 29. 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:
  30. 30. Bicarbonate agar and blood agar plate culturesof Bacillus anthracis
  31. 31. Negative encapsulation: Blood agar and bicarbonateagar plate cultures of Bacillus cereus
  32. 32. • TREATMENT– Penicillin, Ciprofloxacin• IMMUNIZATION–Animals > Live spore vaccine (Sterne strain)– Workers at Risk of Exposure >Anthrax Vaccine Absorbed (AVA) >>―Alum precipitated toxoid‖
  33. 33. II. Bacillus cereus•Food Poisoning.• Clinical Syndromes:i. Severe Nausea &Vomiting.ii. Abdominal Cramps & Diarrhoea.
  34. 34. PATHOGENICITY:>> Due to an Enterotoxin.• Also Causes Disease in Patients with UnderlyingDisease. TREATMENT:>> Tetracycline, Erythromycin.• iii. B. subtilis:• iv. B. stearothermophilus.
  36. 36. 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
  37. 37. 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
  38. 38. Ink Stain of Sporulating Clostridiumsporesappear clear, vegetative cells dark
  39. 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 infectwounds.
  40. 40. I. Clostridium perfringens• Nonmotile• Spores Not Produced in Ordinary Media.• Aerotolerant Anaerobe.• 5 Types: A - E
  41. 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. 42. FOOD POISONING:• Cl. perfringens Type A >> Enterotoxin. > Acute Abdominal Pain and Diarrhoea.
  43. 43. 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.
  44. 44. Gram stain of Clostridium perfringens
  45. 45. Exudate smear of Clostridium perfringens
  46. 46. Tissue smear of Clostridium perfringens
  47. 47. DISEASE:• Clostridial myonecrosis.• Less severe wound infections.• Food poisoning.
  48. 48. 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:
  49. 49. Blood agar plate with Cl. Perfringens characteristicdouble zone of hemolysis
  50. 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. 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. 52. 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.
  53. 53. 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.
  54. 54. 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.
  55. 55. TREATMENT:• Clostridium myonecrosis:i. Surgical removal of all infected and necrotic tissue.ii. Antibiotic and Antitoxin therapy.iii. Adminstration of hyperbaric oxygen.
  56. 56. Clostridia that may be associated with gasgangrene:• Cl. perfringens Type A• Cl. Septicum• Cl. novyi Type A• Cl. Histolyticum• Cl. Sordellii
  57. 57. II. Clostridium tetaniTetanus. > Terminal Spores with drumstick appearance.• Obligate anaerobe.•Gram positive rods
  58. 58. Clostridium tetani
  59. 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 totetanus
  60. 60. 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.
  61. 61. 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.
  62. 62. III. Clostridium botulinum• > Botulism.• > Gram +ve, spore forming bacilli.• > Strict anaerobe.•Gram stain of Cl. botulinum, characteristic longrods
  63. 63. A photomicrograph of Clostridium botulinum type A
  64. 64. Blood Agar plate with C. botulinum
  65. 65. 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).
  66. 66. 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
  67. 67. TREATMENT & PREVENTIONImportant: Specific Treatment should begin asquick as possible.>Polyvalent Antitoxin >>> Immediately.>Physiological support>NEVER Use a swollen or defective can.