1. Medically Important Bacteria – Gram Positive Bacilli Phase I/ Module VII Dr Ekta Chourasia

2. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Learning Objectives Classification of GPBs Classification of GPBs General properties of GPBs General properties of GPBs Discuss the role of toxins and other virulence factors in pathogenesis Discuss the role of toxins and other virulence factors in pathogenesis Describe the mechanism of action of various toxins Describe the mechanism of action of various toxins List pathogenicity List pathogenicity Learning Resources Lecture & Practical Notes Lecture & Practical Notes Textbook Of Microbiology, Ananthanarayan. Ch 26, 27, 28, 29 & 39 Textbook Of Microbiology, Ananthanarayan. Ch 26, 27, 28, 29 & 39 http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/revision_aids.html http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/revision_aids.html http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/revision_aids.html

3. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Medically Important Gram-Positive Bacilli Can be subdivided into three general groups, based on the presence or absence of spores and acid-fastness Can be subdivided into three general groups, based on the presence or absence of spores and acid-fastness 1. Spore-formers 2. Nonspore-formers 3. Irregular shaped and staining properties

4. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Scheme for Differentiating GPBs

5. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Spore Forming Gram Positive Bacilli

6. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Spore Forming GPBs Spore forming gram +ve bacilli: 2 groups Spore forming gram +ve bacilli: 2 groups 1.Aerobic – Bacillus: Important species are Bacillus anthracis – Anthrax Bacillus anthracis – Anthrax Bacillus cereus – food poisoning Bacillus cereus – food poisoning Bacillus stearothermophilus – efficiency testing of an autoclave Bacillus stearothermophilus – efficiency testing of an autoclave 2.Anaerobic – Clostridia: Imp species include Clostridium perfringens – gas gangrene Clostridium perfringens – gas gangrene Clostridium tetani – tetanus Clostridium tetani – tetanus Clostridium botulinum – botulism (food poisoning) Clostridium botulinum – botulism (food poisoning) Clostridium difficile – pseudomembranous colitis Clostridium difficile – pseudomembranous colitis

7. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Bacillus

8. 24.05.09Phase I/ Module VII Dr Ekta Chourasia General Characteristics of the Genus Bacillus Aerobic Aerobic Gram-positive, spore-forming (non bulging spores), bacilli Gram-positive, spore-forming (non bulging spores), bacilli 2 species of medical importance: 2 species of medical importance: – Bacillus anthracis – Bacillus cereus

9. 24.05.09Phase I/ Module VII Dr Ekta Chourasia History of Bacillus anthracis 1 st pathogenic bacterium to be seen under microscope – Pollender, 1849 1 st pathogenic bacterium to be seen under microscope – Pollender, 1849 1 st communicable disease shown to be transmitted by inoculation of infected blood – Davaine, 1850 1 st communicable disease shown to be transmitted by inoculation of infected blood – Davaine, 1850 1 st bacillus to be isolated in pure culture & shown to possess spores – Koch, 1876 1 st bacillus to be isolated in pure culture & shown to possess spores – Koch, 1876 1 st bacterium used for the preparation of an attenuated vaccine – Pasteur, 1881 1 st bacterium used for the preparation of an attenuated vaccine – Pasteur, 1881 Used as Biological Warfare Agent (Bioterrorism) Used as Biological Warfare Agent (Bioterrorism)

10. 24.05.09Phase I/ Module VII Dr Ekta Chourasia B. anthracis - Virulence factors Two virulence factors – Capsular polysaccharide – inhibits phagocytosis Capsular polysaccharide – inhibits phagocytosis Anthrax toxin : made up of 3 fractions Anthrax toxin : made up of 3 fractions 1.Edema factor (EF or Factor I) 2.Protective antigen factor (PA or Factor II) 3.Lethal factor (LF or Factor III) * They are not toxic individually, the whole complex produces local edema & generalised shock. Toxin production is plasmid mediated * They are not toxic individually, the whole complex produces local edema & generalised shock. Toxin production is plasmid mediated

11. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Anthrax toxin

12. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Pathogenicity Anthrax – zoonotic disease of primarily cattle & sheep. Anthrax – zoonotic disease of primarily cattle & sheep. Animals get infected by ingestion of spores present in the soil Animals get infected by ingestion of spores present in the soil Bacilli are shed in discharges from the mouth, nose & rectum - sporulate in the soil. Bacilli are shed in discharges from the mouth, nose & rectum - sporulate in the soil. Human anthrax – contracted from animals, directly or indirectly. Human anthrax – contracted from animals, directly or indirectly.

13. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Human Anthrax The disease may present as The disease may present as 1.Cutaneous anthrax : ‘Hide Porter’s disease’ – inoculation of spores into the skin 2.Pulmonary anthrax : ‘Wool Sorter’s disease’ – inhalation of spores 3.Intestinal anthrax - ingestion of inadequately cooked meat containing anthrax spores * All types lead to fatal septicemia * All types lead to fatal septicemia Human anthrax can be Human anthrax can be 1.Industrial – in meat packing or wool factories 2.Nonindustrial – frequent association with animals like butchers, veterinarians, farmers

14. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Bacillus cereus Readily isolated from soil, vegetables and a wide variety of foods including milk, cereals, spices, poultry & meat. Readily isolated from soil, vegetables and a wide variety of foods including milk, cereals, spices, poultry & meat. Spores survive cooking and reheating Spores survive cooking and reheating Causes foodborne gastroenteritis – 2 patterns of disease (diarrhoeal & emetic); both types are mild & self limited, requiring no specific therapy. Causes foodborne gastroenteritis – 2 patterns of disease (diarrhoeal & emetic); both types are mild & self limited, requiring no specific therapy.

15. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Gastroenteritis Bacillus cereus clinical presentation Chinese fried rice exclusively Incubation period < 6 hours Severe vomiting Lasts 8-10 hours Wide range of foods including cooked meat & vegetables Incubation period > 6 hours Diarrhoea & abdominal pain Lasts 20-36 hours EMETIC FORMDIARRHOEAL FORM

16. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Clostridium

17. 24.05.09Phase I/ Module VII Dr Ekta Chourasia General Characteristics of the Genus Clostridium Gram-positive, spore-forming rods Gram-positive, spore-forming rods Anaerobic and catalase negative Anaerobic and catalase negative Oval or spherical (bulging) spores produced only under anaerobic conditions Oval or spherical (bulging) spores produced only under anaerobic conditions Cause wound infections, tissue infections, and food intoxications Cause wound infections, tissue infections, and food intoxications

18. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Cl. perfringens

19. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Cl. perfringens: Virulence factors Classified into Five strains, A to E - based on the toxins they produce Classified into Five strains, A to E - based on the toxins they produce Produces many toxins: 4 major toxins – alpha, beta, epsilon & iota Produces many toxins: 4 major toxins – alpha, beta, epsilon & iota 1.Alpha toxin – lecithinase C, a phospholipase Damages cell membranes Damages cell membranes Increase capillary permeability Increase capillary permeability Extravasation & increased tension in affected muscles Extravasation & increased tension in affected muscles Lysis of erythrocytes: hemolytic anemia & hemoglobinuria Lysis of erythrocytes: hemolytic anemia & hemoglobinuria 2.Beta, epsilon & iota – necrosis Enzymes – collagenase, hyaluronidase – spread of infection Enzymes – collagenase, hyaluronidase – spread of infection

20. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Cl. perfringens : Pathogenicity Soft tissue infection: Gas gangrene (Type A) Soft tissue infection: Gas gangrene (Type A) Enteric infections: Enteric infections: –Food poisoning (some strains of type A) –Gangrenous appendicitis (A + D) –Necrotising enteritis (C) –Biliary tract infections Other diseases: Other diseases: –Brain abscess & meningitis –Thoracic infections

21. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Gas Gangrene Clostridium perfringens most frequent clostridia involved in soft tissue and wound infections – myonecrosis Clostridium perfringens most frequent clostridia involved in soft tissue and wound infections – myonecrosis Spores found in soil, human skin, intestine, and vagina Spores found in soil, human skin, intestine, and vagina Predisposing factors – crushing injuries, road accidents, gunshot wounds, surgical incisions, compound fractures, diabetic ulcers, septic abortions, puncture wounds Predisposing factors – crushing injuries, road accidents, gunshot wounds, surgical incisions, compound fractures, diabetic ulcers, septic abortions, puncture wounds C/F - high fever, brownish pus, gas bubbles under the skin, skin discoloration, and a foul odor C/F - high fever, brownish pus, gas bubbles under the skin, skin discoloration, and a foul odor

22. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Cl. tetani Gram +ve bacilli with terminal, spherical spore Gram +ve bacilli with terminal, spherical spore Commonly present in soil, GI tracts of animals and inanimate objects like street & hospital dust, cotton wool, bandages, catgut, wall plaster, clothings, etc Commonly present in soil, GI tracts of animals and inanimate objects like street & hospital dust, cotton wool, bandages, catgut, wall plaster, clothings, etc Causes tetanus or lockjaw, a neuromuscular disease Causes tetanus or lockjaw, a neuromuscular disease –Most commonly among geriatric patients and IV drug abusers; neonates in developing countries –Death commonly occurs due to paralysis of respiratory muscles

23. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Mode of transmission Pathogenesis of Tetanus Source of Infection Virulence factor Septic abortion, Cutting umbilical stump Ear boring / circumcision / unsterile injections Tetanospasmin – Neurotoxin Tetanolysin Unhygienic Complication Injury, puncture wounds, wound contamination Contaminated soil / dust

24. 24.05.09Phase I/ Module VII Dr Ekta Chourasia The events in tetanus

25. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Ophisthotonus Spastic Paralysis in Tetanus

26. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Clostridium botulinum Gram +ve bacilli with subterminal, oval spores Gram +ve bacilli with subterminal, oval spores Produces toxin – Botulin (botulinum toxin), a neurotoxin Produces toxin – Botulin (botulinum toxin), a neurotoxin Causes Botulism, a paralytic (flaccid paralysis) disease Causes Botulism, a paralytic (flaccid paralysis) disease –Ingestion of preformed toxins –In preserved (canned) food like meat & meat products, fish & canned vegetables

27. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Effects of botulinum toxin

28. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Clostridium difficile Normal resident of colon, in low numbers Normal resident of colon, in low numbers Causes antibiotic-associated colitis (pseudomembranous colitis) Causes antibiotic-associated colitis (pseudomembranous colitis) –Relatively non-invasive; treatment with broad-spectrum antibiotics kills the other bacteria, allowing C. difficile to overgrow Produces enterotoxins & cytotoxins that damage intestine Produces enterotoxins & cytotoxins that damage intestine Major cause of diarrhea in hospitals Major cause of diarrhea in hospitals

29. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Non spore Forming Gram Positive Bacilli

30. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Gram-Positive Irregular Non-Spore-Forming Bacilli Irregular: pleomorphic, stain unevenly Medically important genera: Corynebacterium Corynebacterium Propionibacterium Propionibacterium Mycobacterium Mycobacterium Actinomyces Actinomyces Nocardia Nocardia

31. 24.05.09Phase I/ Module VII Dr Ekta Chourasia General Features of Corynebacteria Frequently show club shaped swellings – corynebacteria (coryne = club) Frequently show club shaped swellings – corynebacteria (coryne = club) C. diphtheriae : most important member of this genus, causes diphtheria C. diphtheriae : most important member of this genus, causes diphtheria Diphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva Diphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva

32. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Important features of C. diphtheriae Slender Gram positive bacilli, non sporing Slender Gram positive bacilli, non sporing Chinese letter or Cuneiform arrangement Chinese letter or Cuneiform arrangement Stains irregularly, tends to get easily decolorised Stains irregularly, tends to get easily decolorised May show clubbing at one or both ends - Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granules May show clubbing at one or both ends - Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granules Metachromatic Granules: Metachromatic Granules: –made up of polymetaphosphate –Special stains: Albert’s, Neisser’s & Ponder’s Grows aerobically at 37°C Grows aerobically at 37°C

33. 24.05.09Phase I/ Module VII Dr Ekta Chourasia C. diphtheriae - Virulence factor Exotoxin – Diphtheria toxin: Exotoxin – Diphtheria toxin: –Protein in nature –very powerful toxin –Responsible for all pathogenic effects of the bacilli –Produced by all the virulent strains –Two fragments A & B Toxigenicity can be induced by Lysogenic or phage conversion – corynephages (tox + phage) or beta phages Toxigenicity can be induced by Lysogenic or phage conversion – corynephages (tox + phage) or beta phages

34. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Diphtheria toxin – Mechanism of action Fragment A – inhibits polypeptide chain elongation by inactivating the Elongation factor EF 2 in the presence of NAD DT - Acts by inhibition of protein synthesis

35. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Genus Propionibacterium Propionibacterium acnes most common Propionibacterium acnes most common Aerotolerant or anaerobic Aerotolerant or anaerobic Nontoxigenic Nontoxigenic Common resident of pilosebaceous glands Common resident of pilosebaceous glands Causes Acne Causes Acne

36. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Genus Lactobacillus Long, slender rods grow in chains Long, slender rods grow in chains Important member of the vaginal flora of women of reproductive age Important member of the vaginal flora of women of reproductive age Maintains normal vaginal pH Maintains normal vaginal pH Tends to make the environment too acidic for other bacteria to tolerate Tends to make the environment too acidic for other bacteria to tolerate

37. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Mycobacteria: Acid-Fast Bacilli Gram-positive irregular bacilli Gram-positive irregular bacilli Acid-fast staining Acid-fast staining Obligate aerobes Obligate aerobes Possess mycolic acids and a unique type of peptidoglycan Possess mycolic acids and a unique type of peptidoglycan Do not form capsules, flagella, or spores Do not form capsules, flagella, or spores Slow growing, 14-15 hrs generation time Slow growing, 14-15 hrs generation time

38. 24.05.09Phase I/ Module VII Dr Ekta Chourasia

39. 24.05.09Phase I/ Module VII Dr Ekta Chourasia

40. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Mycobacterium Tuberculosis Produces no exotoxins or enzymes that contribute to infectiousness Produces no exotoxins or enzymes that contribute to infectiousness Virulence factors – contain complex waxes and cord factor that prevent destruction by lysosomes or macrophages Virulence factors – contain complex waxes and cord factor that prevent destruction by lysosomes or macrophages

41. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Mycobacterium Leprae: The Leprosy Bacillus Hansen’s bacillus/Hansen’s Disease Hansen’s bacillus/Hansen’s Disease First bacterium to be shown to cause disease in humans First bacterium to be shown to cause disease in humans Humans & Armadillos are the only natural hosts Humans & Armadillos are the only natural hosts Strict parasite – cannot be cultivated “in- vitro”, does not grow on artificial media or tissue culture Strict parasite – cannot be cultivated “in- vitro”, does not grow on artificial media or tissue culture Slowest growing of all species Slowest growing of all species Causes leprosy, a chronic disease that begins in the skin and mucous membranes and progresses into nerves Causes leprosy, a chronic disease that begins in the skin and mucous membranes and progresses into nerves Multiplies within host cells in large packets called globi

42. 24.05.09Phase I/ Module VII Dr Ekta Chourasia Actinomycetes: Filamentous Bacilli Genera Actinomyces & Nocardia are nonmotile filamentous bacteria related to mycobacteria Genera Actinomyces & Nocardia are nonmotile filamentous bacteria related to mycobacteria May cause chronic infection of skin and soft tissues May cause chronic infection of skin and soft tissues Actinomyces israelii – responsible for diseases of the oral cavity, thoracic or intestines – actinomycoses “Sun-ray appearnce” Actinomyces israelii – responsible for diseases of the oral cavity, thoracic or intestines – actinomycoses “Sun-ray appearnce” Nocardia brasiliensis causes pulmonary disease similar to TB Nocardia brasiliensis causes pulmonary disease similar to TB

43. 24.05.09Phase I/ Module VII Dr Ekta Chourasia