Spore-Forming Bacteria PDF

1 The Gram positive bacilli : Bbacillus are aerobic. Clostridum anaerobic ….. Close the window. Bacillus anthracis -------------- pathogenic Bacillus cereus------------------- pathogenic Bacillus subtilis, Bacillus stearothermophilus:- are opportunistic pathogen associated with foreign bodies...

1 The Gram positive bacilli : Bbacillus are aerobic. Clostridum anaerobic ….. Close the window. Bacillus anthracis -------------- pathogenic Bacillus cereus------------------- pathogenic Bacillus subtilis, Bacillus stearothermophilus:- are opportunistic pathogen associated with foreign bodies catheter, and used for autoclave testing b.anthracis :anthrax of the animals and human B.cereus : food poisoning opportunistic Morphology and physiology : Large geam +ve rods have square ends , arranged in long chain , spore forming ,most are saprophytic soil ,water ,air. Bacillus anthracis : - Spore formed 2-3 old culture. - Polypeptide capsule (Ag) consisting of glutamic acid. - polysaccharide cell wall somatic antigen. - Anthrax toxin. - Found in soil and vegetation - Anthrax is a disease primarily of herbivores (cattle, sheep, goats). - Human may be accidentally infected by exposure to contaminated animals or animal products. Three main routes of anthrax infection:- -1Inoculation ---- (95 % of cases) -2Inhalation -3Ingestion ----- (is rare in human but common in animals). -Person to person transmission does not occur. 2 Clinical syndrome:- -1Cutaneous anthrax (skin) 20% mortality -2Pulmonary anthrax (lung) inhalation, woolsorters’disease, respiratory failure & mortality 95%. -3Intestinal anthrax (gut) very rare in human, mortality 95%. Cutaneous lesion - Painless papule at the site of inoculation. - Ulcer form lead to necrotic eschar, hard thick blue black center - Tissue swollen. - Bacteria multiply lead to septicemia. - Malignant pustule. - Patient will die if not treated 3 Bacillus anthracis 1- disease can kills animals and man. 2- 1877, Robert Koch Germany describe the organism. 3- Large Gram +ve rod in short chain or single. 4- Straight side & straight ends. 5- Guinea pig will die 18 – 36 hrs. 6- People work with animal products (wool, hair, hides, bones, skin, farmers) at risk. 7- In the past, sheep wool has been imported from Asia into West Europe, a lot of people died from anthrax. 8- Spores remain viable for years. 9- Spores into tissue vegetative cells after 1-2 days cutaneous anthrax. 10- bacillus cells will die at 56 oC. 11- This is a weapon of war (Biological warfare). General Consideration:- 1- Lab. Personnel should exercise to deal with this organism. 2- Culture should be examined in biological safety cabinet. 3- Work benche should be carefully decontaminated with 5% phenol. 4- Instruments used on infected materials should be autoclaved before cleaning. Laboratory diagnosis Safety rules:- - Film, Blotting paper and culture are danger. - Slide, loopful, culture in the basket & labeled are danger. - Bacilli on slide are still live even when heat the slide to fix the film. - Spores can survive under cover on film for years. - Do not leave loop 4 Laboratory diagnosis - Non hemolytic colonies, grow rapidly with many comma shaped (medusa head). Mcfadyeans reaction:- - for diagnostic of B. anthracis in blood and tissue. - stained with polychrome methylene blue +ve reaction. Treatment, prevention & control - Penicillin is drug of choice. - Animal vaccination in endemic regions. - Burning or burial of animals when died with anthrax. Bacillus cereus: General characteristics:- Gram-positive, spore-forming rods. catalase-positive, isolated strains from food poisoning normally motile Bacillus species are aerobic, sporulating, rod shaped bacteria which are ubiquitous in nature. Gram-stained cells, 1 µm wide, 5-10 µm long, arranged singly or in short chains Bacillus cereus - Cause Gastroenteritis:- mediated by enterotoxins. 1- Heat stable (H. S.) enterotoxin vomiting disease, emetic form 2- Heat labile (H. L.) enterotoxin (diarrheal disease) H. L. enterotoxin similar to enterotoxin of E. coli & Vibrio cholera; induces diarrhea by accumulation of salt and water in the intestinal lumen. Three types of toxins:- 1- Necrotic toxin (heat labile enterotoxin). 2- Cereolysin. 3- Phospholipase C. (lecithinase) eye infection. 5 Clinical syndrome 1- Emetic form:- - Associated with consumption of contaminated rice. - Vegetative bacilli killed during initial cooking but spore survive. - If rice not refrigerated then spores germinate to bacilli and (H.S.enterotoxin) is not destroyed by reheated. - Incubation period 1-6 hrs after ingestion of enterotoxin. - Disease of short duration (less than 24 hrs). - Vomiting, nausea, abdominal cramps. - fever & diarrhea are absent. Diarrheal form:- -2 - Associated with consumption of contaminated meat or vegetables (food poisoning). - Longer incubation period. - Produce H. L. toxin cause (diarrhea, nausea, abdominal cramps B. cereus is the most common cause of traumatic eye infection from either soil or direct inoculation of bacilli (panophthalmitis). Treatment:- Resistance to penicillin & cephalosporins. Sensitive to clindamycin, vancomycin & minoglycoside. Laboratory diagnosis - Food – borne disease (rice, meat, vegetables) should be cultured. Bacillus cereus colonies wide zone of haemolysis around the colonies. 6  B.anthracis B.cereus Gram stain Gram +ve Gram +ve Culture Non haemolytic Haemolytic Motility Non Motile Capsule Capsulated Non Gelatin tes X mas tree Liquefaction Spore Central & Oval Central & Oval How long does it take for Bacillus to Sporulate? In the laboratory spore formation by B. subtilis depend to nutrient exhaustion. Sporulation is started after cells enter the stationary phase in a medium that supports sporulation, and it takes about 8 to 10 h to be completed. How long does it take for bacterial spores to germinate? During the first 30 min of the revival process, the spore undergoes germination (after 15 min, approximately 80% of the spores complete germination) and enters the ripening period. 7 Clostridium close window : Anaerobic sporing bacilli & bulging Found any where (ubiquitous) Methods of anaerobic growth Anaerobic culture methods: 1- Use of media containing reducing substances (Robertson Cooked Meat broth or Thioglycollate broth) 2- Culture away from O2 (deep agar tubes). 3- Chemical exclusion of O2 (anaerobic Gas Pak system). 4- Mechanical exclusion of O2 (anaerobic incubator) By incorporating two mostly reducing agents in the media:- - (a) Thioglycollate broth: Nutrient broth + Sodium thioglycolate used for anaerobic culture for blood. - (b) Robertson’s cooked meat broth. -..Histotoxic Clostridium (toxic to tissue) 1- Cl. welchii, Cl. perfringens 2- Cl. novyi food poisoning & gas gagrene 3- Cl. septicum 4- Cl. histolyticum 5- Cl. sporogenes proteolytic (digest proteins) 6- Cl. difficile -They found world wide, soil, desert, mountain, bottom of the sea Anaerobic chamber Jar gassing system Anaerobic jar 8 1.Clostridium perfringens gram stain: Gram-positive, large, spore forming rods, catalase negative, non-motile - Clostridium perfringens:- - It is inhabitant of GIT of human & animal. - Found in soil, water, contaminated with feces. - Five strains types (A, B, C, D, E) - Production of five toxins:- 1- Alpha toxin, phospholipase C. (lecithinase). - Lyses RBCs, platelets, leukocytes. - Myonecrosis (occurs when the blood supply is inadequate to maintain muscle viability), Gas gangrene, due to type A strain. 2- Beta toxin: - Enteritis necroticans, (Big-Bel), due to type C strain. - Inflammation of intestine with necrosis of the bowel. 3- Epsilon toxin:- - Increase permeability of gastrointestinal wall. Due to Cl. perfringens (types B and D) strains that induces fatal enteric disease of goats, sheep and cattle including humans. 4- Iota toxin:- - Ietal toxin, caused by C. perfringens type E. 5- Enterotoxins:- - Caused by C. perfringens type A strain. - Cytotoxic and enterotoxic - Food poisoning, produced in the colon. - Large number of bacilli must be ingested with the food. 9 Clinical syndrome 1- Bacteremia 2- Myonecrosis, Gas Gangrene. 3- Cellulitis & other soft tissue infection. 4- Food poisoning:- - Short incubation period 8-24hrs. - abdominal cramps & watery diarrhea. - ingestion of meat product with large number of (109organism). - due to enterotoxin produced by type A strains. 5- Enteritis necroticans, (Big-Bel). - abdominal pain & bloody diarrhea. Laboratory diagnosis - Smell, gas in the tissue , toxic patient. 1-Culture:- - Aerobic & anaerobic blood agar - Chocolate agar. - Fast growing, bacteria divide every 8 minutes. - Gentamicin plate (selective media). - Robertson’s cooked meat broth Pink Cl. Perfringens Black Cl. sporogenes Nagler plate (egg yolk agar). 2- Gram film:- Gram positive bacilli. Nagler’s reaction:- to demonstrate lecithinase activity. - Cl. Perfringens inoculated on the medium containing human serum or egg yolk (is a good source of lecithin). - The a-toxin, lecithinase C (phospholipase) hydrolyses phospholipids in the medium producing turbidity ppt. (right). This ppt. is not observed when the organism grown in the presence of antibody against a-toxin (left). 10 Treatment:- 1- surgery 2- Pencillin to kill bacteria. 3- decide when use AGGS (anti gas gangrene serum), (antitoxin vs toxin). 4- patient’s room saturated with O2 several times a day. 5- proper wound care. 2- Clostridium difficile:- 1- It is part of normal intestinal flora. 2- Responsible for antibiotic-associated gastrointestinal disease (diarrhea), pseudomembranous colitis. 3- Inflammation of the large intestine (colon) due to overgrowth of Cl. difficile. This infection is a common cause of diarrhea after antibiotic use. Treatment, stop ampicillin, clindamycin & ciprofloxacin Use metronidazole or vancomycin …………………………………………………………………………………………………………….…………… 11 - Neurotoxic clostridia (toxic to nerve system): 1- Clostridium tetani 2- Costridium botulinum Clostridium tetani Cause tetanus - Terminal spore, - Gram positive bacilli - Drum stick - Anaerobic, difficult to grow in-vitro. - Found every where, soil, in animals gut & humans - in the past, uterine tetanus in the mother “maternal tetanus” & umbilical tetanus in the baby “neonatal tetanus”. Other people get tetanus:- - Trauma; road accident, bullet, stab with knife, pricks from plants. - Surgery: instrument may be cause teteanus. Pathogenesis:-  1- Tetanospasmin (toxin); - Also referred to as tetanus toxin, acts at several sites within the central nervous system, including peripheral motor end plates, the spinal cord, and the brain, and in the sympathetic nervous system. The spastic paralysis induced by the toxin is due to the blockade of neurotransmitter release from spinal inhibitory interneurons 2- Tetanolysin (toxin); It causes local tissue necrosis that may serve to decrease tissue oxygenation and facilitate proliferation of the bacteria. 12 Clinical syndrome:- - Incubation period few days to weeks. 1- Lock jaw; most common seen, Generalized tetanus 2- Opisthtons (back spasm), Generalized tetanus 3- Cephalic tetanus ( head is the primary site of infection) localized tetanus. Laboratory diagnosis:- - Wound specimen - Tissue specimen Culture; - Aerobic, anaerobic blood agar (swarm, swim) - Meat broth - Gram film - Few organism need to cause disease. Treatment:- - Penicillin - Antitoxin (passive immunization with human tetanus Ab) - Vaccination with tetanus toxoid (DPT). Clostridium botulinum: Disease: botulism - Seven botulinum toxins (A, B, C, D, E, F, G). - (A, B, E) associated with human disease. Pathogenesis:- - Toxin blocks neurotransmission at peripheral cholinergic synapses by preventing release of acetylcholine. Clinical syndrome 1- Food-borne botulism; - Associated with home-canned food (canned fish). - Due to type E toxin. - Spore in canned then germinates the toxin produced. - Flaccid paralysis (weakness of the muscles). - Death due to respiratory paralysis. 13 2- Infant botulism; - Associated with honey contaminated with spores. - Ingestion of spores then germinate then toxin produced, in-vivo. -Young infants 1-6 months. - Flaccid paralysis lead to respiratory arrest (sudden infant death syndrome). 3- wound botulism; in-vivo toxin production. Lab. Diagnosis of Clostridium botulinum; - Food borne; culture of feces & remaining of food. - Heat the specimen for 10 minutes at 80C0 to kill vegetative cells & cultured on anaerobic enriched media to germinate the resistant spores. - Serum tested for toxin activity by mouse bioassay: -Toxin + antitoxin; (intraperitoneal) I.P. to mice (protection). - Toxin; I.P. injection to mice (die). Treatment, Prevention & Control Ventilatory support - Gastric lavage - Penicillin therapy - Antitoxin vs (toxin A,B,E). - Maintaining food at acid pH & store food at 4C0 to prevent spore germination. - Heat food for 20 minutes at 80C0 to destruct toxin - Honey should be avoided in infants younger than one year. 14 15