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Medical Microbiology, PGDCR&RAINTRODUCTION: INTRODUCTION Sporing rod shaped bacteria: 2 groups Aerobic – Bacillus Anaerobic – Clostridia Important Bacillus species: Bacillus anthracis Bacillus cereus Bacillus stearothermophilusINTRODUCTION : INTRODUCTION Members of the genus Bacillus are ubiquitous. Present in Soil, Air, Dust, & Water. Frequently isolated as “CONTAMINANTS” in bacteriological culture media. B. anthracis, the causative agent of “ANTHRX”, is the most important pathogen. B. cereus can cause “FOOD POISOINING”. All members are generally “MOTILE” except B. anthracis, which is “NON-MOTILE”.BACILLUS ANTHRACIS : BACILLUS ANTHRACIS Causative agent of “ANTHRX”. A disease primarily of animals, & man gets infected secondarily (Zoonosis).MORPHOLOGY : MORPHOLOGY Gram positive Non-Acid fast Non-Motile Large (3-10 µm X 1-1.6 µm), rectangular Spore forming & retractile, Oval & Central in position & are of the same width as the bacillary body so that they do not cause bulging of vegetative cell.MORPHOLOGY : The bacilli are arranged End to end in long chains. The ends are truncated or often concave & somewhat Swollen so that a chain of Bacilli present a “bamboo-stick” appearance. MORPHOLOGYCULTUR : CULTUR B. anthracis is an aerobe & facultative anaerobe, with a temp. range for growth being 12-45 0 C (optimum 35-37 0 C). NUTRIENT AGAR MEDIA Colonies are round, 2-3 mm in diameter, raised, opaque & grayish white. Under low power microscope, the edge of the colony is found to be composed of long, interlacing chains of bacilli, resembling locks of matted hairs, the so called “Medusa head” appearance.CULTURE: CULTURE BLOOD AGAR MEDIA Colonies are Non - haemolytic, Though occasional strains produce a narrow zone of haemolysis. SELECTIVE MEDIUM A selective medium (PLET) consisting of Heart infusion agar with Polymyxin, Lysozyme, Ethylene diamine tetracetic acid (EDTA) & Thallous acetate has been devised for isolation of B. anthracis from mixtures containing other spore-bearing bacilli.PATHOGENICITY : PATHOGENICITY Naturally anthrax is disease of cattle and sheep, less or more other animals are also susceptible. Subcutaneous Inoculation of G.P. with B. anthracis culture filtrate. Animal dies within 24-72 hrs. Autopsy – Site of inoculation – shows local gelatinous, hemorrhagic edema . Spleen – Extensive subcutaneous congestion, enlarged, dark red, friable spleen is characteristics. Blood – Dark red & coagulates less firmly than normally. Bacilli are found in large number in local lesions, heart, blood, spleenPATHOGENESIS : PATHOGENESIS Bacilli remain attached to interior capillaries, number is more – so obstruction occur in blood flow.VIRULENCE FACTORS: VIRULENCE FACTORS Two major virulence factor – Capsular polypeptide Anthrax toxin Each produced by separate plasmid. Capsular polypeptide – Inhibits phagocytosis. Loss of plasmid (px02) controls production of capsule, leads to loss of virulence. Attenuated anthrax spore vaccine is prepared by this method (Sterne strain).PATHOGENESIS – Animals : PATHOGENESIS – Animals Anthrax is primarily a disease of animals like cattle & sheep, less often of horses & swine. Infected animals discharge large number of bacilli from the mouth, nose & rectum. The bacilli sporulate in soil & remains as the source of infection.HUMAN ANTHRAX : HUMAN ANTHRAX Humans are occasionally secondarily infected from diseased animals. There are three clinical types of disease based on route of infection CUTANEOUS PULMONARY INTESTINAL anthrax. ALL TYPES LEAD TO SEPTICAEMIA.HUMAN ANTHRX – Cutaneous : HUMAN ANTHRX – Cutaneous 95 % of human cases of anthrax. Route of entry: Skin by inoculation. Involves face, neck, hands, arms & back. Papule Vesicles containing colorless or blood stained fluid Malignant Pustule. ‘Malignant pustule’ – Satellite lesions filled with serum or yellow fluid arranged around a central necrotic lesion which is covered by a black Eschar. Also known as ‘Hide Porter’s Disease’. Resolves spontaneously, 10-20% of untreated may develop fatal septicemia or meningitis.HUMAN ANTHRAX – Pulmonary : HUMAN ANTHRAX – Pulmonary Pulmonary anthrax occurs due to inhalation of the dust or filaments of wool from infected animals, particularly in wool factories . This is also called “ Wool – sorter’s Disease” A life- threatening hemorrhagic pneumonia caused by Inhalation of spores.HUMAN ANTHRAX – Gastrointestinal: Intestinal anthrax is a rare in man and is found in those who consume improperly cooked food/ infected meat for e.g.- African Tribes living in Jungle. Human anthrax can be Industrial – in meat packing or wool factories Nonindustrial – frequent association with animals like butchers, veterinarians, farmers HUMAN ANTHRAX – GastrointestinalLABORATORY DIAGNOSIS : LABORATORY DIAGNOSIS A. SPECIMENS – Swabs Fluids or Pus from pustules Sputum & Blood from pulmonary & septicaemic anthrax are generally collected.MICROSCOPY : MICROSCOPY Gram stained smear from the specimen shows often chain of large Gram Positive Bacilli. Capsule appears as a clear halo around the bacterium by India-Ink preparation/ staining. Capsules are produced in the presence of bicarbonates or 10-25% CO 2 Spores are oval and centrally located, non bulging Spores are stained by special stains – Sudan black B. Microscopic features: Staining blood films with polychrome methylene blue: - Pink amorphous material around blue bacillus (M’ Fadyean’s reaction): represents capsular material – used for the presumptive diagnosis of anthrax in animals. Microscopic featuresCULTURE : CULTURE Specimen is inoculated on Nutrient Agar medium & incubated at 37 0 C for overnight. Irregular, round, raised, dull, opaque, grayish white colonies with a frosted glass appearance. Low power – edge of the colony is composed of long, interlacing chains of bacilli, resembling locks of matted hair – “Medusa Head Appearance”. Gelatin stab culture – “inverted fir tree” appearance, with slow liquefaction starting from top.Slide 21: Medusa Head Appearance wavy colonies with small projections Inverted fir treeSlide 22: “String of Pearls reaction” – solid medium containing 0.05-0.5 units of Penicillin/ml, in 3-6 hrs the cells become large, spherical and occur in chains on agar surface, resembling a string of pearls. Selective medium – PLET medium – contains polymyxin, lysozyme, EDTA & thallous acetate : to isolate it from mixtures containing other spore bearing bacilli.ANIMAL INOCULATION : ANIMAL INOCULATION White mouse or Guinea – pigs are inoculated/ injected with exudate or culture. By rubbing contaminated tissue over shaven skin of a guinea pig. Animal dies in 36- 48 hrs. Serology Ascoli’s thermoprecipitation test – to demonstrate anthrax Ag in tissue extracts EIA (using purified anthrax toxin Ag) PCR to detect anthrax contamination of animal & agricultural productsTREATMENT: TREATMENT Bacillus anthracis is sensitive to : - Penicillin - Doxycycline - CiprofloxacinPROPHYLAXIS : PROPHYLAXIS General methods of prevention Improvement of factory hygiene Proper sterilization of animal products Animal carcasses to be buried deep in quicklime or crematedPROPHYLAXIS : PROPHYLAXIS Active immunisation of Domestic animals with live attenuated spore vaccines Persons with occupational risk (butchers, farmers, veterinarians) with a cell- free vaccine containing purified protective antigen as immunogen. 3. 3 doses IM with annual booster injections . * Anthrax infection in humans give life long permanent immunity & secondary infections are very rare.ANTHRAX VACCINES: ANTHRAX VACCINES Original anthrax vaccine – developed by Pasteur – live attenuated bacilli vaccine – strain rendered avirulent by the loss of plasmids which encodes anthrax toxin. Live attenuated anthrax spore vaccines - Sterne vaccine – contains spores of a non - capsulated avirulent mutant strain - loss of plasmid which controls capsule production. Mazucchi vaccine – contains spores of stable attenuated Carbazoo strain.Slide 28: Biological warfare Large epidemics (occasionally) In 1979 – former Soviet Union: due to accidental release of spores from a military facility engaged in biological research In 1980s – Zimbabwe: affected 10,000 persons . In 2001 – USA several died due to mails with spores of B. anthracis. * Hence the need to develop better human vaccine.ANTHRACOID BACILLI : ANTHRACOID BACILLI Aerobic spore bearing bacilli resembling B. anthracis are called “ ANTHRACOID” or “ PSEUDOANTHRAX BACILLI” Some of them are frequent laboratory contaminants & have to be differentiated from B. anthracis. The main differentiating features between anthracoid bacilli & B. anthracis are shown in table.DIFFERENTIATING FEATURES BETWEEN B. ANTHRCIS & ANTHRACOID BACILLI : DIFFERENTIATING FEATURES BETWEEN B. ANTHRCIS & ANTHRACOID BACILLI FEATURES B. anthracis Anthracoid bacilli Motility Non- motile Generally motile Capsule Capsulated Non – capsulated Chain formation Long chains Short chains Colony on Nutrient Agar Medusa Head Colony No such morphology Growth in Broth No turbidity Uniform turbidity Gelatin Stab culture Inverted Fir tree appearance & show gelatin liquefaction Rapid gelatin liquefaction Haemolysis on BA Absent Usually well marked Growth in Penicillin Agar (10 units/ml) No growth Grow usually Growth at 45 0 C No growth Grow usually Susceptibility to Gamma phage Susceptible Not susceptible Pathogenicity test in animals Pathogenic Non-pathogenic Ascoli’s precipitin test Positive Negative Fluorescent Antibody test with anthrax antiserum Positive NegativeBACILLUS CEREUS : BACILLUS CEREUS Cause of FOOD POISOINING. Ubiquitous in nature. Vegetables, milk, cereals, spices, meat & poultry. Some spores survive cooking & germinate into vegetative bacilli which produce ENTEROTOXIN that causes food poisoining.TYPES OF FOOD POISOINING : TYPES OF FOOD POISOINING 1. SHORT INCUBATION PERIOD TYPE (1-5 HRS) Characterized by acute Nausea & vomiting, 1-5 hrs after the meal. Diarrhoea is not common. It is usually associated with consumption of cooked rice, usually fried rice from Chinese restaurants.2. Long incubation period type (8-16) : 2. Long incubation period type (8-16) Characterised by Acute abdominal pain & diarrhoea , 8-16 hrs. after consumption of contaminated food. Vomiting is rare symptom in this type.Slide 34: Gastroenteritis Bacillus cereus clinical presentation Incubation period < 6 hours Severe vomiting Lasts 8-10 hours Incubation period > 6 hours Diarrhoea Lasts 20-36 hours EMETIC FORM DIARRHOEAL FORM Types of Gastroenteritis: Types of Gastroenteritis Type I Wide range of foods including cooked meat & vegetables Diarrhoea & abdominal pain develops 8 –16 hrs after consumption Few bacilli seen in fecal specimens Caused by serotypes 2,6,8,9,10 or 12. Enterotoxin resembles LT of E. coli Type II Chinese fried rice exclusively. Acute nausea & vomiting 1-5 hrs after meals, diarrhoea rare Large no of bacilli in cooked rice & fecal samples. Caused by serotypes 1,3 or 5 Toxin resembles staphylococcal enterotoxinDIAGNOSIS : DIAGNOSIS Suspected food, faeces & vomitus are cultured on ordinary media or a special mannitol – egg yolk – phenol red polymyxin agar (MYPA) medium. Spore bearing Gram Positive Bacilli may be seen on smear from colonies. B. cereus is a motile bacilli, non-capsulated, not susceptible to gamma phage & does not react with fluorescent antibody conjugate.TREATMENT : TREATMENT Disease is mild and self limiting, requiring no specific treatment. Rehydration Antibiotics – in systemic infections You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.