II MBBS Dr Ekta Chourasia Microbiology Bacillus II MBBS Dr Ekta Chourasia Microbiology

Dr Ekta Chourasia, Microbiology Introduction Sporing rod shaped bacteria: 2 groups Aerobic – Bacillus Anaerobic – Clostridia Important Bacillus species: Bacillus anthracis Bacillus cereus Bacillus stearothermophilus 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Bacillus: key words Sporing Gram+ve bacilli Bacillus anthracis Anthrax Hide porter’s disease Wool sorter’s disease Malignant pustule Eschar M’fadyean’s reaction Bamboo stick appearance Medusa head colony String of pearl’s reaction PLET medium Ascoli’s test Duckering Anthrax vaccine Bioterrorism Bacillus cereus Gastroenteritis Bacillus stearothermophilus 28/02/2008 Dr Ekta Chourasia, Microbiology

History of Bacillus anthracis 1st pathogenic bacterium to be seen under microscope – Pollender, 1849 1st communicable disease shown to be transmitted by inoculation of infected blood – Davaine, 1850 1st bacillus to be isolated in pure culture & shown to possess spores – Koch, 1876 1st bacterium used for the preparation of an attenuated vaccine – Pasteur, 1881 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Pathogenicity Anthrax – zoonotic disease primarily involves cattle & sheep. Animals – infected by ingestion of spores present in the soil Large no of bacilli are shed in discharges from the mouth, nose & rectum - sporulate in the soil. Human anthrax – contracted from animals, directly or indirectly. 28/02/2008 Dr Ekta Chourasia, Microbiology

Pathogenicity: virulence factors Two virulence factors – Capsular polysaccharide – inhibits phagocytosis, encoded by a plasmid Anthrax toxin : made up of 3 fractions Edema factor (EF or Factor I) Protective antigen factor (PA or Factor II) Lethal factor (LF or Factor III) * They are not toxic individually, the whole complex produces local edema & generalised shock. Toxin production is plasmid mediated 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Human Anthrax The disease may be Cutaneous Pulmonary, or Intestinal * All types lead to fatal septicemia 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 1. Cutaneous Anthrax 95 % of human cases of anthrax Route of entry: Skin Sites involved – 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 untreaed may develop fatal septicemia or meningitis 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 2. Pulmonary Anthrax Also called ‘Wool Sorter’s disease’ – common in workers in wool factories A life- threatening hemorrhagic pneumonia caused by Inhalation of spores 28/02/2008 Dr Ekta Chourasia, Microbiology

3. Gastrointestinal Anthrax Rare By ingestion of inadequately cooked meat containing B. anthracis spores * Human anthrax can be Industrial – in meat packing or wool factories Nonindustrial – frequent association with animals like butchers, veterinarians, farmers 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Laboratory Diagnosis Specimen Fluid or pus from local lesion, blood, sputum Microscopy Culture In septicemic anthrax, blood culture should be done Serological test Animal inoculation 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Microscopy Large aerobic, non motile, Gm+ve bacilli Arranged singly, in pairs or in short chains, the entire chain is surrounded by a capsule Capsules are produced in the presence of bicarbonates or 10-25% CO2 Spores are oval and centrally located, non bulging Spores are stained by special stains – Sudan black B. 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 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. 28/02/2008 Dr Ekta Chourasia, Microbiology

Cultural Characteristics Grow on blood or nutrient agar, at 37°C Irregular, round, raised, dull, opaque, greyish 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. 28/02/2008 Dr Ekta Chourasia, Microbiology

Medusa Head Appearance Inverted fir tree Medusa Head Appearance wavy colonies with small projections

Cultural Characteristics “String of Pearls reaction” – solid medium containing 0.05-0.5 units of Pn/ ml, in 3-6 hrs the cells become large, spherical and occur in chains on agar surface, resembling a string of pearls. - differentiates B. anthracis from B. cereus Selective medium – PLET medium – contains polymyxin, lysozyme, EDTA & thallous acetate : to isolate it from mixtures containing other spore bearing bacilli. 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Smear from colony Morphology in stained smears from cultures “Bamboo stick appearance” : bacilli arranged end to end in long chains. 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Laboratory Diagnosis Animal inoculation By rubbing contaminated tissues over shaven skin of a guinea pig 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 products 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Resistance Bacilli destroyed at 60°C in 30 mins. Animal carcasses – bacilli remain viable in BM for a wk & in skin for 2 wks. Spores – highly resistant, survive in soil for 60 yrs Spores can be destroyed by 4% KMnO4 in 15 mins ‘Duckering’ – using formaldehyde solution for animal products imported into non endemic countries 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Duckering For disinfection of wool – 2% soln of formaldehyde at 30- 40°C for 20 mins Animal hair & bristles – 0.25% at 60°C for 6 hrs 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Prophylaxis General methods of prevention Improvement of factory hygiene Proper sterilisation of animal products Animal carcasses to be buried deep in quicklime or cremated 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 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 doses IM with annual booster injections. * Anthrax infection in humans give life long permanent immunity & secondary infections are very rare. 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 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 vaccine Sterne vaccine – contains spores of a noncapsulated avirulent mutant strain - loss of plasmid which controls capsule production Mazucchi vaccine – contains spores of stable attenuated Carbazoo strain 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 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. * Hence the need to develop better human vaccine. 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Treatment Bacillus anthracis is sensitive to: - Penicillin - Doxycycline - Ciprofloxacin 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Anthracoid bacilli Belongs to the genus Bacillus Occasionally cause human infections Includes B. cereus, B. subtilis, B. licheniformis & other species. These and a variety of non pathogenic aerobic spore bearing bacilli appear as laboratory contaminants & resemble anthrax bacilli – Pseudoanthrax or Anthracoid bacilli. 28/02/2008 Dr Ekta Chourasia, Microbiology

Differences b/n Anthrax & Anthracoid bacilli Anthrax bacilli Nonmotile Capsulated Grow in long chains Medusa head colony No growth in Pn agar (10units/ml) Weak or no hemolysis Inverted fir tree growth & slow gelatin liquefaction No growth at 45C Anthracoid bacilli Generally motile Noncapsulated Grow in short chains Not present Grow usually Hemolysis well marked Rapid liquefaction Usually grows 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Bacillus cereus Readily isolated from soil, vegetables and a wide variety of foods including milk, cereals, spices, poultry & meat. Causes foodborne gastroenteritis – 2 patterns of disease (diarrhoeal & emetic); both types are mild & self limited, requiring no specific therapy. 28/02/2008 Dr Ekta Chourasia, Microbiology

Bacillus cereus clinical presentation Gastroenteritis DIARRHOEAL FORM EMETIC FORM Incubation period > 6 hours Diarrhoea Lasts 20-36 hours Incubation period < 6 hours Severe vomiting Lasts 8-10 hours

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 enterotoxin 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Diagnosis Primarily depends on clinical diagnosis & food sources Laboratory Diagnosis Specimen – stool, vomitus, food, blood Microscopy – not of much help Culture Test for toxin – to differentiate from staphylococcal food poisoning. 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Culture Blood agar Special MYPA medium: Mannitol - egg yolk - phenol red – polymyxin agar : to isolate B.cereus from feces & other sources. 28/02/2008 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Treatment Rehydration Antibiotics – in systemic infections 28/02/2008 Dr Ekta Chourasia, Microbiology