Bacillus II MBBS Dr Ekta Chourasia Microbiology. 28/02/2008Dr Ekta Chourasia, Microbiology Introduction Sporing rod shaped bacteria: 2 groups 1. Aerobic.

Bacillus

II MBBS

Dr Ekta Chourasia

Microbiology

28/02/2008 Dr Ekta Chourasia, Microbiology

Introduction Sporing rod shaped bacteria: 2 groups

1. Aerobic – Bacillus2. Anaerobic – Clostridia

Important Bacillus species: Bacillus anthracis Bacillus cereus Bacillus stearothermophilus


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


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


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.


Pathogenicity: virulence factors

Two virulence factors – Capsular polysaccharide – inhibits

phagocytosis, encoded by a plasmid Anthrax toxin : made up of 3 fractions1. 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


Human Anthrax The disease may be1. Cutaneous2. Pulmonary, or3. Intestinal

* All types lead to fatal septicemia


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


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


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


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


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.


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.


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.

Medusa Head Appearance-wavy colonies with small

projections

Inverted fir tree


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.


Smear from colony

Morphology in stained

smears from cultures

- “Bamboo stick appearance” : bacilli arranged end to end in long chains.


Laboratory Diagnosis Animal inoculation By rubbing contaminated tissues over

shaven skin of a guinea pig Serology1. Ascoli’s thermoprecipitation test – to

demonstrate anthrax Ag in tissue extracts

2. EIA (using purified anthrax toxin Ag)3. PCR to detect anthrax contamination

of animal & agricultural products


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 by1. 4% KMnO4 in 15 mins2. ‘Duckering’ – using formaldehyde

solution for animal products imported into non endemic countries


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


Prophylaxis General methods of prevention1. Improvement of factory hygiene2. Proper sterilisation of animal products3. Animal carcasses to be buried deep in

quicklime or cremated


Prophylaxis Active immunisation of 1. Domestic animals with live attenuated

spore vaccines

2. 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.


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 1. Sterne vaccine – contains spores of a

noncapsulated avirulent mutant strain - loss of plasmid which controls capsule production

2. Mazucchi vaccine – contains spores of stable attenuated Carbazoo strain


Biological warfare Large epidemics (occasionally)1. In 1979 – former Soviet Union: due to

accidental release of spores from a military facility engaged in biological research

2. In 1980s – Zimbabwe: affected 10,000 persons.

* Hence the need to develop better human vaccine.


Treatment

Bacillus anthracis is sensitive to: - Penicillin - Doxycycline - Ciprofloxacin


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.


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


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.

Gastroenteritis

Gastroenteritis

Bacillus cereus clinical presentation

Incubation period < 6 hoursSevere vomitingLasts 8-10 hours

Incubation period > 6 hoursDiarrhoea

Lasts 20-36 hours

EMETIC FORM DIARRHOEAL FORM


Types of GastroenteritisType 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


Diagnosis Primarily depends on clinical diagnosis

& food sources Laboratory Diagnosis1. Specimen – stool, vomitus, food, blood2. Microscopy – not of much help3. Culture 4. Test for toxin – to differentiate from

staphylococcal food poisoning.


Culture Blood agar

Special MYPA medium: Mannitol - egg yolk - phenol red – polymyxin agar : to isolate B.cereus from feces & other sources.


Treatment Rehydration Antibiotics – in systemic infections

Bacillus II MBBS Dr Ekta Chourasia Microbiology. 28/02/2008Dr Ekta Chourasia, Microbiology Introduction Sporing rod shaped bacteria: 2 groups 1. Aerobic.

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