BACILLUS

Mr. Gunjal Prasad N. Assistant Prof. M.Sc. Medical Microbiology, PGDCR&RA

INTRODUCTION

Sporing rod shaped bacteria: 2 groups

1. 2.

Aerobic Bacillus Anaerobic Clostridia

Important Bacillus species:

Bacillus anthracis Bacillus cereus Bacillus stearothermophilus

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
Causative agent of ANTHRX. A disease primarily of animals, & man gets

infected secondarily (Zoonosis).

MORPHOLOGY
Gram positive
Non-Acid fast Non-Motile

Large (3-10 m X 1-1.6 m), 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.

CULTUR
B. anthracis is an aerobe & facultative anaerobe, with a temp. range for growth being 12-450C (optimum 35-370C). 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
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 sporebearing bacilli.

PATHOGENICITY
o 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, spleen

PATHOGENESIS
Bacilli remain attached to interior capillaries,

number is more so obstruction occur in blood flow.

VIRULENCE FACTORS
o Two major virulence factor o Capsular polypeptide o Anthrax toxin o Each produced by separate plasmid.

o Capsular polypeptide o Inhibits phagocytosis. o Loss of plasmid (px02) controls production of capsule, leads to loss of virulence. o Attenuated anthrax spore vaccine is prepared by this method (Sterne strain).

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
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

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 Porters Disease. Resolves spontaneously, 10-20% of untreated may develop fatal septicemia or meningitis.

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 sorters 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

LABORATORY DIAGNOSIS
A. SPECIMENS Swabs Fluids or Pus from pustules Sputum & Blood from pulmonary & septicaemic anthrax

are generally collected.

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% 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 Fadyeans reaction): represents capsular material used for the presumptive diagnosis of anthrax in animals.

CULTURE
Specimen is inoculated on Nutrient Agar

medium & incubated at 370C 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.

Inverted fir tree

Medusa Head Appearance -wavy colonies with small projections

 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
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
1. Ascolis 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

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

PROPHYLAXIS

General methods of prevention

1. 2. 3.

Improvement of factory hygiene Proper sterilization of animal products 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. 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 vaccines 1. Sterne vaccine contains spores of a non capsulated 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. 3. In 2001 USA several died due to mails with spores of B. anthracis. * Hence the need to develop better human vaccine.

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 FEATURES B. anthracis Anthracoid bacilli Motility Capsule Chain formation Colony on Nutrient Agar Growth in Broth Gelatin Stab culture Haemolysis on BA Growth in Penicillin Agar (10 units/ml) Growth at 450C Susceptibility to Gamma phage Pathogenicity test in animals Ascolis precipitin test Fluorescent Antibody test with anthrax antiserum Non- motile Capsulated Long chains Medusa Head Colony No turbidity Inverted Fir tree appearance & show gelatin liquefaction Absent No growth No growth Susceptible Pathogenic Positive Positive Generally motile Non capsulated Short chains No such morphology Uniform turbidity Rapid gelatin liquefaction Usually well marked Grow usually Grow usually Not susceptible Non-pathogenic Negative Negative

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

1. SHORT INCUBATION PERIOD TYPE (1-5

HRS) Characterized by acute Nausea & vomiting, 15 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)

Characterised by Acute abdominal pain & diarrhoea, 8-16 hrs.

after consumption of contaminated food. Vomiting is rare symptom in this type.

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

Type II Chinese fried rice exclusively.

Acute nausea & vomiting 1-

2,6,8,9,10 or 12. Enterotoxin resembles LT of E. coli

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
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
Disease is mild and self limiting, requiring no

specific treatment.
Rehydration Antibiotics in systemic infections