Bacillus

IT IS HELPFUL TO UNDERSTAND THE BACTERIA BACILLUS

1. BACILLUS

2. INTRODUCTION Gram positive spore forming bacilli belong to two genera: 1. Bacillus: They are obligate aerobes; having non bulging spores. 2. Clostridium: They are obligate anaerobes with bulging spores. Bacillus species are obligate aerobic gram positive spore forming rods. B. anthracis and B. cereus are the only two pathogenic species: other members are universal, present in soil, air, water, dust and also frequently isolated as a contaminant. Bacillus species are generally motile ( peritrichous flagella) and non capsulated except anthrx bacillus, which is non motile and capsulated.

3. BACILLUS ANTHRACIS Bacillus anthracis is the causative agent of an important zoonotic disease called anthrax. It is also gained importance recently because of its ability to be used as biological weapon. MORPHOLOGY They are gram –positive, large rectangular rods (3-10µm x 1-1.6µm) arrange in chains, non motile and capsulated bearing non bulging oval spores. The capsule is polypeptide in nature.

4. Spore are never found in the animal body during life but are formed in culture or in the soil. Sporulation occur under unfavourable conditions. Robert Koch's original photomicrographs of  Bacillus anthracis .  

5. In culture , the bacilli are arranged end to end in long chains. The ends of the bacilli are truncated concave and somewhat swollen so that a chain of bacilli presents in bamboo- stick appearance . Gram stain of Bacillus anthracis

6. HISTORICAL IMPORTANCE Considerable historical interest is attached to anthrax bacillus due to following reasons: It was the first bacteria seen under the microscope. Anthrax was the first communicable disease shown to be transmitted by inoculation of infected blood. It was the first bacterium isolated in pure culture. Anthrax vaccine was the first attenuated bacterial vaccine .

7. CULTURE B.anthracis is an aerobes and facultative anaerobes. Temp: 12-45 C Nutrient agar: Colonies are round ,2-3mm in diameter, raised, opeque and greyish white. Under low power of the microscope , the edge of the colony is found to be composed of long, interlacing chain of bacilli resembling lock of matted hair,so called the medusa head appearance .

8. Medusa head appearance of colony

9. Blood agar media: The colonies are non haemolytic, occasional strains produce a narrow zone of haemolysis . Gelatin stab culture: Gelatin is liquefied mostly at the top due to aerobic environment and gives a charecterstics inverted fir tree appearance. INVERTED FIR TREE APPEARANCE

10. Selective medium:(PLET medium) Consisting of heart infusion agar with polymyxin,lysozyme , ethylene diamine tetracetic acid and thallous acetate. It is used for the isolation of B. anthrasis . PLET MEDIUM showing growth of Anthrax bacilli

11. Solid medium with penicillin: Colonies have a string of pearl appearance look (due to cells becoming larger and spherical because of their weaker cell walls under the action of penicillin, and cells tend to occur in chain on surface of agar.) BIOCHEMICAL REACTIONS Glucose, maltose and sucrose are fermented with acid. Catalase positive Nitrate reduction test positive

12. RESISTANCE Vegetative forms are destroyed – 60 C in 30min. Spore form – viable for years in soil Autoclaving – kills anthrax spores 4% KmNo4 in 15 mts - kills anthrax spores Susceptible to many antibiotics like pencillin etc.,

13. VIRULENCE FACTOR Pathogenesis of anthrax due to two important virulence factor- Antigen- These are three type: Capsular antigen: it is present in virulent strain and consist of polypeptide and act as hapten . Cell wall antigen: polysaccharide Somatic protein antigen: it is heat labile protein present in bacterial body. It stimulate the immune system to produces the antibody which is protective in nature.

14. ANTHRAX TOXIN It is tripartite toxin, consisting of three fragments. 1. Edema factor: is the active fragments; act as adenyl cyclase and increases host cell cAMP . It is responsible for edema and other menifestation seen in anthrax. 2.Protective factor: is the binding fragment that binds to the host cell receptors and facilitates the entry of other fragments into the host cells. 3.Lethal factor: causes cell death, but mechanism of action is not known.

15. These fragments are not toxic individually, but in combination , they produce local edema and generalized shock. Toxin synthesis is controlled by a plasmid (pX01). Loss of plasmid makes the strain avirulent .

16. CLINICAL MANIFESTATION Animal anthrax: Anthrax is primarily a zoonotic disease. Herbivorous animal such as cattle, sheep and less often horses and pig are affected more commonly. Infection occurs in susceptible animals by ingestion of the spore present in the soil. Anthrax in animal is presented as a fetal spticemia . Infected animal discharge large number of bacilli from the mouth, nose and rectum. These bacilli sporulate in soil and remain as the source of infection for man.

17. Human anthrax: Transmission: Cutaneous mode- by spore entering through the abraded skin. Inhalation of spore Inhalation of carcasses of animals drying of anthrax containing spores (manifested as bloody diarrhoea ) Indirectly through fomite (very rare)

18. Clinical types: There are three types of human anthrax: 1.Cutaneous anthrax (most common) 2.Pulmonary anthrax (less common) 3.Intestinal anthrax (very rare)

19. 1.Cutaneous anthrax: Spores enter through abraded skin Germinate & Multiply at the site of entry. Localised lesions - Face, neck & hand. Papule – vesicle – pustular – centrally necrosed – black coloration.

20. PULMONARY ANTHRAX Inhalation of the dust or filaments of wool from infected animals, particularly in wool factories ( wool sorter’s disease) Hemorrhagic pneumonia Bacilli spread by lymphatics or blood, leading to- Bacteremia Meningitis

21. Intestinal anthrax Consumption of improperly cooked infected meat. Causes violent enteritis with bloody diarrhoea . All clinical conditions lead to septicaemic anthrax if not treated early – Highly fatal!!!

22. Epidemiology of Human anthrax Human anthrax is highest in Africa, and central and southern Asia. Human anthrax cases may be of two types- 1. Non industrial cases: it is result from agricultural exposure to animal. 2.Industrial cases: it result from infected animal products such as hides, bristles and wool.

23. LABORATORY DIAGNOSIS Specimen collection- Pus or swab from pustule Sputum in pulmonary anthrax Blood in case of septicemia CSF in hemorrhagic meningitis Gastric aspirate, feces or food in intestinal anthrax Ear lobe from dead animals

24. Direct demonstration: Gram staining- gram positive rectangular bacilli. McFadyean’s reaction- polypeptide casule can be demonstrated by staining with Gurr’s polychrome methylene blue stain for 30 sec. Shows amorphous purple capsule surrounding blue bacilli. McFadyaen’s reaction Gram stain

25. Direct immunofluorescence test – it detect capsular and cell wall polypeptide antigens by using fluorescent tagged monoclonal antibodies. Ascoli’s thermopreciitation test: It is a ring precipitation test, done when the sample is putrid form and bacilli are likely to be non viable. Tissues sample grounded in saline , boiled and filtered. This antigenic extract is layered over the anthrax antiserum on a narrow capillary tube. A ring of precipitate appears at the junction of two liquids within 5min.

26. Culture Nutrient agar: medusa head appearance colony Blood agar: dry wrinkled, non haemolytic colony Gelatin stab agar: Inverted fir tree appearance colony Selective media- Solid medium with penicillin: string of pearl colony PLET medium: inhibit the growth of contaminant

27. Culture smear: Reveals bamboo stick appearance,i.e . long chain bacilli with non bulging spore. Spores: they can be demonstrated by using special stain, such as hot malachite green or 0.25% sulphuric acid (spores are acid fast). Serology: Antibodies appear in convalescent sera and can be detected by ELISA or immunodiffusion in gel method. Molecular diagnosis: PCR with specific primer can be used for further cnfirmation .

28. Treatment: Pencillin Erythromycin Antitoxins – in emergency cases Prophylaxis: ANIMAL VACCINE – Sterne vaccine HUMAN VACCINE – Alum Precipitated Toxoid – 3 doses intramuscularly at intervals of 6 weeks and 6 months Booster dose after one year.

29. Bacillus cereus

30. Introduction: It is normal habitant of soil, also widely isolated from food items such as vegetable, milk, cereals, spices, meat, and poultry. It is an important agent of food poisoning in man.

31. Clinical manifestations: Food poisoning: It produces two type of toxin diarrhoeal toxin (causes diarrheal type of food poisoning) and emetic toxin (causes emetic type of food poisoning).

32. Bacillus cereus Diarrheal type Emetic type Incubation period 8-16 hours 1-5 hours Toxin Secreted in intestine (similar to clostridium perfringens enterotoxin ) Preformed toxin(formed in diet, similar S. aureus ) Heat Heat labile Heat stable Food items contaminated Meat, vegetables, dried beans, cereal Rice ( chinese fried rice) Clinical features Diarrhea , fever , abdominal cramps Vomiting , abdominal cramps Serotype involve 2,6,8,9,10,12 1,3,5 Differences between diarrheal type and emetic type of food poisoning

33. Emetic toxin: It is heat stable preformed toxin resembling S.aureus enterotoxin . It act immediately on intestine so that the incubation period of food poisoning is shorts (1-6 hrs). Diarrheal toxin: Organism secretes this toxin only after entering into the intestine, hence the incubation period is longer (8-16 hrs.).

34. Ocular disease: It causes severe keratitis and panopthalmitis following trauma to the eye that may lead to the loss of vision. Other condition: It rarely causes systemic infections including endocarditis , meningitis, osteomyelitis and pneumonia.

35. Laboratory diagnosis : Bacillus cereus can be isolated from feces by using selective media such as: MYPA ( mannitol , egg yolk, phenol red and agar) PEMBA( polymyxin B, egg yolk, mannitol , bromothymol blue, agar). It is motile, non-capsulated and not susceptible to gamma phage.

36. Treatment: Disease is mild and self limiting, requiring no specific treatment. But bacillus cereus is susceptible to clindamycin , erythromycin, vancomycin , aminoglycosides and tetracycline. It is resistant to penicillin and trimethoprime . Control: Adequate food hygiene is to be maintained while storing cooked food especially rice and reheating should be rapid.

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