Bacillus species

Spore forming aerobic organisms, Causative agent of “ANTHRAX”.
Gram positive bacilli, Bamboo stick appearance. Responsible to cause cutaneous, pulmonary, intestinal anthrax by producing anthrax toxin. M' Fadyean's Reaction presumptive diagnostic method in animals.

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

2. INTRODUCTION Sporogenous OR Spore forming rod shaped bacteria: 2 groups Aerobic – Bacillus – Non Bulging spores . Anaerobic – Clostridia- Bulging spores. Important Bacillus species : Bacillus anthracis Bacillus cereus Bacillus stearothermophilus

3. INTRODUCTION Ubiquitous, present in Soil, Air, Dust, & Water. Frequently isolated as “ LAB CONTAMINANTS”. B. anthracis, the causative agent of an important Zoonotic disease called “ANTHRX”. B. cereus can cause “FOOD POISOINING”. All members are generally “MOTILE” except B . anthracis, which is “NON-MOTILE”. Temp. range for growth 25 -750C. Salt conc. 2% - 25 %. Also gained importance recently because of its abilit y to be used as “Biological Weapon”.

4. Historical Interest First organism observed under microscope, Pollender, 1849. First communicable disease shown to be transmitted by inoculation of infected blood – Davaine, 1850. First bacillus to be isolated in pure culture and shown to possess spores – Koch, 1876. First bacteria to be used for preparation of attenuated vaccine by Pasteur, 1881.

5. BACILLUS ANTHRACIS Causative agent of “ANTHRX”. A disease primarily of animals, & man gets infected secondarily (Zoonosis).

6. MORPHOLOGY Gram positive, Non-Acid fast, Non-Motile. Large (3-10 µm X 1-1.6 µm), rectangular Capsule is made up of polypeptide, polymer of d-glutamic acid. This capsule is Plasmid coded (pX02). It inhibits complement mediated phagocytosis. Capsules not formed under ordinary conditions only if media containing bicarbonate or are incubated at 10 to 25 % CO2. If media contains serum, albumin, charcoal or starch – Capsule formation may occur in absence of CO2.

7. The bacilli are arranged end to end in long chains. The ends are often concave & somewhat swollen so that a chain of bacilli present a “bamboo-stick” appearance. Spore are oval & central in position & are of the same width as the bacillary body so that they do not cause bulging of vegetative cell. Spores are formed in culture and in soil but never in host body. MORPHOLOGY

8. Spores Spores are stain by special methods – Sudan black – B – Fat globules maybe made out within bacilli. “Hot malachite green (Ashby’s method) OR 0.25% Sulphuric acid as spores are Acid fast”. When blood films are stained with polychrome methylene blue for a few seconds and observed – an amorphous purplish material is noticed around the bacilli. This represents the capsular material and is characteristic of anthrax bacillus. This is called as “ Mc Fadyean’s reaction” & used as presumptive diagnosis of Anthrax in animals.

9. CULTURE B. anthracis is an aerobe & facultative anaerobe, with a temp. range for growth being 12-45 C (optimum 35-37 C). NUTRIENT AGAR MEDIA Colonies are irregular, round, 2-3 mm in diameter, dull, raised, opaque & grayish white with frosted glass appearance. 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 .

10. 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.

11. PATHOGENICITY Naturally anthrax is disease of cattle and sheep, less or more other animals are also susceptible (Zoonosis) . 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 - easily broken into pieces or become powder . Blood – Dark red & coagulates less firmly than normally. Bacilli are found in large number in local lesions, heart, blood, spleen (more than 10 8 bacilli / ml)

12. PATHOGENESIS Bacilli remain attached to interior capillaries, number is more – so obstruction occur in blood flow.

13. VIRULENCE FACTORS Two major virulence factor – Capsular polypeptide Anthrax toxin Each produced by separate plasmid. Capsular polypeptide – Composed of poly peptide of a high molecular weight consisting of D-glutamic acid. 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).

14. Anthrax Toxin The toxin is complex of 3 fractions: 1. Edema Factor (OF or Factor I) 2. Protective Antigen Factor (PA or Factor II) 3. Lethal Factor (LF or Factor III) These are nontoxic if act individually. But the complex causes local edema and generalised shock.

15. Anthrax Toxin These three factors have been characterised and cloned. Protective Antigen Factor or PA - It binds the target cell surface receptors and in turns provides attachment sites for Edema factor or OF or Lethal Factor or LF, facilitating their entry inside the target cell. The antibody to PA is protective because it blocks the attachment of PA to target cell surface receptor and inhibit the further action of OF or LF.

16. Edema Factor or OF - It is an Adenyl cyclase which is activated only inside the target cells, leading to intracellular accumulation of cyclic AMP. This is believed to be responsible for the edema and other biological effects of the toxin. Lethal Factor - Entry of LF into the cell causes cell death but the mechanism of action is not known . Anthrax Toxin

17. Loss of the plasmid (px01) which encodes the toxin renders the strain avirulent. This is believed to have been the basis for the original anthrax vaccine developed by Pasteur. The avirulent Sterne vaccine strain is devoid of the plasmid coding for the capsular polysaccharide. Anthrax Toxin

18. PATHOGENESIS – Animals Anthrax is primarily a disease of animals like cattle & sheep, less often of horses & swine. Anthrax in animals presented as a fetal septicemia; however, localized cutaneous lesions may be produced rarely. Infected animals discharge large number of bacilli from the mouth, nose & rectum. The bacilli sporulate in soil & remains as the source of infection.

19. 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.

20. HUMAN ANTHRX – Cutaneous 95 % of human cases are of cutaneous type. 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.

21. 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.

22. 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 animal handlers like butchers, veterinarians, farmers HUMAN ANTHRAX – Gastrointestinal

23. Difference between Cutaneous and Pulmonary Anthrax Features Cutaneous Anthrax Pulmonary Anthrax Other Name Hide porter’s disease (As commonly occurs in dock workers carrying loads of hides and skin on their bare backs) Wool sorter’s disease (As it is seen in workers of wool factories acquire infection by inhalation of dust from infected wool. Transmission Cutaneous exposure to spores (enter through abraded ski) Inhalation of spores Characterized by Malignant pustule – the lesion begins as a papule that evolves into a painless vesicle followed by the development of a coal-black, necrotic eschar surrounded by non-pitting indurated edema. The name anthrax, which means coal, comes from the black colour of the eschar. However, it is a non-malignant condition Haemorrhagic pneumonia Bacillus spread by lymphatics or blood, leading to – Bacteremia Haemorrhagic meningitis

24. Features Cutaneous Anthrax Pulmonary Anthrax Occupational exposure Dock worker, butcher, abattoir and farmer Workers of the wool factory Occurrence Most common (95%) Rare Prognosis Self- limiting, rarely becomes fatal if untreated Fatal Bioterrorism Rarely causes bioterrorism Most common form to cause bioterrorism Difference between Cutaneous and Pulmonary Anthrax

25. LABORATORY DIAGNOSIS A. SPECIMENS – Swabs Fluids or Pus from pustules Sputum & Blood from pulmonary & septicaemic anthrax are generally collected.

26. 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.

27. Staining blood films with polychrome methylene blue: - Pink or purple amorphous material around blue bacillus (M’ Fadyean’s reaction): represents capsular material – used for the presumptive diagnosis of anthrax in animals. Microscopic features

28. CULTURE Specimen is inoculated on Nutrient Agar medium & incubated at 37 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.

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

30. “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.

31. 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 24------------------------------------------------------------ 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 products

32. TREATMENT Bacillus anthracis treatment : - Ciprofloxacin or Doxycycline, plus Clindamycin, and/or Rifampin for 60 days. Antibiotics for post exposure prophylaxis : Ciprofloxacin for 60 days. Doxycycline for 60 days or Amoxicillin for 60days (given if strain is penicillin sensitive). Raxibacumab: It is a monoclonal antibody that neutralizes anthrax toxin (protective antigen). It is intended for the prophylaxis and treatment of inhalation anthrax.

33. PROPHYLAXIS General methods of prevention Improvement of factory hygiene Proper sterilization of animal products Animal carcasses to be buried deep in quicklime or cremated

34. 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 at an interval of 6 weeks with annual booster injections . * Anthrax infection in humans give life long permanent immunity & secondary infections are very rare.

35. 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.

36. Biological warfare Anthrax was a long feared as a potential tool in 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 having enhanced virulence. * Hence the need to develop better human vaccine.

37. 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.

38. 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 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 Negative

39. 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. Ocular disease : It causes severe keratitis and panophthalmitis following trauma to the eye that may lead to loss of vision. Other conditions: Rarely cause systemic infections including – endocarditis, meningitis, osteomyelitis and pneumonia. The presence of medical device or intravenous drug use predispose to these infections.

40. 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.

41. 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.

42. 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

43. Types of Gastroenteritis Bacillus cereus Diarrheal Type Emetic Type Incubation period 8-16 hours 1-5hours Toxin Secreted in intestine ( Cl. perfringens enterotoxin) Performed toxin (formed in diet, similar to Staph. aureus enterotoxin) Heat Heat labile Heat stable Food items contaminated Meat, vegetables, dried beans, cereals Rice (Chinese fried rice) Clinical feature Diarrhoea, fever, abdominal cramps Vomiting , abdominal cramps Serotypes 2,6,8,9,10,12 1,3,5

44. DIAGNOSIS Suspected food, faeces & vomitus are cultured on ordinary media or a special such as – MYPA - Mannitol, Egg yolk, Phenol red, Polymyxin agar . PEMBA – Polymyxin B, Egg yolk, mannitol, bromothymol blue agar. 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.

45. TREATMENT Disease is mild and self limiting, requiring no specific treatment. Rehydration Antibiotics – in systemic infections Bacillus cereus susceptible to Clindamycin, erythromycin vancomycin, aminoglycosides and tetracycline. It is resistant to penicillin (by producing β -lactamase) and trimethoprim.

46. Bacillus thuringinesis It is closely related with B. cereus and may occasionally produce food poisoning. It is also used as Larvicidal agent for mosquito control.

47. Bacillus used as sterilization control B. stearothermophilus & B. subtilis both are used as biological controls for autoclave and plasma sterilization. B. pumilus is used as biological control for ethylene oxide.

48. Industrial use of Bacillus subtilis B. subtilis is used in industries for various purposes – As cleaning agent (Detergent). In paper and textile industries – produces amylase that breaks down starch. For pollution treatment- by breaking down pollutants (Bioremediation). In pesticide industries – for protecting crops against fungi. In food industries – helps in fermentation.

49. Case study Alisha, a 30 yr. old women, was admitted to hospital after a prolonged fever, with chills and night sweats, chest discomfort with blood stained sputum, hospitalized. The doctors performed a Gram’s stain for sputum specimen and found Gram positive rod shaped bacteria arranged in chains. Other tests results showed that bacterium was aerobic and spore bearing. 1. What is the clinical diagnosis and causative agent? 2. Describe the pathogenesis and various forms of clinical presentation of this infection? 3. Mention the laboratory investigation to confirm the diagnosis?

50. Short Notes Malignant pustule. B. cereus food poisoning. Wool sorter’s disease. Hide porter’s disease. Mc Fadyean’s Reaction.

51. MCQ Gram-stain morphology of Bacillus anthrax is : a. Tennis racket b. Drum stick appearance c. Bamboo stick appearance d. Spectacle glass appearance A “Malignant pustule” is a term for: a. An infected malignant melanoma b. A carbuncle c. A rapid spreading rodent ulcer d. Anthrax of the skin. Incubation period for B. cereus food poisoning following consumption of contaminated fried rice? a. 1-6 hrs. b. 8-16 hrs. c. 24 hrs. D. > 24 hrs.