General Characteristics of Bacillus

Transcription

1 Bacillus 1

2 General Characteristics of Bacillus ~ 60 species; Gram-positive or Gram-variable bacilli Large (0.5 x 1.2 to 2.5 x 10 um) Most are saprophytic contaminants or normal flora Bacillus anthracis is most important member Produce endospores Aerobic or facultatively anaerobic Catalase positive (most) Rapidly differentiates from Clostridium Bacillus spp. are ubiquitous Soil, water, and airborne dust Thermophilic (< 75 C) and psychrophilic (>5-8 C) Can flourish at extremes of acidity & alkalinity (ph 2 to 10) 2

3 Bacillus coagulans. Gram stain. Gram-positive or Gram-negative? The cell wall structure of endospore-forming bacteria is consistent with that of Gram-positive bacteria, and young cultures stain as expected. However, many sporeformers rapidly become Gramnegative when entering the stationary phase of growth. 3

4 Endospores Endospores were first described by Cohn in Bacillus subtilis and later by Koch in the pathogen, Bacillus anthracis. Cohn demonstrated the heat resistance of endospores in B. subtilis, and Koch described the developmental cycle of spore formation in B. anthracis. Endospores are so named because they are formed intracellularly, although they are eventually released from this mother cell or sporangium as free spores. Endospores have proven to be the most durable type of cell found in Nature, and in their cryptobiotic state of dormancy they can remain viable for extremely long periods of time, perhaps millions of years. When viewed unstained, endospores of living bacilli appear edged in black and are very bright and refractile. Endospores strongly resist application of simple stains or dyes and hence appear as nonstaining entities in Gram-stain preparations. However, once stained, endospores are quite resistant to decolorization. This is the basis of several spore stains such as the Schaeffer-Fulton staining method which also differentiates the spores from sporangia and vegetative cells. Endospores do not form normally during active growth and cell division. Rather, their differentiation begins when a population of vegetative cells passes out of the exponential phase of growth, usually as a result of nutrient depletion. Typically one endospore is formed per vegetative cell. The mature spore is liberated by lysis of the mother cell (sporangium) in which it was formed. 4

5 Below. Drawing of a cross-section of a Bacillus endospore : The spore protoplast (core) is surrounded by the core (cell) wall, the cortex, and then the spore coat. Depending on the species, an exosporium may be present. The core wall is composed of the same type of peptidoglycan as the vegetative cell wall. The cortex is composed of a unique peptidoglycan that bears three repeat subunits, always contains Diaminopimelic acid (DAP), and has very little cross-linking between tetrapeptide chains. The outer spore coat represents percent of the dry weight of the spore. The spore coat proteins have an unusually high content of cysteine and of hydrophobic amino acids, and are highly resistant to treatments that solubilize most proteins. 5

6 Left. Bacillus thuringiensis phase micrograph. Endospores can be readily recognized microscopically by their intracellular site of formation and their extreme refractility. Right. Bacillus anthracis Crystal violet stain viewed by light microscopy. Endospores are highly resistant to application of basic aniline dyes that readily stain vegetative cells. Below. Spore stain of a Bacillus species. The staining technique employed is the Schaeffer-Fulton method. A fixed smear is flooded with a solution of malachite green and placed over boiling water for 5 minutes. After rinsing, the smear is counterstained with safranine. Mature spores stain green, whether free or still in the vegetative sporangium; vegetative cells and sporangia stain red. 6

7 Important Bacillus species: Bacillus Bacillus Bacillus Bacillus Bacillus anthracis cereus stearothermophilus mycoides thuringiensis 7

8 Diseases Associated with Bacillus 8

9 Bacillus anthracis 9

10 Pathogenicity: virulence factors 10

11 Bacillus anthracis 11

12 Epidemiology of Anthrax in Animal and Human Hosts 12

13 Human Anthrax The disease may be 1. Cutaneous 2. Pulmonary 3. Intestinal * All types lead to fatal septicemia 13

14 1. Cutaneous Anthrax 95 % of human cases of anthrax Route of entry: Skin Sites involved face, neck, hands, arms & back 1 to 5 days after contact Papule Vesicles containing colorless or blood stained fluid Malignant Pustule Mali a l satellite lesions filled with serum or yellow fluid arranged around a central necrotic lesion which is covered by a black eschar Also known as Hi P i a Infection may spread to lymphatics w/ local adenopathy Resolves spontaneously, 10-20% of untreaed may develop fatal septicemia or meningitis 14

15 Pulmonary Inhalation Anthrax A life- threatening hemorrhagic pneumonia caused by Inhalation of spores Also called Wool Sorter s disease common in workers in wool factories Virtually 100% fatal (pneumonic) Meningitis may complicate cutaneous and inhalation forms of disease Pharyngeal anthrax Fever Pharyngitis Neck swelling 15

16 Gastrointestinal (Ingestion) Anthrax Rare By ingestion of inadequately cooked meat containing B. anthracis spores Virtually 100% fatal Abdominal pain Hemorrhagic ascites Paracentesis fluid may reveal gram-positive rods * Human anthrax can be Industrial in meat packing or wool factories Nonindustrial frequent association with animals like butchers, veterinarians, farmers 16

17 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 17

18 Laboratory Diagnosis 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/Schaeffer Fulton stain. 18

19 Laboratory Diagnosis Microscopic features Staining blood films with polychrome methylene blue: - Pink amorphous material around blue bacillus (M F y ): represents capsular material used for the presumptive diagnosis of anthrax in animals. Morphology in stained smears from cultures : bacilli arranged end to end in long chains. 19

20 FA stain of the capsule of Bacillus anthracis. Negative stain (India Ink outline) of the capsule of Bacillus anthracis. 20

21 Laboratory Diagnosis 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 H of matted hair M Gelatin stab culture appearance, with slow liquefaction starting from top. 21

22 Colonial growth of Bacillus anthracis. When lifted by an inoculation loop, colonies, in this case on sheep blood agar, show a tenacity that allows them to be pulled up and stay upright with a texture similar to egg whites beaten to a stiff peak. Encapsulation test for Bacillus anthracis. The difference in appearance of colonies on bicarbonate agar (smooth colonies on the left) and rough colonies (sheep blood agar on the right) is indicative of capsule 22

23 Bacillus colonies on BA 23

24 Laboratory Diagnosis Inverted fir tree Medusa Head Appearance wavy colonies with small projections 24

25 Cultural Characteristics S i P al a i solid medium containing 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. Serology to demonstrate anthrax Ag in tissue extracts 2. EIA (using purified anthrax toxin Ag) PCR used to detect anthrax contamination of animal & agricultural products 1. 25

26 Treatment & Prophylaxis Treatment Penicillin is drug of choice Erythromycin, chloramphenicol acceptable alternatives Doxycycline now commonly recognized as prophylactic Vaccine (controversial) Laboratory workers Employees of mills handling goat hair Active duty military members 26

27 Resistance Bacilli destroyed at 60 C in 30 mins. Animal carcasses bacilli remain viable in bone marrow BM for a wk & in skin for 2 wks. Spores highly resistant, survive in soil for 60 yrs Spores can be destroyed by 1. 4% KMnO4 in 15 mins 2. D using formaldehyde solution for animal products imported into non endemic countries For disinfection of wool 2% soln of formaldehyde at C for 20 mins Animal hair & bristles 0.25% at 60 C for 6 hrs 27

28 Prophylaxis General methods of prevention 1. Improvement of factory hygiene 2. Proper sterilisation of animal products 3. Animal carcasses to be buried deep in quicklime or cremated 28

29 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. 29

30 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. 30

31 Differences between Anthrax & Anthracoid bacilli Anthrax bacilli Nonmotile Capsulated Grow in long chains Medusa head colony No growth in Pn (penicillin) 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 31

32 Bacillus cereus 32

33 Bacillus cereus 33

34 Bacillus cereus clinical presentation DIARRHOEAL FORM Incubation period > 6 hours Diarrhoea Lasts hours Gastroenteritis EMETIC FORM Incubation period < 6 hours Severe vomiting Lasts 8-10 hours 34

35 Foodborne Diseases of B. cereus (Intoxication) (Foodborne Infection) 35

36 Diagnosis Primarily depends on clinical diagnosis & food sources Laboratory Diagnosis Specimen stool, vomitus, food, blood Microscopy not of much help Culture: Blood agar Special MYPA medium: Mannitol - egg yolk - phenol red polymyxin agar : to isolate B.cereus from feces & other sources. 4. Test for toxin to differentiate from staphylococcal food poisoning. Treatment Rehydration Antibiotics in systemic infections 36