GPB AEROBIC SPORE Bacillus etc Hari pankaj Vanam

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GPB Aerobic Sporebearing BACILLUS :

HARI PANKAJ VANAM ASSISTANT PROFESSOR DEPARTMENT OF MICROBIOLOGY GPB Aerobic Sporebearing BACILLUS

Classification of Bacillus:

Classification of Bacillus Class: Bacilli Order: Bacillales Family: Bacillacea Genus : Bacillus –Large genus over 90spp. GPB AEROBIC SPORE

General characteristics:

General characteristics Defining –Aerobic endospore forming New discoveries: Bacillus infernus and B. arseniciselenatis  strictly anaerobic Non sporing  B. halodenitrificans , B. infernus etc., Gram positive- get decolorized easily Motile- peritrichous flagella Non pathogenic bacillus termed as Anthracoides

Habitat:

Habitat Soils of all kinds, acid to alkaline, hot to cold, and fertile to desert Water columns and bottom deposits all waters spores resistant to Radiation Heat Disinfectants, and desiccation ASB!!! Troublesome genus Contaminats in OTs On surgical dressings In pharmaceutical products, and in foods.

Positive- clinical /health importance :

Positive- clinical /health importance Antibiotics produced: Bacitracin from B. licheniformis or B. subtilis Gramicidin from Brevibacillus brevis Polymyxin from Paenibacillus polymyxa Vitamins: Vitamins B 12 and B 2 from B. megaterium , Biotin and riboflavin from B. subtilis Synthesis of enzymes

Sterilization validation: :

Sterilization validation: In the validation of disinfectants ( B. cereus ) Monitoring of fumigation ( B. subtilis ) Heat sterilization ( G. stearothermophilus ) Radiation ( B. pumilus ) basis of antibiotic assays As probiotics in supplements Bacillus thuringiensis - biopesticide

Bacillus anthracis- Pollender bacilli The history maker:

Bacillus anthracis - Pollender bacilli The history maker Was discovered in 1877 by Robert Koch first pathogenic bacterium to be seen under microscope-Pollender,1849 first communicable disease to be transmitted by inoculation of infected blood-Davaine,1850 first bacterium to be isolated in pure culture and shown to possess spores-Koch,1876 first bacterium used for preparation of attenuated vaccine (Pasteur,1881)

Properties of B.anthracis:

Properties of B.anthracis Large 1 - 1.2µm x 3 - 5µm Facultatively anaerobic, Gram-positive, sporeforming rod- Spore Position: oval, refractile and central. Capsule –polypeptide in nature-polymer d- glutamic acid non-motile Spores readily formed only under aerobic conditions and are not found in tissues or circulating blood. But if an infected animal’s blood is spilled during an autopsy, bacilli exposed to air rapidly spores are formed.Handling animal carcasses The temperature range for growth is 12-45°C (optimum 35°C) 8

Mc’Fadyeans reaction:

Mc’Fadyeans reaction When Blood films containing anthrax bacilli stained with polychrome methylene blue amorphous purplish material seen around –capsular material used in the presumptive diagnosis of anthrax in animals. 9

Gram stains from culture::

Gram stains from culture: In cultures, bacilli arranged end to end in long chains Ends of bacilli –truncated or concave and swollen- bamboo stick appearance

Cultural characteristics:

Cultural characteristics Aerobe and facultative anaerobe Optimum temp-35-37 0 C Optimum temp – sporulation 25-30 0 C Growth occurs on ordinary media Virulent capsulated strains form rough cultures Avirulent strains form smooth colonies On agar plates-irregularly round colonies 2-3 mm diameter, raised dull opaque greyish white frosted glass appearance Blood agar media- non haemolytic colonies

Cultural characteristics contd.:

Cultural characteristics contd. Medusa head appearance Under low power ( 10X)- edge of colony –long interlacing chains of bacilli-resembling locks of matted hair - Medusa head appearance On gelatin stab culture -inverted fir tree appearance with slow liquefaction commencing from the top

:

In broth occurs as floccular deposit with no turbidity When Bacillus anthraxis grown on solid medium containing 0.05-0.50 u of penicillin/ ml,in 3-6 hrs cells become large spherical and occur in chains – string of pearls reaction String of pearls reaction Selective PLET media Polymyxin , lysozyme , EDTA, Thallous acetate added heart infusion agar- isolate Bacillus anthracis from mixtures of other spore bearing bacilli

Biochemical reactions:

Biochemical reactions Glucose,maltose,sucrose are fermented producing acid, no gas Nitrate reduced to nitrite Catalase is formed

Physical properties:

Physical properties Spores highly resistant -In dead animals, bacilli remain viable in bone marrow for 1 wk, skin for 2 wks. Bacilli killed at 60 0 C in 30 mins Spores 100 0 C X 10 minutes 4% KMno 4 kills in 15 minutes 4% Formaldehyde kills spores Hypochlorite ( 0.5%) commercially available kills spores endospores can remain viable over 60 -100 years

Duckering :

Duckering Destruction of spores in animal products when imported in to non endemic countries Formaldehyde 2%, 30-40 0 C,20 min  wool 0.25%, 60 0 C , 6 hrs  hairs, bristles

Epidemiology:

Epidemiology Anthrax is a disease of great antiquity-Virgil’s Georgics (36–29 BC). Derived from-black eschar Greek word for coal. Primarily Zoonotic -All animals are susceptible to anthrax An outbreak in Iraq in 1945 killed 1 million sheep. Direct human-to-human transmission is exceptionally rare.

Epidemiology: India:

Epidemiology: India Carnivores relatively resistant Herbivores highly susceptible- Sudden death in any herbivore should be treated with suspicion!!! Omnivores intermediate level of resistance- I ndia Largest live stock population in the world Incidence is not accurately known-Sporadic Pondicherry ( JIPMER) - 30 human cases reported ( Mostly Cutaneous , Septicemic or Meningeal ) Vellore ( CMC)- 49 human cases Chittor ( Rajasthan)- 30 human cases Tirupati ( Andhra )- 25 human cases Midnapur ( WB)- 22 human cases

Pathogenesis:

Pathogenesis Anthrax - zoonosis - a disease of animals transmissible secondarily to man. Human beings relatively resistant to infection Men suffer from anthrax occasionally due to close contact with infected animal or animal products Transmission: Inhalation,Ingestion , enter body by abrasions

VIRULENCE FACTORS:

VIRULENCE FACTORS Two imp virulent factors!!! I-Anthrax Toxin – Tripartite toxin combine to form  two binary toxins, Complex of proteins ( all the components thermolabile ) A. Protective antigen PA B. Edema factor EF C. Lethal Factor LF II-Protein capsule – Poly D Glutamic acid capsule – Inhibits phagocytosis ( Unencapsulated strains – nonpathogenic)

Bacillus anthracis -Anthrax toxin:

Bacillus anthracis - Anthrax toxin Protective antigen : Binds plasma membrane of target cells Cleaved to 2 fragments ( cellular trypsin or proteases) Larger fragment is attached to cell surface – binding domain for LF & EF Specific receptor mediated endocytosis of LF & EF

PowerPoint Presentation:

EDEMA FACTOR Calmodulin dependent adenyl cyclase Increased cellular Camp Edema Impaired Neutrophil function Depletes ATP from Macrophages LETHAL FACTOR Zinc metallo proteases that inactivates protein kinases Stimulates Macrophages – TNF alpha and IL – 1 beta – Shock & Death Sudden and unexpected Death . due to oxygen depletion, secondary shock, increased vascular permeability, respiratory failure and cardiac failure.

Virulence of Anthrax bacillus is due to presence of two plasmids :

Virulence of Anthrax bacillus is due to presence of two plasmids px01 – Toxin encoding plasmid-temperature-sensitive plasmid px02 - Capsule encoding plasmid synthesis of poly glutamic acid capsule Both plasmids are required for virulence - loss of either  attenuation - genes expressed only in vegetative state Pasteur strain - Encapsulated Sterne strain – Non encapsulated

PowerPoint Presentation:

Pathogenesis Endospores infectious forms (Abrasion, inhalation, ingestion) Death Introduced Septicemia Phagocytosed by Macrophages 10 7 to 10 8 /ml Regional LNs Blood stream Multiply in Lymphatics Germinate inside Macrophages Release Vegetative Forms

Clinically three forms of Human anthrax occur :

Clinically three forms of Human anthrax occur Cutaneous anthrax Pulmonary anthrax Intestinal anthrax Broadly can be classified into Non Industrial/Agricultural ( Through infected animals): Cutaneous anthrax Rarely intestinal anthrax Industrial Anthrax ( Through animal products): Mostly through animal products( wools, hair, hides, bones) Likely to develop Cutaneous and pulmonary anthrax ( inhalation)

I-CUTANEOUS ANTHRAX-Common type:

I-CUTANEOUS ANTHRAX-Common type OCCUPATIONAL- Veterinarian, butcher, Zoo keeper Spores infect skin (FACE,NECK,HANDS,BACK )- gelatinous edema develops at the site  Papule  Vesicle -Primary lesion  Malignant Pustule- Necrotic ulcer-Black Escher) 80-90% heal spontaneously ( 2-6wks) 0-20% progressive disease – develop septicemia 95-99% of all human anthrax occur as cutaneous anthrax Coagulation necrosis of the centre of the pustule results in the formation of a dark-coloured eschar

II. Pulmonary/inhalation Anthrax:

II. Pulmonary/inhalation Anthrax Require very high infective dose ( > 10,000 spores) Stable at lyophilized powder form. Acquired through inhalation of spores ( Bioterrorism BW Agent class I - aerosol) Present with symptoms of severe respiratory infection( High fever & Chest pain) sometimes incorrectly referred to as pneumonic anthrax, the disease does not develop as a bronchopneumonia Haemorrhagic mediastinitis Progress to septicemia very rapidly Mortality rate is very high > 95%

PowerPoint Presentation:

Hide porters disease: Cutaneous anthrax The disease used to be common in dock workers carrying loads of hides and skin on their bare backs Wool sorter , s disease cases of inhalational anthrax persons working in industries handling animal skins, hides and wool

III. Intestinal anthrax:

III. Intestinal anthrax Occurs among pastoralists  eat dead infected animals. Extremely high mortality rate . Meningitis from any of the forms of anthrax Outbreaks of primary anthrax meningoencephalitis have been reported from India and elsewhere

LABORATORY DIAGNOSIS :

LABORATORY DIAGNOSIS IMP POINTS Anthrax is not highly contagious. Cutaneous anthrax is not lethal and is readily treated with common antibiotics. ID for human pulmonary / intestinal infection is > 10,000 spores Specimen: Fluid aspirated from the surrounding vesicles, more likely to yield anthrax bacilli. Specimens taken before antibiotic therapy Clinical diagnosis easy -characteristic appearance and occupational exposure

SPECIMEN TO COLLECT ( HUMAN ANTHRAX) :

SPECIMEN TO COLLECT ( HUMAN ANTHRAX) Universal precautions : Disposable gloves, masks, overalls, boots, head gear and dust mask,Disposable items – Autoclave and incinerate Cutaneous anthrax : Vesicular exudate – swabs and capillary tube aspirate Intestinal anthrax: - Stool sample - isolate – guinea pig inoculation - Blood( venipuncture ) smear examination for bacilli - Peritoneal fluid for culture - Paired sera for Ab Pulmonary anthrax: If mild disease ( No sample) Severely ill – Blood , sputum, serum samples for Ab

MICROSCOPY & CULTURE:

MICROSCOPY & CULTURE Gram's stain may show typical large Gram-positive bacill . Limited dx value Indian ink- capsule appears as clear halo Should have acumen to differentiate spores from inclusion granules. Properties: Blood agar-Medusa head colonies-Non heamolytic non-motility, gelatin liquefaction- inverted fir tree Best isolation: Polymyxin lysozyme EDTA- thallous acetate (PLET) agar

Important Bacillus identification:

Important Bacillus identification ( i ) on blood agar plates to test for γ phage and penicillin susceptibility (ii) directly or subsequently in blood to look for capsule production using M’Fadyean’s stain or by indirect fluorescent antibody capsule staining.

PowerPoint Presentation:

Serological assay ELISA: based on anthrax toxin ( PA, LF and EF) for routine confirmation and vaccine response) Molecular techniques ( Only in the referral laboratories): - RFLP - PCR Fingerprinting Animal Inoculation : Guinea pig and mice inoculation

Culture is confirmed by gamma phage lysis ( PlyG lysin enzyme- g phage) :

Culture is confirmed by gamma phage lysis ( PlyG lysin enzyme- g phage) confirmation

Treatment :

Treatment Antibiotics should be given to unvaccinated individuals exposed to inhalation anthrax. Penicillin, tetracyclines and fluoroquinolones are effective if administered before the onset of lymphatic spread or septicemia Corticosteroides once dissemination with toxin. Antibiotic treatment is effective in cutaneous anthrax at least 60days-reason spores can be generated to vegitative forms!!! INITIAL THERAPY OPTIMAL THERAPY Adults Ciproflox Penicillin G 4 mu iv qdsX60days ( 400mg iv BDX60days) Doxycycline 100mg iv BDX60 days Children Ciproflox 20-30mg/ kgbodywt ivX60days Penicllin G 50,000 u/kg X 60 days Alternatives – Amox , Tetracycline, Chloramphenicol , Erythromycin, Streptomycin

Prevention: Vaccine against Anthrax :

Prevention: Vaccine against Anthrax Killed bacilli and/or capsular antigens produce no significant immunity. A nonencapsulated toxigenic strain (Sterne Strain) has been used effectively in livestock.1930 Vaccine for humans: preparation of the protective antigen (PA) V770-NP1-R & BioThrax IM ( avirulent and non encapsulated) sublethal amounts of the toxin produced Who are to be vaccinated? Professionals ( Veternarians , butcher, Zoo keeper, Wild life workers, Forest guards) Military personnels

Anthrax bioterrorism:

Anthrax bioterrorism The possibility of creating aerosols containing anthrax spores has made B. anthracis a chosen weapon of bioterrorism. Iraq, Russia and as many as ten nations have the capability to load spores of B. anthracis into weapons. Accidental outbreak: accident at Soviet Union biological weapons factory in the 1980s  release of spores into the air. In the town of Sverdlosk 79 cases of human inhalation recorded - 6 weeks after exposure 68 deaths. 39

Spores in powder form:

Spores in powder form Usually drug resistant strains

ANTHRACOID BACILLI:

ANTHRACOID BACILLI Aerobic spore bearing bacilli similar- B.anthracis-ASB Some are frequent lab contaminants B.subtilis  opportunistic pathogen Eye infections Septicemia

Bacillus cereus:

Bacillus cereus Large Gram-positive bacillus broad cells with ellipsoidal subterminal spores, Resembles B. Anthracis Motile and lacks the glutamic acid capsule Saprophyte Soil water vegetation Blood smears do not show the characteristic pink capsule with McFadyean's stain

Bacillus cereus:

Bacillus cereus Cause of food poisoning Seen in Soil Vegetables Milk Cereals Spices meat poultry Dried tea !! 43

Pathogenesis- Food poisoning :

Pathogenesis- Food poisoning A: Diarrheal syndrome Associated with meat, poultry, and soups occurring 8-24 h after ingestion  2,6,8,9,10,12h similar to enteritis caused by Escherichia coli or Salmonella enterica serotype & also similar to those of C. perfringens food poisoning. are heat labile enterotoxin LT formed in the intestine. abdominal cramps, profuse watery diarrhea and rectal tenesmus . Occasionally there is fever and vomiting. Resolves within 24 hours

B. Emetic form :

B. Emetic form Associated with fried rice vomiting, occurring within 6 h of ingestion- rice-1,3,5h It is caused by preformed toxin- low molecular weight, heat- and acid-stable peptide ST withstand intestinal proteolytic enzymes. Abdominal cramps and vomiting Resolves in 9 hours

Ocular pathogen & Infections in compromised pts.:

Ocular pathogen & Infections in compromised pts. Ocular pathogen: Panophthalmitis following penetrating trauma of the eye (commonly by a metal fragment in an environment such as a farm or a garage), and intraocular surgery. Immunocompromised Hosts: Meningitis, septicemia, and osteomyelitis Found as contaminants in drug paraphernalia

LABORATORY DIAGNOSIS:

LABORATORY DIAGNOSIS suspected food available for testing. 10 8 organisms/g -make the diagnosis in the absence of other food-poisoning bacteria . Anthracoid colonies on blood agar - B. cereus . Food reference laboratories are able to confirm identification and type if necessary. PLET no growth + ve egg-yolk reaction acid-from- mannitol Negative pyruvate and Negative polymyxin

Treatment :

Treatment Emetic and diarrhoeal syndromes  short lived No specific treatment is needed. Acute symptoms last less than 24 h and recovery on a reduced diet and fluids is rapid. Antibiotic regimens for anthrax would be equally effective for the treatment of disease caused by atypical strains of B. cereus strains.

CONTROL:

CONTROL Food poisoning caused by B. cereus is easily prevented by proper cooling and storage of food. Ideally, all dishes should be freshly prepared and eaten. DONOT REHEAT RICE!!! Rice, in particular, should not be stored for long periods above 10°C.

Special thanks to “The Web world for enormous resources” and corresponding authors of every aspect of literature”…its sure a propriety of u all!!!..Thanks for inspiring me to teach!!!:

Special thanks to “The Web world for enormous resources” and corresponding authors of every aspect of literature”…its sure a propriety of u all!!!..Thanks for inspiring me to teach!!! Thank you vanam.haripankaj@facebook.com

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