BACILLUS General Features • • • • Obligate Aerobes Gram-positive spore forming bacilli Non-bulging spores Generally motile with peritrichous flagella & noncapsulated (Except B.anthracis) • Pathogenic species - B. anthracis and B. cereus BACILLUS ANTHRACIS History • 1849 - Pollender - First pathogenic bacterium seen under microscope • A nthrax was the first communicable disease shown to be transmitted by inoculation of infected blood • 1876 -Robert Koch - First bacterium to be isolated in pure culture • Koch’s postulates were based on B. anthracis • 1881 - Louis Pasteur - Anthrax vaccine was the first live attenuated bacterial vaccine prepared Virulence Factors and Pathogenesis • Anthrax Toxin 1. Edema factor - Active fragment - Acts as adenylyl cyclase increases host cell cAMP 2. Protective factor - Binding fragment. Binds to the host cell receptors and facilitates the entry of other fragments into the host cells. 3. Lethal factor - Causes cell death - Acts by cleaving host cell MAPK (mitogen-activated protein kinases). Virulence Factors and Pathogenesis • Toxin 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 (Basis of original anthrax vaccine prepared by Pasteur) • Anthrax Capsule - Polypeptide capsule (polyglutamate) - C apsule is plasmid (pX02) coded - Inhibits complement mediated phagocytosis Clinical Manifestations • Transmission - Cutaneous mode—spores entering through the abraded skin - Inhalation of spores - Ingestion of carcasses of animals dying of anthrax containing • Clinical Types 1. Cutaneous anthrax 2. Pulmonary anthrax 3. Intestinal anthrax – rare, occurs due to ingestion of spores Cutaneous v/s Pulmonary Anthrax Cutaneous anthrax Pulmonary anthrax Transmission Cutaneous exposure to spores Inhalation of spores Clinical Features Malignant pustule • Begins as a papule painless vesicle coal-black, necrotic eschar surrounded by non-pitting indurated edema Prognosis Self-limiting Hemorrhagic pneumoniaBacilli spread by lymphatics or blood  • Bacteremia • Hemorrhagic mediastinitis • Hemorrhagic meningitis Fatal Bioterrorism Rarely causes bioterrorism MC form for bioterrorism Also called as Hide porter’s disease Wool sorter’s disease Malignant Pustule Animal Anthrax • Anthrax is primarily a zoonotic disease • Animals affected – Herbivores -cattle, sheep and less often horses & pigs • Acquired by ingestion of spores present in soil. Direct spread from animal to animal is rare • Presentation - fatal septicemia • Infective materials - Discharges from mouth, nose & rectum. Bacilli sporulate in soil Epidemiology • Animal anthrax: Progressive global reduction in livestock anthrax due to effective preventive measures - Enzootic (endemic) & Epizootic (epidemic) forms - Prevalent in Andhra-Tamil Nadu border, foci in Karnataka & West Bengal • Human anthrax • Incidence highest in Africa, central & southern Asia. 1. Non-industrial cases – Agricultural exposure to animals 2. Industrial cases - Infected animal products such as hides, hair, bristles and wools. Laboratory Diagnosis • High risk of laboratory acquired infection • Specimen Collection - Pus or swab from malignant pustule - Sputum in pulmonary anthrax - Blood (in septicemia) - CSF (in hemorrhagic meningitis) - Gastric aspirate, feces or food (in intestinal anthrax) - Ear lobes from dead animals. Laboratory Diagnosis…Specimen Microscopy • Gram staining - Gram-positive, large rectangular rods - Spores are usually not seen in clinical samples Laboratory Diagnosis…Specimen Microscopy • McFadyean’s reaction - Gurr’s polychrome methylene blue Capsule appears as amorphous purple material surrounding blue bacilli Laboratory Diagnosis…Specimen Microscopy - Direct immunofluorescence test (direct-IF) - Capsular and cell wall polypeptide antigens detected - Useful during bioterrorism outbreaks • Ascoli’s thermoprecipitation test - It is a ring precipitation - Done when sample is received in putrid form & bacilli are non-viable CULTURE • Aerobic, non-fastidious • Sporulation -25–30°C, distilled water, 2% NaCl, oxalate and oxygen • Nutrient agar – Frosted glass - Medusa Head Appearance CULTURE • Blood agar - dry wrinkled, non-hemolytic • Gelatin stab agar - Inverted fir tree appearance • Selective media: - Solid medium with penicillin - string of pearl appearance - PLET medium (Polymyxin, lysozyme, EDTA and thallous acetate in heart infusion agar) Culture Smear • Gram-staining - Bamboo stick appearance • Spores: - Hot malachite green (Ashby’s method) - 0.25% sulfuric acid (spores are acid fast) • Lipid granules - Sudan black B (Burdon’s method). Molecular Diagnosis • PCR with specific primers - BA pX01 (encoding protective Ag) - BA pX02 (encoding capsular polysaccharide) • Molecular typing - useful for epidemiological studies - MLVA (Multiple locus variable number of tandem repeat analysis) - AFLP (Amplified fragment length polymorphisms). TREATMENT • Antibiotic regimen for treatment - Ciprofloxacin/doxycycline + clindamycin, and/or rifampin- 60 days • Antibiotics for postexposure prophylaxis - Ciprofloxacin for 60 days + Doxycycline for 60 days or Amoxicillin for 60 days (given if strain is penicillin sensitive). Raxibacumab - Monoclonal antibody that neutralizes anthrax • toxin (protective antigen) - For prophylaxis & treatment of inhalational anthrax Prevention • General control measures - Disposal of animal carcasses by burning or by deep burial in lime pits - Decontamination (usually by autoclaving) of animal products - Protective clothing and gloves for handling potentially infectious materials. Prevention - Immunoprophylaxis • Live Attenuated, Non-capsulated Spore Vaccine (Stern Vaccine) - For animals. Protective for 1 year - Not safe for human use. • Adsorbed (Alum Precipitated) Toxoid Vaccine - Prepared from the protective antigen - Safe & effective for human use - Indicated for pre exposure and post exposure prophylaxis Anthrax Vaccines Indication Route Dosing Schedule Pre-exposure prophylaxis for persons at high risk of exposure Intramuscular (0.5 mL/dose) Primary series: 0,1, and 6 months Boosters: at 6 and 12 month after primary series and then yearly Post-exposure prophylaxis following exposure to suspected or confirmed case Subcutaneous (0.5 mL/dose) 0, 2, and 4 weeks postexposure combined with antimicrobial therapy Anthrax bacilli v/s Anthracoid bacilli Anthrax bacilli Anthracoid bacilli Motility Non motile Motile Capsule Present Absent Bacilli In long chain In short chain Under low power microscope Medusa head colony seen Not seen Blood agar No hemolysis Hemolytic colony Broth Turbidity absent Usually turbid Anthrax bacilli v/s Anthracoid bacilli Anthrax bacilli Salicin Not fermented Gamma phage Susceptible Gelatin stab Inverted fir tree appearance agar seen. Gelatin liquefaction slow Solid medium with String of pearls appearance penicillin At 450C No growth Virulence Pathogenic Anthracoid bacilli Fermented Resistant Not seen Rapid gelatin liquefaction No growth Usually grows Mostly non-pathogenic BACILLUS CEREUS Bacillus cereus • Normal habitant of soil • Widely isolated from vegetables, milk, cereals, spices, meat & poultry • Food poisoning • Diarrheal toxin (causes diarrheal type of food poisoning) • Emetic toxin (causes emetic type of food poisoning) • Ocular disease - Severe keratitis & panophthalmitis following trauma to the eye Bacillus cereus Food Poisoning B.cereus Incubation period Toxin Food items contaminated Clinical feature Diarrheal type 8-16 hours Secreted in intestine (Similar to Clostridium perfringens enterotoxin) Heat labile Meat, vegetables, dried beans, cereals Diarrhea, fever , rarely nausea Serotype involved 2,6,8,9,10,12 Emetic type 1-5 hours Preformed toxin (formed in diet, similar to S.aureus enterotoxin) Heat stable Rice (Chinese fried rice) Vomiting, abdominal cramps 1,3,5 Laboratory Diagnosis & Treatment • • - Sample – feces Culture isolation MYPA (mannitol, egg yolk, polymyxin, phenol red and agar) PEMBA (polymyxin B, egg yolk, mannitol, bromothymol blue, agar) • M otile, non-capsulated & not susceptible to gamma phage • Treatment of Bacillus cereus - Susceptible to clindamycin, erythromycin, vancomycin, aminoglycosides and tetracycline - Resistant to penicillin (by producing β-lactamase) and trimethoprim Bacillus thuringiensis - Closely related to B. cereus - Occasionally produce food poisoning - Used as larvicidal agent for mosquito contro Bacillus spores as Biological controls • Geobacillius stearothermophilus - Autoclave, H2O2 gas plasma sterilization & liquid acetic acid sterilizer • Bacillus atropheus - Ethylene oxide sterilizer and dry heat sterilization