BACILLUS

Sporogenous Bacilli

Aerobic Bacilli Anaerobic Clostridia

1.Bacillus anthracis 1. Cl.perfringens 2.Bacillus cereus 2. Cl. tetani 3. Anthracoid bacilli 3. Cl.botulinum

Bacillus anthracis

-First pathogenic bacteria to be observed under microscope-First bacilli to be isolated in pure culture-First bacilli in which spores were demonstrated -First bacterium used for preparation of attenuated vaccine-First bacterium to be shown cause of disease-First bacterium that evolved as a potent weapon in bioterrorism

OVERVIEWMorphologyCultural characteristicsBiochemical reactionsResistancePathogenicityLab diagnosisEpidemiologyProphylaxisTreatment

BIOTERRORISM

MORPHOLOGY-One of the largest pathogenic bactera;4-8x1-1.5 mm in size-Gram +ve, rod shaped, non-motile and non acid-fast-Bamboo stick appearance of long chains-Polypeptidal capsule-Central or sub terminal oval spores

CULTURAL CHARECTERISTICS-Aerobic ,Facultative anaerobic-On NA : Colonies are round, grayish white,irregular,raised with “frosted glass” appearance,2-3 mm in diameter.Medusa head /Barrister's wig appearance of colonies under low magnification-On BA: Non-hemolytic colonies-Gelatin stab culture: “Inverted fir tree" appearance-PLET medium: for selective isolation

BIOCHEMICAL REACTIONS-Glucose, Maltose, Sucrose fermented with ACID production -Nitrate reduction test-Gelatin liquefaction test All positive-Catalase test

RESISTANCE-Spores survive for many years(dry state & soil)

-Moist heat kills : -vegetative cells 60 0C x 30 min. -spores 1000C x 10 min. -4% FD & KMnO4 kills sporesDuckering: 2% formaldehyde at 30-40 0C for 20 min. disinfects wool & 0.25% at 60 0C for 6 hrs. for animal hair & bristles.

PATHOGENICITYVirulence factors:1.Capsular polypeptide – inhibits phagocytosis2.Anthrax toxin –a complex of 3 fractions:-Edema factor(OF or Factor I) whole complex

-Protective antigen factor(PA or Factor II) produces local edema

-Lethal factor(LF or Factor III) & generalized shock

Edema factor + protective antigen = Edema toxin Lethal factor + protective antigen = Lethal toxin

Clinically three forms of Human anthrax occur1.Cutaneous anthrax2.Pulmonary anthrax3.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

1.Cutaneous Anthrax(95-99% human anthrax)

•Mainly in professionals( Veterinarian, butcher, Zoo keepers, persons handling carcasses/hides/hair, loading skin on bare backs-hide porters disease .

• Spores infect skin- a characteristic gelatinous edema & congestion develops at the site (Papule- pustule-black ulcer(eschar) .Later a ring of vesicle containing serous fluid surrounds eschar termed as Malignant pustule.

•Face ,neck,arms and back are common sites

• 80-90% heal spontaneously ( 2-6wks)

• 10-20% progressive disease – develop fatal septicemia, death

Different locations & morphology of lesion in cutaneous anthrax

2.Pulmonar Anthrax- wool sorter's/Ragpicker’s disease:

-Acquired with inhalation of spores(bioterrorism-aerosol)-Requires very high infective dose(10,000 t0 20,000 spores)-Inflammatory reaction occurs in trachea, bronchi with hemorrhagic bronchospasm-Presents initially with non -specific symptoms but later with symptoms of severe respiratory infection & severe respiratory collapse-Hemorrhagic meningitis sometimes occurs as complication-Progress to septicemia very rapidly-Mortality rate is very high

3.Intestinal Anthrax

-Rare in man and is found in primitive communities eating dead carcasses of infected animal-Presents with serious severe enteritis, bloody or hemorrhagic diarrhoea,bloody vomit -Fatality rate is 25-60% depending upon treatment

EPIDEMIOLOGY(INDIA)

-Anthrax is enzootic in India

-An epizootic of anthrax in sheep active AP,TN borders-Largest live stock population in the world

-Pondicherry ( JIPMER) - 30 human cases reported ( Mostly Cutaneous, Septicemic or Meningeal)

-Vellore ( CMC)- 49 human cases

-Chittor ( Rajasthan)- 30 human cases

-Tirupati ( Andhrapradesh)- 25 human cases

-Midnapur ( WB)- 22 human cases

LABORATORY DIAGNOSISA. Hematological investigation-not significantB.Bacteriological investigation:Specimen- swabs, pus, pustules, blood ,sputum, feces etc.1.Microscopy:Gram staining & spore staining Characteristic Mc’ Fadyean’s reaction Immunofluorescent microscopy

2.Culture: Suitable culture media for selective isolation3.Animal inoculation : done in guinea pigs, mice & rabbits. 3.Serology(Ascoli’s thermoprecipitation test)4.Molecular methods : PCR with specific primers5.Molecular typing: MLVA & AFLL for epidemiological studies

PROPHYLAXISHumans protected by preventing disease in animals

-Veterinary supervision-Trade restrictions-Proper sterilization & disinfection of animal

products-Deep burial or cremation of carcasses Improved industry standards

Safety practices in laboratories Post-exposure antibiotic prophylaxis

VACCINATION:-Active immunization with Pasteur’s anthrax vaccine-Salvo immune serum in serious toxic cases-Cell free vaccine in high risk groups-Inactivated /killed vaccine for veterinarians & Agri-workers-Sterne vaccine used effectively in livestock's-Alum precipitated toxoid found safe in professionals 3 doses given intramuscularly

TREATMENTEffective antibiotics in humans:Sulphonamides,ciprofloxacin,penicillin,erythromycin,Vancomycin,doxycyline,chloramphenicolFDA -approved ciprofloxacin,doxycycline and penicillin

BIOTERRORISM-anthrax as a bioweapon

-Anthrax was used by Scandinavian rebels against Russians -Operation vegetarian by Royal Air Force against Germany in 1944 ,an anti-livestock operation-In 1997-accidental release of anthrax spores from biological weapons complex in Russia infected 94 people ,68 died-In Oct.2001 anthrax attacks in USA termed Amerithrax(FBI) 22 cases- 11 inhalation(5 deaths),11 cutaneous(no deaths)

Bacillus cereus-Important cause of food poisoning-Distributed widely in nature(soil, vegetables & foods)-Non-Capsulated but Motile (few non-motile strains )-Two patterns of food borne disease are produced:1.Diarrhoeal type: -caused by serotypes 2,6,8,9,10 & 12 -associated with wide range of foods -characterized by diarrhea & abdominal pain2.Emetic type(fried rice syndrome) caused by STs 1,3 & 5 - associated with consumption of cooked rice - characterized by acute nausea & vomitingFor isolation MYPA medium is used10,00000 bacilli/gram of stool is significant

ANTHRACOIS /PSEUDOANTHRAX-Saprophytic ,spore-forming, non-pathogenic species-They are most common laboratory contaminants (e.g. B.subtilis contaminating blood transfusion bottles-They are opportunistic & may cause septicemia