CHAPTER 16Non-branching, Catalase +, Gram + BacilliB. anthracisB. subtilisB. cereusB. megateriumB. mycoidesOthers:B. circulansBrevibacillus brevisB. licheniformisPaenibacillus spp.

GENERAL CHARACTERISTICS:Bacillus previously phenotypically classified Catalase + Recognized based on 16rRna sequence analysis 53 genera Largest genus within this group Most clinically medically relevant organismsBacillusGram +, aerobicBrevibacillusfacultative anaerobePaenibacillusspore-forming rods

Bacillus anthracis bioterrorist event large Gram + aerobic rods typical nonhemolytic medusa head or ground glass colonies observe in 5% SBA Non-motile

Epidemiology Anthrax disease of wild and domestic animals (sheep, goat, horse, cattle) A disease that affect warm blooded animals Normally found in soil and can cause disease to Herbivores. Humans gets infected when inoculated with spores, traumatic introduction, inhalation, ingestion Hides Woolsorters diseaseENDOSPORE highly resistant to heat and desiccationSpores - remain viable in dormant state until favourable for growth are again encountered. Agent for biologic warfareNice to know

Process of spore: (6-28 hrs.)Vegetative cell actively sporangium SPORE

Endospore for survival; hard and thick; destroyed by incineration; high conc. of Calcium

Pathogenesis Most highly virulent spp. Causative agent of Anthrax 3 forms of disease:(1) Cutaneous form contact with infected animal products. Infection via close contact and inoculation of endospores through a break in the skin.

2-6 days incubation periodSmall papule progress to a ring of vesicles then develop to ulceration

Eschar black necrotic lesion* low mortality rate

(2) Ingestion anthrax (GIT) ingestion of spores via:Oral or oropharyngeal lesion in the buccal cavity, tongue, tonsils or pharyngeal mucosa

Symptoms: sore throat, lymphadenopathy, edema of throat and chest

GIT anthrax lesions developing in the GIT.Symptoms: abdominal pain, bloody diarrhea, hematemesis*high mortality rate

(3) Pulmonary (inhalation) anthrax aka Woolsorters disease- Due to inhalation of spores- Ingested by macrophage taken to the lymph nodes

Symptoms:Develops flu-like symptomsedema, cyanosis, shock, death, abnormal chest x-ray with pleural effusion

Woolsorters disease and Ragpickers disease respiratory infection result from exposure to endospore during the handling of animal hides, hair or fibers and other animal products.Nice to know

Patients often develop Meningitis within 6 days.Recovery results in long-term immunity to subsequent infections.

Virulence:Anthrax toxin consist of 3 protein Protective antigen (PA) facilitates the transport of other 2 protein into the cell Edema factor (EF) responsible for edema Lethal factor (LF) primarily responsible to death.

Bacillus cereus Clinically relevant spp. worthy of identification Penicillin resistant Beta-hemolytic, motile Produce wide zone of lecithinase on Egg yolk agar

Epidemiology Very closely relative to B. anthracis, is also found within the soil. Opportunistic pathogen Foodborne illness

Pathogenesis food poisoning Ingestion of wide variety of foods such as meats, vegetables, deserts, sauces, and milk. fried rice bacteria Higher incidence seen by ingestion of rice dishes Serious pathogen of the eye causing endophthalmitis Symptoms:Diarrhea and abdominal pain (8-16hrs.)Nausea and vomiting (1-5 hrs.)Nice to know

Several toxin (Diarrheal symptoms) including: Hemolysin BL (Hbl) nonhemolytic enterotoxin (Nhe) cytotoxin K (CytK)

* these 3 toxin act synergistically, with Nhe responsible for the major symptoms in the diarrheal presentation of the infection.

*Emetic form of illness is associated with:Heat-stableProteolysisAcid resistant toxinCereulide

which are produced in food

Bacillus thuringiensis harboring the genes of B. cereus-associated enterotoxins. Occupational exposure to insecticides and pesticides Rare cases: wound, burn, pulmonary and ocular infectionsNice to know

It can be ingested by insectsIndustrial bacteria

Bacillus subtilis Identified in clinical specimen which causes:Pneumonia, bacteremia, septicaemia, surgical wounds, meningitis following head trauma and other surgical infections. Epidemiology Most other Bacillus spp. opportunistic pathogens of low virulence, Immunocompromised patients following exposure to contaminated materials

Pathogenesis Spore of bacillus ubiquitous in natureNice to know

B. coagulans main cause of tomato juice spoilageB. subtilis source of BacitracinP. polymyxa produces polymyxin

*Polymixin B and E produce colistin that is toxic to kidney

SPECIMEN PROCESSINGLeak-proof containers specimen collectedSecondary container with B. anthracis

Cutaneous anthrax Collected from underneath the eschar 2 specimens from vesicular fluid collected from underneath the lesion with a swab Punch of biopsy for histochemical testingInhalation anthrax Specimens are: Blood cultures, pleural fluid, and serum specimen (serology) Biopsy of bronchial or pleural tissueAdd notes:Plating solid media may include alcohol or shockFormaldehyde disinfectant used Glutaraldehydebefore disposingHydrogen peroxidespecimen with sporesPeraacetic acid

GRAM STAINBacillus spp. Large gram + rods Singles or pairs or serpentine changes Aerobic organisms Produce spores in the presence of Oxygen Sporulation is inhibited by high conc. of CO2**** Others please refer to the book (Under Direct detection methods)

CULTIVATION

ALL Bacillus Grow well on 5% SBA Chocolate agar Routine blood culture media Nutrient brothsIsolates susceptible to nalidixic acid will not grow on CNA

PEA is useful for removing contaminating organism and isolation of Bacillus spp.

Polymyxin-lysozyme-EDTA-thallous-acetate (PLET) can be used for selection and isolation from contaminated specimen.

Colonies appear: creamy white, domed, circular colonies

Bicarbonate agar induce B. anthracis capsule appear large and mucoidB. cereusFor specific isolation and identification of organism: mannitol, egg yolk, polymyxin B agar (MEYP) polymyxin B, egg yolk, mannitol, Bromthymol blue (PEMBA) B. cereus medium (BCM)** Phospholipase C positive rxn on Egg yolk agar**no production of acid from Mannitol**incorporation of pyruvate or polymixin as selective agents

Heat shock treatment utilize the growth and enhancement of endosporeKill vegetative cells and retaining spores for most Bacillus spp.70C for 30mins / 80C for 10mins

B. anthracis heat treatment62C to 65C for 15-20mins.

OrganismLecithinaseVPMotilePenicillin SensitivityParasporal Crystals

B. anthracis(+)(-)(+)(-)

B. cereus(+)(+)(-)(-)

B. thuringiensis(+)(+)(-)(+)

B. mycoides(+)(-)(-)(-)

For most speciesDetectable growth: within 24 hrs.Incubation: 35C in ambient air or in 5% CO2Bicarbonate agar requires incubation in CO2Add notes:B. anthracisB. cereus

Hemolysis-+

String of pearl+-

Gelatin hydrolysis-+

Growth in PEA-+

Serodiagnostic methods are not used to diagnose infections caused by other opportunistic Bacillus spp.Serodiagnosis of B. anthracis is typically available for the detection of Anthrax toxin Anaerobic black granules of wound with pungent odorPrevention:Cell-free inactivated vaccineChemoprophylaxis with ciprofloxacin

basa, rhoann m. / microbiology3