Bacillus Anthrax & Clostridium

Bacillus Anthracis

First pathogenic bacteria which was:

• Seen under microscope

• To have Live attenuated vaccine

• Isolated in culture media.

It is the largest pathogenic bacterium

Category (A) Bioterrorism agent

virulence factor

Plasmid Coded

Pxo1 plasmid

• Anthrax toxin (Tripartite)

• (only toxin which has 3 parts)

  • Edema factor: increases cAMP (MOA)

  • Protective factor: attachment of bacteria

  • Lethal factor: cause death of cells

Pxo2 plasmid

• Capsule

  • Polypeptide made of polyglutamate

  • (all other capsules are polysaccharides)

Loss of plasmid → Loss of virulence → Useful in making vaccine (Sterne vaccine)

C/F & Pathogenesis

• It is a zoonotic disease by : Cutaneous (M/C) , Inhalation , Ingestion

• Infectious forms: Spores

Cutaneous Anthrax

• Hide Porter's disease

• Lesions seen on neck.

• Referred to as Malignant Eschar (black color)

Pulmonary Anthrax

• Wool Sorter's disease

• Results in Hemorrhagic mediastinitis

• Complications

  • Pericarditis

  • Septicemia

• Form associated with Bioterrorism.

Intestinal Anthrax

• Caused by eating undercooked meat.

• Hemorrhagic enteritis

Complications

• CNS/ Meningitis/ Meningoencephalitis

• Hemorrhagic CSF

• Hemorrhagic mediastinitis

• Hemorrhagic enteritis

Diagnosis

Specimen

• Skin

• Blood

• CSF

• Sputum

• Stool

Autopsy never performed in an animal with anthrax; alternatively blood sample/ cut one ear, taken and kept in biosafety cabinets (BSL II/III cabinets).

Microscopy

Bamboo stick appearance or box car appearance

There are holes inside which are spores (Gram poor)

• For spores: Schaeffer & Fulton stain is used

McFadyean Reaction

• Smear purple material around organism (indicates capsular material).

Blood culture morphologies

1.Blood agar

Blood agar + Penicillin

String of Pearls appearance

Agar + 0.5 - 0.50 u/ml of Penicillin

Cells: Spherical

3. Gelatin Liquefaction

• B. anthrax are obligate aerobes (require oxygen)

• At surface of tube there is more oxygen than bottom, so it will have more energy at surface

• Gelatin liquefaction occurs maximally at surface and keep on diminishing as moving down.

• Shows inverted fir tree appearance.

PLET

PLET

• Selective media

• Polymyxin Lysozyme EDTA Thallous acetate.

Serology

• Ascoli's ring thermo precipitin test

• ELISA (used commonly)

CDC guidelines

• Any Gram +ve bacillus,

• Non-Motile,

• Non-Hemolytic,

• Catalase +ve.

Gives presumptive diagnosis of Anthrax

Precautions

Biosafety

• Sanitization of factory

• No autopsy for anthrax died animals.

• Buried deep in quicklime / cremated to prevent soil contamination.

Duckering: Disinfection of wool is done by: 2% formaldehyde at 30-40° C for 20 minutes.

Treatment

• Vaccination

• Rx

Bacillus cerus

Lab Diagnosis

• Motile, Hemolytic

• Non encapsulated

• Selective media

  • MYPA: Mannitol, Yolk Polymyxin, Agar

  • PEMBA: Polymyxin, Mannitol, Bromothymol blue, Agar

C. Perfringens

• Non-motile

• Encapsulated

• Gram +ve Bacilli

• Subterminal spores

• Earlier known as C. Welchii

virulence factor

Invasive + Toxigenic

4 major toxins:

• Beta (P)

• Epsilon (8)

• Iota (I)

• Alpha (a) or Lecithinase or Phospholipase C

8 minor toxins:

• Gamma (v),

• Delta (0),

• Theta (0) ,

• Кара (к),

• Lambda (2),

• Eta (n),

• Meu (M),

• Veu (v)

Enzymes:

• Histaminase,

• Neuraminidase

Features of Gas Gangrene

• Foul smelling, black, dirty looking tissues

• Pain

• Discharge

• Gas bubbles (Crepitus)

• Shock

Incubation Period of Organisms causing Gas Gangrene

C. perfringens: 10-48 hrs

C. septicum: 2-3 days

C. novyi: 5-6 days

Treatment of Gas Gangrene

• Surgical Debridement - Removal of Dead muscles

• IV Penicillin + Clindamycin for 10-14 days

• Hyperbaric oxygen

• Passive Immunization can be given in some cases: Anti- gangrene serum

Others clinical features

2. Food Poisoning

• Because of heat resistant Spores

• Can be caused due to cold/ warmed up meat.

3. Gangrenous appendicitis

• Caused by Type A

4. Necrotizing enteritis

• Caused by Type C

5. PIGBEL

• Common in European countries.

• Abdominal pain & diarrhea

• Caused by consumption of pork and sweet potato together.

• Sweet potato has trypsin inhibitors which prevents the breakdown of beta toxin of pork in the intestine.

• Rx: IV Penicillin + Metronidazole

Lab Diagnosis

Specimen: For gangrene case, we will take dead tissues such as-

• Necrotic tissue

• Muscle fragments

Microscopically

• C. Perfringens shows: Subterminal spores.

• C. Septicum shows: Citron body

Media: Robertson Cooked meat broth (Red/ Saccharolytic)

Litmus Milk: Stormy clot formation on litmus milk.

Naglar Reaction

• Nagler plate has Egg yolk (lecithin) based agar

• Medium is divided into two parts and patient's serum is put

• C. perfringens has Lecithinase & egg yolk has lecithin which will break and opaque area is seen

• On the other side, no opacification is seen as there is antitoxin which neutralizes.

Target Hemolysis

Double zone of hemolysis.

Inner complete part is due to theta toxin

Outer incomplete part is due to alpha toxin

Reverse Camp Test +ve

Horizontal line: C. perfringes.

Arrow head formation at junction: S. agalactae.

Clostridium tetani

Causes: Tetanus (muscle spasm)

has terminal spore (drumstick appearance)

Transmission

By Injury: Unsterile RTA

No person to person transmission

Incubation Period- 6-10 days (shorter IP → worse)

Tetanus is also known as 8th day disease

Clinical features

symptom is increased tone of masseter muscle → Lockjaw / Trismus

With progression of disease:

• Limb Spasm

• Descending spastic paralysis

• Deep tendon reflexes exaggerated.

• Autonomous disturbance (2nd week; Increased BP, PR, Sweating)

• M/c cause of death- Respiratory failure

Complications

Risus Sardonicus (looks like patient is smiling but it's muscle contraction)

Opisthotonos

Specimen: Necrotic Tissue

Microscope examination

• Gram +ve Bacilli

• Spores: Terminal (drumstick appearance)

On blood agar: Concentric movement is seen- Swarming

Gelatin Stab: Fir tree appearance(as organism will work maximally at the bottom).

Robertson's Cooked Meat Broth: Black color; proteolytic reaction.

Virulence Factors of botulism toxin

Botulinum toxin: Produced intracellularly appears in medium only after cell death.

Serotype: 9 (A,B,C1,C2,D,E,F,G,FA hybrid)

0 A, B, E- cause infections in humans

• A- most severe

• All are chromosomal mediated

All are chromosomally coded except C1, C2, D which is phage coded exception: C2 is an enterotoxin

Clinical manifestations

No contraction of muscle (floppy muscles) → flaccidity is noted.

Types of botulism

Food borne botulism

• Bottled and canned food (Heat labile toxins)

• IP: 12-36 hrs

• Results in

  • • Diplopia

  • • Dysphagia

  • • Dysarthria

  • • Descending flaccid paralysis.

  • • Dilated pupils.

  • • GI symptoms are also present.

Wound botulism

• IP: 7-10 days

• No GI symptoms

Infant botulism

• Bottle fed / honey / baby food

• IP: 1-2 days

• First symptom: Constipation.

• Floppy baby syndrome → flaccid muscles

Surgeries

• Iatrogenic botulism

Lab diagnosis & Treatment of C. botulinum

Lab diagnosis

• • Gram +ve Bacillus

• • Spores seen

  • • Subterminal

  • • Oval

  • • Bulging

• • Anaerobe

Treatment

• • Toxoid Antiserum

Clostridium difficale

Normally present in the gut

Causes: pseudomembranous enterocolitis.

Virulence factors

• • ToxinA: Enterotoxin (gut attached)

• • Toxin B: Cytotoxin

mechanism

• Toxin A & B entering the system → attack the GTP binding proteins (Rho, Rac, CDC 42) - damage to actin cytoskeletal → cell death.

• Toxins will stimulate ILS production → neutrophils recruitment → form dirty yellow necrotic membrane present on mucosa.

• The dead cells will burst out like a volcano eruption (in microscopic vision)

Pseudomembranous Enterocolitis

• Risk factor: Long term usage of antibiotics (3rd generation cephalosporins)

• Clinical feature: Watery diarrhea

diagnosis

Colonoscopy: 100% specificity, 50% sensitivity.

Toxin: Tissue culture Assay; ELISA; PCR.

Media:

• CCFA: Cefoxitin Cycloserine Fructose Agar

• CCYA: Cefoxitin Cysteine Yeast Extract Agar

HPE: Volcano like eruption is seen.

treatment

• Earlier oral vancomycin was used but now Fidaxomycin is used.

• Recommended treatment: Oral vancomycin (500 mg four times daily) + intravenous metronidazole and rectal vancomycin enemas in cases of severe ileus (Fulminant CDI).