Gram Positive Organisms Part 1A & 1B

What are the Gram-Positive Organisms?

• Cocci

  • Staphylococcus

  • Streptococcus

  • Enterococcus

• Bacilli

  • Bacillus

  • Clostridium spp

  • Listeria spp

  • Corynebacterium spp

A 53-year-old farmer presents for evaluation of a growth on his arm. This began as a blister about a week ago and then ruptured developing into an ulcer. He also had fever along with the blister. He has had no ill contacts. He takes care of the cows, buffaloes and sheep on his farm. On examination he has a 4.5-cm circular black eschar surrounded by vesicles and edema. He also has tender lymph nodes in his arm pit. A gram stain of the eschar pus shows Gram Positive Bacilli.

What is this disease likely to be?

Anthrax

A 53-year-old farmer presents for evaluation of a growth on his arm. This began as a blister about a week ago and then ruptured developing into an ulcer. He also had fever along with the blister. He has had no ill contacts. He takes care of the cows, buffaloes and sheep on his farm. On examination he has a 4.5-cm circular black eschar surrounded by vesicles and edema. He also has tender lymph nodes in his arm pit. A gram stain of the eschar pus shows Gram Positive Bacilli.

What led to the infection?

contact with animals

A 53-year-old farmer presents for evaluation of a growth on his arm. This began as a blister about a week ago and then ruptured developing into an ulcer. He also had fever along with the blister. He has had no ill contacts. He takes care of the cows, buffaloes and sheep on his farm. On examination he has a 4.5-cm circular black eschar surrounded by vesicles and edema. He also has tender lymph nodes in his arm pit. A gram stain of the eschar pus shows Gram Positive Bacilli.

Describe the likely pathogen?

Bacillus anthracis

A 53-year-old farmer presents for evaluation of a growth on his arm. This began as a blister about a week ago and then ruptured developing into an ulcer. He also had fever along with the blister. He has had no ill contacts. He takes care of the cows, buffaloes and sheep on his farm. On examination he has a 4.5-cm circular black eschar surrounded by vesicles and edema. He also has tender lymph nodes in his arm pit. A gram stain of the eschar pus shows Gram Positive Bacilli.

What is the virulence factor responsible?

capsular polypeptide and anthrax toxin

A 53-year-old farmer presents for evaluation of a growth on his arm. This began as a blister about a week ago and then ruptured developing into an ulcer. He also had fever along with the blister. He has had no ill contacts. He takes care of the cows, buffaloes and sheep on his farm. On examination he has a 4.5-cm circular black eschar surrounded by vesicles and edema. He also has tender lymph nodes in his arm pit. A gram stain of the eschar pus shows Gram Positive Bacilli.

What is the mode of action of the virulence factor?

inhibiting phagocytosis and activation of neutrophils

Anthrax and Bacillus anthracis

• Infections of ______

  • _____ infection

• Infections of herbivores

  • zoonotic infection

Anthrax and Bacillus anthracis

• Possible _____ agent

• Possible bioterrorism agent

Anthrax and Bacillus anthracis

• Gram ______

• Gram-positive rod

Anthrax and Bacillus anthracis

• Spore-forming

  • _________ of bacterial cell

    • ______ survival under harsh conditions

• Protective, dormant form of bacterial cell

  • increases survival under harsh conditions

B. anthracis

• Microscopy

  • Gram stain= ______

  • Capsule = _____

  • Spores = _____

• Gram stain= long GPR/ GPB; single paired or long serpentine chains (box cars)

• Capsule= only in clinical specimens; made of polypeptides

• Spores= old culture- not in clinical specimens; malachite green stain

B. anthracis

• Culture = _____

• Culture= Blood agar

  • large nonhemolytic adherent colonies

Cutaneous anthrax

• 90-95% of cases

• Eschar produced at entry site

Inhalation anthrax

• Woolsorter’s disease

• Inhalation of spores from animal hair and wool

• Bioterrorism event

Gastrointestinal anthrax

• Rare in humans

• Common in herbivores

B. anthracis Virulence Factors:

• Spore= ___

• Capsule = ____

• Exotoxins= _____

Spore = the infectious particle

• Capsule - poly-D-glutamic acid

  • antiphagocytic

  • capsule genes on plasmid

• Exotoxins- Anthrax toxin is a tripartite toxin.

  1. edema factor (EF): adenylate cyclase

  2. lethal factor (LF): kills cells

  3. protective antigen (PA): mediates entry of EF or LF into eukaryotic cells

• nontoxic individually, but pathogenic when combined

  • PA + EF = edema toxin Æ edema

  • PA + LF = lethal toxin Æ tissue necrosis

• all toxin genes are on pX01 plasmid

How anthrax toxins work?

• EF= _____

• EF = adenylate cyclase —> ↑ intracellular cAMP —→ ↑ efflux of fluids and ions —→ Edema

How anthrax toxins work?

• LF= _____

• EF = mitogen-activated protein kinase (MAPKK) protease —> ↓ disrupts cell signaling —→ Cell death and tissue necrosis

Anthrax-Diagnosis:

• Microscopy:

  • Gram stain = _____

  • Spores = ____

  • Capsule= ____

• Gram stain= GPR, boxcars

• Special stains for Capsule

• Spores: seen in malachite green stain

Anthrax-Diagnosis:

• Culture on blood agar plate:

  • ________

• Large, non-hemolytic adherent colonies

Anthrax-Diagnosis:

• Molecular assays:

  • _____

• PCR

Anthrax treatment =

Ciprofloxacin

Anthrax prevention:

• Toxoid vaccine used for high-risk persons

• Vaccination of animals to control disease

• Eradication unlikely because of spore production

What is the causative agent for Botulism?

Clostridium botulinum

What is Botulism?

• rare poisoning caused by toxins by Clostridium botulinum

• Can be fatal and requires emergency medical care

  • can occur in infants, be spread in food, or infect a wound

• Symptoms:

  • difficulty swallowing (dysphagia) or speaking (dysarthria), facial weakness and paralysis

Botulism- Clostridium botulinum

• Gram stain = ___

• Large GPR

• Strict anaerobic

• Spore forming

• Found in soil, river and sea water; Animal gut

Botulism- Clostridium botulinum

• Common sources = ____

• For older children and adults- contaminated canned vegetables/ canned meats (canned foods with low acidity)

• For infants- contaminated honey/ formula feed

Which type of botulism is best described below:

• Due to ingestion of foods contaminated with spores of C. botulinum

a) Food botulism

b) Infant botulism

c) Wound botulism

d) Iatrogenic botulism

e) Inhalation botulism

b)

Which type of botulism is best described below:

• Due to ingestion of foods contaminated with preformed toxins of C. botulinum

a) Food botulism

b) Infant botulism

c) Wound botulism

d) Iatrogenic botulism

e) Inhalation botulism

a)

Which type of botulism is best described below:

• Due to wound contamination with soil containing spores of C. botulinum

a) Food botulism

b) Infant botulism

c) Wound botulism

d) Iatrogenic botulism

e) Inhalation botulism

c)

Which type of botulism is best described below:

• Rare, due to inadvertent injections of medications contaminated with spores (Botox therapy)

a) Food botulism

b) Infant botulism

c) Wound botulism

d) Iatrogenic botulism

e) Inhalation botulism

d)

Which type of botulism is best described below:

• Rare, due to inhalation of soil containing spores of C. botulinum

a) Food botulism

b) Infant botulism

c) Wound botulism

d) Iatrogenic botulism

e) Inhalation botulism

e)

In the image, what occurs at:

• A

a) Spread in general circulation

b) Entry into peripheral nerve terminals

c) Transcytosis across intestinal epithelium

c)

In the image, what occurs at:

• B

a) Spread in general circulation

b) Entry into peripheral nerve terminals

c) Transcytosis across intestinal epithelium

a)

In the image, what occurs at:

• C

a) Spread in general circulation

b) Entry into peripheral nerve terminals

c) Transcytosis across intestinal epithelium

b)

Botulism:

• Is there constipation or diarrhea?

a) Constipation, no diarrhea

b) Diarrhea, no constipation

a)

Botulism:

• Flaccid paralysis and ultimately death due to respiratory paralysis

a) True

b) False

a)

Floppy Baby Syndrome

• Ingestion of C. botulinum spores = Infant botulism

• Features include:

  • weak cry

  • constipation

  • inability to hold head or feed well, failure to thrive

C. Botulinum Exotoxin:

• C. botulinum forms ____

• Neurotoxin

  • Mode of action = _____

• Forms a very potent heat labile toxin coded for by lysogenic prophage

• Neurotoxin

  • Mode of action=

    • Blocks release of acetylcholine from peripheral nerves

      • Leads to flaccid paralysis, double/ blurred-vision, muscle weakness

Clostridium tetani Exotoxin:

• Intracellular-acting toxins = ____

  • Mode of Action = ____

• Intracellular-acting toxins= Neurotoxin

  • Mode of action=

    • blocks release of inhibitory neurotransmitters Æ unopposed firing of the motor neurons —→ constant contraction

Food Botulism

• Lab diagnosis = ______

• toxin detection in food

Infant Botulism

• Lab diagnosis = ______

• toxin detection in serum or feces

Botulism

• Treatment = ______

• Treatment = Mechanical ventilatory support, antitoxin

Food Botulism

• Prevention = ______

• Prevention= Proper sterilization of canned foods

Infant Botulism

• Prevention = ______

• Prevention= avoid honey in children < 1 year of age

Listeriosis

• serious infection caused by the ingestion of food contaminated with the bacterium Listeria monocytogenes

Listeria monocytogenes:

• Characteristics= _____

• Small, nonsporting gram-positive bacilli or cocco-bacilli

• Facultative intracellular pathogen

• Grow at 4°C (cold enrichment) -35°C

• Show characteristic tumbling motility at 25°C

• Weak beta hemolysis on Blood agar, Catalase positive

• Found in animal gut and colonizes human gut or genital tract

Listeria monocytogenes:

• Pathogenesis = ______

• Virulence factors = _____

• Lab test = ____

• Pathogenesis =

  • Attach to host cell receptors like Enterocytes, M cells, phagocytes, etc.

• Virulence factors =

  1. Internalins= attachment and entry into nonphagocytic cells

  2. Listeriolysin O= Beta-hemolysin (pore forming exotoxin) allowing escape from phagosome into cytoplasm

  3. ActA= actin polymerization —> lateral propulsion and involvement of adjacent cells

• Lab test =

  • Gram stain and culture of body fluids, such as CSF or placenta

• Treatment=

  • Antibiotics- Ampicillin/ Ampicillin + Gentamicin

• Prevention=

  • Avoid unpasteurized cheese/ unwashed salads esp. in pregnancy and old age

Which virulence factor of Listeria monocytogenes is best described?

• Attachment and entry into nonphagocytic cells

a) Internalins

b) Listeriolysin O

c) ActA

a)

Which virulence factor of Listeria monocytogenes is best described?

• Beta-hemolysin (pore forming exotoxin) allowing escape from phagosome into cytoplasm

a) Internalins

b) Listeriolysin O

c) ActA

b)

Which virulence factor of Listeria monocytogenes is best described?

• Actin polymerization —> lateral propulsion and involvement of adjacent cells

a) Internalins

b) Listeriolysin O

c) ActA

c)

What is the principle of the Coagulase Test:

Coagulase enzyme converts plasma fibrinogen into fibrin clots

In the image, which is the positive Coagulase test?

a) Left

b) Right

a)

Which of the following is best described below?

• Normal flora of skin and mucous membrane

• May cause disease (clinically significant)

• Hands and nares of 20-40% of population (higher in hospital personnel)

• Most clinically significant staphylococcus species

• Gram-positive cocci, catalase positive, coagulase positive

• Beta-hemolytic with golden pigment

a) Staphylococcus epidermidis

b) Staphylococcus aureus

c) Streptococcus pyogenes

d) Streptococcus pneumoniae

b)

Which of the following is best described below?

• Gram-positive cocci, catalase positive, coagulase positive

• Beta-hemolytic with golden pigment

• Pathogenesis:

  • Forms a host of virulence factors and causes a variety of diseases

a) Staphylococcus epidermidis

b) Staphylococcus aureus

c) Streptococcus pyogenes

d) Streptococcus pneumoniae

b)

S. Aureus causes diseases through _______ or ______ (2)

Direct invasion -with abscess formation or Production of exotoxins

Staphylococcus aureus —→ Direct invasion-with abscess formation —→ ______

Pyogenic disease:

• Folliculitis, furuncles

Staphylococcus aureus —→ Production of exotoxins—→ ______

Toxin-mediated diseases:

• Toxic shock syndrome

• Food poisoning

• Scalded skin syndrome

What are the virulence factors for:

• Staphylococcus aureus (Pyogenic Disease)

• Capsule: produced by some strains

• Adhesion and Protection:

  • Surface adhesins

  • Coagulase and Clumping factor

    • fibrinogen —> fibrin clot —> abscess

  • Protein A- binds Fc receptor of IgG —> prevents antibody-mediated immune clearance

• Damage:

  • Enzymes: Lipases (degrades skin lipids), hyaluronidases

  • Teichoic acids: inflammatory response

  • Cytolytic toxins:

    • Alpha toxin= pore forming toxins

    • Panton-Valentine leuckocidin= lyses white blood cells

Of the following cytolytic toxins given which is found in Staphylococcus aureus (Pyogenic Disease)*

a) Protein A

b) Pyrogenic exotoxins

c) Panton-Valentine Leukocidin

d) Pneumolysin

c)

Which of the following is best described below:

• ____ = pore-forming toxin

a) Teichoic acids

b) Panton-Valentine leukocidin

c) Lipases

d) Alpha toxin

d)

Which of the following is best described below:

• ____ = lyses white blood cells

a) Teichoic acids

b) Panton-Valentine leukocidin

c) Lipases

d) Alpha toxin

b)

Which of the following best describes:

• Coagulase and Clumping factor = _________

a) binds Fc receptor of IgG —> prevents antibody-mediated immune clearance

b) fibrinogen —> fibrin clot —> abscess

b)

Which of the following best describes:

• Protein A = _________

a) binds Fc receptor of IgG —> prevents antibody-mediated immune clearance

b) fibrinogen —> fibrin clot —> abscess

a)

20-yr old Jane is brought to the hospital by her friend as she complained of fever, a headache, diarrhea and weakness. There was a steady deterioration in her condition over the last 24 hours and this morning she was confused and very ill. On examination she has high temperature and a low blood pressure. There is a widespread erythematous rash over the trunk and peeling of skin on the palms and soles. Further questioning reveals that she was menstruating and usually used tampons.

• What condition is Jane likely suffering from?

Toxic shock syndrome

20-yr old Jane is brought to the hospital by her friend as she complained of fever, a headache, diarrhea and weakness. There was a steady deterioration in her condition over the last 24 hours and this morning she was confused and very ill. On examination she has high temperature and a low blood pressure. There is a widespread erythematous rash over the trunk and peeling of skin on the palms and soles. Further questioning reveals that she was menstruating and usually used tampons.

• What led to Jane’s infection?

Contaminated tampons

20-yr old Jane is brought to the hospital by her friend as she complained of fever, a headache, diarrhea and weakness. There was a steady deterioration in her condition over the last 24 hours and this morning she was confused and very ill. On examination she has high temperature and a low blood pressure. There is a widespread erythematous rash over the trunk and peeling of skin on the palms and soles. Further questioning reveals that she was menstruating and usually used tampons.

• Describe the likely pathogen?

GPC in clusters

20-yr old Jane is brought to the hospital by her friend as she complained of fever, a headache, diarrhea and weakness. There was a steady deterioration in her condition over the last 24 hours and this morning she was confused and very ill. On examination she has high temperature and a low blood pressure. There is a widespread erythematous rash over the trunk and peeling of skin on the palms and soles. Further questioning reveals that she was menstruating and usually used tampons.

• What is the virulence factor responsible?

TSST- a superantigen

What is TSST-1?

• Virulence Factor- Toxic Shock Syndrome Toxin

  1. TSST-1 binds directly to MHCII on macrophages

  2. Interact with T cell receptors

  3. Nonspecific proliferation of T cells and massive release of cytokines

What is best described below:

• Sudden onset: fever, vomiting, diarrhea

• Red rash resembling a sunburn and desquamation 1-2 weeks later

• Occasional deaths

a) Listeriosis

b) Botulism

c) Toxic Shock Syndrome

d) Anthrax

c)

Staphylococcus aureus can also cause food poisoning

a) True

b) False

a)

Explain how Staphylococcus aureus can also cause food poisoning?

Enterotoxin

• Intoxication usually associated with potato salad, dairy products, and ham

• No detectable odor of food appearance change

• Nausea, vomiting, abdominal cramping and diarrhea within 1-6 hours of ingestion

• Resolves within 24 hrs

• No fever

S. aureus Infections-Treatment

• Superficial lesions and food poisoning are usually _______

self-limiting

S. aureus Infections-Treatment

• ______ and ______ for extensive or deeper infections

a) Superficial lesions and food poisoning

b) Surgical drainage and superficial lesions

c) Surgical drainage and food poisoning

d) Surgical drainage and antibiotic therapy

d)

S. aureus Infections-Treatment

• Antibiotic resistance is a big problem

  • Give example of S. aureus that is resistant to an antibiotic = _______

Methicillin-resistant S. aureus (MRSA)

S. aureus Infections-Treatment

• Antibiotic resistance is a big problem

  • Methicillin-resistant S. aureus (MRSA)

  • Explain the mechanism?

Organism acquires a gene, mecA, that alters binding site for penicillins

• PBP2 —> PBP2A

What is the treatment for Methicillin-resistant S. aureus (MRSA)

Vancomycin or linezolid

Which of the following is best described below?

• A few species are normal flora of skin and mucous membranes, especially in the oral cavity

• May cause disease (clinically significant)

• GPC in chains

• Catalase negative

• Growth on blood agar

a) Staphylococcus

b) Streptococcus

c) Enterococcus

b)

How are Streptococci classified?

• Alpha hemolytic strep

• Beta hemolytic strep

• Gamma hemolytic strep

_____ hemolytic strep = Partial

a) Alpha hemolytic strep

b) Beta hemolytic strep

c) Gamma hemolytic strep

a)

_____ hemolytic strep = Complete

a) Alpha hemolytic strep

b) Beta hemolytic strep

c) Gamma hemolytic strep

b)

_____ hemolytic strep = None

a) Alpha hemolytic strep

b) Beta hemolytic strep

c) Gamma hemolytic strep

c)

Which of the following is best described below:

• Group A strep, Beta Strep

• Beta-hemolytic colonies

  • Small, pinpoint colony

  • Big zone of hemolysis

• GPC, catalase neg

• Bacitracin susceptible

a) Staphylococcus epidermidis

b) Staphylococcus aureus

c) Streptococcus pyogenes

d) Streptococcus pneumoniae

c)

What are the physiological characteristics of:

• Bacitracin disc (A disc)

• Bacteria streaked onto plate

• Filter paper disc containing bacitracin dropped

• Incubate and observe for growth inhibition

In the Bacitracin disc (A disc) image, which shows resistance?

a) A

b) B

a)

In the Bacitracin disc (A disc) image, which shows Susceptible?

a) A

b) B

b)

What are the virulence factors of:

• Streptococcus pyogenes

Adhesion & Protection

• M-protein– binds to fibronectin and is antiphagocytic

• F-protein – binds fibronectin

• Hyaluronic acid capsule– non-immunogenic, antiphagocytic

Spreading factors & Damage

• Exoenzymes – DNase, hyaluronidase, hemolysins

• Teichoic acids – Inflammatory response

Toxins

• Streptococcal pyrogenic exotoxins – superantigens

• Streptolysin O and S pore forming —> hemolysins

Which virulence factor of Streptococcus pyogenes is best described:

• _______ = binds to fibronectin and is antiphagocytic

a) F-Protein

b) A- Protein

c) M- Protein

d) Hyaluronic acid capsule

c)

Which virulence factor of Streptococcus pyogenes is best described:

• _______ = binds fibronectin

a) F-Protein

b) A- Protein

c) M- Protein

d) Hyaluronic acid capsule

a)

Which virulence factor of Streptococcus pyogenes is best described:

• _______ = non-immunogenic, antiphagocytic

a) F-Protein

b) A- Protein

c) M- Protein

d) Hyaluronic acid capsule

d)

Strep Throat diagnosis =

• Diagnosis from throat swab

  • Rapid strep enzyme immunoassay

  • Culture

Sequelae

• Scarlet Fever

• Cross-reacting antibodies

  • Acute streptococcal glomerulonephritis

  • Rheumatic fever

Which of the following is best described below:

• Also k/a Pneumococcus

• Lancet-shaped GP diplococci

• Alpha hemolytic colonies

• Catalase negative

• Susceptible to Optochin

a) Staphylococcus epidermidis

b) Staphylococcus aureus

c) Streptococcus pyogenes

d) Streptococcus pneumoniae

d)

What are the virulence factors found in:

• Streptococcus pneumoniae

Polysaccharide capsule

• 90 pneumococcal serotypes, 23 responsible for most diseases

IgA protease

• Colonization

Pneumolysin

• Pore-forming toxin

Pneumococcal Pneumonia = Typical Lobar Pneumonia

• Rapid onset

• Chills & fever

• Chest pain

• Productive cough + blood in the sputum (rusty sputum)

• Damage of epithelium stimulates outpouring of fluid, red blood cells, and leukocytes from alveoli Æ productive cough with blood

Treatment for Streptococcal infections

• Penicillin or Amoxycillin

• Ceftriaxone in very ill patients of pneumonia

• Drug sensitivity is good

Prevention for Streptococcal infections

• Avoiding contact with sick patients

• Handwashing and cough etiquette