Identification Of Gram Positive Bacteria

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An antibiotic isolated from streptomyces niveus. It has a chemical structure similar to coumarin. It binds to DNA gyrase, and blocks adenosine triphosphatase (ATPase) activity.

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Novobiocin (albamycin/cathomycin)

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Novobiocin test

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Used to differentiate S. saprophyticus (resistant) to S. epidermidis (sensitive).

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A chemical used in cell culture techniques for the presumptive identification of Streptococcus pneumoniae, which is optochin-sensitive, from other alpha-hemolytic streptococci such as Streptococcus viridans, which are resistant.

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Optochin (ethylhydrocupreine)

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Is a mixture of related cyclic polypeptides produced by the licheniformis group of Bacillus subtilis var Tracy. Its unique name derives from the bacillus producing it was first isolated from a knee scrape from a girl named Margaret Tracy.

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Bacitracin

Useful for differentiating β-hemolytic Group A (sensitive) streptococci from β-hemolytic non-Group A streptococci (resistant).

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Alpha-hemolytic bacteria

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Form green ring around colonies on blood agar.
Includes:
Streptococcal pneumonia (catalase negative and optochin sensitive)

Viridans streptococci (Catalase negative and optochin resistant)

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Beta-hemolytic bacteria

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Form clean area of hemolysis on blood agar.

Includes:

Staphylococcus aureus (catalase and coagulase positive)

Streptococcus pyogenes - group A strep (catalase negative and bacitracin resistant)

Streptococcus agalactiae - group B strep (catalase negative and bacitracin negative)

Listeria monocytogenes (tumbling motility, meningitis in newborn, unpasteurized milk)

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Used to differentiate staphylococci(positive)

from streptococci(negative).

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Catalase test(gram positive cocci)
Catalase is produced by bacteria that respire oxygen, and protects them from the toxic by-products of oxygen metabolism.Catalase degrades hydrogen peroxide before it can be converted to microbial products by the enzyme myeloperoxidase.

Catalase producing microbes easily degrade what little hydrogen peroxideis present in people with chronic granulomatous disease (NADPH oxidase deficiency) thereby causing recurrent infections

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Myeloperoxidase (MPO)

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The 150-kDa MPO protein is a dimer consisting of two 15-kDa light chains and two variable-weight glycosylated heavy chains bound to a prosthetic heme group. MPO produces hypochlorous acid (HOCl) from hydrogen peroxide(H2O2) and chloride anion (Cl-) (or the equivalent from a non-chlorine halide) during the neutrophil's respiratory burst.

Myeloperoxidase is a peroxidase enzyme that in humans is encoded by the MPO gene.Myeloperoxidase is most abundantly expressed in neutrophil granulocytes (a subtype of white blood cells).

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Identifies whether an organism is capable of activating fibrin which causes blood plasma to clot.
S. aureus make coagulase whereas S. epidermidis and S. saprophyticus don't

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Coagulase test
Organisms that produce coagulase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents.

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Staphylococcus aureus

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Protein A(virulence factor) binds to Fc-IgG, inhibiting complement fixation and phagotcytosis.

Causes:

1. Inflammatory disease - skin infections

2. Toxin-mediated disease - toxic shock sydrome (TSST-1 toxin; is a superantigen that binds to MHC II and T-cell receptor, resulting to polyclonal T-cell activation)scalated skin syndrome(exfoliative toxin), rapid-onset food poisoning (enterotoxin)

3. MRSA(methicillin-resistant S. aureus) infection - important cause of serious nosocomial and community acquired infections. Resistant to ß- lactams due to altered PBP

4. Causes acute bacterial endocarditis and osteomyelitis

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Exotoxins released by Staph aureus.

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Exfoliatin
Enterotoxin
TSST-1

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Powerful defenses of Staph aureus.

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Protein A
Coagulase
Hemolysins
Leukocidins
Penicillinase
Novel PBP

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Staph aureus' enzymes that allow them to tunnel through tissues

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Hyaluronidase (spreading factor)
Staphylokinase
Lipase
Protease

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Also known as the spreading factor. Breaks down proteoglycans

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Hyaluronidase

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An enzyne of Staph aureus responsible for breaking down formed fibrin clots.

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Staphylokinase (like streptokinase)

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Diseases cauaed by Staph aureus by direct organ invasion

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Pneumonia, Meningitis, Osteomyelitis, Acute endocarditis, Septic arthritis and skin infections.

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The "accidental bacteria". Infects prosthetic devices and IV catheters by producing adherent biofilms.

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Staphylococcus epidermidis

Component of normal skin flora; contaminates blood cultures

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Most common cause of:

Meningitis, Otitis media (in children), Pneumonia, and Sinusitis.

alpha-hemolytic, sensitive to optochin

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Streptococcus pneumoniae

Lancet shape, diplococcis, encapsulated, has IgA protease.

"Pneumococcus" associated with "rusty" sputum, sepsis in sickle cell anemia and splenectomy

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How is meningitis diagnosed?

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Lumbar puncture

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Classical sign of meningitis in adults which is not seen in neonatal meningitis

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Stiffness of the neck

in babies: Fever, vomiting, poor feeding, irritability

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The first pnuemococcal vaccine which has 25 of the most common capsular polysaccharide antigens

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Pneumovax

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Viridans group streptococci

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alpha-hemolytic. They are normal flora of the orapharynx and cause dental caries(Strep. mutans) and subacute endocarditis (Strep. sanguis)

alpha-hemolytic, resistant to optochin.

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Viridans is from viridis meaning

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Green

*alpha hemolytic (green)

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Streptococcus pyogenes (group A streptococci)

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Causes:

1. Pyogenic - pharyngitis, cellulitis, impetigo.

2. Toxigenic - scarlet fever, toxic shock-like sydrome

3. Immunologic - rheumatic fever, acute glomerulonephritis.

Beta-hemolytic. Bacitracin sensitive.

ASO titer detects recent infection.

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Antibodies to M protein (virulence factor for S. pyogenes) enhance host defences against this bacteria but can give rise to rheumatic fever. What cells produce such antibodies.

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Plasma B cells by opsonization.

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Oxygen-labile enzyme produced by group A streptococci which is responsible for reaction its on BAP and is also antigenic

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Streptolysin O

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Oxygen-stable enzyme produced by group A streptococci which can also hemolyze RBC'sbut is not antigenic

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Streptolysin S

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Strains of group A drep that can cause scarlet fever have this toxin,

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Pyrogenic toxin (erythrogenic toxin)

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Enzyme present in group A strep that activates plasmin which breaks down fibrin clots

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Streptokinase

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Diseases caused by group A strep via local invasion/exotoxin release

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1. Streptococcal pharyngitis "Strep throat"

2. Streptococcal skin infections: folliculitis, cellulitis, impetigo and Necrotizing faciitis (aka Flesh-eating streptococcus; can infect muscle and cause myositis; can also be caused by Staph and Clostridium)

3. Scarlet fever

4. Stretococcal toxic shock syndrome

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Disease caused by group A strep via delayed antibody-mediated disease

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1. Rheumatic fever

2. Glomerulonephritis

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Symptoms of Scarlet fever

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Fever
Myocarditis
Joint swelling
Chorea
Subcutaneous nodules
Erythema marginatum

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A delayed antibody-mediated disease caused by nephritogenic strains of group A strep infecting the pharynx or skin. Characterized by darker urine(due to hematuria) with hypervolemia secondary to fluid retention.

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Acute post-streptococcal glomerulonephritis

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Beta-hemolytic. Bacitracin resistant. Produces cAMP factor, which enlarges the area of hemolysis by S. aureus. (CAMP - author of the test)

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Streptococcus agalactiae (group B streptococci)

Screen pregnant women in 35-37 weeks. Patient with positive culture receive intrapartum penicillin prophylaxis

Colonizes vagina; causes pneumonia, meningitis, and sepsis mainly in babies.

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3 main types of infection caused by Viridans strep.

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1. Dental infections (esp. s mutans; binds to teeth and ferments sugar)

2. Endocarditis (SBE; bacteria produces an extracellular dextran and clings to cardiac valves usually previously damaged heart valve)

3. Abscesses (subgroup of viridans, streptococcus intermidius. Includes S. intermidius, S. constellatus, S. anginosus)

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Are normal colonic flora that are penicillin G resistant and subacute endocarditis.

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Enterococci (group D streptococci)

Lancefield group D includes the enterococci and the nonenterococcal group D streptococci. Lancefield group is based on differences in the C carbohydrate on the bacterial cell wall. Variable hemolysis. VRE (vancomycin-resistant enterococci) are an important cause of nosocomial infection.

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Group D streptococci are divided into two subgroups. What are these groups?

* can be alpha-hemolytic or gamma-hemolytic

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Enterococci (E. faecali and E. faecium)

and

Non enterococci (S. bovis and S. equinus)

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Group D strep that grow well on in 40% bile and 6.5% NaCl. Not as virulent as S. pyogenes. Causes UTI and biliary tract infections, bacteremia and SBE

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Enterococci

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What antibiotics are enterococci resistant to?

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Ampicillin and Vancomycin

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A new class of drug used to treat VRE, which causes painful arthralgias and venous irritation.

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Pristinomycins (dalfopristin & quinupristin)

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A group D non-enterococci that is associated with colon cancer

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S. bovis

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Colonizes the gut. Can cause bacteremia and subacute endocarditis in colon cancer patients.

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Streptococcus bovis (group D streptococci)

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Causes diphtheria via exotoxin coded by beta-prophage.

Potent exotoxin inhibits protein synthesis via ADP ribosylation of EF-2

Lab diagnosis based on gram-positive rods with metachromic(blue and red) granules

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Corynebacterium diphtheriae.

Symptomps include psuedomembranous pharyngitis (grayish-white membrane) with lymphadenopathy.

Toxoid vaccine prevents diphtheria

"Coryn" = club shaped

Grown on tellurite media

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Spore-forming bacteria found in soil.

"only certain gram-positive rods form spores when nutrients are depleted (end of stationary phase)"

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Bacillus anthracis, Clostridum perfringens, C. tetani.

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Spore-formers not found in soil.

"only certain gram-positive rods form spores when nutrients are depleted (end of stationary phase)"

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B. cereus, C. botulinum

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Structure of bacteria; higly resistant todestruction by heat and chemicals. have dipicolinic acid in their core. Have no metabolic activity

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Spores

Autuclave is done to kill spores by steaming at 121 degree celcius for 15 mins.

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Gram positive, spore-forming, obligate anaerobe bacilli

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Clostridia

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Clostrida with exotoxins include

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C. tetani, C. botulinum, C. perfringens, C. difficile

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Produces tetanospasmin

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An exotoxin causing tetanus

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Produces a preformed, heat-labile toxin that inhibits Ach release at the neuromuscular junction causing botulism.

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Clostridium botulinum.

In adults, ingestion of preformed toxin.

In babies, ingestion of bacterial spores in honey causes disease (floppy baby syndrome)

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Ingestion of C. botulinum toxin causes what symptoms first?

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Cranial nerve palsies leading to diplopia. Difficulty in swallowing.

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Produces alpha-toxin("lecithase", a phospholipase) that can cause mynecrosis (gas gangrene) and hemolysis

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C. perfringens

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Types of infection with C. perfringens

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Cellulitis/ wound infection.
Clostridial Myonecrosis

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Produces 2 toxins. Toxin A and Toxin B (pseudomembranous colitis). Often secondary to antibiotic use, esp. clindamycin or ampicillin. Diagnosed by detection of 1 or both toxins in stool.

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C. difficile
Toxin A, enterotoxin, binds to the brush border of the gut.
Toxin B, cytotoxin, destroys the cytoskeletal structure of enterocytes, causing pseudomembranous colitis.

Also causes diarrhea, treatment is metronidazole.

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A toxin causing paralysis from Renshaw cells by blocking the release of glycine and GABA release(inhibitory transmitter) in spinal cord. Causing spastic paralysis, trismus (lockjaw) and risus sardonicus (continues spasm of facial muscle that appears to produce grinning)

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Tetanus

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Acquired from bad bottles of food and honey.

Causes flaccid paralysis.

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Botulinum

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Perforates a gangrenous leg

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Perfringens

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A gram-positive ; spore-forming rod that produces anthrax toxin and the only bacterium with a polypeptide capsule (contains D-glutamate)

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Bacillus anthracis

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Anthrax

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Caused by B. anthracis.

Types:

Cutaneous anthrax(contact) - produces black skin lesions(black eschar; painless ulcer) surrounded by edematous ring; can progress to bacteremia and death. Caused by lethal factor and edema factor.

Pulmonary anthrax(inhalation of spores from contaminated wool) - aka woolsorter's disease. flu-like symptoms that rapidly progress to fever, pulmonary hemorrhage, mediastinitis, and shock

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The release of exotoxin is the major reason why anthrax carries such high mortality rate. These exotoxin are encoded on what plasmid?

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pxO1

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3 proteins present in anthrax which are nontoxic but together ellicits their effects.

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Edema factor (active a component)
Protective antigen
Lethal factor

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Component of anthrax exotoxin which is calmodulin-depenent adenylate cyclase

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Edema factor

*increases cAMP, inhibits neutrophil function, causes massive edema

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Subunit of anthrax toxin which promotes entry of EF in phagocytic cells

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Protective antigen

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Subunit of anthrax exotoxin which is a zinc metalloprotease that inactivates protein kinase

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Lethal factor

*Stimulates macrophage to release TNF and IL-1

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Plasmid responsible for encoding 3 genes necessary for poly-glutamyl capsule

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pxO2

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Enterotoxins produced by B. cereus

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Heat labile(resembles E. coli)
Heat stable(resembles Staph aureus

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Facultative intracellular microbe; acquired by ingestion of unpasteurized milk/cheese. and deli meats or by vaginal transmission during birth. Form "actin rockets" by which they move from cell to cell. Characteristic tumbling mobility.

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Listeria monocytogenes.

Can cause amnionitis, septicemia, and spontaneous abortion in women; granulomatosis infantiseptica; neonatal meningitis; meningitis in immunocompromised patients; mild gastoenteritis.

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3 step treatment for diptheria toxin.

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1. Antitoxin (inactivates circulating toxin)
2. Penicillin or Erythromycin
3. DPT vaccine

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Gram positive forming long branching filaments resembling fungi. An anaerobe, causes oral/facial abscesses that may drain through sinus tracts in skin. Normal oral flora. Forms yellow"sulfur granules" in sinus tracts

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Actinomyces israelii

Penicillin

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Gram positive forming long branching filaments resembling fungi. Weakly acid-fast aerobe in soil. causes pulmonary infection in immunocompromised patients.

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Nocardia asteroids.

Treatment: TMPS

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Mycobacterium infections: "All mycobacterium are acid-fast"

TB granulomas( Ghon focus/lesion; usually in lower lobes + lobar and perihilar lymph node involvement). Reflect primary infection or exposure

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Ghon complex

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Mycobacterium infections: "All mycobacterium are acid-fast"

TB, symptoms include fever, night sweats, weight loss, and hemoptysis, often resisitant to multiple drugs

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Mycobacterium tuberculosis

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Mycobacterium infections: "All mycobacterium are acid-fast"

pulmonary TB-like symptoms

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Mycobacterium kansasii

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Mycobacterium infections: "All mycobacterium are acid-fast"

Causes disseminated disease in AIDS; often resistant to multiple drugs. Prophylactic treatment with azithromycin

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Mycobacterium avium-intracellulare

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Mycobacterium infections: "All mycobacterium are acid-fast"

Caused by Mycobacterium leprae(infects skin and superficial nerves) whcih likes cool temperatures and cannot be grown in vitro.

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Leprosy (Hansen's disease).

Has two forms: Lepromatous and Tuberculoid

M. leprae reservoir in US: armadillos

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Mycobacterium infections: "All mycobacterium are acid-fast"

Form of Hansen's disease which presents diffusely over skin and is communicable (pateints with weak T-cell mediated immunity). It can be lethal.

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Lepromatous

*Lionlike face, saddle nose

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Mycobacterium infections: "All mycobacterium are acid-fast"

Form of Hansen's disease which is limited to a few hypoesthetic skin nodules (patients with intact T-cell response).

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Tuberculoid

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Mycobacterium infections: "All mycobacterium are acid-fast"

Primary treatment and alternative treatment for M. leprae infectoin.

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Long term oral dapsone(toxicity is hemolysis and methemoglobinemia)

Alternative: include rifampin and cobination of clofazimine and dapsone.

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Cord factor

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mycoside formed by 2 mycolic acids with a disaccharide (trehalose). Found in virulent strains of mycobacterium tuberculosis

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Caseous necrosis

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Activated macrophage attack local lung tissue. Looks like granular creamy cheese.

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Lymph node tuberculosis

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Scrofula

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Patient will have red and white blood cells in urine, but no bacteria is seen on gram stain or grow in culture

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Sterile pyuria