UNIT 12. CATALASE-POSITIVE, GRAM-POSITIVE COCCI

• Staphylococci

• Micrococci

CATALASE-POSITIVE, GRAM-POSITIVE COCCI usually refers to the two genera

ability of microorganism to produce catalase to breakdown hydrogen peroxide into water and oxygen = EFFERVESCENCE

principle involved in Catalase test

“staphle” = bunches of grapes

etymology of “Staphylococci”

capablity of producing coagulase to form clumping or clot in the presence of plasma

principle of Coagulase test

• Slide Method = detect clumping factor (bound coagulase)

• Tube Method = detect free coagulase

• Slide Method = detect _

• Tube Method = detect _

Staphylococcus aureus

Couagulase test is usually specific for what specie?

• Staphylococcus delphini

• Staphylococcus intermedius

• Staphylococcus pseudintermidius

ENUMERATE the Staphylococcus species (seen in animals) other the S. aureus that may give a positive coagulase test

Staphylococcus aureus

What is/are the catalase-positive, coagulase-positive, gram-positive staphylococci

• Staphylococcus epidermidis

• Staphylococcus haemolyticus

• Staphylococcus saprophyticus

• Staphylococcus lugdunensis

• Staphylococcus schleiferi

• Staphylococcus capitis

• Staphylococcus caprae

• Staphylococcus warneri

• Staphylococcus auricularis

• Staphylococcus hominis

• Staphylococcus nohnii

• Staphylococcus xylosus

• Staphylococcus simulans

• Micrococcus spp. and related genera

• Alloiococcus

What is/are the catalase-positive, coagulase-negative, gram-positive staphylococci

Staphylococcaceae

Family of Staphylococcus

aerobic or facultative anaerobic;

S. aureus subsp. anaerobius;

S. sacchrolyticus;

obligate anaerobes

Staph are generally ___ (O2 requirement) except ___ and ___ which are ___

F

They are nonmotile, non spore-forming

T or F
Staph are motile and non spore forming

• spherical

• 0.5-1.5 um

• singly, in pair, in clusters

Shape, size, and arrangement of Staph

• skin

• mucous membranes

• intestines

normal location of Staph

community-acquired

Staphylococcus aureus nature of infection is _

nosocomial infection

Other Staphylococci (coagulase-negative) causes _

• medium sized (4-8 mm)

• cream-colored, white, or rarely light gold,

• “buttery-looking”

• other spp. may have gray colonies

• some may be B-hemolytic (S. aureus)

Describe the appearance and morphology of Staph colonies grown in BAP

fermenter

Staph result in OF medium

oxidizer

Micrococci result in OF medium

Staphylococcus aureus

true coagulase positive and most virulent species of staphylococci

NA and TSB

S. aureus grow well on most routine media like __ and __

• golden yellow color

• B-hemolytic

describe S. aureus grown in BAP

• round,

• smooth,

• opaque,

• butyrous

describe S. aureus grown in solid media

• Protein A

• Peptidoglycan

• Teichoic Acid

• Clumping Factor

• Capsular Polysaccharide

[PePeT Cum Cum]

ENUMERATE

(Virulent factors) Antigenic Structure of S. aureus

• Peptidoglycan

• Teichoic Acid

(VIRULENT FACTORS) ANTIGENIC STRUCTURE OF S. AUREUS

• it protects the bacteria from lysis and probably aids in adherence

Clumping Factor

(VIRULENT FACTORS) ANTIGENIC STRUCTURE OF S. AUREUS

• component on cell wall responsible for clumping of the whole Staphylococci in the presence of plasma

Protein A

(VIRULENT FACTORS) ANTIGENIC STRUCTURE OF S. AUREUS

• group specific antigen unique to S. aureus

Capsular Polysaccharide

(VIRULENT FACTORS) ANTIGENIC STRUCTURE OF S. AUREUS

• protects for phagocytosis

• COAGULASE (STAPHYLOCOAGULASE)

• HYALURONIDASE (SPREADING FACTOR)

• STAPHYLOKINASE (FIBRINOLYSIN)

• LIPASE (FAT-SPLITTING ENZYME)

• DEOXYRIBONUCLEASE (DNASE) AND PHOSPHATASE

• PROTEASE

• GELATINASE

• B-LACTAMASE

ENUMERATE

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

COAGULASE (STAPHYLOCOAGULASE)

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

• coagulates fibrinogen in the plasma

• promotes fibrin layer formation around the staphylococcal abscess protecting the bacteria from phagocytosis

• Cell-bound Coagulase of Clumping Factor

• Unbound or Free Coagulase

ENUMERATE

2 TYPES OF STAPHYLOCOAGULASE

Cell-bound Coagulase of Clumping Factor

2 TYPES OF STAPHYLOCOAGULASE

• bound the cell wall and clots human, rabbit, or pig plasma

Unbound or Free Coagulase

2 TYPES OF STAPHYLOCOAGULASE

• extracellular enzymes not bound to the cell wall and cause clot formation when bacterial cells are incubated with plasma

HYALURONIDASE (SPREADING FACTOR)

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

• hydrolyzes hyaluronic acid present in the intracellular ground substance, permitting the spread of infection

STAPHYLOKINASE (FIBRINOLYSIN)

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

• fibrinolytic activities by dissolving fibrin clot

LIPASE (Fat-Splitting Enzyme)

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

• act on lipids present on the surface of the skin, particularly fats and il secreted by the sebaceous glands

LIPASE (Fat-Splitting Enzyme)

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

• important in the formation of furuncles, carbuncles, and boils

T

T or F

LIPASE (Fat-Splitting Enzyme) are produced by both coagulase (+) and coagulase (-) staphylococci

DEOXYRIBONUCLEASE (DNASE) AND PHOSPHATASE

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

• lowers viscosity of exudates giving the pathogen more mobility

• destroys DNA

B-LACTAMASE

(VIRULENT FACTORS) ENZYMES OF S. AUREUS

• breaks down Penicillin and B-lactam drugs

1. CYTOTOXIC TOXINS

   • Alpha (α)-hemolysin

   • Beta (β)-hemolysin

   • Delta (δ)-hemolysin

   • Gamma (γ)-hemolysin

   • Staphylococcal (Panton-Valentine) Leukocidin

2. ENTEROTOXINS

3. TOXIC SHOCK SYNDROME TOXIN-1 (TSST-1)

4. EXFOLIATIVE TOXIN

ENUMERATE

(VIRULENT FACTORS) TOXINS OF S. AUREUS

• Alpha (α)-hemolysin

• Beta (β)-hemolysin

• Delta (δ)-hemolysin

• Gamma (γ)-hemolysin

ENUMERATE

4 Types of Hemolysins

Alpha (α)-hemolysin

4 Types of Hemolysins

• damage RBC, platelets, and macrophages and cause severe tissue damage

Alpha (α)-hemolysin

4 Types of Hemolysins

• predominant hemolysin of S. aureus

• Beta (β)-hemolysin / Sphingomyelinase C

4 Types of Hemolysins

• acts on sphingomyelin in the plasma membrane of RBC

• Beta (β)-hemolysin / Sphingomyelinase C

4 Types of Hemolysins

• aka “hot-cold” lysine

• enhanced hemolytic activity in incubation at 37 C (heat labile) and subsequent exposure to cold (4 C)

Delta (δ)-hemolysin

4 Types of Hemolysins

• produced by all S. aureus strain that cause RBC injury in culture and produce edematous lesions

• less toxic to cells than either a-hemolysin of B-hemolysin

Gamma (γ)-hemolysin

4 Types of Hemolysins

• associated with Panton-Valentine Leukocidin (PVL)

Staphylococcal Leukocidin / Panton-Valentine Leukocidin

exotoxin lethal to polymorphonuclear leukocytes ;

associated with community-acquired staphylococcal infections

Staphylococcal Leukocidin / Panton-Valentine Leukocidin

pore forming exotoxin that suppress phagocytosis and associated with severe cutaneous infections and necrotizing pneumonia

100° C

30 minutes

ENTEROTOXINS are heat-stable exotoxin, able to grown at __ for _

gastric and jejunal enzymes

ENTEROTOXINS resistant to hydrolysis by _

ENTEROTOXINS

act as neurotoxins that stimulate vomiting through the vagus nerve

30-5

ENTEROTOXINS are produced by _% of S. aureus isolates

ENTEROTOXINS A, B, and D

Staphylococcal food poisoning

Usually causes nausea, vomiting, abdominal cramps, and diarrhea.

Enterotoxins B and C (sometimes G and I)

Enterotoxins that causes Toxic Shock Syndrome (TSS)

Enterotoxin B

causes Staphylococcal Pseudomembranous Enterocolitis (from contaminated milk products)

• ENTEROTOXIN F

• PYROGENIC EXOTOXIN C

TOXIC SHOCK SYNDROME TOXIN-1 (TSST-1) aka?

TOXIC SHOCK SYNDROME TOXIN-1 (TSST-1)

• menstruating-associated TSS = TSS associated with tampon use

• chromosomal-mediated toxin

SUPERANTIGEN;

T-cell

cytokines

TSST-1 acts as a ___ stimulating __ proliferation and production of a large amount of _

• EPIDERMOLYTIC TOXIN A and B

• EXFOLIATIN serotypes A and B

EXFOLIATIVE TOXIN aka?

EXFOLIATIVE TOXIN

serine protease that divides the intracellular bridges of the epidermis and causes excessive sloughing of the epidermis (stratum granulosum)

Staphylococcal Scalded Skin Syndrome (Ritter’s Disease);

Bullous Impetigo

Exfoliative toxin causes ____ (___) and is implicated in ___

Folliculitis

mild inflammation of a hair follicle or oil gland; infected area is raised and red

raised, domed pustules form around hair follicles

Furuncles (Boils)

focal suppurative lesions which has resulted from an infection (folliculitis) that extend into subcutaneous tissue; large, raised, superficial abscesses

large abscess forms around a hair follicle

Carbuncles

larger, more invasive lesions develop from multiple furuncles, which can progress into deeper tissues; present with fever and chills, indicating systemic infection

multilocular abscess around several hair follicles

Bullous Impetigo

larger pustules surrounded by a small zone of erythema

highly contagious infection that spread by direct contact, fomites, or autoinoculation

Impetigo

superficial cutaneous infection commonly seen in newborns and young children characterized by the formation of encrusted pustules surrounded by red border

Scalded Skin Syndrome

bullous exfoliative dermatitis that occurs primarily in newborns and previously healthy young children

causes the skin to peel off in sheets

likely to occur in children under age 2.

• few blisters

• pemphigus neonatorum

• Ritter disease

Localized form of SSS

• cutaneous erythema

• profuse peeling of the epidermis

Generalized form of SSS

Toxic Epidermal Necrolysis (TEN)

clinical manifestation with multiple causes;

symptoms are due to hypersensitivity reaction

TOXIC SHOCK SYNDROME

rare but potentially fatal, multisystem disease characterized by sudden onset of fever, chills, vomiting, diarrhea, muscle aches, and rash, which can quickly progress to hypotension and shock

FOOD POISONING

intoxication resulting from ingestion of a toxin formed outside the body

symptoms appear rapidly (2 to 8 hours after ingestion) and resolve within 24 to 48 hours

nausea, vomiting, abdominal pain, and severe cramping, diarrhea

• Enterotoxin A (78%)

• Enterotoxin D (38%)

• Enterotoxin B (10%)

enterotoxins causing food poisoning

FOOD POISONING

perfuse and watery diarrhea due to water and electrolyte loss

food won’t appear or taste tainted

• Enterotoxin A

• LukE

• LukP

Virulence factors causing ENTEROCOLITIS

STAPHYLOCOCCAL BACTEREMIA

leads to secondary pneumonia and endocarditis observed among IV drug users

STAPHYLOCOCCAL OSTEOMYELITIS

secondary to bacteremia

STAPHYLOCOCCAL PNEUMONIA

• secondary to influenza virus infection

• multiple abscesses and focal lesions in the pulmonary parenchyma

SEPTIC ARTHRITIS

frequent in children and occur in patients with a history of rheumatoid arthritis or IV drug abuse

• Pus

• Purulent Fluids

• Sputum

• Urine

• Blood

Specimens used for S. aureus diagnosis

Gram (+) cocci in irregular clusters

Gram stain reaction of S. areus

Columbia Colistin-Nalidixic Acid (CNA)

purulent exudates of S. aureus should be grown in what type of medium?

MSA and PEA

heavily contaminated specimens of S. aureus should be grown in what type of medium?

CHROM Agar

growth medium for selective-differential for MRSA

Coagulase Test

best single criterion of recognition and pathogenicity of S. aureus

Slide Method

Coagulase test methods:

• rapid screening test

• detects cell-bound coagulase of clumping factor

Tube Method

Coagulase test methods:

• sensitive but definitive

• confirm all slide negative results

• detects extracellular or free coagulase

1-4 hours

35-37 C

Tube Method: incubate for ___ at ___

room temperature

20 hours

Tube Method: incubate for 1-4 hours at 35 C - 37 C; if no clot forms after 4 hours reincubate at ___ for additional ___

4 hours

false (-)

FIBRINOLYSIN

Coagulase test result should be read within ___ to prevent ___ reaction due to ___ which lyses the clot formed

Causes False (+) result for Pseudomonas and Enterococci

• use citrate and release calcium forming clot in the absence of coagulase

Why is coagulase plasma with citrate not suitable?

mannitol

(7.5-10 %)

S. aureus can ferment ___ and can tolerate high salt concentrations ___

positive result: yellow-colored colonies surrounded by a yellow halo

Mannitol Fermentation Test results for S. aureus

JET-BLACK COLONIES

S. aureus reduce tellurite (in Tellurite Glycine Agar) producing ___

• S. aureus = resistant

• other Staphylococci = susceptible

Polymyxin B Sensitivity results

• S. aureus = sensitive

• Micrococci = resistant

Lysostaphin Sensitivity Test (2 ug/mL) Results

S. intermedius

Voges-Proskauer (VP) test differentiate S. aureus (+) from _ (-)

positive

S. aureus = DNAse _