MT 37 (LEC): Gram-positive cocci 1

Micrococcaceae, Staphylococcaceae

Cocci Classifications (Family)

MICROCOCCI

• gram + cocci in the family: micrococcaceae

• Normal flora: skin, mucosa, and oropharynx

• contaminants of clinical specimens

• rarely implicate as cause of infections in humans

MICROCOCCI

• May cause brain abscess, meningitis, pneumonia, and endocarditis in immunocompromised patients

MICROCOCCI

Gram-positive cocci in packets of four (tetrads) or eight cocci

M. Flavus, M. Luteus, M. Lylae, M. Roseus, M. Dentrificans, M. Varians, M. Freudenreichii

Species from the genus Microccocus

M. roseus

Micrococcus Found in the dust of the air

M. dentrificans

Micrococcus found in soil

M. colpogenes

Micrococcus found in marine waters

M. flavus

• Microccocus in Skin, skin glands, or skin-gland secretions of vertebrates

• Usually found in humans

M. luteus, M. varians, and M. freudenreichii

• “Milk micrococci”

• Cause spoilage of milk

M. lylae

microccocus found on the skin, dust, and water

STAPHYLOCOCCI

• Growth requires supplementation with various amino acids and other growth factors (Enriched Media)

• NF: skin, mucous membrane, and intestine

STAPHYLOCOCCI

Facultative anaerobe & catalase (+)

Facultative anaerobe

• It can thrive with or without the presence of oxygen

• with oxygen = aerobic respiration

• without oxygen = fermentation to produce ATP

Catalase

• enzyme that detoxifies hydrogen peroxide to prevent bacterial cell wall disruption

• Results to the “bubble” reaction if positive

• produced by Staphylococci

Positive catalase reaction

• bubble forms due to the conversion of hydrogen peroxide (H2O2) into H2O + O2

Staphylococci

• Nonmotile, non-spore-former, and glucose fermenter

Staphylococci

Grows in 7.5% NaCl (halotolerant)

Staphylococci

Resistant to heat and drying, and thus can persist for long periods on fomites (inanimate objects)

Staphylococci

• Species are initially differentiated by coagulase test

Coagulase test

• enzyme causes coagulation by converting fibrin to fibrinogen

• Coagulation in tube test

• Agglutination in plasma (slide test)

Blood Agar Plate (BAP)

Culture media for STAPHYLOCOCCI

Staphylococci in BAP

• 4-8mm colonies,

• creamy, white to light gold,

• “buttery-looking” (unique charact.)

• some species are grayish & ßhemolytic

Buttery-looking

Unique characteristic of staphylococcus growing in BAP

Alpha hemolysis, beta hemolysis, gamma (non-hemolytic)

Hemolytic Reactions in SBA

Alpha hemolysis

• green zone around colony

• Narrow or wide

• Partial lysis of RBCs

Beta hemolysis

• clear zone around colony

• Narrow or wide

• Complete lysis of RBCs

Gamma (non-hemolytic)

• no zone of hemolysis

• No lysis

Staphylococcus aureus

• Normal flora: Anterior nares, nasopharynx, perineal area, skin, and colonizer of mucosa

Staphylococcus aureus

• Endogenous strain: sterile site by traumatic introduction

• Direct contact: person-to-person, fomites

• Indirect contact: aerosolized

Staphylococcus epidermidis

• Normal flora: Skin, mucous membranes

Endogenous strain, Direct Contct

MOT of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis

Staphylococcus saprophyticus

Normal flora: skin, genitourinary tract, mucosa

Staphylococcus saprophyticus

• Endogenous strain: sterile urinary tract, notably in young, sexually active females

• Common cause UTI

Staphylococcus haemolyticus, Staphylococcus lugdunensis

• Normal flora: skin, mucous membranes (low numbers)

Staphylococcus aureus

• Most clinically significant species of staphylococci

• the most virulent species of staphylococcus

False, true-coagulase positive

S. aureus is true coagulase-negative (TRUE/FALSE)

Facultatively anaerobic

What type of anaerobe is S. aureus?

False, it is non-motile and non-spore-forming

S. aureus is motile, spore-forming (TRUE/FALSE)

Positive; negative

S. aureus is catalase ___ and oxidase ___

7.5%

S. aureus: Tolerance to ____ sodium chloride

Mannitol

What does S. aureus ferments what sugar alcohol?

Hyaluronidase

What enzyme does S. Aureus produce?

Capsule, slime layer, peptidoglycan, techoic acid, Protein A

Enumerate the VIRULENCE FACTORS

Virulence factors

• Helps the bacteria evade the immune system

Capsule

• Inhibits chemotaxis and phagocytosis;

• inhibits proliferation of mononuclear cells

Neutrophils, Macrophages, Phagocytes

Wbcs that perform phagocytosis

Biofilm

a thin, slimy film of bacteria that adheres to a surface.

Slime layer

• Facilitates adherence to foreign bodies;

• inhibits phagocytosis

• Prevents penetration of antibiotics

Slime layer

• Prevents penetration of antibiotics

Peptidoglycan

• Provides osmotic stability;

• inhibits phagocytosis

Peptidoglycan

• leukocyte chemoattractant (abscess formation);

Peptidoglycan

• stimulates production of endogenous pyrogen (endotoxin-like activity);

Abscess formation

• difficult to treat with antibiotics due to failure to penetrate

• Made by S. aureus

• can be removed through drainage (if deep within tissue)

Boils, furuncles, carbuncles

Examples of abscess formations due to S. aureus

Peptidoglycan

• thick layer in the cell wall in S. aureus

• What contains the crystal violet within the cell wall

Teichoic acid

• for adherence

• Binds to fibronectin

Protein A

• Inhibits antibody-mediated clearance by binding IgG, IgG2, and IgG4 Fc receptors;

• anti-complementary by creating a pore in the cell wall or cell-membrane

Protein A

• leukocyte chemoattractant;

Staphylocoagulase/Coagulase, Hyaluronidase, Lipase, DNAse and phosphatase, β-lactamase

Virulence factors: Enzymes and toxins produced by S. aureus

Staphylocoagulase/Coagulase

• Coagulates fibrinogen in plasma (causes abnormal clotting)

• Promotes the formation of a fibrin layer around the staphylococcal abscess thereby protecting the bacteria from phagocytosis

Cell-bound coagulase or clumping factor, Unbound or free coagulase

2 types of coagulase

Cell-bound coagulase or clumping factor

• Bound to the cell wall and clots human, rabbit, or pig plasma by directly converting fibrinogen into fibrin

• Detected by slide method

Slide method

• Detects Cell-bound coagulase or clumping factor

• Faster method compared to tube method

Unbound or free coagulase

• An extracellular enzyme that is not bound to the cell wall;

• Performed by tube method because it requires incubation

Tube method

• Detects Unbound or free coagulase

• Requires incubation (18-24 hours)

Unbound or free coagulase

• causes clot formation when bacterial cells are incubated with plasma

Hyaluronidase

• Spreading factor enzyme

• Helps the bacteria reach more places to infect

• Enhances invasion and survival of S. aureus in the tissue and skin

Hyaluronidase

Breaks down hyaluronic acid that is present in the intracellular ground substance of connective tissues, resulting in the spread of bacteria

fibrinolysin

Staphylokinase Is Also known as?

Staphylokinase

• Causes fibrinolytic activities by dissolving fibrin clots

• Proteolytic enzyme

• Helps S. aureus establish skin infections

Staphylokinase, Hyaluronidase

Enzymes that Help S. aureus establish skin infections

Lipase

• Produced by both coagulase-negative and coagulase-positive staphylococci

Fat-splitting enzyme

Lipase Is aka?

Lipase

• Essential for bacterial survival in the sebaceous areas of the body

• Found in armpits, hair follicles, and other areas that are rich in oil and fats

Lipase

Important in the formation of furuncles, carbuncles, and boils

DNAse and phosphatase

• Lower the viscosity of exudates giving the pathogen more mobility

Exudates

• mass of cells in fluid (nana)

• Viscous

DNAse and phosphatase

Destroys DNA

β-lactamase

• produced in response to antibiotics

• Breaks down penicillin and other β-lactam drugs

β- lactam drugs

• Inhibits cell wall synthesis like hindering peptidoglycan synthesis

Enterotoxins, Leukocidin, Exfoliatin serotypes A and B or Epidermolytic toxins A and B, Hemolysin, Toxic Shock Syndrome Toxin-1 (TSST-1)

VIRULENCE FACTORS: Toxins produced by S. aureus

Enterotoxins

• heat-stable at 100°C for 30 minutes

30 minutes

Enteroxins are heat-stable at 100°C for?

30% to 50%

Enterotoxins are Produced by ___% to ___% of S. aureus isolates

Enterotoxins

Resistant to hydrolysis by gastric and jejunal enzymes

Enterotoxins A, B, and D

• Enterotoxins responsible for food poisoning with A being the most common cause

Enterotoxins B, C, G, and I

• Enterotoxins associated with TSS (Toxic shock syndrome)

Enterotoxin B

• enterotoxin associated with pseudomembranous enterocolitis and is often found in contaminated milk products

Pseudomembranous enterocolitis

• mainly cause by Clostridium difficile and sometimes by S. aureus

Enterotoxin

• Neurotoxin

• stimulates the vagus nerve (a nerve responsible for nusea center of brain)

• if ingested by host, host keeps vomiting

Leukocidin

• Attacks and kills white blood cells (PMN, macrophage, and monocytes)

Panton-Valentine Leukocidin (Cytolytic toxin)

Leukocidin is aka?

Leukocidin

Pore-forming exotoxin that suppresses phagocytosis

Leukocidin

Responsible for severe cutaneous infections and necrotizing pneumonia

Exfoliatin serotypes A and B (superantigens)

• aka Epidermolytic toxins A and B

Exfoliatin serotypes A and B (superantigens)

• Divides the intracellular bridges of the epidermis resulting to extensive sloughing to produce burn-like effect

Superantigens

• overstimulates the immune system

• Causes the host to Excessively produce cytokines

• Eventually results to organ failure

Stratum granulosum

Exfoliatin serotypes A and B (superantigens) destroys what layer of the skin/

Bullous impetigo

Exfoliatin serotypes A and B (superantigens) Has been implicated in What disese?