Gram Positive Cocci Part 1 PDF

Gram Staining: Review It separates bacteria into two large groups (Gram-positive and Gram-negative) based Heat fixation on their different cell wall constitue...

Gram Staining: Review It separates bacteria into two large groups (Gram-positive and Gram-negative) based Heat fixation on their different cell wall constituents. Crystal Violet (primary stain) REMEMBER: VIAS Gram’s Iodine (mordant) Acetone-alcohol (decolorizer) Safranin (counter stain) Bacterial Shapes: Review Three Basic Shapes of Bacteria Coccus Bacillus Spiral Spherical or round Rod shape Spiral shape shape Gram-Positive Cocci Arrangements of Cocci Bacteria (Microscopic Morphology) Diplococci Streptococci Staphylococci Cocci in pairs Cocci in chains Cocci in clusters Tetrad Sarcina Cocci in fours Cocci in eights or octet (cube) Gram-Positive Bacteria  They are initially differentiated by the The catalase test is used to differentiate staphylococci (+) catalase test. from streptococci (–).  This test determines the presence of the enzyme catalase  Catalase is an enzyme that breaks down the harmful substance hydrogen peroxide into water and oxygen.  If an organism can produce catalase, it will produce bubbles of oxygen when hydrogen peroxide is added to it.  Positive result: effervescence  Catalase-positive: Staphylococci  Catalase-negative: Streptococci Gram-Positive Bacteria  The catalase enzyme is a significant virulence factor (disease-causing factor) for Staphylococcus spp.  Staphylococcal catalase protects intraphagocytic microbes by destroying hydrogen peroxide produced by the phagocyte  The catalase test detects the presence of this enzyme Gram-Positive Bacteria A positive catalase test is indicated by effervescence Positive result: effervescence or formation of bubbles Catalase-positive: Staphylococci Catalase-negative: Streptococci Genera to be Considered  Staphylococcus aureus (coagulase-positive)  Coagulase-negative staphylococci (most common ones)  Staphylococcus epidermidis  Staphylococcus haemolyticus  Staphylococcus saprophyticus  Staphylococcus lugdunensis  Micrococcus spp.  Rothia, Aerococcus, Alloiococcus spp. Overview  Gram-positive cocci are those that stain positive with the Gram staining method (purple color)  The peptidoglycan layer is substantially thicker in Gram- positive bacteria  Common isolates in the clinical microbiology laboratory.  Most Gram-positive cocci are members of the indigenous microbiota (normal flora)  Some species are pathogenic (disease-causing) Staphylococci  Gram-positive cocci  They produce the enzyme catalase  Exhibit spherical shapes (0.5 to 1.5 µm) that may appear singly, in pairs, and in clusters.  The microscopic appearance is NOT specific or characteristic for staphylococci  They resemble some members of the family Micrococcaceae, such as the genus Micrococcus Staphylococci  They are non-motile  They are non-spore-forming  They may be aerobic or facultatively anaerobic, except for Staphylococcus saccharolyticus (obligate anaerobe)  Colonies (after 18-24 hours of incubation at 37oC): medium-sized (4-8 mm) and appear cream-colored, white or rarely light gold, and buttery-looking  Some are -hemolytic  They are one of the most commonly isolated organisms Reference: Tille, Patricia. (2014). Staphylococcus, Micrococcus, and Similar Organisms. Bailey & Scotts Diagnostic Microbiology, 13th Edition (page 233). St. Louis, Missouri: Elsevier-Mosby. Staphylococci Staphylococcus aureus (Blood Agar Plate) Staphylococcus aureus (Gram stain) Notice the -hemolytic pattern (clear zone Notice the Gram-positive spherical bacterial around the bacterial colony) on sheep blood cells. They may appear singly, in pairs, or in agar plate. Colonies are medium-sized and clusters. buttery-looking. Staphylococci  They are initially differentiated by the coagulase test.  This test determines the presence of the enzyme staphylocoagulase  This enzyme is a virulence factor for Staphylococcus aureus staphylocoagulase Coagulase-reacting factor (CRF) Coagulase-CRF complex Coagulase-CRF Fibrinogen Fibrin (clot) complex  The positive result is the formation of clot in plasma  Staphylococci that are able to produce this enzyme are called coagulase-positive staphylococci Staphylococci Free, extracellular coagulase (staphylocoagulase) Remember! Coagulase Test Bound or cell-bound coagulase Slide = Bound (clumping factor) Tube = Free Slide Tube Slide Tube (bound coagulase) (free coagulase) (bound coagulase) (free coagulase) Positive Coagulase Test Negative Coagulase Test Organism: Staphylococcus aureus Organism: Staphylococcus epidermidis Notice the formation of clot in plasma Notice the absence of clot in plasma Staphylococci Coagulase-positive staphylococci (CoPS):  Staphylococcus aureus – most commonly isolated, most virulent and clinically significant CoPS Animal-associated species (less frequently isolated from human samples):  Staphylococcus intermedius  Staphylococcus delphini  Staphylococcus lutrae  Some strains of Staphylococcus hyicus Staphylococci Coagulase-negative staphylococci (CoNS):  Clinically significant and commonly recovered CoNS:  Staphylococcus epidermidis – nosocomial infection  Staphylococcus saprophyticus – urinary tract infection  Occasional isolates and can be significant pathogens:  Staphylococcus haemolyticus – wound, septicemia (blood), UTI, native valve infections  Staphylococcys lugdunensis – catheter-related bacteremia and endocarditis  Staphylococci Coagulase as a virulence factor Bound coagulase (clumping factor) Fibrin capsule Coagulase-positive staphylococci coagulase Fibrin-encapsulated staphylococci The fibrin capsule may protect the bacterium from Coagulase-positive staphylococci showing phagocytosis and isolate it from other defenses of bound coagulase (clumping factor) on the the host. The fibrin coat or capsule can therefore bacterial cell wall. make the bacteria more virulent. Staphylococcus aureus  Most clinically significant species of staphylococci  Can be recovered from almost any specimen  An important cause of nosocomial (healthcare-acquired) and community-acquired infections  Associated with suppurative skin and wound infections  Abscess filled with pus and surrounded by necrotic tissue and damaged leukocytes  Examples: folliculitis, furuncles (boils), carbuncles (multiple furuncles with fever and chills), and bullous impetigo (a highly contagious infection; larger postules surrounded by small zone of erythema) Staphylococcus aureus  Staphylococcus: Greek „staphle‟ means bunches of grapes  aureus: Latin „aurum‟ means „golden‟ On microscopy of Gram-stained bacterial smears, S. aureus cells occur in grape-like clusters, may occur singly or in pairs. Colonies produce a golden pigment staphyloxanthin Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Enzymes: catalase, staphylocoagulase, clumping factor, hyaluronidase, protease, lipase, nuclease, fibrinolysin  Enterotoxins  Toxic shock syndrome toxin-1 (TSST-1)  Exfoliative toxin  Cytolytic toxin  Protein A Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Enzymes: coagulase, hyaluronidase, lipase, protease  Coagulase (Staphylocoagulase)  Bound coagulase (or clumping factor) – bound to cell wall of the bacterium  Has the ability to stimulate clot formation in plasma  Facilitates in the conversion of fibrinogen to fibrin in the immediate vicinity of the bacterium as means of self- protection  fibrin capsule  inhibits phagocytosis  Mainly produced by S. aureus; coagulase is also produced by other coagulase-positive staphylococci and Yersinia pestis Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Enzymes: coagulase, hyaluronidase, lipase, protease  Hyaluronidase  This enzyme hydrolyzes hyaluronic acid that makes up your connective tissues  Damage to connective tissues will permit the spread of bacteria during infection Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Enzymes: coagulase, hyaluronidase, lipase, protease  Lipase  This enzyme is produced by both coagulase-positive and coagulase-negative staphylococci  Lipase acts on the lipids present on the surface of the skin, particularly the fats and oil secreted by the sebaceous glands  Acting together with protease and hyaluronidase, these enzymes are capable of destroying tissue and may facilitate the spread of infection to adjoining tissues Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Enterotoxins  Staphylococcal enterotoxins are heat-stable exotoxins (stable at 100oC for 30 minutes)  reheating contaminated food does not prevent disease or infection  Cause symptoms such as vomiting and diarrhea  Staphylococcal food poisoning is most commonly caused by enterotoxins A, B, and D  BAD enterotoxins  Enterotoxins B and C (sometime G and I) are associated with toxic shock syndrome  Enterotoxin B is associated with staphylococcal pseudomembranous enterocolitis Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Toxic Shock Syndrome Toxin-1 (TSST-1)  Causes nearly all cases of menstruating-associated toxic shock syndrome  Previously called enterotoxin F  A superantigen that stimulates T-cell proliferation and subsequent production of cytokines that are responsible for the symptoms  At low concentrations, TSST-1 causes leakage by endothelial cells  It is cytotoxic to these cells at higher concentrations Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Exfoliative Toxin  Also called epidermolytic toxin  It causes the epidermal layer of the skin to slough off  Associated with scalded skin syndrome (SSS), which is sometimes referred to as Ritter disease; it is a bullous exfoliative dermatitis primarily in newborns and previously healthy young children  Also has been implicated in bullous Scalded skin impetigo syndrome Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Cytolytic Toxin  An extracellular protein that affect red blood cells and white blood cells  Hemolysins – affects red blood cells; cause hemolysis!  Leukocidin (loo-kow-side-in) – affects white blood cells  S. aureus produces four (4) hemolysins: alpha-, beta-, gamma-, and delta-hemolysin  Beta-hemolysin (sphingomyelinase C, “hot-cold” lysin) – acts on the sphingomyelin in the RBC plasma membrane  Gamma-hemolysin is associated with the Panton-Valentine Leukocidin (PVL), which is lethal to neutrophils and contributes to the invasiveness of the organism by suppressing phagocytosis; associated with severe skin infections and necrotizing pneumonia Staphylococcus aureus Disease-Causing Factors (or Virulence Factors):  Protein A  Also identified in the cell wall of Staphylococcus aureus  Its most significant role is its binding ability to the Fc portion (crystallizable fragment) of immunoglobulin G (IgG) antibodies.  Binding IgG inhibits opsonization, which is an important step in phagocytosis and negates the protective effect of IgG antibody Opsonization Reference: Tille, Patricia. (2014). Staphylococcus, Micrococcus, and Similar Organisms. Bailey & Scotts Diagnostic Microbiology, 13th Edition (page 233). St. Louis, Missouri: Elsevier-Mosby.  Identification Scheme for Staphylococci: Proceed to Gram staining Gram Stain *MSA – mannitol salt agar Beta-hemolytic colonies after 18-24 MSA-negative: hours of incubation Other staphylococci Gram-positive cocci Gram-negative Proceed to catalase test if Gram (+) Catalase-negative: Catalase-positive: Streptococci Staphylococci Proceed to coagulase test if catalase (+) Coagulase-negative: – + Coagulase-positive: MSA-positive: Other staphylococci Staphylococcus aureus Staphylococcus aureus  Catalase-negative: Catalase-positive: Streptococci Staphylococci Proceed to coagulase test if catalase (+) – + Coagulase-negative: Coagulase-positive: Other staphylococci Staphylococcus aureus Proceed to novobiocin sensitivity test if coagulase-negative Novobiocin-sensitive: Novobiocin-resistant: S. epidermidis S. saprophyticus S. aureus “resistant” is indicated by a “sensitive” is indicated by the small or absence of zone of presence of a zone of inhibition of colonies around inhibition of colonies around the antimicrobial disk the antimicrobial disk  Key Tests for the Identification of the Most Clinically Significant Staphylococcus Species Affecting Humans Test S. aureus S. epidermidis S. saprophyticus Gram staining Gram (+) Gram (+) Gram (+) Colony pigment Positive Negative Negative Staphylocoagulase Positive Negative Negative Clumping factor Positive Negative Negative Mannitol fermentation Positive Negative Negative Novobiocin susceptibility testing Sensitive Sensitive Resistant