Staphylococcus & Other Related Organisms PDF

BROKENSHIRE COLLEGE, INC BACTERIOLOGY STAPHYLOCOCCUS & OTHER RELATED MEDICAL LABORATORY SCIENCE...

BROKENSHIRE COLLEGE, INC BACTERIOLOGY STAPHYLOCOCCUS & OTHER RELATED MEDICAL LABORATORY SCIENCE ORGANISM GRAM POSITIVE BACTERIA CLAASIFICATION BACITRACIN (0.04U) AND PROPERTIES PRINCIPLE:  The antibiotic bacitracin inhibits the synthesis of bacterial cell walls by interfering the peptidoglycan synthesis of bacteria.  A disk impregnated with a small amount of bacitracin (0.04 units) is placed on an agar plate, allowing the antibiotic to diffuse into the medium and inhibit the growth of susceptible organisms. GENERAL CHARACTERISTICS OF STAPH GROUP  Catalase producing gram (+) cocci that belong to the family Staphylococcaceae  Non-motile, non-spore forming, aerobic or facultative anaerobic except for S. aureus subsp. anaerobius and S. sacchrolyticus which are obligate anaerobes  Spherical cells (0.5 to 1.5 um) that appear singly, in FURAZOLIDONE SUSCEPTIBILITY TEST (100ug) pairs and in clusters and normal inhabitants of skins, mucous membranes and intestines.  BAP- colonies are medium sized (4-8 mm) and PRINCIPLE: appear cream colored white or rarely gold and  Furazolidone (furoxone) susceptibility test is buttery-looking, other spp may have gray colonies; performed as a disk susceptibility procedure using some may be Beta hemolytic (S. aureus) commercially available furazolidone disks.  Gram positive cocci  Staphylococci are inhibited by furazolidone, but ( from Greek staphyle, micrococci and related species are resistant. This is means bunch of grapes ) one of the commonly that occur singly and in used methods for pairs, short chains and differentiating irregular grape-like staphylococci from clusters. micrococci. Micrococci usually grow right up to the edge of the 6-mm furazolidone disk. DIFFERENCE TEST BETWEEN Staphylococci AND Micrococci LYSOSTAPHIN SENSITIVITY TEST (200ug/ml) PRINCIPLE:  The peptidoglycan Staphylococcus contain an interpeptide bridge consisting of glycine-rich peptides.  Lysostophin is an endopeptidase that cleaves these peptide linkages, rendering the cells susceptible to osmotic lysis (lysostaphin susceptible).  The interpeptide bridge of Micrococcus does not contain glycine Since the presence of glycine is essential for the action of lysostaphin, the Micrococcus cells are not affected and are lysostaphin resistant. BAUTISTA 3C  Staphylococci  Genera and Species to be considered:  Staphylococcus aureus  Coagulase Negative Staphylococci: (Most Commonly Encountered)  Staphylococcus epidermidis  Staphylococcus haemolyticus  Staphylococcus saprophyticus  Staphylococcus schleiferi  Coagulase Negative Staphylococci: (Less MODIFIED OXIDASE TEST Commonly Encountered)  Staphylococcus capitis  Staphylococcus caprae PRINCIPLE:  Staphylococcus warneri  Bacteria that produce cytochrome C produce a  Staphylococcus xylosus dark-blue end product when reacted with the  Staphylococcus auricularis modified oxidase reagent  Staphylococcus cohnii  Micrococcus organism possess cytochrome C in the  Staphylococcus simulans cytochrome oxidase system. Staphylococci do not. REAGENT:  6% tetramethylphenylenediamine hydrochloride in GENERAL CHARACTERISTICS OF Staphylococcus dimethyl sulfoxide or Microdase Disk (with auerus impregnated Mod Oxidase Reagent) EXPECTED RESULT:  45 species and 24 subsp  Positive: Dark Blue  Common inhabitant of the skin and mucous  Negative: No color Change membranes  Spherical cells arranged in irregular clusters  Gram-positive  Lack spores and flagella  May have capsules  Facultative anaerobe  Withstands high salt, extremes in pH, and high temperatures  Produces many virulence factors  Staphylococcus species Oxidizer Fermentation Test (OF Test) / Glucose contain teichoic acids in their Utilization Test cell walls  Cell walls of Micrococcus & Rothia species do not contain teichoic acid  Glucose fermenter: when acid production is detected on both tube LABORATORY DIAGNOSIS since fermentation can occur with or without oxygen  GRAM STAINED SMEAR  Glucose Oxidizer: acid  CULTURE MEDIA is detected by the open  BIOCHEMICAL TEST aerobic tube  CATALASE TES  Nonutilizer: some bacteria do not use glucose as a  COAGULASE TEST substrate  and Streptococcus mitis are a-hemolytic (the GRAM STAINING REACTION picture in the middle below shows the a-hemolysis of S. mitis).  Gram Positive Cocci in (Cluster)  If no hemolysis occurs, this is termed gamma-hemolysis. There are no notable zones around the colonies, gamma- hemolytic. CULTURE MEDIA i. Mannitol Salt Agar  Selective and Differential Media  Inhibitor: 7.5% NaCl ( S. aureus can tolerate high CULTURE MEDIA (Blood Agar) salt concentration )  Carbohydrate Source: Mannitol  pH indicator: Phenol Red  Both Differential and enriched Medium (5%  Original Color: RED- Pink defibrinated Sheep Blood)  S. Aureus - a light to golden yellow pigment,  S. epidermidis has a white pigment  S. saprophyticus either a bright yellow or white pigment.  On blood agar, S. aureus is usually beta-hemolytic, S. epidermidis and S. saprophyticus are almost ii. Tellurite Lysine Agar always nonhemolytic.  Differential and Selective Media for the isolation of C. diphtheriae; however, a few strains of streptococci and staphylococci are able to grow on this medium; C. diphtheriae (also Staphylococcus) produces gray to black colonies because the tellurite is reduced intracellularly to tellurium.  S. aureus: “jet black” colonies  Other Staphylococcus - inhibited but if there is a growth occurs as “gray colony” iii. Columbia Colistin-Nalidixic Acid  Selective medium used in the isolation of gram-positive cocci from clinical and non-clinical materials  Differential- Blood hemolysis  Antibiotic (Inhibit Gram Negative):  Colistin and Nalidixic Acid  Quadrant 1: growth on the plate indicates this organism, Enterococcus faecalis, is resistant BLOOD AGAR PLATE (BAP) to the antibiotics colistin and naladixic acid and is Gram-positive  Quadrant 2: absence of growth on the plate  Beta-hemolysis is complete indicates the organism, Enterobacter hemolysis. It is characterized by a aerogenes, is sensitive to the antibiotics and is clear (transparent) zone surrounding Gram-negative the colonies. Staphylococcus  Quadrant 3: absence of growth on the plate aureus, Streptococcus pyogenes indicates the organism, Escherichia coli, is and Streptococcus agalactiae are sensitive to the antibiotics and is b-hemolytic Gram-negative  Quadrant 4: growth on the plate indicates this  Partial hemolysis is termed organism, Staphylococcus aureus, is alpha-hemolysis. Colonies typically resistant to the antibiotics and is are surrounded by a green, opaque Gram-positive zone. Streptococcus pneumoniae  iv. Phenylethyl Alcohol vi. CHROM Agar  selective isolation of gram-positive cocci from  CHROMagar™ MRSA is a selective and differential clinical specimens containing gram-negative chromogenic medium for the qualitative direct organisms. detection of colonization by methicillin resistant Staphylococcus aureus (MRSA) to aid in the prevention and control of MRSA in healthcare ORGANISM EXPECTED RESULT settings PEA AGAR BIOCHEMICAL TEST Streptococcus pyogens Growth i. Catalase Test ATCC 19615  Catalase is an enzyme produced by microorganisms that live in oxygenated Staphylococcus aureus Growth environments to neutralize the bactericidal effects of ATCC 25923 toxic forms of oxygen metabolites such as hydrogen peroxide (H2O2).  Principle: Catalase mediates the breakdown of Proteus mirabilis Partial Inhibition hydrogen peroxide (H2O2) into oxygen and water. ATCC 12453 PEA ANAEROBIC AGAR Bacteroides fragilis Growth ATCC 25285 Streptococcus pyogens Growth ATCC 25285 Proteus mirabilis Partial Inhibition ATCC 12453 Procedure: SLIDE CATALASE 1) Transfer a small amount of bacterial colony to a surface of a clean, dry glass slide using a loop or v. Chapman Stone Agar sterile wooden stick (be sure the colony is visible to the naked eye on the slide).  A selective media used for the isolation of food 2) Place a drop of 3% H2O2 onto the slide and mix. poisoning staphylococci. Foods commonly 3) A positive result is the rapid evolution of oxygen contaminated with S. aureus included synthetic (within 5-10 seconds), as evidenced by bubbling. creams, custards and high-salted food. It is 4) A negative result is no bubbles or only a few selective, due to the relatively high salt content, and scattered bubbles.* is differential due to pigmentation, mannitol 5) Dispose of your slide in fermentation and the presence or absence of gelatin the biohazard glass liquefaction. disposal container. Interpretation Procedure: TUBE CATALASE  After incubation, cream to golden yellow colonies 1) Add 4 to 5 drops of 3% H2O2 to a test tube surrounded by clear zones are presumptively 2) Using a wooden applicator stick, collect a small identified as S.aureus. White or non-pigmented amount of organism from a colonies, with or without a clear zone, are well-isolated 18 to 24-hour presumptively identified as S. epidermidis. colony and place it into the Coagulase activity should be performed to confirm test tube (Note: Be careful the findings. Enterococci and/or Group D not to pick up any agar streptococci may exhibit growth on the medium and (especially if using Blood show slight mannitol fermentation. The colonies, Agar). however, are tiny and can easily be differentiated 3) Place the tube against a dark background and from staphylococci by gram stain and the catalase observe for immediate bubble formation (O2 + water test. = bubbles) at the end of the wooden applicator stick.  RESULTS develop, but re-examine any slow reacting  Catalase positive reaction: Evident by strains by the tube coagulase test. immediate effervescence (bubble formation)  Catalase negative reaction: No (effervescence)  OBSERVATION bubble formation or a few bubbles after 20  Coagulase positive: Macroscopic clumping in seconds. 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop.  Catalase Positive Organisms: Staphylococci,  Coagulase-negative: No clumping in either Micrococci, Bacillus species, Propionibacterium drop acnes, Helicobacter pylori, Haemophilus influenzae, Moraxella species, Neisseria species,  INTERPRETATION Pasteurella species, Brucella species  Slide coagulase test is the main method used to identify S. aureus in clinical laboratories but  Catalase Negative Organisms: Streptococci (For it has some limitations. example, Streptococcus agalactiae,  About 15% of ordinary strains of S. aureus and Streptococcus pneumoniae, Streptococcus many more of MRSA give negative reactions. pyogenes), Enterococcus species, Clostridium  Few species of coagulase-negative species (For example, Clostridium perfringens, staphylococci give positive reactions. Clostridium septicum, Clostridium tertium), Haemophilus ducreyi, Ekinella  Note: All coagulase-negative slides must be corrodens, Kingella species confirmed using a tube coagulase test as the definitive test for S. aureus.  Note: Some bacteria possess enzymes other than catalase that can decompose peroxide, a few tiny iii. Tube Coagulase Test bubbles forming after 20-30 seconds is not considered a positive test.  Detects free coagulase (staphylocoagulase) which reacts with coagulase-reacting factor (CRF). CRF is ii. Slide Coagulase Test a thrombin-like molecule. Staphylocoagulase and CRF combine to indirectly convert fibrinogen to  Done to detect “bound coagulase” or “clumping fibrin. A suspension of the organism is suspended factor” and incubated with plasma at 37°C. Clot formation  The clumping factor is a fibrinogen binding cell within 4 hours indicates a positive test. surface receptor present in the cell walls of most,  The coagulase clot thus formed can be destroyed but not all, S. aureus. by S. aureus fibrinolysin or staphylokinase, a  As the clumping factors are not present in all S. plasmid-carried enzyme that is more active at 35°C aureus, some strains will give a negative slide than at 25°C. So the tube coagulase tubes must be coagulase test. In addition, examined periodically (1, 2, and 4 hours) for clot clumping factors can be formation. Negative tubes should be held overnight masked by cell surface at room temperature. capsular polysaccharides. So, the negative slide PROCEDURE: tests must be confirmed 1) 0.5ml of diluted plasma, emulsify growth from by tube coagulase test. isolated colonies and incubate for 1-4 hours at 35-37 degree Celsius 2) Observe for the presence of a gel or a clot  Slide Coagulase Test Procedure 3) If no clot forms after 4 hours, reincubate at room temperature overnight 1) Emulsify a staphylococcal colony in a drop of water on a clean and grease-free glass slide  NOTE: Result should be read within 4 hours to with a minimum of spreading (If the isolate prevent a false (-) reaction because most strains of does not form a smooth, milky suspension, do S. aureus will produce a clot within this time and not proceed with the test). some strains produce a fibrinolysin which lyses the 2) Make similar suspensions of control positive clot formed within 4 hours. and negative strains to confirm the proper  Plasma with citrate is not reactivity of the plasma. suitable for this test because 3) Dip a flamed and cooled straight inoculating organism such as wire into the undiluted plasma at room Pseudomonas and temperature, withdraw, and stir the adhering Enterococci use the citrate traces of plasma (not a loopful) into the and release calcium, thus staphylococcal suspension on the slide. Flame forming a clot in the the wire and repeat for the control suspensions. absence of coagulase (false (+) result). 4) Read as positive a coarse clumping of cocci visible to the naked eye within 10 seconds. Read as negative the absence of clumping or any reaction taking more than 10 seconds to  vii. DNA Hydrolysis Test/DNAse Test PRINCIPLE:  The test is used to determine the ability of an organism to hydrolyze DNA. DNase agar is a differential medium that tests the ability of an organism to produce an exo-enzyme, called deoxyribonuclease. DNase are extracellular endonucleases that cleave DNA and release free nucleotides and phosphate.  DNase agar contains nutrients for the bacteria, DNA, and mostly methyl green as an indicator. PROCEDURE: 1) Using a sterile loop, inoculate the DNase agar with iv. NOVOBIOCIN SENSITIVITY TEST the organism to be tested on the test area. 2) Incubate the plate at 35-37°C for 24 hours. PRINCIPLE: 3) After incubation observe the color change in DNase  S. saprophyticus is second only to E. coli as the with methyl green. most frequent causative organism of uncomplicated 4) In DNase agar without indicators: urinary tract infections (UTIs) in young sexually 5) Flood the surface of agar with 1N HCL solution. Tip active women. So when an organism that looks like off the excess acid. staphylococci is isolated from a woman of 6) Allow the reagent to absorb into the plate. reproductive age coagulase test is done. If the 7) Observe for clear zone around the colonies within 5 organism is coagulase-negative i.e. CONS the minutes. laboratory must further identify that isolate and find out if this is a true pathogen (Staphylococcus  Expected Results saprophyticus-novobiocin resistant) or a  Positive: Medium is colorless around the test contaminant (i.e. Staphylococcus organism. epidermidis-novobiocin sensitive).  Negative: If no degradation of DNA occurs, the medium remains green.  PROCEDURE: 1) Allow containers to come to room temperature before use. 2) Using a pure 18-24 hour culture, prepare a suspension of the organism; equivalent to a McFarland 0.5 opacity standard; to be identified in tryptic soy broth, sterile water, or brain heart infusion (BHI) broth 3) Inoculate Mueller Hinton Agar, 5% blood agar, or tryptic soy agar plate with a sterile swab to obtain confluent growth. 4) Aseptically apply one 5ug novobiocin disk onto the inoculated agar surface and lightly press down to ensure full contact with the medium. 5) Incubate plate aerobically for 18 to 24 hours at 35 to 37°C. 6) Measure (in millimeters) the diameter of the DNAse Positive Organism zone of inhibition around the novobiocin disk, and record it as susceptible or resistant.  Used to differentiate Staphylococcus aureus  INTERPRETATION which produces the enzyme deoxyribonuclease  Resistant – zone size of < 12 mm from other Staphylococci which do not produce  Sensitive – zone size greater or equal to 16 DNase. mm.  It is also used to distinguish Serratia (positive)  Staphylococcus saprophyticus – growth < from Enterobacter sp. 12mm or uniform growth up to the edge of the  Moraxella catarrhalis (positive) from Neisseria disk  Staphylococcus epidermidis – Zone of inhibition >16 mm or larger Beta (β) Lactamase Test CULTURE MEDIA Objective:  To detect the enzyme beta-lactamase, which 8) Observe for a pink-red color at the surface within 30 confers penicillin resistance to various bacterial min. Shake the tube vigorously during the 30-min organisms. period. Principle:  INTERPRETATION:  Beta-Lactamase Test rapidly detects the presence  Positive of beta-lactamase enzyme produced by strains of Reaction: A pink-red Staphylococcus aureus, Neisseria gonorrhoeae, color at the surface Branhamella catarrhalis, and Haemophilus  Negative influenzae. These enzymes confer resistance to a Reaction: A lack of a number of penicillin antibiotics by cleaving the pink-red color beta-lactam ring of penicillin and cephalosporin antibiotics, resulting in the inactivation of these drugs. This mode of action forms the basis of the beta-Lactamase test reaction. Pyrrolidonyl Arlamidase (PYR) Test  Differentiate coagulase (+) Staphylococci by slide method  Principle: PYR is a rapid method for presumptive identification of bacteria based on the pyrrolidonyl arylamidase enzyme. The enzyme L-pyrrolidonyl arylamidase hydrolyzes the Pyroglutamyl-β Naphthylamide (L-pyrrolidonyl- β-naphthylamide) substrate to produce a L-pyrrolidone and β-naphthylamine. The β-naphthylamine can be  Result Interpretation of Beta (β)-Lactamase Test detected in the presence of N,N-methylaminocinnamaldehyde reagent by the  Positive reaction: yellow to red color change production of a bright red precipitate. on the area where the culture is applied.  Following hydrolysis of the substrate by the  Note: For most bacterial strains a positive peptidase, the resulting b-naphthylamide produces result will develop within 5 minutes. However, a red color upon the addition of 0.01% positive reactions for some staphylococci may p-dimethylaminocinnamaldehyde reagent. When a take up to 1 hour to develop, and color change visible inoculum of microorganism is rubbed onto a does not usually develop over the entire disk. small area of a disk impregnated with the substrate,  Negative reaction: no color change on the the hydrolysis occurs within 2 min, at which time the disc cinnamaldehyde reagent is added to detect the reaction by a color change to purple. Procedure of PYR Test: Voges-Proskauer (VP) Test  Broth Method 1) Inoculate PYR broth with 3-5 colonies from  Differentiate S. aureus from S. intermedius 18-24 hours pure culture. 2) Incubate the tube aerobically at 35-37°C for 4 PRINCIPLE: hours.  used to determine if an organism produces 3) Add 2-3 drop of PYR reagent and observe for acetylmethyl carbinol from glucose fermentation. color change. 4) Observe for the red color development within Culture Medium: 1-2 minutes.  VP broth with 5 % Glucose  Disk Method (Rapid)  Reagent: Alpha-Naphthol and KOH 1) Wet the PYR test disc on the strip with 10 µl sterile distilled water or deionized water. PROCEDURE: 2) Note: Do not flood the disk. 1) Prior to inoculation, allow medium to equilibrate to 3) Put 5-10 colonies of the tested strain from room temperature. 18-24 hours culture on the surface of the disc 2) Using organisms taken from an 18-24 hour pure with a loop and smear them lightly on it. culture, lightly inoculate the medium. 4) Incubate the disc for 1-2 minutes at room 3) Incubate aerobically at 37 degrees C. for 24 hours. temperature. 4) Following 24 hours of incubation, aliquot 2 ml of the 5) After incubation, add 1 drop of N, broth to a clean test tube. N-dimethylaminocinnamaldehyde. 5) Re-incubate the remaining broth for an additional 24 6) Observe for red color development within 1-2 hours. minutes. 6) Add 6 drops of 5% alpha-naphthol, and mix well to aerate.  INTERPRETATION: 7) Add 2 drops of 40% potassium hydroxide, and mix well to aerate.  Positive: Bright pink or cherry-red color within 1-2  Medically Important Staphylococcus spp minutes.  Examples: Group A Streptococci (Streptococcus GENERAL CHARACTERISTICS: pyogenes), Group D Enterococci (Enterococcus  Grows well on most laboratory media like NA or faecalis and Enterococcus faecium), Coagulase TSA negative Staphylococcus species such as S.  Sheep Blood Agar - used for primary isolation from hemolyticus, S. lugdunensis, S. Clinical Specimen schleiferi., Enterobacter, Citrobacter, Klebsiella,  Colonies on solid media are round, smooth, opaque Yersinia and Serratia, Aerococcus, Gamella, and butyrous Lactococcus, most Corynebacterium  Facultative anaerobes and exhibits golden yellow (Arcanobacterium) hemolyticum. pigmentation due to the action of the pigment  Negative: No color change or a blue color due to a STAPHYLOXANTHIN positive indole reaction.  Blood agar- a zone of beta hemolysis surrounds the  Examples: Group B Streptococci (Streptococcus colonies agalactiae), Streptococcus mitis, S. bovis, S.  Ubiquitous, can grow in 7.5-10% NaCl, reduces equinus, S. milleri. nitrate to nitrite Summary of Biochemical Reactions for S. aureus Pathogenic factors from a whole cell and S. intermedius group S. aureus The Gram-Positive Cocci of Medical Importance  Staphylococci : 45 species and 24 subsp. General Characteristics  Common inhabitant of the skin and mucous membranes  Spherical cells arranged in irregular clusters  Gram-positive  Lack spores and flagella  May have capsules  Facultative anaerobe  Withstands high salt, extremes in pH, and high temperatures  Produces many virulence factors Staphylococci Coagulase-positive  S. aureus – coagulates plasma and blood; produced by 97% of human isolates; diagnostic Staphylococci Coagulase-negative staphylococcus; frequently involved in nosocomial and opportunistic infections  S. epidermidis – lives on skin and mucous ANTIGENIC STRUCTURE: membranes; endocarditis, bacteremia, UTI  Polysaccharide Capsule  S. hominis – lives around apocrine sweat glands  Peptidoglycan  S. capitis – live on scalp, face, external ear  Teichoic acid  All 3 may cause wound infections by penetrating  Protein A through broken skin  Clumping Factor  S. saprophyticus – infrequently lives on skin, intestine, vagina; may cause UTI CAPSULAR Staphylococcus aurues  capsular antigens are surface-associated, limited in antigenic specificity, and highly conserved among clinical isolates. Capsule inhibits phagocytosis,  promotes adherence of the organism to host cells  MSCRAMM ( Microbial Surface Components and in prosthetic devices. Recognizing Adhesive Matrix Molecules”) Transmission electron micrographs of S. aureus cells cultured on agar plates. TEICHOIC ACID PROTEIN A  WTAs are required for ß-lactam resistance in methicillin-resistant S. aureus (MRSA), and they modulate susceptibility to cationic antibiotics in  Has the ability to bind to Fc several organisms. portion of Immunoglobulin.  The Fc portion of the antibody IgG, the portion that would normally binds to Fc receptors on phagocytes, instead binds to Protein A on Staphylococcus aureus.  Thus the bacterium becomes coated with a protective coat of antibodies that do not allow for opsonization Freely secreted Protein A binds the Fc regions of antibody (Ab) thereby can prevent phagocytosis Freely secreted Protein A can manipulate B Cells  In addition to its Fc Binding capacity, Protein A binds Fab regions of the B-cell receptor (membrane-anchored IgM)  Teichoic acids contribute to staphylococcal adhesion and colonization, cell division, and biofilm formation. Overexpression of teichoic acid increases the virulence of S. aureus.  In addition, D-alanine (D-Ala) residues on teichoic acids contribute to resistance to cationic antimicrobial peptides such as defensins or cathelicidins, and to glycopeptide antibiotics such as vancomycin or teicoplanin. Find more about Teichoic acids (TA). PEPTIDOGLYCAN  Give rigidity to the cell wall, activates complement. Find more about peptidoglycan   Act on lipids present on the surface of the skin particularly fats and oils secreted by the sebaceous glands  Important in the formation of furancles, carbuncles and boils iii. Gelatinase  allows the organisms that produce it to break down gelatin into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the organism. When gelatin is broken down, it can no longer solidify. If an organism can break down gelatin, the areas where the organism has grown will remain liquid even if the gelatin is refrigerated. Clumping Factors iv. Hyaluronidase (Spreading Factor)  Hydrolyzes hyaluronic acid present in the intracellular ground substance, permitting the  Component of CW of S.aureus spread of infection  is a fibrinogen-binding surface protein of S. aureus. v. Deoxyribonuclease (DNAse) and Phosphatase  Acts as a virulence factor in certain infections by  Lowers viscosity of exudates giving the inhibiting phagocytosis, as well as promoting pathogens more mobility destroys DNA adhesion to fibrin and fibrinogen. vi. Staphylokinase (Fibrinolysin)  activates human plasminogen (h-plg) into plasmin. Plasmin in turn digests fibrin clots and many components of extracellular matrix and basal membranes, enhance staphylococcal escape from the fibrin clots and facilitate systemic bacterial dissemination from the infection site vii. Protease  Breakdown of peptide bond in amino acids of protein molecule. c) Toxins i. Cytolytic Toxins  2 Types (Hemolysin & Leukocidin) 1) Hemolysin  Alpha Toxins (Alpha Hemolysin) - penetrates host cell membranes causing osmotic swelling, rupture, DETERMINANTS OF PATHOGENICITY lysis and subsequently cell death.  Beta Toxin ( Staphylococcal sphingomyelinase) - toxic to a variety a) Surface Antigens of cells, including erythrocytes, fibroblasts, leukocytes, and i. Polysaccharides: enables organism to resist macrophages. Susceptible cells are phagocytosis subject to lysis of exposed ii. Protein receptor: (e.g Fibrinectin, Fibrinogen, IgG sphingomyelin on their membrane and C1q) surfaces. Produce “hot-cold lysis” iii. Peptidoglycan and teichoic acids - aids  Delta Toxin - less toxic to cells, organism’s attachment to mucous memebranes, produced by all S. aureus strain that enables organism to resist unfavorable condition/ cause RBC injury in culture and prevent from cell lysis produce edematous lesions  Gamma Toxin - play a role in the b) Extracellular Enzymes pathogenesis of toxic shock syndrome (TSS) together with toxic i. Coagulase shock syndrome toxin 1 (TSST-1)  binds to prothrombin, together they become enzymatically active and intiate fibrin 2) Leukocidin (Phanton Valentine polymerization; may coat neutrophil to protect Laukocidins (PVL)) organism from phagocytosis.  lysis neutrophils and macrophages; ii. Lipase (Fat-Splitting Enzyme) inhibit phagocytes; lytic activity is due  Produce by both Coag (+ and -) Staphylococci to an alteration of the activity of the Na+/K+ pump,   associated with community acquired  Carbuncles staphylococcal infection  larger, more invasive lesions develop from multiple furuncles, which can progress into ii. Enterotoxins deeper tissues  Heat stable exotoxin: 100 degree Celsius for 30  present with fever and chills, indicating minutes. systemic infection.  Resistant to hydrolysis by gastric and jejunal enzymes  Bullous Impetigo  Act as neurotoxins that stimulate vomiting through  larger pustules surrounded by a small zone of the vagus nerve erythema  Group of seven heat stable proteins: A,B,C, C2, D,  highly contagious infection that spread by E and F direct contact, fomites, or autoinoculation  A-E (Food Poisoning)- heat stable and they act to stimulate neural receptors in the GI tract causing  Impetigo pain, vomiting, diarrhea within 6 hours of ingestion  superficial cutaneous infection  Toxic Shock Syndrome: TSST-1 (Enterotoxin F/  commonly seen in newborns and young Pyrogenic Exotoxin C) children characterized by the formation of  Menstruating-associated TSS= TSS associated encrusted pustules surrounded by red border with tampon use  Chromosomal –mediated toxin  Scalded Skin Syndrome  Superantigen stimulating T-cell production and  Bullous exfoliative dermatitis that occurs production of large amount of cytokines primarily in newborns and previously healthy  Low concentrations= leakage by endothelial young children cells  Localized skin lesions:  Higher concentrations= cytotoxic  Few blisters  Pemphigus neonatorum iii. Exfoliative Toxin (Epidermolytic Toxin A and B  Ritter Von Ritterschein Disease or Exfoliatin serotype A and B  Generalized from cutaneous erythema  hydrolyze tissue through cleavage of stratum  Profuse peeling of the epidermis granulosum, causes Staphylococcal Scalded Skin Syndrome, Toxic Shock Syndrome  implicated in Bullous Impetigo  Rare but potentially fatal, multisystem disease  Preformed, heat-resistant enterotoxin mediates characterized by sudden onset of fever, chills, staphylococcal food poisoning (symptoms in 2-6 vomiting, diarrhea, muscle aches, and rush, which hours; usually self-limiting) can quickly progress to hypotension and shock  Exfoliative toxins A and B results in staphylococcal scalded skin Food Poisoning  Enterotoxin A (78%), D (38%) and B (10%) iv. Beta-lactamase (Penicillinase)  Intoxication resulting from ingestion of a toxin  Hydrolysis and inactivation of penicillin antibiotic formed outside the body through breakdown of beta lactam ring in penicillin  Symptoms appear rapidly (2-8 hour after ingestion) molecule and resolve within 24 to 48 hours  Profuse and watery diarrhea due to water and v. Slime Production electrolyte loss  extracellular glycoconjugate that helps the organism  Food won’t appear or taste tainted to adhere to smooth surface and is produced by  Death: intoxication rather than infection CoNS as well as S. aureus  Allows colonization of indwelling catheters, a major Staphylococcal Bacteremia problem in hospitalization patients ( Nosocomial  Large to secondary pneumonia and endocarditis Infection) observed among IV drug users Staphylococcal Osteomyelitis CLINICAL INFECTION  Secondary to bacteremia Staphylococcal Pneumonia Cutaneous Infection  Secondary to influenza virus infection  Multiple abscesses and focal lesions in the  Folliculatis pulmonary parenchyma  mild inflammation of a hair follicle or oil gland  Infected is raised and red Septic Arthritis  Frequent in children and occur in patient with a  Furuncles (boils) history of rheumatoid arthritis or IV drug abuse  focal suppurative lesions which has resulted from an infection (folliculitis) that extend into Acute Bacterial Endocarditis UTI subcutaneous tissue  SAU clinical infection  large, raised, superficial abscesses