Micro-1-Chapter-14.pdf

Source  Textbook of Diagnostic Microbiology 5th ed. By Mahon, Lehman, and Manuselis  Bailey and Scott’s Diagnostic Microbiology 13th ed. Objectives  Identify and differentiate Staphylococcus from Streptococcus in terms of general properties, pathogenesis, morphology, culture m...

Source  Textbook of Diagnostic Microbiology 5th ed. By Mahon, Lehman, and Manuselis  Bailey and Scott’s Diagnostic Microbiology 13th ed. Objectives  Identify and differentiate Staphylococcus from Streptococcus in terms of general properties, pathogenesis, morphology, culture media, colony characteristics, biochemical tests, and serological tests.  CHAPTER 14: STAPHYLOCOCCI GENERAL CHARACTERISTICS  Gram (+) cocci arranged in pairs, tetrads or clusters  Non‐motile  Non‐spore formers  Facultative anaerobes  Catalase (+); Oxidase (‐)  Grows in 7.5‐10% NaCl GENERAL CHARACTERISTICS  Colonies are medium sized (4 to 8 mm) and appear cream-colored, white or rarely light gold, and “buttery-looking.”  Some species are β-hemolytic. CLINICALLY SIGNIFICANT SPECIES 1. Staphylococcus aureus 2. Staphylococcus epidermidis 3. Staphylococcus saprophyticus 4. Staphylococcus lugdunensis 5. Staphylococcus haemolyticus  Staphylococcus aureus A. Virulence factors I. Enterotoxins Heat Stable exotoxins: A‐E and G‐J II. Toxic Shock Syndrome Toxin‐1 Formerly known as Enterotoxin F (Superantigens) Causes menstruating‐associated TSS III. Exfoliative Toxin Epidermolytic toxin Causes Staphylococcal Scalded Skin Syndrome (Ritter disease) and Bullous impetigo  Staphylococcus aureus A. Virulence factors  Cytolytic Toxins (hemolysins and leukocidins)  α, β (sphingomyelinase C), δ, ϒ hemolysins  Enzymes  Coagulase (Staphylocoagulase)  Fibrinolysin (Staphylokinase)  Protease  Staphylococcus aureus A. Virulence factors  Enzymes  Hyaluronidase  Lipase  Beta Lactamase (Penicillinase)  Protein A  Binds in the Fc portion of IgG  Epidemiology  The primary reservoir for staphylococci is the human naris, with colonization also occurring in the axillae, vagina, pharynx, and other skin surfaces.  Transmission of S. aureus may occur by direct contact with unwashed, contaminated hands and by contact with inanimate objects (fomites). Staphylococcus aureus Infections & Associated Diseases  Skin and Wound Infections FOLLICULITIS FURUNCLES CARBUNCLES Staphylococcus aureus Infections & Associated Diseases SSS TSS TEN  Staphylococcus aureus  Food Poisoning  enterotoxins A, D, and B  ingestion of a toxin formed outside the body  The enterotoxins do not cause any detectable odor or change in the appearance or taste of the food. Staphylococcus epidermidis Exopolysaccharide “slime” or biofilm Enhances organisms' adhesion to implanted medical device and provides refractoriness to host defense B. Infections & Associated Diseases  Health care-acquired UTIs  Prosthetic valve endocarditis Staphylococcus saprophyticus Adheres to the epithelial cells lining the urogenital tract than other CoNS. A. Infections & Associated Diseases  Causes UTIs in sexually active young females Staphylococcus lugdunensis  can be more virulent and can clinically mimic S. aureus infections  contain the gene mecA that encodes oxacillin resistance  Infections & Associated Diseases  infective endocarditis  septicemia, meningitis, skin and soft tissue infections, UTIs, and septic shock Staphylococcus haemolyticus  commonly isolated CoNS  Vancomycin resistance exists in some S. haemolyticus isolates.  Infections & Associated Diseases  wounds, bacteremia, endocarditis, and UTIs LABORATORY DIAGNOSIS A. Isolation and Identification 1. Specimen: a) Aspirate or swabs 2. Culture Media a. Blood Agar b. Colistin‐Nalidixic Acid c. Phenylethyl Alcohol d. Mannitol Salt Agar LABORATORY DIAGNOSIS  Cultural Characteristics  Staphylococci produce round, smooth, white, creamy colonies on SBA after 18‐24 hrs Staphylococcus aureus Beta hemolytic, creamy, butter‐ looking colonies LABORATORY DIAGNOSIS Organism Coagulase DNAse MSA Novobiocin Fermentation (5µg) S.aureus + + + S.epidermidis (‐) (‐) (‐) S S.saprophyticus (‐) (‐) (‐) R LABORATORY DIAGNOSIS 1. Catalase Test Test the ability of the organism to breakdown hydrogen peroxide into oxygen and water. Positive  Copious Bubble Formation Negative  No Bubble formation  LABORATORY DIAGNOSIS 2. Coagulase test  Detects the ability of bacteria to convert fibrinogen to fibrin.  2 types:  cell-bound coagulase (clumping factor)  Staphylocoagulase or free coagulase  LABORATORY DIAGNOSIS A. Coagulase Slide Test B. Coagulase Tube Test  Detects bound coagulase  Detects Free coagulase  Positive:  Positive: Macroscopic clumping Clot of any size  Negative:  Negative: No clumping No clot  Tube Coagulase test Note! The clot formed in the tube may undergo autolysis (owing to fibrinolysin), giving the appearance of a negative result. Laboratory scientists should look for clot formation after 4 hours of incubation at 37° C. If no clot appears, the tube should be left at room temperature and checked the following day. LABORATORY DIAGNOSIS 3. Microdase Test  for the detection of oxidase enzyme Positive: Dark Blue/purple Negative: No color change LABORATORY DIAGNOSIS 4. Bacitracin Susceptibility  Test for the susceptibility to 0.04U Bacitracin Susceptible:  Zone diameter > than 10mm Resistant:  No zone inhibition LABORATORY DIAGNOSIS 5. Novobiocin Susceptibility  Test for the susceptibility to Novobiocin Susceptible:  Zone diameter >16mm Resistant:  Zone diameter < to 16mm LABORATORY DIAGNOSIS LABORATORY DIAGNOSIS Dnase Test  Used to detect the ability of the organism to hydrolyze DNA Positive:  Hydrolysis of the surrounding medium (clear zone) Negative:  No clearing observed  LABORATORY DIAGNOSIS Mannitol Salt Agar  Test for the ability to ferment mannitol  Components:  High salt content 7.5%, D‐mannitol and Phenol Red Growth with fermentation:  Yellow halos surrounding growth Growth without fermentation:  Plate remains pink to red Antimicrobial Susceptibility  Testing of CoNS depends on source and determination if the isolate is a contaminant or a likely pathogen.  antimicrobial susceptibility testing of S. saprophyticus from urine not required  Serious infections with S. aureus and S. lugdunensis require susceptibility testing.  Methicillin-Resistant Staphylococci  isolates that are resistant to nafcillin or oxacillin  Vancomycin remains the treatment of choice for MRSA  The latest CLSI M100 document recommends cefoxitin be used to detect methicillin resistance.  An oxacillin-salt agar plate can be used to screen for MRSA in clinical samples.  Methicillin-Resistant Staphylococci  oxacillin resistance is due to the gene mecA  mecA codes for an altered penicillin-binding protein (PBP) called PBP2a, also designated PBP2′  The “gold standard” for MRSA detection is the detection of the mecA gene by using molecular nucleic acid probes or PCR amplification. Macrolide Resistance modified double disk diffusion test (D-zone test) THANK YOU!