Microbiology and Parasitology Testing for Staphylococci (PDF)

MICROBIOLOGY AND PARASITOLOGY TESTING FOR STAPHYLOCOCCI Hannah Leian T. So, RMT, MD | September 18, 2024 STAPHYLOCOCCUS SPP. General Characteristics of Staphylococcus spp.: Gram-positive cocci arranged in grape-like c...

MICROBIOLOGY AND PARASITOLOGY TESTING FOR STAPHYLOCOCCI Hannah Leian T. So, RMT, MD | September 18, 2024 STAPHYLOCOCCUS SPP. General Characteristics of Staphylococcus spp.: Gram-positive cocci arranged in grape-like clusters “Staphyle” = bunch of grapes, “kokkos” = berry Nonmotile, non-spore forming Ferment carbohydrates – produce lactic acid but NOT GAS Relatively resistant to: → drying → heat (can withstand 500C for 30 minutes) Halotolerant – can live in environments with up to 9% Impetigo (Bullous) Usually caused by NaCl autoimmune disease. Not related to Staph infection. Some are normal flora of the skin and mucous membranes Impetigo (Non-bullous) Caused by S. aureus is found on skin, in the nostrils, Staphylococcus aureus armpits or Streptococcus May cause suppurative (pus-forming) infections and pyogenes. It begins as tiny septicemia blisters that eventually burst and leave small wet COMMON SUPPURATIVE DEFINTION: patches of red skin that INFECTIONS CAUSED BY may weep fluid. Then, it STAPHYLOCOCCI: forms the characteristic honey-colored crust. Folliculitis Superficial infection involving the hair follicle CLINICALLY IMPORTANT SPECIES S. aureus- causes pyogenic skin and soft tissue infections, pneumonia, bacteremia, infective endocarditis, toxin-mediated illnesses S. epidermidis- infection from skin contamination and implants/prosthetic materials (CRBSI, prosthetic and native valve endocarditis) → catheter-related / any prosthetics inserted in the body S. lugdunensis- infection from skin contamination and implants, native valve endocarditis, osteomyelitis Furuncle / Boil Deeper than folliculitis, S. saprophyticus – UTI in young women; part of normal more extensive, more flora in women painful, but still centers around the hair follicle STAPHYLOCOCCUS SPP. ON CULTURE Grow fastest at 370C BUT produce pigment best at room temperature (20-250C) Form round, raised, smooth, shiny colonies; different species vary in pigment: → S. lugdunensis, S. epidermidis, and S. saprophyticus – cream to whitish-gray colonies Abcess Have a wall formation around the pus Carbuncle Accumulation of boils/ furuncles → Erysipelas Superficial layer of the skin STAPHYLOCOCCUS AUREUS ON CULTURE is affected. STAPHYLOCOCCUS AUREUS can produce golden There is a layer that is yellow colored colonies. “pushed up”. → Doc Hannah: But it depends– not all strains can produce really pigmented golden yellow colonies Cellulitis Deeper layer of the skin is But in general, they are able to do so because of the affected. No pushing up is pigment known as staphyloxanthin skin. Margin of involved area is less distinct. ON NUTRIENT AGAR: - S. aureus produces cream, or golden yellow colonies due to staphyloxanthin Transcribed by: NMD 2027  Specimen Collection ON MANNITOL SALT AGAR: - S. aureus turns red agar to yellow due to fermentation of mannitol (S. epidermidis on DNAse Agar) - They will produce lactic acid upon fermentation, thereby lowering the pH, STAPHYLOCOCCUS AUREUS changing the pH indicator (phenol red) VIRULENCE FACTORS color of the agar from red to yellow. → Catalase – protects intraphagocytic microbes by destroying hydrogen peroxide produced by the ON BLOOD AGAR PLATE: phagocyte - S. aureus creates golden yellow colonies and a → Coagulase – formation of fibrin clot around this halo of clear beta-hemolysis bacteria likely protects it from phagocytosis and can function as a biofilm scaffold ON DNAse AGAR: → Hyaluronidase – spreading factor; allows - Only S. aureus has DNAse! This is what makes it dissemination of bacteria through tissue unique compared to other Staph species. → Staphylokinase – plasminogen activator; converts plasminogen to plasmin thus allowing lysis of fibrin and metastasis of bacteria to other sites → Protein A – binds to the Fc region of IgG to disrupt opsonization - On the agar above, there is DNA compounded with methyl green: ▪ - A: S. aureus- there is clearing since S. aureus have → Panton-Valentine leukocidin - cytotoxin causes DNAse. leukocyte lysis and tissue necrosis - B: Serratia marcescens- also has DNAse → Exofoliatin - exotoxin that causes a blistering of the - C: S. epidermidis skin as Staphylococcal Scalded Skin Syndrome. ▪ exfoliation or desquamation of the skin - This has no methyl green, so you have to add → Enterotoxin - leads to gastroenteritis with emesis, hydrochloric acid. nausea and diarrhea - HCl will precipitate the DNA present in the ▪ Something to do with the gastrointestinal tract which agar. can cause GI symptoms in cases of S. Aureus food - Note that the rest of the agar is cloudy poisoning. except for the area of clearing (since there → TSST-1 - superantigen; produces Staphylococcal is no DNA, there is nothing for HCl to bind Toxic Shock Syndrome to). GRAM POSITIVE BACTERIA TESTING - Compare this to the image below which is cloudy all throughout, since it is S. epidermidis on the plate, which has no DNAse. 2 of 4  Specimen Collection Catalase Testing: staphylococcus (CoNS – Coagulase-Negative Staphylococci - catalase positive Staphylococci) Streptococci - catalase negative Coagulase is an enzyme-like protein that causes plasma y to clot by converting fibrinogen to fibrin\ CATALASE TESTING All staphylococcal species give a positive catalase test, which differentiates it from Streptococcus Principle: → Production of water and oxygen from hydrogen peroxide through the enzyme catalase S. aureus produces 2 types of coagulase → Bound coagulase ▪ Cell wall bound coagulase (i.e. clumping factor) is detected by the slide coagulase test ▪ Reacts directly with fibrinogen in plasma Positive result: bubbling or effervescence → Free coagulase → this is because of the release of oxygen from the ▪ Detected by the tube coagulase test hydrogen peroxide ▪ Requires the presence of a coagulase-reacting Steps factor (CRF) – which may be prothrombin – to form 1. Inoculate bacteria onto a microscope slide staphylothrombin 2. Add a drop of hydrogen peroxide onto the bacteria − The staphylothrombin will now react with 3. If bubbles are formed, the organism is fibrinogen to form fibrin clot catalase-positive SLIDE COAGULASE TEST Detects bound coagulase (clumping factor) Add one drop heavy bacterial suspension + one drop of plasma on a clean slide Mix well and observe for clumping, clotting, agglutination, coagulation after 10 secs Advantage: → Rapid diagnosis Disadvantage: → Less accurate After knowing that the unknown isolate is staphylococcus, the next thing you need to know if it’s S. aureus or coagulase negative staphylococci > then do coagulase testing this time. IF NEGATIVE TO BOUND COAGULASE PROCEED TO TUBE COAGULASE TUBE COAGULASE Detects free coagulase Steps: 1. Transfer 0.5mL of reconstituted plasma into the test After coagulase testing, the S. Aureus is coagulase tube. positive the other 3 is coagulase negative. To further 2. Select 2-3 isolated colonies of bacteria to be tested. differentiate you do Novobiocin Susceptibility Test 3. Emulsify the bacteria in the plasma and place them → In Novobiocin Susceptibility Test in the incubator at 37⁰C ▪ You inoculate the unknown microbe on the agar then 4. Note the time the test started. Observe the culture you place a disc of Novobiocin (antibiotic) at regular intervals over the next four hours for the − If no growth around the novobiocin - the bacteria presence of a clot. Any clot formation is a positive is susceptible. So it can be S. Epidermidis or S. result Lugdunensis − If there is still growth - then it is novobiocin resistant, then it is S. Saprophyticus COAGULASE TEST The test is used to differentiate between Staphylococcus aureus, which is coagulase positive, vs other species of 3 of 4  Specimen Collection Advantage: → More accurate (all isolates of S. aureus are positive) – definitive test for coagulase Disadvantage: → More time-consuming (final observation is at 24 hrs) FREEDOM WALL 4 of 4