General Considerations 530 different Bacterial Species or taxa reported by clinical laboratory 95% of Isolated reported are distributed among 27 different taxa More than 90% of Isolated reported are distributed among 16 different taxa CATAlASE-POSITIVE GRAM-POSITIVE COCCI Staphylococci Micrococcaceae Staphylococci have been traditionally differentiated from micrococci on the basis of oxidation-fermentation (O/F) reactions produced in O/F glucose medium Staphylococci ferment glucose, whereas micrococci fail to produce acid under anaerobic conditions. Bacitracin & Furazolidone Susceptible Bacitracin = or > 10 mm Furazolidone = or > 15 mm Differentiation Among Gram- Positive, Catalase-Positive Cocci Staphylococci Bacitracin = R ,some strains show opposite reaction Microccocus / 48-72 hrs. Some micrococci produce a yellow pigment CAP > BAP Microccocus Staphylococci Staphylococci is currently composed 35 species and 17 subspecies Gram positive Cocci Catalase Positive Nonmotile Spherical cells (0.5 to 1.5 Micron) that appear singly, in pairs, and in clusters Clusters of Staphylococci G + cocci in cluster After antimicrobial therapy & older cells easily decolorized Presumptive report: Gram-Positive Cocci resembling Staphylococci Staphylococci Colonies produced after 18 to 24 hours of incubation appear cream-colored, white or rarely light gold, and buttery-looking in 1 to 3 mm in diameter and 3 to 8 mm by 3 days Hold plates for 72 h can result in selection of more than one species or strain Some species are B-hemolytic on sheep blood agar at 18 to 24 hours Staphylococci Colonies should be allowed to developed on primary isolation medium for 3 to 4 days at 35 to 37°C and then for 2 days at room temperature for identify colonial morphology of strains in staphylococcal species Staphylococcus aureus B-hemolytic, creamy, buttery-looking colonies Catalase Test Catalase enzyme that hydrolyzes hydrogen peroxide into water and oxygen (bubbles) Hydrogen peroxide (3%, aqueous). Dilute 30% (v/v) hydrogen peroxide 1:10 in sterile distilled water • Store at 4 °C in a dark bottle Catalase Test 30% H2O2 can be used for catalase test but it is more hazardous 30% H2O2 is extremely caustic to skin If contact occurs ,wash immediately with 70% Ethyl Alcohol , NOT WATER Catalase Test / Procedure 1-Touch the center of well-isolated young colony (18-24 hrs.) with a wooden stick to transfer to a clean ,dry glass slide. • Older cultures may give false-negative result Place 1 drop of reagent and observe immediately bubbles • Do not reverse ; False Negative Results can occur. Catalase Test / Procedure Be careful to avoid picking up any of the agar medium RBCs contain catalase and Blood agar may produce false-positive reaction due to peroxidase • CHOC dose not interfere with the assay Catalase Test / Interpretation Positive reaction : immediate appearance of bubbles • QC : S. aureus ATCC 25923- Catalase Positive Negative reaction : No bubbles, or the slow elaboration of a few bubbles after 20 seconds • Some strain of Enterococcus faecalis growth on SBA may appear weakly Positive / Pseudocatalase test Catalase Test / Limitations False-positive : some metal bacteriological loop materials Platinum loops do not yield false-positive results Catalase Test / Precautions Rapid evolution of oxygen may generate droplet or aerosol formation The catalase reaction does not affect organism viability Coagulase Test / Clumping factor Formerly referred to as cell-bound coagulase, causes agglutination in human , rabbit plasma Clumping factor on the surface of the bacterial cells directly converts fibrinogen to fibrin, which precipitates onto the cell surface, causing agglutination. Coagulase Test / Clumping factor A heavy suspension of the suspected organism is prepared on a glass slide in water to give a milky suspension and spread over 10mm area of the circle Colonies that are less than 24h old Hemolytic Colonies on fresh BAP at 18h Coagulase Test / Clumping factor Stirring the mixture to a homogeneous so as not to confuse clumping with autoagglutination Autoagglutination / Sticky organism ; Perform Tube Coaglulase Coagulase Test / Clumping factor Adding 1 drop of plasma, and observing for clumping within 10 sec. False-positive result may appear with reaction times longer than 10 sec. Coagulase Test / Clumping factor Coagulase Test / Clumping factor False-positive ; Autoagglutination ,colonies from media containing high concentrations of salt e.g., Mannitol Salt Agar Coagulase Test / Clumping factor Isolates can also be occasionally confused as coagulase-positive staphylococci because of the presence of clumping factor • S. lugdunensis • S. schleiferi subsp. Schleiferi Reaction is more efficient if human plasma is used rather than rabbit plasma Coagulase Test / Clumping factor Because about 10-15% (previously 5% )of S. aureus ,especially MRSA, do not produce clumping factor MRSA : low level Bound Coagulase & Protein A & mask the cell wall with capsular polysacchaides Coagulase Test / Clumping factor Negative slide coagulase test result, must be confirmed with the tube method, which detects free coagulase • Especially if it is from normally sterile body site , blood joint isolates • Methicillin Resistant Staphylococci Coagulase Test / Clumping factor +Protein A Some strains of ; S. saprophyticus ( Protein A) / nonhemolytic colonies S.warneri ( Protein A) S.capitis ( Protein A ) S. lugdunensis (Clumping factor ) / PYR + S. schleiferi (Clumping factor ) / PYR + • may produce positive tests with this method, but they would be tube coagulase negative Tube Coagulase Test Free Coagulase Staphylocoagulase reacts with a thermostable, thrombinlike molecule called coagulase-reacting factor (CRF) to form coagulase-CRF complex. The complex resembles thrombin and indirectly converts fibrinogen to fibrin Tube Coagulase Test Staphylocoagulase producing (coagulase positive) staphylococci are S. aureus in human & animal • VP + & hemolytic at 18hrs S. intermedius in Dog & very rarely found in human ( Dog bite) / VP - & nonhemolytic at 18hrs Some strains of S. hyicus in Pig &very rarely found in human / VP - & nonhemolytic at 18hrs Frequency of Isolation of Staphylococcus intermedius from Humans We collected 3,397 consecutive isolates of coagulase- positive staphylococci from various specimens of hospitalized patients. All were retrospectively classified as Staphylococcus aureus, except two which were identified as S. intermedius = 0.06 % or 6 / 10000 coagulase-positive staphylococci One isolated from the nasal flora of a healthy carrier and the other isolated from pleural fluid, probably as a sample contaminant Tube Coagulase Test Sterile rabbit Plasma containing EDTA most satisfactory • Rehydrated reagent expires after 1 month if stored at -20°C or 5 days if stored at 2 to 8°C • Do not use citrated plasma ,a false –positive result can occur Emulsify several colonies in 0.5 ml of rabbit plasma with EDTA Tube Coagulase Test Incubate at 37° C Look for clot formation hourly up to 4 hours by slowly tilting it 90° from vertical If no clot appears the tube should be left at room temperature to incubate overnight and checked the following day • Rare S. aureus strains require >4 hours to clot the tube coagulase reagent Tube Coagulase Test Tube Coagulase Test Some strains of S. aureus produce a staphylokinase , a plasmid –carried enzyme ,that dissolves the clot, giving a false-negative result Staphylokinase is less active at 25°C Tube Coagulase Test Flocculent or fibrous precipitate is not a true clot and should be recorded as negative result Some strains of S. intermedius and most coagulase-producing strains of S. hyicus require more than 4 h (12 to 24 hours) for positive Coagulase Test Staphylococcus aureus Gram-positive cocci in clusters Catalase-positive Staphylococcus aureus Cultural Characteristics Round, smooth, white, creamy colonies on SBA after 18 to 24 hours of incubation at 35° to 37° C May produce hemolytic zones around the colonies May exhibit pigment production (yellow) with extended incubation Staphylococcus aureus Small –Colony Variants ( SCVs) Small –Colony Variants ( SCVs) of S.aureus with large capsule & grows slowly and produce small , glistening, wet , convex colonies . SCVs are most common in patient with unusually persistent infections, such as chronic osteomylitis and who are chronically exposed to aminoglycosides and CO-timoxazole Mannitol Salt Agar (MSA) NaCl ; 7.5% Incubation for at least 48 to 72 hours S.saprophyticus resemble S.aureus Odor ? Staphylococcus aureus Rapid thermonuclease test Positive result in the rapid (four-hour) thermonuclease test accurately identifies S.aureus • S. Schleiferi & S.intermedius : positive Several colonies in to broth Boiling for 15 min Punch a hole in TBO DANase agar Fill well with 2 drops of cooled broth Incubate at 35 for 3 h, and observe for color change Coagulase-negative Staphylococci (CoNS) CoNS Coagulase-negative Staphylococci (CoNS) 35 recognized species of coagulase- negative staphylococci The most clinically significant species in this group are • S. epidermidis • S. saprophyticus Staphylococci Staphylococcus epidermidis Infections caused by S. epidermidis are predominantly hospital acquired Biofilm production is a key component in bacterial pathogenesis S. epidermidis has been linked to important nosocomial infections, often associated with foreign body implants Staphylococcus epidermidis Small to medium , most colonies nonhemolytic , slime-producing strains are extremely