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 sticky and adhere to the agar surface Polymyxin B Resistance

 Polymyxin B 300-U  Sheep BAP or MHA  Polymyxin B resistance inhibition zone diameter of < 10 mm  S.aureus : R  S.epidermidis : R  S.saprophyticus : S Staphylococcus saprophyticus

 This species adheres more effectively to the epithelial cells lining the urogenital tract than other coagulase-negative staphylococci

 Novobiocin susceptibility using a 5 Microgram novobiocin disk .S.saprophyticus is resistant to novobiocin Staphylococcus saprophyticus Cultural Characteristics

Larger colonies

About 50% of the strains producing a yellow pigment after 24 hrs

Novobiocin Resistance Staphylococcus saprophyticus Novobiocin Resistance

CLSI Methods

MHA 0.5 McFarland Incubate for 18 h at 35° C in non-CO2 Zone of = or < 16 mm Staphylococcus saprophyticus Novobiocin Resistance

Hebert method

SBA 1 McFarland Incubate for 24 h at 35° C in non-CO2 Zone of = or < 12 mm

CATAlASE- NEGATIVE GRAM- POSITIVE COCCI Enterococci Enterococcus species

Gram stain : positive cocci or coccobacilli in pairs and chains Colonies >1 mm Non-beta hemolytic on sheep blood agar Catalase-negative Pyrrolidonyl arylamidase (PYR) : Positive

Bile Esculin test

Bile esculin test is a two-step;

 Bacteria must grow in the presence of 40% bile

 Hydrolyze esculin to produce a positive reaction.

 Hydrolysis of esculin results in esculetin, which reacts with ferric citrate or ferric ammonium citrate in the medium to form a black precipitate Bile Esculin test

 Pick one or two isolated colonies from the sheep blood agar plate and inoculate to bile esculin agar medium  Incubate at 35° C for 18 to 24 hours, cap loosely

 Positive result is often seen within 4 hours  A negative result should be incubated for an additional 24 h Salt Tolerance

Organisms positive for bile esculin are separated into group D streptococci or Enterococcus by the salt tolerance

Growth in 6.5% sodium chloride broth is used to identify Enterococcus and Aerococcus organisms. Salt Tolerance

 Some species of Pediococcus and Leuconostoc spp. grow in 6.5% NaCI broth when incubated for 24 hours  Pediococcus and Leuconostoc are vancomycin resistant < 15 mm

 Group D streptococci ,do not grow in a 6.5% NaCI broth Salt Tolerance

Pick one or two isolated colonies from the blood agar plate and lightly inoculate 5 mL of NaCI broth Incubate at 35° C for 3 days

 Check for growth daily

Enterococci Enterococci /BEA Positive - Negetive Enterococci /BEA Wood Lamp

CATAlASE- NEGATIVE GRAM- POSITIVE COCCI

Streptococci , Enterococci and similar organism

Streptococcus pyogenes

 Gram-positive cocci in pairs and chains

 Catalase-negative

 Beta-hemolytic colonies >0.5 mm in diameter on sheep BAP after 24 hours incubation

 Colonies are usually dry, peaked, or convex with a sharp periphery to the zone of hemolysis Streptococcus pyogenes

Positive PYR test identifies S. pyogenes • Limitation: Beta-hemolytic enterococci are also PYR-positive

Serogrouping by particle agglutination approaches 100% accuracy Group A streptococci (GAS) on sheep blood agar Group A streptococci (GAS)

 Bacitracin : S/rare R & SXT : R

 Pyrrolidonyl arylamidase (PYR): +

Group A streptococci (GAS) Bacitracin 0.04 S > or = 12mm

CATAlASE- NEGATIVE GRAM- POSITIVE COCCI

Streptococcus agalactiae (Group B) Streptococcus agalactiae (Group B)

 Gram-positive cocci in pairs and chains  Catalase-negative  Narrow zone of beta hemolysis with a soft periphery on sheep BAP  Rapid (two to four hours) hippurate hydrolysis tests  Commercial particle agglutination tests approach 100% accuracy

Group B streptococci are not associated with pharyngitis Group A streptococci (GAS) vs. Group B streptococci Group B streptococci Bacitracin :R rare s & SXT : R CAMP test (Christie, Atkins, and Munch-Petersen)

B-Hemolysin (sphingomyelinase C) acts on sphingomyelin in the plasma membrane of erythrocytes

Lecithin:Sphingomyelin Ratio • Human 3 : 2 • Sheep 1 : 12 CAMP

hot-cold lysin ; an enhanced hemolytic activity on incubation at 37° C and subsequent exposure to cold (4° C) CAMP

 Sheep blood agar plates must be prewarmed before use to avoid hot-cold lysis

 Test plates should be read as soon as possible ; if held at room temperature (25 C ) for any period of time , interpretation is difficult because of hot-cold lysis of sheep RBC CAMP Test Principle

S. agalactiae produces a diffusible protein (CAMP factor) that acts synergistically with the beta-lysin elaborated by S. aureus (e.g : S. aureus ATCC 25923 )to produce a zone of enhanced hemolysis

Limitation

Some group A Streptococci will be CAMP positive if incubated in a candle jar ,in a CO2 atmosphere ,or under anaerobic conditions

Rapid Hippurate Hydrolysis Test Principle

 Hydrolysis of sodium hippurate by Group B streptococci produces benzoic acid and glycine

 When ninhydrin (a protein detector) is added to hydrolyzed sodium hippurate, it reacts with the amino acid glycine and produces a deep blue color

 Ninety-nine percent (99%) of Group B streptococci hydrolyze hippurate while other groups of beta streptococci do not Rapid Hippurate Hydrolysis Test Sodium Hippurate (1% w/v)

 Add 1 gram sodium hippurate to 100 mL distilled water  Mix well to dissolve completely  Dispense in capped tubes in 0.4-mL amounts  Freeze at –20 oC until needed  Shelf life: until quality control no longer performs appropriately Rapid Hippurate Hydrolysis Test Ninhydrin

 Mix 50 mL acetone and 50 mL 1-butanol thoroughly in a dark glass bottle

 Add 3.5 g ninhydrin, mix, and store at room temperature • Ninhydrin : cancerogene

 Caution: Flammable Rapid Hippurate Hydrolysis Test Procedure

 Defrost one tube containing 0.4 mL sodium hippurate reagent for each organism to be tested

 Use a wooden stick or bacteriological loop to inoculate the sodium hippurate with a heavy inoculum of the suspected organism from a fresh subculture on blood agar

 Take care not to pick up pieces of agar, as the protein present will cause a weak positive reaction Rapid Hippurate Hydrolysis Test Procedure

Emulsify the organism in the substrate

Incubate tubes for two hours in a 37 oC

Add 0.2 mL of the ninhydrin solution and mix gently • do not shake or vigorously agitate the tubes Rapid Hippurate Hydrolysis Test Procedure

 Return tubes to the heating block or water bath for ten minutes

 Deep blue color (about the color of crystal violet), indicating a positive result

 Negative reaction results in a colorless broth or faint tinge of purple in the broth Group B streptococci Hippurate Hydrolysis : +

CATAlASE- NEGATIVE GRAM- POSITIVE COCCI

Streptococcus pneumonia Streptococcus pneumonia

 Gram-positive cocci in pairs and chains

 Catalase-negative

 Alpha hemolytic on sheep BAP

 Colonies are usually transparent, slightly mucoid, or flattened (resemble a checkers playing piece), not peaked S. pneumoniae S. pneumoniae Optochin Susceptibility

Disk containing optochin (ethylhydrocuprein hydrochloride)

SBA plate

Plate is incubated overnight at 35° C in CO2 Optochin Susceptibility

A zone of inhibition greater than 14 mm with a 6-mm disk

A zone of inhibition greater than 16 mm with a 10-mm disk are considered susceptible and a presumptive identification of S. pneumoniae Streptococcus pseudopneumoniae 2004

A newly Streptococcus pseudopneumoniae discovered organism

S. pseudopneumoniae strains do not have pneumococcal capsules Streptococcus pseudopneumoniae 2004

 Streptococcus pseudopneumoniae are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO2

 But are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres ; False Positive S. Pneumoniae

Optochin susceptibility test > 14 or > 16 mm Streptococcus pneumonia Bile Solubility Test

The bile solubility test is more specific than the optochin test for identification of S. pneumoniae

Limitation: Some S. pneumoniae may not be bile soluble Rapid Bile Solubility Test Principle

The active autocatalytic enzyme of Streptococcus pneumoniae is enhanced by bile or sodium deoxycholate

10% reagent may yield more rapid reactions, but both 2% and 10% have been used successfully Rapid Bile Solubility Test Reagents

 Add 1 g sodium deoxycholate to 9.0 mL of sterile, distilled water (10% solution), or dilute this solution 1:5 in water to make a 2% solution

 Store at 4 °C in a dark bottle

 Reagent is good for one year from the date of preparation  New lot should be prepared if it becomes cloudy or contaminated Rapid Bile Solubility Test Plate Method

 Place a drop of the bile reagent directly onto an isolated colony to be tested

 Without tipping the plate, incubate at room temperature or at 35 °C for 15 minutes or until the liquid has evaporated or adsorbed into the medium

 Observe carefully for a flattening of the colony ( positive reaction)  Be certain that the colony did not simply float away Rapid Bile Solubility Test Tube Method

 Place 0.5 mL of bile reagent into a small, sterile tube

 Place 0.5 mL sterile saline into another tube for a control

 Inoculate enough test organism into each tube to create a slightly turbid suspension Rapid Bile Solubility Test Tube Method

Incubate at room temperature or at 35 °C for 5 to 15 minutes

Watch for a decrease in turbidity in the tube containing the bile salt suspension relative to the control tube Rapid Bile Solubility Test Tube Method / Interpretation

 Decreased turbidity or clearing of the suspension in the bile-containing tube is considered positive

 No decrease in turbidity of the suspension in the bile-containing tube is considered a negative • QC : non-pneumoniae viridans streptococcus S. Pneumoniae The bile solubility 2% sodium deoxycholate