laboratory identification of gram-positive cocci &...

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 glucoseferment 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

StaphylococciBacitracin = R ,some strains show opposite

reaction

Microccocus / 48-72 hrs.Some micrococci produce a yellow pigment

CAP > BAP

Microccocus

Staphylococci

Staphylococci is currently composed 3535 species and 1717 subspecies

Gram positive CocciCatalase PositiveNonmotile Spherical cells (0.5 to 1.5 Micron) that appear

singly, in pairs, and in clusters

Clusters of Staphylococci

G + cocci in clusterAfter 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 8mm 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°Cand 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 7070%% Ethyl Alcohol , NOT WATEREthyl 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 humanhuman , rabbit 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 suspensionand spread over 10mm area of the circle

Colonies that are less than Colonies that are less than 2424hh oldold

Hemolytic Colonies on fresh BAP at Hemolytic Colonies on fresh BAP at 1818hh

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 10sec.

False-positive result may appear with reaction times longer than longer than 10 10 secsec.

Coagulase Test /Clumping factor

Coagulase Test /Clumping factor

False-positive ; AutoagglutinationAutoagglutination ,colonies from media containing high concentrations of salt e.g., Mannitol Salt AgarMannitol 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 MRSAMRSA, 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 TestFree 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. aureusS. aureus in human & animal • VP + & hemolytic at 18hrs

S. intermediusS. intermedius in Dog & very rarely found in human ( Dog bite) / VP - & nonhemolytic at 18hrs

Some strains of S. hyicusS. 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 = = 00..06 06 % or % or 6 6 / / 10000 10000 coagulasecoagulase--positive positive staphylococci staphylococci

One isolated from the nasal flora of a healthy carriernasal flora of a healthy carrierand the other isolated from pleural fluidpleural 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° CLook 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 temperatureroom 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 precipitateFlocculent 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 more than 4 4 hh (12 to 24 hours) for positive Coagulase Test

Staphylococcus aureus

Gram-positive cocci in clustersCatalase-positive

Staphylococcus aureusCultural 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 chronic osteomylitis andosteomylitis and who are chronically exposed to aminoglycosidesaminoglycosides and COCO--timoxazole timoxazole

Mannitol Salt Agar (MSA)

NaCl ; 7.5%

Incubation for at least 48 to 72 hours

S.saprophyticus resemble S.aureus

Odor ?

Staphylococcus aureusRapid 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)

CoNSCoNS

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 : SS

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 saprophyticusCultural Characteristics

Larger colonies

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

Novobiocin Resistance

Staphylococcus saprophyticus Novobiocin Resistance

CLSI Methods

MHA0.5 McFarlandIncubate for 18 h at 35° C in non-CO2 Zone of = or < 16 mm

Staphylococcus saprophyticus Novobiocin Resistance

Hebert method

SBA1 McFarlandIncubate 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 chainsColonies >1 mmNon-beta hemolytic on sheep blood agarCatalase-negativePyrrolidonyl 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 twoone 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 hoursA 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 brothIncubate at 35° C for 3 days

Check for growth daily

Enterococci

Enterococci /BEAPositive - Negetive

Enterococci /BEAWood 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 agalactiaeStreptococcus agalactiae(Group B)

Streptococcus agalactiae (Group B)

Gram-positive cocci in pairs and chainsCatalase-negativeNarrow zone of beta hemolysis with a soft

periphery on sheep BAPRapid (two to four hours) hippurate hydrolysis

testsCommercial particle agglutination tests

approach 100% accuracy

Group B streptococciGroup B streptococciare are notnot associated with associated with

pharyngitispharyngitis

Group A streptococci (GAS) vs.Group B streptococciGroup B streptococci

Group B streptococciGroup B streptococciBacitracinBacitracin ::R rare s R rare s && SXTSXT : R: 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 an enhanced hemolytic activity on hemolytic activity on incubation at incubation at 3737°° C and C and subsequent exposure to cold subsequent exposure to cold ((44°° C)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 TestPrinciple

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 TestPrinciple

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 TestSodium Hippurate (1% w/v)

Add 1 gram sodium hippurate to 100 mL distilled water

Mix well to dissolve completelyDispense in capped tubes in 0.4-mL amountsFreeze at –20 oC until needed Shelf life: until quality control no longer

performs appropriately

Rapid Hippurate Hydrolysis TestNinhydrin

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 : cancerogeneNinhydrin : cancerogene

Caution: Flammable

Rapid Hippurate Hydrolysis TestProcedure

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 TestProcedure

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 TestProcedure

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 streptococciGroup B streptococciHippurate Hydrolysis : 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 Plate is incubated overnight at 3535°° C C in in COCO22

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 pseudopneumoniae2004

A newly Streptococcus pseudopneumoniae discovered organism

S. pseudopneumoniae strains do not have pneumococcal capsules

Streptococcus pseudopneumoniae2004

Streptococcus pseudopneumoniae are resistantto 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. PneumoniaeOptochin susceptibility test > 14 or > 16 mm

Streptococcus pneumoniaBile 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 TestPrinciple

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 TestReagents

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 TestPlate 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 TestTube 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 TestTube 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 TestTube 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. PneumoniaeThe bile solubility 2% sodium

deoxycholate