Laboratory Identification of Gram-Positive Cocci &...
Transcript of Laboratory Identification of Gram-Positive Cocci &...
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General Considerations
530 different Bacterial Species or taxa reported by clinical laboratory
95% of Isolated reported are distributed among 27 different taxa
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More than 90% of Isolated reported are distributed among 16 different taxa
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CATAlASE-POSITIVE GRAM-POSITIVE
COCCI
Staphylococci
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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.
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Bacitracin & Furazolidone Susceptible
Bacitracin = or > 10 mm
Furazolidone = or > 15 mm
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Differentiation Among Gram-Positive, Catalase-Positive Cocci
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StaphylococciBacitracin = R ,some strains show opposite
reaction
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Microccocus / 48-72 hrs.Some micrococci produce a yellow pigment
CAP > BAP
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Microccocus
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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
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Clusters of Staphylococci
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G + cocci in clusterAfter antimicrobial therapy & older
cells easily decolorized
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Presumptive report: Gram-Positive Cocci resembling
Staphylococci
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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
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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
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Staphylococcus aureus B-hemolytic, creamy, buttery-looking colonies
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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
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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
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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.
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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
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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
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Catalase Test / Limitations
False-positive : some metal bacteriological loop materials
Platinum loops do not yield false-positive results
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Catalase Test / Precautions
Rapid evolution of oxygen may generate droplet or aerosol formation
The catalase reaction does not affect organism viability
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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.
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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
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Coagulase Test /Clumping factor
Stirring the mixture to a homogeneous so as not to confuse clumping with autoagglutination
Autoagglutination / Sticky organism ; Perform Tube Coaglulase
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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.
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Coagulase Test /Clumping factor
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Coagulase Test /Clumping factor
False-positive ; AutoagglutinationAutoagglutination ,colonies from media containing high concentrations of salt e.g., Mannitol Salt AgarMannitol Salt Agar
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Tube Coagulase Test
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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
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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
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Staphylococcus aureus
Gram-positive cocci in clustersCatalase-positive
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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
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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
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Mannitol Salt Agar (MSA)
NaCl ; 7.5%
Incubation for at least 48 to 72 hours
S.saprophyticus resemble S.aureus
Odor ?
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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
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Coagulase-negative Staphylococci (CoNS)
CoNSCoNS
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Coagulase-negative Staphylococci (CoNS)
35 recognized species of coagulase-negative staphylococci
The most clinically significant species in this group are • S. epidermidis • S. saprophyticus
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Staphylococci
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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
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Staphylococcus epidermidis
Small to medium , most colonies nonhemolytic , slime-producing strains are extremely sticky and adhere to the agar surface
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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
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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
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Staphylococcus saprophyticusCultural Characteristics
Larger colonies
About 50% of the strains producing a yellow pigment after 24 hrs
Novobiocin Resistance
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Staphylococcus saprophyticus Novobiocin Resistance
CLSI Methods
MHA0.5 McFarlandIncubate for 18 h at 35° C in non-CO2 Zone of = or < 16 mm
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Staphylococcus saprophyticus Novobiocin Resistance
Hebert method
SBA1 McFarlandIncubate for 24 h at 35° C in non-CO2 Zone of = or < 12 mm
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CATAlASE-NEGATIVE GRAM-POSITIVE COCCI
Enterococci
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Enterococcus species
Gram stain : positive cocci or coccobacilli in pairs and chainsColonies >1 mmNon-beta hemolytic on sheep blood agarCatalase-negativePyrrolidonyl arylamidase (PYR) :
Positive
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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
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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
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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.
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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
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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
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Enterococci
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Enterococci /BEAPositive - Negetive
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Enterococci /BEAWood Lamp
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CATAlASE-NEGATIVE GRAM-POSITIVE COCCI
Streptococci , Enterococci and similar organism
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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
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Streptococcus pyogenes
Positive PYR test identifies S. pyogenes• Limitation: Beta-hemolytic enterococci are
also PYR-positive
Serogrouping by particle agglutination approaches 100% accuracy
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Group A streptococci (GAS)on sheep blood agar
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Group A streptococci (GAS)
Bacitracin : S/rare R & SXT : R
Pyrrolidonyl arylamidase (PYR): +
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Group A streptococci (GAS)Bacitracin 0.04 S > or = 12mm
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CATAlASE-NEGATIVE GRAM-POSITIVE COCCI
Streptococcus agalactiaeStreptococcus agalactiae(Group B)
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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
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Group B streptococciGroup B streptococciare are notnot associated with associated with
pharyngitispharyngitis
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Group A streptococci (GAS) vs.Group B streptococciGroup B streptococci
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Group B streptococciGroup B streptococciBacitracinBacitracin ::R rare s R rare s && SXTSXT : R: R
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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
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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)
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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
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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
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Limitation
Some group A Streptococci will be CAMP positive if incubated in a candle jar ,in a CO2 atmosphere ,or under anaerobic conditions
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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
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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
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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
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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
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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
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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
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Group B streptococciGroup B streptococciHippurate Hydrolysis : Hippurate Hydrolysis : ++
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CATAlASE-NEGATIVE GRAM-POSITIVE COCCI
Streptococcus pneumonia
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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
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S. pneumoniae
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S. pneumoniae
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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
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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
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Streptococcus pseudopneumoniae2004
A newly Streptococcus pseudopneumoniae discovered organism
S. pseudopneumoniae strains do not have pneumococcal capsules
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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
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S. PneumoniaeOptochin susceptibility test > 14 or > 16 mm
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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
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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
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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
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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
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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
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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
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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
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S. PneumoniaeThe bile solubility 2% sodium
deoxycholate
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