Diagnosis of Diagnosis of streptococcistreptococci

Compiled by Thamer HamdanCompiled by Thamer Hamdan

M.Sc. Clinical Microbiology and M.Sc. Clinical Microbiology and ImmunologyImmunology

StreptococciStreptococci

Characters of StreptococciCharacters of Streptococci– Gram positive cocciGram positive cocci– 11µµm in diameterm in diameter– Chains or pairsChains or pairs– Usually capsulatedUsually capsulated– Non motileNon motile

– Non spore formingNon spore forming– Facultative anaerobesFacultative anaerobes– FastidiousFastidious– Catalase negative Catalase negative (Staphylococci are catalase (Staphylococci are catalase

positive)positive)

Classification of Classification of StreptococciStreptococci

Streptococci can be classified according Streptococci can be classified according to:to:– Oxygen requirementsOxygen requirements

Anaerobic (Anaerobic (PeptostreptococcusPeptostreptococcus))

Aerobic or facultative anaerobic (Aerobic or facultative anaerobic (StreptococcusStreptococcus))

– Serology (Lanciefield Classification)Serology (Lanciefield Classification)– Hemolysis on Blood Agar (BA)Hemolysis on Blood Agar (BA)

Serology: Lanciefield Serology: Lanciefield ClassificationClassification

Streptococci classified into many groups from A-K & H-VStreptococci classified into many groups from A-K & H-VOne or more species per groupOne or more species per groupClassification based on C- carbohydrate antigen of cell Classification based on C- carbohydrate antigen of cell wallwall– Groupable streptococciGroupable streptococci

A, B and D (more frequent)A, B and D (more frequent)C, G and F (Less frequent)C, G and F (Less frequent)

– Non-groupable streptococciNon-groupable streptococciS. pneumoniaeS. pneumoniae (pneumonia) (pneumonia)viridans streptococciviridans streptococci

– e.g. e.g. S. mutansS. mutans– Causing dental carriesCausing dental carries

Streptococci

Group AS. pyogenes

Group BS. agalactiae

Group CS. equisimitis

Group DEnterococcus

Lanciefield classification

Other groups (E-U)

Classification of Streptococci Classification of Streptococci Based on Hemolysis on Blood Based on Hemolysis on Blood

AgarAgarHemolysis on BAHemolysis on BA -hemolysis-hemolysis

Partial hemolysisPartial hemolysisGreen discoloration around the coloniesGreen discoloration around the coloniese.g. non-groupable streptococci (e.g. non-groupable streptococci (S. pneumoniaeS. pneumoniae & & S. S. viridansviridans))

-hemolysis-hemolysisComplete hemolysisComplete hemolysisClear zone of hemolysis around the coloniesClear zone of hemolysis around the coloniese.g.e.g. Group A & B (Group A & B (S. pyogenesS. pyogenes & & S. agalactiaeS. agalactiae))

-hemolysis-hemolysisNo lysisNo lysise.g. Group D (e.g. Group D (Enterococcus sppEnterococcus spp))

Streptococci

-hemolysis -hemolysis -hemolysis

Hemolysis on Blood agarHemolysis on Blood agar

-hemolysis

-hemolysis

-hemolysis

Differentiation between Differentiation between --hemolytic streptococcihemolytic streptococci

The following tests can be used to differentiate The following tests can be used to differentiate between between -hemolytic streptococci-hemolytic streptococci– Lanciefield ClassificationLanciefield Classification– Bacitracin susceptibility TestBacitracin susceptibility Test

Specific for Specific for S. pyogenesS. pyogenes (Group A) (Group A)

– CAMP testCAMP testSpecific Specific for S. agalactiae (Group B)(Group B)

– Hippurate hydrolysisHippurate hydrolysisDifferentiates Group B streptococci from other Differentiates Group B streptococci from other beta hemolytic streptococcibeta hemolytic streptococci

Group B streptococci hydrolyzes sodium hippurate Group B streptococci hydrolyzes sodium hippurate

Bacitracin sensitivityBacitracin sensitivityPrinciple:Principle:– Bacitracin test is used for Bacitracin test is used for

presumptive identification of group presumptive identification of group AA

– To distinguish between To distinguish between S. pyogenesS. pyogenes (susceptible to B) & non group A such (susceptible to B) & non group A such as as S. agalactiaeS. agalactiae (Resistant to B) (Resistant to B)

– Bacitracin will inhibit the growth of Bacitracin will inhibit the growth of gp A gp A Strep. pyogenesStrep. pyogenes giving zone of giving zone of inhibition around the diskinhibition around the disk

Procedure:Procedure:– Inoculate BAP with heavy suspension Inoculate BAP with heavy suspension

of tested organismof tested organism– Bacitracin disk (0.04 U) is applied to Bacitracin disk (0.04 U) is applied to

inoculated BAPinoculated BAP– After incubation, any zone of After incubation, any zone of

inhibition around the disk is inhibition around the disk is considered as susceptibleconsidered as susceptible

CAMP testCAMP test

Principle:Principle:– Group B streptococci produce extracellular protein (CAMP Group B streptococci produce extracellular protein (CAMP

factor)factor)– CAMP act synergistically with staph. CAMP act synergistically with staph. -lysin to cause lysis of -lysin to cause lysis of

RBCsRBCs

Procedure:Procedure:– Single streak of Single streak of StreptococcusStreptococcus to be tested and a to be tested and a Staph. Staph.

aureusaureus are made perpendicular to each other are made perpendicular to each other– 3-5 mm distance was left between two streaks3-5 mm distance was left between two streaks– After incubation, a positive result appear as an arrowhead After incubation, a positive result appear as an arrowhead

shaped zone of complete hemolysisshaped zone of complete hemolysis– S. agalactiaeS. agalactiae is CAMP test positive while non gp B is CAMP test positive while non gp B

streptococci are negativestreptococci are negative

CAMP testCAMP test

– Trimethoprim Trimethoprim sulfamethoxazolsulfamethoxazole (SXT)e (SXT)

Inhibits beta-Inhibits beta-hemolytic hemolytic streptococcal groups streptococcal groups other than A and Bother than A and B

Group A streptococcus growing in the presence of SXT

SXT testSXT test

Differentiation between Differentiation between --hemolytic streptococcihemolytic streptococci

The following definitive tests used to The following definitive tests used to differentiate between differentiate between S. pneumoniae & S. pneumoniae & viridans streptococciviridans streptococci

– Optochin TestOptochin Test– Bile Solubility TestBile Solubility Test– Inulin FermentationInulin Fermentation

Optochin Susceptibility TestOptochin Susceptibility Test

Principle:Principle:– Optochin (OP) test is presumptive test that is used to Optochin (OP) test is presumptive test that is used to

identify identify S. pneumoniaeS. pneumoniae– S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml) giving a inhibition zone ≥14 mm in diameter.

Procedure:Procedure:– BAP inoculated with organism to be testedBAP inoculated with organism to be tested– OP disk is placed on the center of inoculated BAPOP disk is placed on the center of inoculated BAP– After incubation at After incubation at 37oC for 18 hrs, accurately measure , accurately measure

the diameter of the inhibition zone by the rulerthe diameter of the inhibition zone by the ruler– ≥14 mm zone of inhibition around the disk is considered mm zone of inhibition around the disk is considered

as positive and ≤13 mm is considered negativeas positive and ≤13 mm is considered negative

S. pneumoniaeS. pneumoniae is positive (S) while is positive (S) while S. viridansS. viridans is is negative (R)negative (R)

Optochin Susceptibility TestOptochin Susceptibility Test

Optochin susceptibleS. pneumoniae

Optochin resistantS. viridans

Bile Solubility testBile Solubility test

Principle:Principle:– S. pneumoniae produce a self-lysing enzyme to

inhibit the growth– The presence of bile salt accelerate this

processProcedure:Procedure:– Add ten parts (10 ml) of the broth culture of the Add ten parts (10 ml) of the broth culture of the

organism to be tested to one part (1 ml) of 2% Na organism to be tested to one part (1 ml) of 2% Na deoxycholate (bile) into the test tubedeoxycholate (bile) into the test tube

– Negative control is made by adding saline instead of bile Negative control is made by adding saline instead of bile to the cultureto the culture

– Incubate at 37oC for 15 min– Record the result after 15 minRecord the result after 15 min

Bile Solubility testBile Solubility test

Results:Results:– Positive test appears as Positive test appears as

clearing in the presence of bile clearing in the presence of bile while negative test appears as while negative test appears as turbidturbid

– S. pneumoniaeS. pneumoniae soluble in bile soluble in bile whereas whereas S. viridansS. viridans insoluble insoluble

Group D streptococciGroup D streptococci

Bile Esculin hydrolysisBile Esculin hydrolysis– Ability to grow in Ability to grow in

40% bile and 40% bile and hydrolyze Esculin are hydrolyze Esculin are features of features of streptococci that streptococci that possess Group D possess Group D antigenantigen

Growth in 6.5% NaCl Growth in 6.5% NaCl brothbroth– Differentiates Group Differentiates Group

D streptococci from D streptococci from enterococcienterococci

PYR hydrolysisPYR hydrolysis– Substrate L-Substrate L-

pyrrolidonyl-pyrrolidonyl-napthlyamide (PYR) napthlyamide (PYR) is hydrolyzed by is hydrolyzed by Group A Streptococci Group A Streptococci and and EnterococcusEnterococcus sp. sp.

– As specific as 6.5% As specific as 6.5% NaCl broth for NaCl broth for EnterococcusEnterococcus sp. sp.

– More specific than More specific than Bacitracin for Group Bacitracin for Group A streptococciA streptococci

PYR test for Group A streptococci and enterococci. Both are positive for this test (right); left is a negative result

Differentiation between Differentiation between -hemolytic -hemolytic streptococcistreptococci

HemolysisHemolysisBacitracin Bacitracin

sensitivitysensitivityCAMP testCAMP test

S. pyogenesS. pyogenesSusceptibleSusceptibleNegativeNegative

S. S. agalactiaeagalactiae

ResistantResistantPositivePositive

HemolysiHemolysiss

OptochiOptochin n

sensitivisensitivityty

Bile Bile solubilitsolubilit

yy

Inulin Inulin FermentatioFermentatio

nn

S. S. pneumoniaepneumoniae

Sensitive Sensitive (≥ 14 (≥ 14 mm)mm)

SolubleSolubleNot fermentNot ferment

Viridans Viridans strepstrep

ResistantResistant

(≤13 (≤13 mm)mm)

InsolubleInsolubleFermentFerment

Differentiation between Differentiation between -hemolytic -hemolytic streptococcistreptococci

Outline of differentiation Outline of differentiation between Gram-Positive coccibetween Gram-Positive cocci

e.g. S. epidermidis