Streptococcus -biochemical Reactions 22191p

Streptococci Biochemical reactions By Dr. Nabil El Aila Assistant Professor of Molecular Microbiology Medical Technology Department Al -Aqsa University Dr. Nabil El Aila Diagnostic Microbiology

Streptococci • Characters of Streptococci – – – – –

Gram positive cocci 1µm in diameter Chains or pairs Usually capsulated Non motile

– – – –

Non spore forming Facultative anaerobes Fastidious Catalase negative (Staphylococci are catalase positive) Dr. Nabil El Aila Diagnostic Microbiology

Hemolysis on Blood agar -hemolysis

-hemolysis

-hemolysis

Dr. Nabil El Aila Diagnostic Microbiology

Classification

Based on O2 Anaerobes

Aerobes

Peptostreptococci

Growth on BA α hemolysis

β hemolysis

Incomplete hemolysis (green color)

Complete hemolysis

γ hemolysis α / β / no hemolysis

Lancefield grouping

Strep. viridans

specific C carbohydrate Ag on cell wall

Strep. pneumoniae

18.05.09 Group A S. pyogenes

Group B S. agalactiae

Enterococcus fecalis

Group A – U (21 groups)

Group C S. equisimitis

Group D Enterococcus

4

Other groups (E-U)

Differentiation between -hemolytic streptococci • The following tests can be used to differentiate between -hemolytic streptococci – Lanciefield Classification – Bacitracin susceptibility Test • Specific for S. pyogenes (Group A)

– CAMP test • Specific for S. agalactiae (Group B)

Dr. Nabil El Aila Diagnostic Microbiology

Laboratory Diagnosis: Group A Streptococcus • Colony morphology – Transparent, smooth, and well-defined zone of complete or b- hemolysis

Dr. Nabil El Aila

Laboratory Diagnosis: Group A Streptococcus • Identification – Catalase-negative – Bacitracin-susceptible – PYR-positive – Bile-esculin–negative – 6.5% NaCl-negative

Group A streptococci is susceptible to Bacitracin disk (left); The right shows resistance Dr. Nabil El Aila

Bacitracin sensitivity • Principle: – Bacitracin test is used for presumptive identification of group A – To distinguish between S. pyogenes (susceptible to B) & non group A such as S. agalactiae (Resistant to B) – Bacitracin will inhibit the growth of gp A Strep. pyogenes giving zone of inhibition around the disk

• Procedure: – Inoculate BAP with heavy suspension of tested organism – Bacitracin disk (0.04 U) is applied to inoculated BAP – After incubation, any zone of inhibition around the disk is considered as susceptible

Pyrrolidonyl arylamidase Test (PYR) I.Principle • Some bacteria like group A streptococci and Enterococcu s species produce pyrrolidonyl arylamidase which hydrolyzes the substrate Lpyrrolidonyl -β-naphthylamide to form β-naphthylamine. • A pink to red color forms when p-dimethylaminocinnam-aldehyde (PYR reagent) is added to β-naphthylamine. II. Inoculum • Strains are grown on blood agar plates overnight at 35°C in CO 2 . More than 1 day of incubation may be necessary for more fastidious genera such as the gemellae, alloiococci, and helcococci. The strains to be tested are grown on a blood agar plate until sufficient growth is seen to heavily inoculate the disks. III. Reagents and Materials • PYR disk (Remel) - PYR reagent • Loops - Deionized Sterile water Dr. Nabil El Aila

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IV. Procedure

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The procedure that is used in the Streptococcus laboratory is modified from the package insert. The LAP test is usually done simultaneously.

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1. Place the disks on blood agar plate in an area of little or no growth or on a slide. The moisture from the plate is usually sufficient to rehydrate the disk. If the disk is placed on a slide, then a tiny drop of sterile deionized water is added. (DO NOT OVERSATURATE THE DISK).

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2. Using a loop or wooden stick, inoculate the disks heavily. Using two or more loop-fulls of culture is necessary for satisfactory results.

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3. Leave the plates with the disks on the bench at room temperature for 10 minutes.

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4. Add the detection reagent and read after 3 minutes.

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V. Reading and Interpretation

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The development of a red color within 3 minutes is positive. No change in color or a yellow color is negative. The color develops immediately. Discard the test after 10 minutes.

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VI. Limitations

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False negative reactions may result if too little inoculum is used.

PYR Negative

Dr. Nabil El Aila

PYR positive

Laboratory Diagnosis: Group B -Hemolytic Streptococcus • Colony morphology – Grayish-white, mucoid, creamy, narrow zone of bhemolysis • Presumptive Identification tests – Catalase-negative – Bacitracin-resistant

Dr. Nabil El Aila

Laboratory Diagnosis: Group B b-Hemolytic Streptococcus • Presumptive identification tests – Bile-esculin-hydrolysis– negative – Does not grow in 6.5% NaCl – CAMP-test–positive – Hipppurate Hydrolysis

S. agalactiae shows the arrow-shaped hemolysis near the staphylococcus streak, showing a positive test for CAMP factor Dr. Nabil El Aila

CAMP Test I. Principle • Some bacteria produce CAMP factor (a diffusible extracelluar protein) that synergistically acts with the beta-lysin of Staphylococcus aureus and enhances the lysis of red blood cells. The purpose of the CAMP test is to aid in the identification of nonhemolytic group B streptococci and other ß-hemolytic streptococci. II. Specimen Growth from a blood agar plate or any solid media. III. Reagents and Materials TSA-sheep blood agar Dr. Nabil El Aila

IV. Procedure 1. The CAMP test is performed on TSA-sheep blood agar. A single streak of ß-lysin producing S. aureus made across the center of the plate. Strain SS-695 (Strep. Lab number is a ß-lysin producing strain of S. aureus. 2. A single colony of the unknown strain (beta hemolytic streptococci) is picked up with an inoculating loop and used to make a single streak perpendicular but not touching the S. aureus streak. A 2-3 mm space should remain between the streaks. 3. Incubate the inoculated plate normal atmosphere overnight at 35ΕC. Group B streptococci and a few other beta-streptococci produce an enhancement of the ßlysin activity of the S. aureus strain. V. Reading and Interpretation This enhanced activity is in the shape of an arrowhead at the juncture of the two streaks, with the widest portion of the arrowhead on the group B side. VI. Limitations Do not incubate in an anaerobic environment or under CO 2. Some S. pyogenes strains will give a positive reaction when incubated in CO 2.

CAMP test

Dr. Nabil El Aila

Hipppurate Hydrolysis Test I. Principle • Some bacteria like group B streptococci produce the enzyme hippurate hydrolase which hydrolyzes sodium hippurate to form benzoic acid and glycine. The addition of ferric chloride to benzoic acid forms an insoluble brown ferric benzoate precipitate. II. Inoculum • An overnight culture in Todd Hewitt broth incubated at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture from a blood agar plate may also be used. III. Reagents and Materials 1. Hippurate broth commercial suppliers. 2. Ferric choride (FeCl3) commercial supplies. Labeled as TDA if purchased for bioMereiux

IV. Procedure 1. The hippurate broth is inoculated with one drop of a fresh (16-20 h) ToddHewitt broth culture. 2. The broth is incubated for up to 7 days or until turbid growth is seen at 35C°. 3. The tube of broth is then centrifuged to sediment the bacteria. 4. Pipette 0.8 ml the clear supernatant to a small clear tube (13 x 100). 5. Add 0.2 ml of ferric chloride reagent to the supernatant. Mix well. V. Results and Interpretation A heavy precipitate that does not clear within 10 minutes indicates a positive test. A clear golden-brown liquid indicates a negative test. VI. Limitations Growth should be turbid before testing. Some fastidious organisms may show poor growth Dr. Nabil El Aila

Laboratory Diagnosis: Streptococcus Group D and Enterococcus Species • Identification tests – Catalase: may produce a weak catalase reaction – Hydrolyze bile esculin – Differentiate Group D from Enterococcus sp. with 6.5% NaCl or PYR test

Dr. Nabil El Aila

Laboratory Diagnosis: Streptococcus Group D and Enterococcus Species • Microscopic morphology – Cells tend to elongate

• Colony morphology – Most are nonhemolytic, although some may show or, rarelyhemolysis – Possess Group D antigen

Dr. Nabil El Aila

Bile Esculin Test I.Principle • A selective and differential medium used in the identification of catalase-negative bacteria. The selective agent bile, inhibits most gram positive bacteria. The enterococci and Streptococcus bovis will grow. • Esculin in the medium is hydrolyzed to esculetin and dextrose. The esculetin reacts with ferric chloride in the media to form a black-brown color. II. Inoculum • An overnight culture in Todd Hewitt broth incubated over night at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture may also be used. III. Reagents and Materials • 1. Bile esculin slant (Remel) Dr. Nabil El Aila

IV. Procedure 1. Inoculate tube with 1 drop of inoculum allowing drop to run down slant. Alternatively, the slant may be inoculated with a loopful of growth from a blood agar plate. 2. The slant is then incubated at 35ΕC for 2 days in ambient air. Fastidious organisms may be held up to 14d. V. Reading and Interpretation The bile esculin test is positive when a black color forms over one-half or more of the slant. If no blackening occurs the test is negative. VI. Limitations Do not incubate medium in a carbon dioxide atmosphere. The increase in C0 2 will cause the viridans streptococci to grow better and increase the likelihood of a positive BE reaction. Streptococcus bovis and enterococci do not require C02 for good growth.

Dr. Nabil El Aila

Identification Schema

Schema to differentiate Group A and B from other b-hemolytic streptococci

Identification Schema

Schema to differentiate Enterococcus and Group D streptococci from other nonhemolytic streptococci

Laboratory Diagnosis: Streptococcus pneumoniae • Colony morphology – Smooth, glistening, wetlooking, mucoid – -Hemolytic – CO2enhances growth

Dr. Nabil El Aila

Laboratory Diagnosis: Streptococcus pneumoniae • Identification – Catalase negative – Optochinsusceptibility-test– susceptible – Bile-solubility-test– positive

Dr. Nabil El Aila

Differentiation between -hemolytic streptococci • The following definitive tests used to differentiate between S. pneumoniae & viridans streptococci – Optochin Test – Bile Solubility Test – Inulin Fermentation

Dr. Nabil El Aila

Optochin Susceptibility Test • Principle: – Optochin (OP) test is presumptive test that is used to identify S. pneumoniae – S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml) giving a inhibition zone ≥14 mm in diameter. • Procedure: – BAP inoculated with organism to be tested – OP disk is placed on the center of inoculated BAP – After incubation at 37oC for 18 hrs, accurately measure the diameter of the inhibition zone by the ruler – ≥14 mm zone of inhibition around the disk is considered as positive and ≤13 mm is considered negative • S. pneumoniae is positive (S) while S. viridans is negative (R)

Optochin Susceptibility Test Optochin resistant S. viridans Optochin susceptible S. pneumoniae

Dr. Nabil El Aila

Bile Solubility Test I.Principle • The purpose of the bile solubility test is to aid in the differentiation of S. pneumoniae from all other alpha-hemolytic streptococci. Sodium deoxycholate (2%) acts on the cell wall of pneumococci resulting in lysis. II. Inoculum • An overnight culture grown on blood agar incubated 35°C in CO2. III. Reagents and Materials 1. 2% deoxycholate (CDC Central Services Laboratory, formula #5333) 2. physiologic saline pH 7.0 3.13 X 100mm glass tube

Dr. Nabil El Aila

IV. Procedure 1. Make a 1.0 ml saline suspension of cells from growth on an agar plate. A turbidity equal to that of 1.0 to 2.0 McFarland density standard should be used. 2. After a satisfactory density is achieved, divide the suspension into 2 tubes with approximately 0.5 ml in each. 3. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube and 0.5 ml saline to the other tube. Mix by vigorous shaking. 4. Incubate the tubes at 35-37C° for up to 2 h. V. Reading and Interpretation • Examine for clearing of the turbidity periodically. • A clearing of the turbidity in the bile tube but not in the saline control tube indicates a positive test, i.e., the pneumococcal cells have lysed ("solubilized"). • If the tube containing the cells and bile have not cleared the test is negative. VI. Limitations • The turbidity must be sufficient to detect a difference in the saline control tube.

Dr. Nabil El Aila

Dr. Nabil El Aila

Identification Schema

Schema to differentiate S. pneumoniae from other hemolytic streptococci

Dr. Nabil El Aila

Differentiation between -hemolytic streptococci CAMP test

Bacitracin sensitivity

Hemolysis

Negative

Susceptible



S. pyogenes

Positive

Resistant



S. agalactiae

Differentiation between -hemolytic streptococci Inulin Fermentatio n

Bile solubilit y

Optochi Hemolysi n s sensitivi ty

Not ferment

Soluble

Sensitive (≥ 14 mm)

Ferment

Insoluble

Resistant



S. pneumoniae

Outline of differentiation between Gram-Positive cocci

e.g. S. epidermidis