How to identify Streptococcus pneumoniae?

Streptococcus pneumoniae (pneumococci) is a part of the normal nasopharyngeal and oropharyngeal flora. It is an important etiological agent of upper and lower respiratory tract infections (URTI and LRTI), bacteremia, and septicemia. Streptococcus pneumoniae is also associated with otitis media, sinusitis, meningitis, and endocarditis.

Lanceolate diplococci. Image source: microbiologyinpictures.com

Pneumococci are Gram positive lancet shaped diplococci (intracellularly or extracellularly), non-motile, and encapsulated. They occur in pairs with the broader end opposed, hence called Gram positive diplococci. S. pneumoniae is a fastidious bacterium, which grows best at at 35-37°C with ~5% CO2 (or in a candle-jar).

Table of Contents

Laboratory diagnosis

Laboratory diagnosis of Streptococcus pneumoniae infection is based on finding characteristics shape of the organism in the sample, characteristic colony morphology, biochemical reactions, susceptibility to certain diagnostic discs, and latex agglutination test.

Sample

Specimens used for the laboratory diagnosis of Streptococcus pneumoniae of may be

Specimens from respiratory tract: Sputum, lung aspirate, pleural fluid
Body fluids e.g., Blood/ cerebrospinal fluid
Exudates from the joint, middle ear, other sites

Sample Collection

Sputum:
Collect > 1.0 ml expectorated sputum in a sterile screw-capped container.
Lung aspirate/ pleural fluid
Collect > 1.0 ml by percutaneous needle aspiration in a sterile screw-capped tube.
Blood:
Clean the venipuncture site with 70% alcohol and iodine, allow it to evaporate and collect blood aseptically in a culture broth with an anticoagulant. In case of adults, collect 5-10 ml blood in culture bottle, for children < 12 year old, collect 1.5-2.0 ml blood. Mix the blood and broth by rotating gently to avoid clotting.
Cerebrospinal fluid (CSF):
Clean the skin over L3-L4 inter-space with 70% alcohol and iodine. Collect > 1.0 ml CSF in a sterile screw-capped tube. Keep the CSF in an incubator at 35-37 degree centigrade, if it is not processed immediately.
Exudates from joints/middle ear
Collect > 1.0 ml by aspiration in a sterile screw-capped tube or add directly in a culture broth used for blood culture.

Note: DO NOT REFRIGERATE THE SAMPLE

Sample Transport

Streptococcus pneumoniae is a fastidious bacteria. Care must be taken during transport of specimen. Specimens must be transported promptly to the laboratory preferably within 1-2 hours.

Blood can only be transported after collecting in a culture broth containing appropriate anticoagulant. The inoculated medium can be held at room temperature (20°C– 25 °C) for 4 – 6 hours before incubation at 37 °C. The samples during transportation should be protected from extremes of temperature (less than 18 °C, more than 30 °C) and direct sunlight.

Culture and identification during suspected Streptococcus pneumoniae infection

Flow chart for identification and characterization of a S. pneumoniae isolate — Flow chart for identification and characterization of a *S. pneumoniae* isolate

Microscopy and Staining

Perform Gram staining of the sample (sputum/CSF)
Gram staining shows Gram positive lanceolate shaped diplococci

gram positive cocci streptococcus — Gram positive diplocci: *Streptococcus* spp

2. Culture and Sensitivity

Inoculate sample onto blood agar and chocolate agar plate.
Incubate at 37°C with 5-10% CO₂ for 24 – 48 hours.

Colony morphology

Colonies on blood agar plate are small (0.5 mm), round, transluscent or mucoid with alpha-hemolysis (A green discolouration of the agar around the colonies). Alpha-hemolytic property differentiates S. pneumoniae from many species, but not from the commensal alpha-hemolytic (viridans) streptococci.
Young alpha-hemolytic colonies appear raised, and in 24 – 48 hours colonies are flattened with depressed centre and is called draughtsman colony. It is due to partial autolysis (these colonies are tentatively identified as Pneumococci).
Streptococcus viridans also produces alpha-hemolytic colonies but does not produce draughstman colony.

Alpha-hemolytic colonies are further identified by the following confirmatory tests.

**Identification of Streptococcus pneumoniae by biochemical reactions.**

Mnemonics: “Streptococcus pneumoniae is a BOSS” i.e. Bile Soluble, Optochin Sensitive

A. Optochin test (6 mm disc with 5µg).

Inoculate blood agar plate with suspected alpha-hemolytic isolates.
Apply commercially available optochin discs on the streaked blood agar plate
Incubate plates at 37°C with 5-10% CO₂ for 18-24 hours.

Alpha hemolysis in Blood Agar by *Streptococcus* pneumoniae, Observe the zone of inhibition around Optochin disk.

Observe the zone of growth inhibition around the disc and interpret as:

A zone size > 14mm indicates susceptibility which is diagnostic of Streptococcus pneumoniae.
alpha-haemolytic colonies with zone of inhibition between 9 and 13 mm should be tested for bile solubility.

B. Bile solubility test.

alpha-haemolytic colonies showing zone of inhibition around optochin disc between 9 to 13 mm should be tested for bile solubility.
Prepare 1.0 ml of saline suspension of the organism from blood agar plate. The turbidity of the suspension should be equivalent to 0.5 McFarland standard.

Bile solubility test: Streptococcus pneumonie colonies are lysed by bile — Bile solubility test: *Streptococcus pneumoniae* colonies are lysed by bile

Inoculate 0.5 ml of the suspension into two tubes.
Add an equal amount (0.5 ml) of 2% sodium deoxycholate in one tube marked as test and 0.5ml of saline into the second tube marked as control.
Shake gently and incubate the tubes at 37°C for 2 hours.

Reading

Positive reaction: Clearing of the tube or loss in turbidity in the presence of deoxycholate due to disruption of cells.
Negative reaction: Persistence of turbidity.

Note: This test can also be done directly onto the colony and the colony is lysed by the addition of bile solution.

Interpretation of Optochin and Bile solubility test

Zone inhibition of growth around optochin 14mm is definitely Pneumococci.
A definite inhibition zone around optochin disc < 14mm and if the isolate is bile soluble, the isolate is considered as Streptococcus pneumoniae (If not bile soluble, it is not Streptococcus pneumoniae).
Strains with < 14 mm zone of inhibition to optochin or no zone at all and the isolate is not bile soluble it is not pneumococci. It is probably Streptococcus viridans.

Antimicrobial susceptibility

Perform antimicrobial susceptibility test against a selected group of antimicrobials by disk-diffusion method

Reporting of results: Streptococcus pneumoniae isolated and resistance patterns with tested antibiotics

3. Detection of the antigen

C-carbohydrate antigen of the Streptococcus pneumoniae can be detected in the urine (Read:Pneumococcal Urinary Antigen Testing (UAT): Principle, Procedure and Results ) for the diagnosis of pneumonia and in CSF for the diagnosis of pneumococcal meningitis.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.