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.

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).

Laboratory diagnosis

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

Culture and  identification during suspected Streptococcus pneumoniae infection

1. Microscopy and Staining 

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

2. Culture and Sensitivity 

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• 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.

      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.

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.

  

• 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 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).Mneomonics: Remember the sentence “Streptococcus pneumoniae is a BOSS” i.e. Bile Soluble, Optochin Sensitive
• 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.