Magnetic bead-based separation of pneumococcal serotypes

Abstract

The separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed an assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A, and 15A) present at ∼0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of known pneumococcal serotypes from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. This method may have further laboratory or clinical applications when the selection of specific serotypes is required.

Keywords: Streptococcus pneumoniae; capsular polysaccharide; capsule switch; pneumococcus; saliva; serotypes; transformation.

Graphical abstract

Figure 1

An overview of the magnetic bead-based separation (MBS) method To a dual serotype mixture (1), antisera specific for the desired serotype are added. (2) Following brief wash steps, IgG-specific secondary antibody conjugated to a magnetic bead is incubated (3), and finally, the desired cells are extracted using the KingFisher Flex Purification System (4). Created with BioRender.com.

Figure 2

Efficiency of minority strain enrichment using the MBS method (A) Percentage minority serotype present prior to MBS (Pre) and after MBS (Post) for six serotypes in three initial serotype pairs. The average percentage minority in the post sample is presented above the data points. Minority and majority serotypes are displayed on the x axis in the following format: minority (majority). Triplicate results are shown for each of three serotype pairs where each serotype of the pair was tested as the minority serotype. (B) Comparison of percentage minority serotype present after MBS (Post) as determined by qPCR and colony counting methods. (C) Averages of triplicate data are shown for 23F (when with a majority of 12F), and averages of duplicate data were plotted for 23F (when with a majority of 3SCV and 3Muc). Minority and majority serotypes are displayed on the x axis in the following format: minority (majority). (D) Averages of triplicate data are shown for 3SCV (when with a majority of 14), and averages of duplicate data were plotted for 3SCV and 3Muc (when with a majority of 23F). Percentage minority from both colony counting and qPCR methods is shown. Minority and majority serotypes are displayed on the x axis in the following format: minority (majority). See also Tables S4 and S5.

Figure 3

Efficiency of minority enrichment using the MBS method on eight serotypes (A) Percentage minority serotype present prior to MBS (Pre) and after MBS (Post). (B) Percentage minority 23F present after MBS (Post) as determined by qPCR and colony counting. Minority and majority serotypes are displayed in the following format: minority (majority). Results shown are singlicate data points only. See also Tables S4 and S6.