Hislop, M. | bioRxiv Science

bioRxiv2024

An abundance of aliC and aliD genes were identified in saliva using a novel multiplex qPCR to characterize group II non-encapsulated pneumococci with improved specificity

BackgroundSurveillance of pneumococcus is reporting increasing prevalence of non-encapsulated pneumococci (NESp). NESp are an important reservoir for genetic exchange among streptococci, including for antimicrobial resistance (AMR), and are increasingly implicated in disease. Disease-associated NESp commonly carry the virulence genes pspK, or aliC and aliD in their cps locus instead of capsule genes. While molecular methods targeting the cps region are widely used for serotyping encapsulated strains, there are few assays available for classification of NESp, meaning it is not widely undertaken. Therefore, we exploited these genes as targets for a novel qPCR assay for detecting and classifying NESp strains with improved efficiency and specificity. MethodsWe conducted bioinformatic analysis on sequences from 30 NESp and 23 other mitis-group streptococcal sequences and developed a multiplex-qPCR, targeting pspK, aliD and two regions of aliC. The assay was validated using 11 previously characterised, and 5 uncharacterised NESp isolates. We then applied the assay to DNA extracted from culture-enriched saliva, and isolated and characterised suspected NESp colonies, with confirmation by whole genome sequencing. ResultsBioinformatic analyses demonstrated that previously published primers for aliC and aliD had low pneumococcal-specificity but indicated that targeting two regions of aliC would improve species-specificity, without compromising sensitivity. Our novel multiplex assay accurately typed all isolates. When screening saliva, we found a high prevalence of aliC and aliD, even in samples negative for pneumococcal genes lytA and piaB. Isolated colonies which were aliC and aliD positive could be differentiated as non-pneumococcal streptococci using our assay. ConclusionOur multiplex-qPCR assay can be used to efficiently screen even highly polymicrobial samples, such as saliva, for NESp genes, to detect and differentiate potentially pathogenic NESp clades from closely related mitis-group streptococci. This will allow for a better understanding of the true prevalence of NESp, and their impact upon pneumococcal carriage, disease, and AMR.

microbiology↗

bioRxiv2021

Magnetic Bead-Based Separation (MBS) of Pneumococcal Serotypes

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 a known pneumococcal serotype from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. Moreover, this method may have further laboratory or clinical applications when the selection of specific serotypes is required.

microbiology↗