An Abundance of aliC and aliD Genes Was Identified in Saliva Using a Novel Multiplex qPCR to Characterize Group II Non-Encapsulated Pneumococci With Improved Specificity

BACKGROUND: Surveillance 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. METHODS: We 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. RESULTS: Bioinformatic 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. CONCLUSION: Our 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.