Storage stability of non-encapsulated pneumococci in saliva is dependent on null-capsule clade, with strains carrying aliC and aliD showing a competitive disadvantage during culture enrichment

Abstract

BackgroundNon-encapsulated Streptococcus pneumoniae (NESp) represent up to 19% of circulating pneumococci and exhibit high rates of antimicrobial resistance. Saliva is increasingly used as a pneumococcal carriage study specimen, and we recently developed a qPCR assay to enhance carriage surveillance and characterization of NESpn in saliva. Previous work has established that pneumococci remain viable in unsupplemented saliva for extended periods under various conditions, however these findings may not be applicable to NESp. Therefore, to ensure the robustness of NESp detection in saliva-based carriage studies we evaluated the impact of transport and storage conditions of saliva samples on NESp detection. MethodsSix NESp strains from two clinically relevant NESp null capsule clades (NCC), NCC1 (carrying pspK) and NCC2 (carrying aliC and aliD), were spiked into lytA-negative saliva and incubated through various temperatures and freeze-thaw conditions. Endpoints were processed using either culture-enrichment and DNA extraction (CE-DNA), or an extraction-free method without CE, before testing for lytA using qPCR. Detection stability was assessed using regression modelling over temperature, time and freeze-thaws. ResultsFollowing CE-DNA, detection of NESp remained stable for [≤]24 or [≤]72 hours when stored at room temperature or 4{degrees}C, respectively, and over 2 freeze-thaw cycles (-80{degrees}C), with glycerol-supplementation providing slight benefits. Stability of detection when using CE-DNA depended on NCC; detection of NCC2 strains was lower, and less stable than NCC1. Compared to CE-DNA, extraction-free detection was more stable, with no significant loss over 72 hours at room temperature and over 3 freeze-thaw cycles. With extraction-free detection, there were also negligible diderences in detection between NCC1 and NCC2. Additionally, extraction-free detection of NCC1, and less so NCC2, increased over the first 24 hours when stored at 20-30{degrees}C, suggesting growth in saliva. Testing of{Delta} aliCaliD and{Delta} pspK mutants revealed these genes increased in vitro viability of NCC2 and NCC1, respectively, but did not significantly alter competitive fitness during CE. ConclusionNCC1 NESp strains exhibit similar stability patterns in unsupplemented saliva as encapsulated pneumococci. NCC2 strains, however, are less resilient during CE, likely due to competition with other oral microbes. Therefore, recovery of NCC2 NESp may be impacted by transport and storage conditions, leading to an underestimation of carriage prevalence when tested using CE-based methods. For the reliable carriage surveillance of NESp, samples should be stored at 4{degrees}C soon after collection and at -80{degrees}C within 72 hours. Methods which directly detect DNA without CE may provide a less biased accounting of NCC2 strains.