# 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

## TL;DR

Non-encapsulated pneumococci in saliva show varying stability depending on their genetic makeup, with some strains being less detectable under certain storage conditions.

## Contribution

The study reveals that non-encapsulated pneumococci strains with aliC and aliD are less stable during culture enrichment, affecting their detection in saliva-based studies.

## Key findings

- NCC2 strains (with aliC and aliD) showed lower and less stable detection compared to NCC1 strains during culture enrichment.
- Extraction-free detection methods provided more stable results for NESp in saliva, especially for NCC2 strains.
- NCC2 strains may be underrepresented in carriage studies due to reduced resilience during culture-based detection.

## Abstract

Non-encapsulated Streptococcus pneumoniae (NESp) represent up to 19% of circulating pneumococci and exhibit high rates of genetic exchange and 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 NESp 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. Six NESp strains from two clinically relevant NESp null-capsule clades (NCCs), NCC1 (carrying pspK) and NCC2 (carrying aliC and aliD), were spiked into pneumococcus (lytA)-negative saliva and incubated through various temperatures and freeze-thaw conditions. Endpoints were processed using either culture enrichment (CE) and DNA extraction (CE-DNA), or an extraction-free method without CE, before testing for lytA using qPCR. Detection stability was assessed using linear regression modelling over temperature, time and freeze-thaws. Following CE-DNA, detection of NESp remained stable for ≤24 or ≤72 h when stored at room temperature or 4 °C, respectively, and over two freeze-thaw cycles (−80 °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 h at room temperature and over three freeze-thaw cycles, and negligible differences in detection between NCC1 and NCC2 strains. Additionally, extraction-free detection of NCC1, and less so NCC2, increased over the first 24 h when stored at 20–30 °C, suggesting growth of the NESp strains in saliva. Testing of ΔaliCaliD and ΔpspK mutants revealed that these genes increased in vitro viability when cultured in broth but did not significantly alter competitive fitness during saliva CE. The NCC1 NESp strains tested exhibited similar stability patterns in unsupplemented saliva as encapsulated pneumococci. However, the NCC2 strains tested here were 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 °C soon after collection and at −80 °C within 72 h. Methods which directly detect DNA without CE may provide a less biassed accounting of NCC2 strains.