Biology subjects

Allicock, O. M.

Publications and source records attributed to Allicock, O. M..

3 recordsLinked to original sources

bioRxiv2025

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

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.

microbiology↗

bioRxiv2024

Tiled Amplicon Sequencing Enables Culture-free Whole-Genome Sequencing of Pathogenic Bacteria From Clinical Specimens

Pathogen sequencing is an important tool for disease surveillance and demonstrated its high value during the COVID-19 pandemic. Viral sequencing during the pandemic allowed us to track disease spread, quickly identify new variants, and guide the development of vaccines. Tiled amplicon sequencing, in which a panel of primers is used for multiplex amplification of fragments across an entire genome, was the cornerstone of SARS-CoV-2 sequencing. The speed, reliability, and cost-effectiveness of this method led to its implementation in academic and public health laboratories across the world and adaptation to a broad range of viral pathogens. However, similar methods are not available for larger bacterial genomes, for which whole-genome sequencing typically requires in vitro culture. This increases costs, error rates and turnaround times. The need to culture poses particular problems for medically important bacteria such as Mycobacterium tuberculosis, which are slow to grow and challenging to culture. As a proof of concept, we developed two novel whole-genome amplicon panels for M. tuberculosis and Streptococcus pneumoniae. Applying our amplicon panels to clinical samples, we show the ability to classify pathogen subgroups and to reliably identify markers of drug resistance without culturing. Development of this work in clinical settings has the potential to dramatically reduce the time of diagnosis of drug resistance for multiple drugs in parallel, enabling earlier intervention for high priority pathogens.

genomics↗

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↗