Biology subjects

Wyllie, A. L.

Publications and source records attributed to Wyllie, A. L..

10 recordsLinked to original sources

bioRxiv2025

SeroBA(v2.0) and SeroBAnk: a robust genome-based serotyping scheme and comprehensive atlas of capsular diversity in Streptococcus pneumoniae

The unprecedented number of Streptococcus pneumoniae (the pneumococcus) genomes sequenced in recent years has accelerated the discovery of novel serotypes and highlighted the genetic diversity both between and within each serotype. A novel serotype should demonstrate a distinct cps locus, capsular structure, and serological profile. In only the past four years, nine new serotypes have been identified. Accurate and timely serotyping of pneumococcal isolates is key to understanding its global distribution, evolution, and the response of the bacterial population to vaccination. However, current bioinformatics serotyping tools are infrequently updated, and struggle to accommodate the rapid discovery of new serotypes in a timely manner. To address these limitations, we built a comprehensive and curated library (SeroBAnk) encompassing all known pneumococcal serotypes; this resource is presented as an atlas on a dedicated publicly accessible webpage (https://www.pneumogen.net/gps/#/serobank). Building upon this resource, we developed SeroBA(v2.0), a tool with an easy-to-update database that can accurately identify 102 of 107 known pneumococcal serotypes (except for serotypes 24B, 24C, 24F, 7D and 6H) and 18 genetic subtypes within serotypes 6A, 6B, 11A, 19A, 19F and 33F. We validated SeroBA(v2.0) on 26,306 genomes from the Global Pneumococcal Sequencing project, reference isolates and simulated reads derived from the reference genetic sequences of capsular polysaccharide biosynthetic (cps) locus and showed that SeroBA(v2.0) can reliably detect the nine recently discovered serotypes. Additionally, we show that in silico serotypes inferred by SeroBA(v2.0) had high concordance with phenotypic serotypes determined by either Quellung or latex agglutination at the serotype level (88.9%; 15,945/17,933), and at the serogroup level (91.9%; 16,480/17,933). Finally, we propose a community-contribution based approach to ensure that SeroBA(v2.0) is maintained and updated as novel serotypes continue to be discovered. The global community can submit putative novel serotypes through our public repository on GitHub (https://github.com/GlobalPneumoSeq/seroba/issues). The submitted putative novel serotypes will be curated based on the genetic sequence of cps region, capsular structure and serological profile by people of relevant expertise in the field. SeroBA(v2.0) can be accessed at https://github.com/GlobalPneumoSeq/seroba. Data summaryGenome sequences are available in the European Nucleotide Archive (ENA) and are also available alongside metadata on the Monocle Database available at https://data.monocle.sanger.ac.uk/. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Impact StatementThe polysaccharide capsule has been an effective vaccine antigen against diseases caused by Streptococcus pneumoniae (the pneumococcus). The pneumococcal conjugate vaccine has been estimated to have halved pneumococcal-related childhood mortality over 15 years (2000-2015). We collated the genetic locus and capsular structure of each known capsule type (serotype), alongside with pneumococcal vaccine formulation and licensure history, into a single webpage (SeroBAnk), providing a valuable resource for basic research and vaccine development. With increasing use of whole genome sequencing in clinical and public health laboratories, we also provided a fast and accurate bioinformatics tool, SeroBA(v2.0), to identify 102 pneumococcal serotypes, alongside a proposed system to expand SeroBA(v2.0) to include new serotypes as they are discovered, ensuring that the tool remains valuable to the global research community in the long-term.

bioinformatics↗

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

The capsule and genetic background, rather than specific loci, strongly influence pneumococcal growth kinetics

Bacterial growth characteristics intrinsic to each strain can impact and influence gene expression, antibiotic susceptibility, and disease pathogenesis. However, little is known about specific genomic variations that influence these bacterial growth features. Here, we investigate the impact of Streptococcus pneumoniae genetics on its in vitro growth features to shed light on genes that may be important targets in the development of vaccines and therapeutics. We use statistical models to estimate growth features and demonstrate that they varied significantly across capsular serotypes and lineages, were strongly correlated with phylogeny, and showed high heritability, highlighting a strong genetic basis. Despite this, genome-wide association studies revealed no specific genomic loci statistically associated with the growth features independently of the genetic background, including those in the locus responsible for capsular polysaccharide synthesis. Our findings suggest that the serotype and lineage, as well as a combination of genomic loci, influence intrinsic pneumococcal growth kinetics, which may have implications for pneumococcal disease pathogenesis.

microbiology↗

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↗

bioRxiv2021

Pneumococcal genetic variability influences age-dependent bacterial carriage

The pneumococcal conjugate vaccine (PCV) primarily reduces disease burden in adults through a reduction in carriage prevalence of invasive serotypes in children. Current vaccine formulations are the same for both adults and children, but tailoring these formulations to age category could optimize vaccine efficacy. Identification of specific pneumococcal genetic factors associated with carriage in younger or older age groups may suggest alternative formulations and contribute to a better mechanistic understanding of immunity. Here, we used whole genome sequencing to dissect pneumococcal variation associated with age. We performed genome sequencing in a large carriage cohort, and conducted a meta-analysis with an existing carriage study. We compiled a dictionary of pathogen genetic variation including serotype, sequence cluster, sequence elements, SNPs, burden combined rare variants, and clusters of orthologous genes (COGs) for each cohort - all of which used in a genome-wide association with host age. Age-dependent colonization had some heritability, though this varied between cohorts (h2 = 0.10, 0.00 - 0.69 95% CI in the first; h2 = 0.46, 0.33 - 0.60 95% CI in the second cohort). We found that serotypes and genetic background (strain) explained most of the heritability in each cohort (h2serotype = 0.06 and h2GPSC = 0.04 in the first; h2 serotype = 0.20 and h2 GPSC = 0.23 in the second cohort). We found one candidate association (p = 1.2x10-9) upstream of an accessory Sec-dependent serine-rich glycoprotein adhesin. Overall, association with age was highly cohort and strain dependent, supporting proposals for a future vaccination strategy which is primarily targeted using serotypes rather than proteins, and is tailored towards specific pathogen populations.

microbiology↗

bioRxiv2020

Detection of SARS-CoV-2 RNA by multiplex RT-qPCR

The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. To simplify testing and reduce the volume of required reagents, we developed a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the CDC, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the singleplex assay adapted for research purposes. Low copies (>500 copies / reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current singleplex diagnostics by saving reagents, costs, time and labor.

microbiology↗

bioRxiv2018

Response of pneumococcus to changes in temperature and oxygen varies by serotype, lineage, and site of isolation

BackgroundPneumococcus, a bacterium that typically resides in the nasopharynx, is exposed to a variety of temperature and oxygen levels in the upper respiratory tract and as it invades the lung, tissues, and blood. The response to these variations likely varies by strain and could influence the fitness of a strain and its virulence. We sought to determine the effect of environmental variability on the growth characteristics of pneumococcus and to evaluate correlations between variability in growth characteristics between strains and biological and epidemiological characteristics. MethodsWe evaluated the effect of temperature and oxygen on the growth of 256 pneumococcal isolates representing 53 serotypes, recovered from healthy carriers and from disease patients. Strains were grown at a range of temperatures anaerobically or in ambient air with and without catalase and were monitored by reading the optical density. Regression models were used to evaluate bacterial and environmental factors associated with characteristics of the growth curves. ResultsMost isolates grew to the maximal density at the temperature of the nasopharynx (~33C) and under aerobic conditions (with catalase). Maximum density achieved was positively associated with the presence of N-acetylated sugars in the capsule and negatively associated with the presence of uronic acids. Reaching a greater density at an early time point was positively associated with the prevalence of serotypes among healthy carriers in the pre-vaccine period. DiscussionEnvironmental variability affects the growth of pneumococcus, with notable differences between isolates and by serotype. Such variability could be influenced by characteristics of the capsule and might affect virulence and transmissibility.

microbiology↗

bioRxiv2018

Joint sequencing of human and pathogen genomes reveals the genetics of pneumococcal meningitis

Streptococcus pneumoniae is a common nasopharyngeal colonizer, but can also cause life-threatening invasive diseases such as empyema, bacteremia and meningitis. Genetic variation of host and pathogen is known to play a role in invasive pneumococcal disease, though to what extent is unknown. In a genome-wide association study of human and pathogen we show that human variation explains almost half of variation in susceptibility to pneumococcal meningitis and one-third of variation in severity, and identified variants in CCDC33 associated with susceptibility. Pneumococcal variation explained a large amount of invasive potential, but serotype explained only half of this variation. Newly developed methods identified pneumococcal genes involved in invasiveness including pspC and zmpD, and allowed a human-bacteria interaction analysis, finding associations between pneumococcal lineage and STK32C.

genomics↗