The routine use of genomics for disease surveillance provides the opportunity for high-resolution bacterial epidemiology. Current whole-genome clustering and multilocus typing approaches do not fully ...exploit core and accessory genomic variation, and they cannot both automatically identify, and subsequently expand, clusters of significantly similar isolates in large data sets spanning entire species. Here, we describe PopPUNK (
ulation
artitioning
sing
ucleotide
-mers), a software implementing scalable and expandable annotation- and alignment-free methods for population analysis and clustering. Variable-length
-mer comparisons are used to distinguish isolates' divergence in shared sequence and gene content, which we demonstrate to be accurate over multiple orders of magnitude using data from both simulations and genomic collections representing 10 taxonomically widespread species. Connections between closely related isolates of the same strain are robustly identified, despite interspecies variation in the pairwise distance distributions that reflects species' diverse evolutionary patterns. PopPUNK can process 10
-10
genomes in a single batch, with minimal memory use and runtimes up to 200-fold faster than existing model-based methods. Clusters of strains remain consistent as new batches of genomes are added, which is achieved without needing to reanalyze all genomes de novo. This facilitates real-time surveillance with consistent cluster naming between studies and allows for outbreak detection using hundreds of genomes in minutes. Interactive visualization and online publication is streamlined through the automatic output of results to multiple platforms. PopPUNK has been designed as a flexible platform that addresses important issues with currently used whole-genome clustering and typing methods, and has potential uses across bacterial genetics and public health research.
(pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines (PCVs) targeting pneumococcal ...capsules has greatly reduced infections by the vaccine types but increased infections by nonvaccine serotypes. Herein, we report a new and the 100th capsule type, named serotype 10D, by determining its unique chemical structure and biosynthetic roles of all capsule synthesis locus (
) genes. The name 10D reflects its serologic cross-reaction with serotype 10A and appearance of cross-opsonic antibodies in response to immunization with 10A polysaccharide in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D
has three large regions syntenic to and highly homologous with
loci from serotype 6C, serotype 39, and an oral streptococcus strain (
SK145). The 10D
region syntenic to SK145 is about 6 kb and has a short gene fragment of
α at the 5' end. The presence of this nonfunctional
α fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. Since oral streptococci have a large repertoire of
loci, widespread PCV usage could facilitate the appearance of novel serotypes through interspecies recombination.
The polysaccharide capsule is essential for the pathogenicity of pneumococcus, which is responsible for millions of deaths worldwide each year. Currently available pneumococcal vaccines are designed to elicit antibodies to the capsule polysaccharides of the pneumococcal isolates commonly causing diseases, and the antibodies provide protection only against the pneumococcus expressing the vaccine-targeted capsules. Since pneumococci can produce different capsule polysaccharides and therefore reduce vaccine effectiveness, it is important to track the appearance of novel pneumococcal capsule types and how these new capsules are created. Herein, we describe a new and the 100th pneumococcal capsule type with unique chemical and serological properties. The capsule type was named 10D for its serologic similarity to 10A. Genetic studies provide strong evidence that pneumococcus created 10D capsule polysaccharide by capturing a large genetic fragment from an oral streptococcus. Such interspecies genetic exchanges could greatly increase diversity of pneumococcal capsules and complicate serotype shifts.
Vaccines are powerful agents in infectious disease prevention but often designed to protect against some strains that are most likely to spread and cause diseases. Most vaccines do not succeed in ...eradicating the pathogen and thus allow the potential emergence of vaccine evading strains. As with most evolutionary processes, being able to capture all variations across the entire genome gives us the best chance of monitoring and understanding the processes of vaccine evasion. Genomics is being widely adopted as the optimum approach for pathogen surveillance with the potential for early and precise identification of high-risk strains. Given sufficient longitudinal data, genomics also has the potential to forecast the emergence of such strains enabling immediate or pre-emptive intervention. In this review, we consider the strengths and challenges for pathogen genomic surveillance using the experience of the Global Pneumococcal Sequencing (GPS) project as an early example. We highlight the multifaceted nature of genome data and recent advances in genome-based tools to extract useful information relevant to inform vaccine strategies and treatment options. We conclude with future perspectives for genomic pathogen surveillance.
Full text
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Genomic evolution, transmission and pathogenesis of Streptococcus pneumoniae, an opportunistic human-adapted pathogen, is driven principally by nasopharyngeal carriage. However, little is known about ...genomic changes during natural colonisation. Here, we use whole-genome sequencing to investigate within-host microevolution of naturally carried pneumococci in ninety-eight infants intensively sampled sequentially from birth until twelve months in a high-carriage African setting. We show that neutral evolution and nucleotide substitution rates up to forty-fold faster than observed over longer timescales in S. pneumoniae and other bacteria drives high within-host pneumococcal genetic diversity. Highly divergent co-existing strain variants emerge during colonisation episodes through real-time intra-host homologous recombination while the rest are co-transmitted or acquired independently during multiple colonisation episodes. Genic and intergenic parallel evolution occur particularly in antibiotic resistance, immune evasion and epithelial adhesion genes. Our findings suggest that within-host microevolution is rapid and adaptive during natural colonisation.
Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods.
serotyping from WGS has not yet been applied to long-read sequences ...produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (
= 17; 14.3%), 14 (
= 10; 8.4%), and 15B/C (
= 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.
Streptococcus pneumoniae can produce a wide breadth of antigenically diverse capsule types, a fact that poses a looming threat to the success of vaccines that target pneumococcal polysaccharide (PS) ...capsule. Yet, many pneumococcal capsule types remain undiscovered and/or uncharacterized. Prior sequence analysis of pneumococcal capsule synthesis (
) loci suggested the existence of capsule subtypes among isolates identified as "serotype 36" according to conventional capsule typing methods. We discovered these subtypes represent two antigenically similar but distinguishable pneumococcal capsule serotypes, 36A and 36B. Biochemical analysis of their capsule PS structure reveals that both have the shared repeat unit backbone →5)-α-d-Gal
-(1→1)-d-Rib-ol-(5→P→6)-β-d-Man
NAc-(1→4)-β-d-Glc
-(1→ with two branching structures. Both serotypes have a β-d-Gal
branch to Ribitol. Serotypes 36A and 36B differ by the presence of a α-d-Glc
-(1→3)-β-d-Man
NAc or α-d-Gal
-(1→3)-β-d-Man
NAc branch, respectively. Comparison of the phylogenetically distant serogroup 9 and 36
loci, which all encode this distinguishing glycosidic bond, revealed that the incorporation of Glc
(in types 9N and 36A) versus Gal
(in types 9A, 9V, 9L, and 36B) is associated with the identity of four amino acids in the
encoded glycosyltransferase WcjA. Identifying functional determinants of
-encoded enzymes and their impact on capsule PS structure is key to improving the resolution and reliability of sequencing-based capsule typing methods and discovering novel capsule variants indistinguishable by conventional serotyping methods.
Abstract
Summary
Homologous recombination is an important evolutionary process in bacteria and other prokaryotes, which increases genomic sequence diversity and can facilitate adaptation. Several ...methods and tools have been developed to detect genomic regions recently affected by recombination. Exploration and visualization of such recombination events can reveal valuable biological insights, but it remains challenging. Here, we present RCandy, a platform-independent R package for rapid, simple and flexible visualization of recombination events in bacterial genomes.
Availability and implementation
RCandy is an R package freely available for use under the MIT license. It is platform-independent and has been tested on Windows, Linux and MacOSX. The source code comes together with a detailed vignette available on GitHub at https://github.com/ChrispinChaguza/RCandy.
Supplementary information
Supplementary data are available at Bioinformatics online.
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