Posibilities for use of whole genome sequencing (WGS) for the analysis of Streptococcus pneumoniae isolates.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus causing both non-invasive and invasive infectious diseases. Pneumococcal diseases are vaccine preventable. Invasive pneumococcal diseases (IPD) meeting the international case definition are reported nationally and internationally and are subject to surveillance programmes in many countries, including the Czech Republic. An important part of IPD surveillance is the monitoring of causative serotypes and their frequency over time and in relation to ongoing vaccination programmes. In the world and in the Czech Republic, whole genome sequencing (WGS) is increasingly used for pneumococci, which allows for serotyping from sequencing data, precise analysis of their genetic relationships, and the study of genes present in their genome. Whole-genome sequencing enables the generation of reliable and internationally comparable data that can be easily shared. Sequencing data are analysed using bioinformatics tools that require knowledge in the field of natural sciences with an emphasis on genetics and expertise in bioinformatics. This publication presents some options for pneumococcal analysis, i.e., serotyping, multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analysis, assignment to Global Pneumococcal Sequence Cluster (GPSC), and identification of virulence genes and antibiotic resistance genes. The WGS strategies and applications for Europe and WGS implementation in practice are presented. WGS analysis of pneumococci allows for improved IPD surveillance, thanks to molecular serotyping, more detailed typing, generation of internationally comparable data, and improved evaluation of the effectiveness of vaccination programmes.

Detection of superantigens in Streptococcus pyogenes isolates based on whole genome sequencing data.

Streptococcus pyogenes causes a variety of human diseases ranging from uncomplicated respiratory tract and skin infections to severe invasive diseases possibly involving toxic shock syndrome. Besides the emm gene-encoded M protein, important virulence factors are pyrogenic exotoxins, referred to as superantigens. The National Reference Laboratory for Streptococcal Infections has newly introduced bioinformatics tools for processing S. pyogenes whole genome sequencing data. Using the SRST2 software and BV-BRC platform, WGS data of 10 S. pyogenes isolates from patients with invasive disease were analysed, and emm type, sequence type, and superantigen encoding gene profiles were determined. The Unicycler assembly pipeline with the SPAdes de novo assembler was used to assemble genome sequences from short reads.

Molecular genotyping of Streptococcus agalactiae isolates with non-typeable serotype, Czech Republic, 2008­2020

The NRL for Streptococcal Infections performed serotyping of 1038 isolates of Streptococcus agalactiae from 972 patients by the latex agglutination method in 2008­2020. Forty-three isolates (4.4%) whose serotyping failed were classified as non-typeable. The aim of the present study was to determine the genotype of these non-typeable isolates using multiplex polymerase chain reaction (mPCR). Genotyping was successful in the entire set of 43 non-typeable isolates. The most common genotype was V (41.9%), followed by Ia (20.9%). The isolates serotyped by latex agglutination were predominantly assigned to serotype III (29.2%) and V (26.2%). Complete data were obtained on the prevalence of S. agalactiae serotypes/genotypes in the Czech Republic in 2008­2020. Monitoring the serotype and genotype distribution of the pathogen is a prerequisite for the introduction of a potential vaccine against S. agalactiae into clinical practice.