Project description:Diagnostic primer extension assay to serotype Streptococcus pneumoniae. Assay validation. Background: Monitoring of Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. To simplify S. pneumoniae serotyping, a novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Results: Autolysin (lytA), pneumolysin (ply) and eight genes located in the capsular operon (cps) were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system that transforms genetic typing data into capsular serotype identification. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. In addition, the assay was tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitidis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. The assay presented with no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. Conclusion: This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype prevalence changes after new conjugate vaccines introduction.
Project description:Diagnostic primer extension assay to serotype Streptococcus pneumoniae. Assay validation. Background: Monitoring of Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. To simplify S. pneumoniae serotyping, a novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Results: Autolysin (lytA), pneumolysin (ply) and eight genes located in the capsular operon (cps) were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system that transforms genetic typing data into capsular serotype identification. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. In addition, the assay was tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitidis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. The assay presented with no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. Conclusion: This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype prevalence changes after new conjugate vaccines introduction. 166 quellung serotyped strains and two negative controls
Project description:Mouse lung RNAseq after infection with Influenza A virus (H1N1, PR/8/34, mouse-adapted) and Streptococcus pneumoniae (serotype 19F, strain BHN 100) Results: Differentially expressed genes were observed after single and co-infection Project: COST_mouse_2021_lung
Project description:Mouse blood transcriptome after infection with Influenza A virus (H1N1, PR/8/34, mouse-adapted) and Streptococcus pneumoniae (serotype 19F, strain BHN 100) Results: Differentially expressed genes were observed after single and co-infection Project: COST_mouse_2021
Project description:Genome-wide screens have discovered a large set of essential genes in the human pathogen Streptococcus pneumoniae. However, the function of many essential genes is still unknown, hampering vaccine and drug development programs. Based on results from transposon-sequencing (Tn-Seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting all 391 potentially essential genes using CRISPR interference (CRISPRi). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We identified SPD1416 and SPD1417 (named to MurT and GatD, respectively) as essential peptidoglycan synthesis proteins and we show that SPD1198 and SPD1197 (named to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. Our CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets. This RNA-Seq dataset is aimed to show that induction of the CRISPRi system very selectively represses its target gene, firefly luciferase, without other observable transcriptional effects.
Project description:Transcriptional profiling of primary human alveolar macrophages (AMs) comparing control untreated AMs with AMs exposed with Serotype 14 Streptococcus pneumoniae (NCTC11902) strain (MOI 10) for 4 hours) Two-condition experiment, control AMs vs. infected AMs. Biological replicates: 3 control replicates, 3 infected replicates MOI 10.
Project description:Transcriptional profiling of aortic sinus atherosclerotic plaque macrophages obtained by laser capture microdissection from male ApoE deficient mice infected with 5x10(5) cfu serotype 4 Streptococcus pneumoniae via intranasal instillation (n=9) as compared with mice mock infected with PBS (n=11). Mice were culled 2 weeks post infection/mock infection.