Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.

Respiratory syncytial virus (RSV) is the primary cause for acute lower respiratory syndrome in children younger than 5 years. Research on B cell repertoires and antibodies binding the RSV fusion protein (RSV F) is of major interest in the development of potential vaccine candidates and therapies. B cell receptors (BCRs) which have higher affinities for a specific antigen are preferentially selected for B cell clonal expansion in germinal center reactions. Consequently, antigen-specific BCR repertoires share common features, as for instance preferential variable gene usage, variable region mutation levels or lengths of the heavy chain complementarity-determining region 3. Since RSV repeatedly infects every person throughout life, memory B cells (MBC) expressing RSV F-binding BCRs circulate in the blood of healthy adults. This dataset of BCR variable region sequence features was derived from single cell-sorted RSV F-directed MBCs of a healthy adult blood donor [1]. The dataset was produced with publicly available data analysis software programs and scripts, which facilitates integration or comparison with antibody sequence repertoire data of different individuals derived with the same or comparable data analysis approaches and tools.

Streptococcus agalactiae (Group B Streptococcus, GBS) causes life-threatening infections in newborns and adults with chronic medical conditions. Serotype IV strains are emerging both among carriers and as cause of invasive disease and recent studies revealed two main Sequence Types (STs), ST-452 and ST-459 assigned to Clonal Complexes CC23 and CC1, respectively. Whole genome sequencing of 70 type IV GBS and subsequent phylogenetic analysis elucidated the localization of type IV isolates in a SNP-based phylogenetic tree and suggested that ST-452 could have originated through genetic recombination. SNPs density analysis of the core genome confirmed that the founder strain of this lineage originated from a single large horizontal gene transfer event between CC23 and the hypervirulent CC17. Indeed, ST-452 genomes are composed by two parts that are nearly identical to corresponding regions in ST-24 (CC23) and ST-291 (CC17). Chromosome mapping of the major GBS virulence factors showed that ST-452 strains have an intermediate yet unique profile among CC23 and CC17 strains. We described unreported large recombination events, involving the cps IV operon and resulting in the expansion of serotype IV to CC23. This work sheds further light on the evolution of GBS providing new insights on the recent emergence of serotype IV.

The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies.

Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre-existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell-sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F-protein (cross-binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross-binder nAbs used a wide array of gene rearrangements, while preF-binder nAbs derived mostly from the expansion of B-cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F-protein, identifying an important site of pathogen vulnerability.

The Neisseria meningitidis (Nm) chromosome shows a high abundance of simple sequence DNA repeats (SSRs) that undergo stochastic, reversible mutations at high frequency. This mechanism is reflected in an extensive phenotypic diversity that facilitates Nm adaptation to dynamic environmental changes. To date, phase-variable phenotypes mediated by SSRs variation have been experimentally confirmed for 26 Nm genes.