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Affinity labeling of biomacromolecules is vital for bioimaging and functional studies. However, affinity probes recognizing glycans with high specificity remain scarce. Here we report the development of glycan recombinant affinity binders (GRABs) based on mutant bacterial sialidases, which are enzymatically inactive but preserve stringent specificity for sialoglycan substrates. By mutating a key catalytic residue of Streptococcus pneumoniae neuraminidase A (SpNanA) and Ruminococcus gnavus neuraminidase H (RgNanH), we develop GRAB-Sia and GRAB-Sia3 recognizing total sialoglycans and α2,3-sialosides, respectively. The GRABs exhibit strict substrate and linkage specificity, and tetramerization with streptavidin substantially increases their avidity. The GRABs and tetrameric GRABs (tetra-GRABs) are effective tools for probing sialoglycans in immunoblotting, flow cytometry, immunoprecipitation, and fluorescence imaging. Furthermore, multiplex analysis with tetra-GRABs uncovers spatially distinct sialoglycans in the various mouse organs. This work provides a versatile toolkit for labeling and analyzing sialoglycans with high specificity, sensitivity, and convenience., Competing Interests: Competing interests: A Chinese patent application (application no. 202311031342.5) covering the sequence and use of GRAB-Sia and GRAB-Sia3 has been filed in which Peking University is the applicant with X.C., P.D., S.L., and Q.T. being the inventors. The remaining authors declare no competing interests., (© 2025. The Author(s).)

Streptococcus pneumoniae is an important human pathogen that normally resides in the human nasopharynx. Competence-mediated bacteriocin expression by S. pneumoniae plays a major role in both the establishment and persistence of colonization on this polymicrobial surface. Over 20 distinct bacteriocin loci have been identified in pneumococcal genomes, but only a small number have been characterized phenotypically. In this work, we demonstrate that three-fourths of S. pneumoniae strains contain a highly conserved scb locus that encodes an active lactococcin 972-like bacteriocin called streptococcin B. In these backgrounds, the scbABC locus is part of the early competence cascade due to a ComE binding site in the promoter region. Streptococcin B producing strains target both members of the population that have failed to activate competence and the 25% of the population that carry a naturally occurring deletion of the ComE binding site and the functional bacteriocin gene. The ComR-type regulator found directly upstream of the scb locus in S. pneumoniae strains can activate scb expression independent of the presence of the ComE binding site but only when stimulated by a peptide that is encoded in the scb locus of Streptococcus pseudopneumoniae , a closely related bacterium that also inhabits the human nasopharynx. Given the co-regulation with competence and the phenotypic confirmation of activity, streptococcin B represents a previously unrecognized fratricide effector that gives producing strains an additional advantage over the naturally occurring deleted strains during colonization., Importance: Streptococcus pneumoniae is a common cause of pneumonia, meningitis, sinusitis, and otitis media. In order to successfully colonize humans, a prerequisite to the development of invasive disease, S. pneumoniae must compete with other bacterial inhabitants of the nasal surface for space and nutrients. Bacteriocins are small antimicrobial peptides produced by bacteria that typically target neighboring bacteria by disruption of the cell surface. S. pnuemoniae encodes a large number of potential bacteriocin, but, for most, their role in competitive interactions has not been defined. This work demonstrates that isolates that produce the bacteriocin streptococcin B have an advantage over non-producers. These observations contribute to our understanding of the competitive interactions that precede the development of S. pneumoniae disease., Competing Interests: The authors declare no conflict of interest.

Background: Pneumococcal carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is not well understood., Methods: Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by polymerase chain reaction (PCR). Pneumococcal carriage in adults 18-44 years was compared with carriage among pneumococcal conjugate vaccine-vaccinated children aged 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and national IPD data, 2014-2019. Age group-specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for ≥65 years., Results: Overall, 98 isolates (97 carriers) were identified (3 solely by PCR) from 1631 ≥18 years adults (standardized carriage prevalence 6.4%). Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R = 0.9). Serotypes 3, 37, and 8 represented a higher proportion of adult carriage than expected. Predicted carriage serotype distributions for ≥65 years aligned closest with the young adult carriage serotype distribution., Conclusions: Nasal wash technique is highly sensitive. For some serotypes carried by adults aged ≥65 years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered., Competing Interests: Potential conflicts of interest. M. D. S. has been an investigator on behalf of the University of Oxford for studies funded or otherwise supported by vaccine manufacturers including GlaxoSmithKline, Janssen, Pfizer, Novavax, and MCM vaccines. Since 2022, M. D. S. has been employed by Moderna Biotech Distributors UK and holds equity in Moderna Inc. N. K. F. and D. L.'s laboratory has received grant funding from vaccine manufacturers for investigator-led research on pneumococcal carriage and disease. J. H. has received project grants (all payments to institution) from Pfizer. A. J. P. is Chair of the UK Department of Health and Social Care Joint Committee on Vaccination and Immunisation. B. G., J. C., C. T., E. B., and M. L. are employees of Pfizer and may hold stock and/or stock options. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

A current challenge in the rational design of biomolecular sensors is the ability to custom design binding affinities and detection mode in silico. To this end, we re-engineered a previously reported computationally-designed fluorescent maltooligosaccharide (MOS)-detecting biosensor to both alter its ligand-binding affinity and to analyse the underlying sensing mechanism. The dynamic range of the biosensor was expanded through the computer aided introduction of a series of amino acid substitutions in the starting protein scaffold (MalX from Streptococcus pneumoniae), which generated a biosensor set with binding affinities spanning over five orders of magnitude. The impact of the introduced substitutions on the underlying mode of signal generation was assessed in silico using our previously reported Computational Identification of Non-disruptive Conjugation sites (CINC) pipeline. CINC utilizes molecular dynamics simulations and an in-house developed algorithm to examine and exploit the structural dynamics of a protein at amino acid-level resolution. Using CINC, we demonstrate that re-engineering of the MOS-detecting biosensor set resulted in sensors with two distinct output modes which differed based on local conformational changes at the fluorescently modified reporter position. These output modes were classified as "ligand-sensing"-type biosensors (readout based on the tool sensing a unique conformation in the ligand-bound state), and "apo-sensing"-type biosensors (readout based on the tool sensing a unique conformation in the apo state). Together, these results demonstrate that structural dynamics at the individual amino acid residue level can be used as an engineer-able feature to rationally alter the fluorescence reporting properties of a biosensing device. Moving forward, the CINC workflow can also be adapted for the rational design of protein dynamic properties maximizing its utility as an in silico design platform for custom biomolecular tools., Competing Interests: Declaration of competing interest DDS, DWA, and HJW are Joint Inventors on International PCT Patent Application No. CA2021/05/01/10 (Filed January 30, 2021) which describes the CINC biosensor design process., (Crown Copyright © 2025. Published by Elsevier Inc. All rights reserved.)

Background: The introduction of the 10-valent pneumococcal conjugate vaccine (PCV10) for nationwide childhood immunization in 2010 led to a significant reduction in colonization and invasive pneumococcal disease (IPD) by vaccine serotypes in young. However, non-vaccine serotypes have emerged, and serotype 19A is now the leading cause of IPD in Brazil., Methods: We analyzed 32 serotype 19A isolates of Streptococcus pneumoniae recovered from children and adults who attended different health facilities in the state of Rio de Janeiro, Brazil, between 2010 and 2023. The capsular types of the isolates were determined by sequential multiplex PCR or by cpsB gene sequencing. All isolates were subjected to antimicrobial susceptibility testing and MLST., Results: Of the 32 serotype 19A isolates, 29 (90.6 %) isolates were recovered from children aged ≤5 years and three (9.4 %) isolates were recovered from adults. Nineteen (59.4 %) isolates were associated with colonization, and 13 (40.6 %) isolates were from diseases. All isolates were susceptible to chloramphenicol, levofloxacin, linezolid, rifampin, and vancomycin. The highest frequencies of non-susceptibility (intermediate + resistant) were observed for sulfamethoxazole-trimethoprim (n = 30; 93.8 %), penicillin (n = 24; 75 %), and erythromycin (n = 23; 71.9 %). Twenty-two (68.8 %) isolates were multidrug resistant (MDR). MICs for penicillin among penicillin-non-susceptible pneumococci (PNSP) ranged from 0.12 to 8.0 μg/mL. MICs for erythromycin ranged from 0.064 to >256 μg/mL. MICs for ceftriaxone ranged from 0.023 to 4 μg/mL. The most common genetic lineages were ST733 (n = 7; 21.9 %), mostly found before and in the early years of PCV10 introduction, and CC320 (n = 25; 78.1 %), mostly found in the late-PCV10 period. All 25 isolates within CC320 were PNSP and mostly (n = 22; 88 %) MDR., Conclusions: We observed a shift in antimicrobial susceptibility profiles and genetic lineages after long-term use of PCV, mostly PCV10, for routine childhood immunization, characterized by clonal expansion of the MDR lineage CC320., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) family transporters are essential in glycan synthesis, flipping lipid-linked precursors across cell membranes. Yet, how they select their substrates remains enigmatic. Here, we investigate the substrate specificity of the MOP transporters in the capsular polysaccharide (CPS) synthesis pathway in Streptococcus pneumoniae . These capsule flippases collectively transport more than 100 types of capsule precursors. To determine whether they can substitute for one another, we developed a high-throughput approach to systematically examine nearly 6000 combinations of flippases and substrates. CPS flippases fall into three groups: relaxed, type-specific, and strictly specific. Cargo size and CPS acetylation affect transport, and we isolated additional gain-of-function flippase variants that can substitute for the peptidoglycan flippase YtgP (MurJ). We also showed that combining flippase variants in a single cassette allows various CPS precursors to be flipped, which may aid glycoengineering. This study reveals that MOP flippases exhibit broad specificity, shaping the evolution of glycan synthesis.

Background: Purulent meningitis poses a significant clinical challenge with high mortality. We present the case of a 54-year-old female transferred to our emergency department with suspected bacterial meningitis, later diagnosed as an Austrian syndrome., Case Presentation: The patient exhibited subacute somnolence, severe headache, nausea and fever. Initial antibiotic therapy was initiated without successful lumbar puncture. Upon arrival at our hospital, she presented with septic shock, meningism, and respiratory symptoms. Lumbar puncture revealed cloudy cerebrospinal fluid with elevated cell count, protein, and low glucose. While blood and CSF cultures remained negative, multiplex PCR for Streptococcus pneumoniae was positive even 10 h after beginning of effective antibiotic therapy. Subsequent echocardiogram revealed mitral valve endocarditis and the patient underwent valve replacement., Conclusion: Altogether, bacterial meningitis presents with cardinal clinical signs only half of cases. Lumbar puncture remains crucial, and our patient's CSF findings aligned with bacterial meningitis. Multiplex PCR aided in diagnosis, even after antibiotic treatment. The case highlights the importance of prompt lumbar puncture despite antibiotic pre-treatment. The patient's Austrian syndrome, characterized by meningitis, endocarditis, and pneumonia, emphasizes the need for vigilance regarding skin lesions, early cerebral infarctions, and iritis. This case emphasizes the complexity of bacterial meningitis diagnosis and the utility of multiplex PCR, especially in prolonged antibiotic-treated patients. However, PCR cannot replace cultures when it comes to adapting therapy based on the antibiotic sensitivity of the causative pathogen. Awareness of Austrian syndrome's diverse manifestations is crucial for timely recognition and appropriate management., Competing Interests: Declarations. Ethics approval and consent to participate: No ethics board approval was required for this case report. Consent for publication: Written informed consent was obtained from the patient for the publication of this manuscript. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

We used polymerase chain reaction (PCR) to identify bacterial infections in culture-negative pleural fluid specimens from Alaska Native children hospitalized with empyema. PCR identified ≥1 organism in 11 (79%) of 14 specimens. Streptococcus pneumoniae serotype 3 was detected in 6 specimens; all 6 participants had received 13-valent pneumococcal conjugate vaccine., (Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society 2024.)

Homologous recombination (HR) is a universally conserved mechanism of DNA strand exchange between homologous sequences, driven in bacteria by the RecA recombinase. HR is key for the maintenance of bacterial genomes via replication fork restart and DNA repair, as well as for their plasticity via the widespread mechanism of natural transformation. Transformation involves the capture and internalization of exogenous DNA in the form of single strands, followed by HR-mediated chromosomal integration. In the human pathogen Streptococcus pneumoniae , transformation occurs during a transient, stress-induced differentiation state called competence. RecA and its partner DNA branch migration translocase RadA are both well conserved and cooperate in HR during transformation and certain genome maintenance pathways. Both recA and radA genes are basally expressed and transcriptionally induced during competence. In this study, we explored the importance of competence induction of recA and radA in transformation and genome maintenance. We confirmed that competence induction of recA , but not radA, was important for transformation. In contrast, we uncovered that the competence induction of both genes was required for optimal tolerance faced with transient exposure to the lethal genotoxic agent methyl methanesulfonate. However, this was not the case for another DNA-damaging agent, norfloxacin. These results show that competence induction of HR effectors is important for the increased tolerance to genotoxic stress provided to competent pneumococci. This reinforces the finding that pneumococcal competence is a stress-sensing mechanism, transiently increasing the expression of some genes not to optimize transformation but to improve survival faced with specific lethal stresses.IMPORTANCEHomologous recombination (HR) is a mechanism of DNA strand exchange important for both the maintenance and plasticity of bacterial genomes. Bacterial HR is driven by the recombinase RecA along with many accessory partner proteins, which define multiple dedicated pathways crucial to genome biology. Thus, a main mechanism of genome plasticity in bacteria is natural genetic transformation, which involves uptake and chromosomal integration of exogenous DNA via HR. In the human pathogen Streptococcus pneumoniae , transformation occurs during a transient, stress-induced physiological state called competence. RecA and the helicase RadA are key for both genome maintenance and transformation, and both are over-produced during competence. Here, we explore the importance of this over-production for transformation and genome maintenance, quantified by tolerance to genotoxic stress. While over-production of RecA was important for both processes, over-production of RadA was required only for genotoxic stress tolerance. This highlights the importance of competence as a stress-responsive mechanism, with induction of HR genes important for genotoxic stress tolerance., Competing Interests: The authors declare no conflict of interest.

Upon entry into the upper respiratory tract (URT), Streptococcus pneumoniae (Spn) upregulates neuraminidases (NA) that cleave sialic acid (SA) from host glycans. Because sialylation is thought to contribute to the physical properties that determine mucus function, we posited that Spn directly alters host mucus through NA activity. By directly imaging the colonized URT, we demonstrated NA-mediated alterations to the characteristics and distribution of mucus along the respiratory epithelium, where colonizing bacteria are found. Mucus exposed to NA showed increased localization within goblet cells and lining the glycocalyx. By contrast, NA-naïve mucus was more likely to be observed sloughing away from the epithelial surface. We also visualized Spn in the URT and observed that NA promoted efficient bacterial localization to the firm mucus layer overlying the glycocalyx, whereas NA-deficient Spn was associated more with loose mucus. By facilitating tighter association with the glycocalyx, NA promoted increased Spn colonization density. The magnitude of the NA-mediated effect on colonization was widened during late colonization by increased evasion of host-mediated clearance mechanisms. Thus, Spn-encoded NAs directly modify the host environment by desialylating mucus, which allows close interaction with mucus at the epithelium, and this is associated with enhanced bacterial colonization., Importance: Although severe illness and death caused by Spn result from secondary invasive diseases including pneumonia, sepsis, and meningitis, stable colonization of the upper respiratory tract (URT) is a prerequisite to invasive disease. Therefore, understanding host-Spn dynamics during asymptomatic colonization of the URT is warranted with respect to the pathogenesis of Spn disease. In this study, we found that Spn NA activity directly alters mucus characteristics that result in increased density and duration of URT colonization. Therefore, targeting Spn NA activity during URT colonization may be a viable strategy to mitigate Spn infection., Competing Interests: The authors declare no conflict of interest.

DNA transformation is key for phenotypic diversity and adaptation of Streptococcus pneumoniae including in the emergence of multidrug resistance (MDR). Under laboratory conditions, DNA transformation is facilitated by the artificial triggering of competence by the competence stimulating peptide (CSP). In ongoing DNA transformation work, we observed that exogenous CSP was dispensable depending on the combination of strains and culture media. Here, we carried out a chemogenomic screen to select for S. pneumoniae mutants capable of natural transformation in medium that normally would not sustain natural transformation. Our chemogenomic screen relied on chemical mutagenesis followed by selection of mutants with increased DNA transformation capacities. Sequencing the genome of these mutants revealed an abundance and diversity of mutated genes proven experimentally to increase natural transformation. A genome wide association study between MDR and sensitive clinical isolates revealed gene mutations associated with MDR, many of which intersected with those pinpointed by our chemogenomic screens and that were proven to increase natural transformation. S. pneumoniae has adopted DNA transformation as its lifestyle and can select for mutations facilitating DNA transformation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Streptococcus pneumoniae is an opportunistic human pathogen responsible for high morbidity and mortality rates. Extensive genome sequencing revealed its large pangenome, serotype diversity, and provided insight into genome dynamics. However, functional genome analysis has lagged behind, as that requires detailed and time-consuming manual curation of genome annotations and integration of genomic and phenotypic data. To remedy this, PneumoBrowse was presented in 2018, a user-friendly interactive online platform, which provided the detailed annotation of the S. pneumoniae D39V genome, alongside transcriptomic data. Since 2018, many new studies on S. pneumoniae genome biology and protein functioning have been performed. Here, we present PneumoBrowse 2 (https://veeninglab.com/pneumobrowse), fully rebuilt in JBrowse 2. We updated annotations for transcribed and transcriptional regulatory features in the D39V genome. We added genome-wide data tracks for high-resolution chromosome conformation capture (Hi-C) data, chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), ribosome profiling, CRISPRi-seq gene essentiality data and more. Additionally, we included 18 phylogenetically diverse S. pneumoniae genomes and their annotations. By providing easy access to diverse high-quality genome annotations and links to other databases (including UniProt and AlphaFold), PneumoBrowse 2 will further accelerate research and development into preventive and treatment strategies, through increased understanding of the pneumococcal genome., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Streptococcus pneumoniae causes invasive pneumococcal disease (IPD) in adults. The introduction of pneumococcal conjugate vaccines (PCVs) has reduced vaccine serotypes but has also led to the rise of non-vaccine serotypes. The aim of this study was to analyse pneumococcal lineages and their association with recent changes in IPD among adults in Spain., Methods: Data from adult IPD cases (≥18 years) were collected from six Spanish hospitals in 2019-2021. Strains were serotyped, tested for antibiotic susceptibility and subjected to whole genome sequencing (WGS). Findings were compared with data from previous periods (2008-2016)., Results: A total of 655 IPD episodes were examined. Pneumonia was the main focus (515/655), and 366 episodes occurred in adults over 64 years. Although IPD incidence decreased during COVID-19 pandemic, the burden of disease caused by PCV13 serotypes was significant. Notably, serotype 3 persisted (GPSC12-ST180 and GPSC83-ST260), and a new serotype 4 lineage emerged (GPSC162-ST13022). Among non-PCV13 serotypes, serotype 8 expanded (GPSC3-ST53) and a new serotype 12F lineage emerged (GPSC55-ST8060). Most serotypes presented a dominant Global Pneumococcal Sequencing Cluster (GPSC) like GPSC16-ST67 of 9N or GPSC19-ST433 of 22F. Nevertheless, some GPSCs were associated with several serotypes, the most numerous were GPSC3 (serotypes 8, 11A, and 33F) and GPSC6 (serotypes 11A and 14). The overall penicillin non-susceptibility rate was 17.0 %, 14.6 % resistance for meningitis and 1.6 % for pneumonia (15.1 % susceptible at increased exposure [SIE]). Serotypes 11A and 14 (GPSC6-ST156/6521) and 19A (GPSC1-ST320) had penicillin MICs above 1 mg/L. Acquired resistance genes associated with macrolide and/or tetracycline resistance were present in 19.4 % of isolates, particularly among serotypes 6C (GPSC47-ST386/4310) and 19A (GPSC1-ST320)., Conclusions: The burden of PCV13 serotypes in adult IPD remains significant, and serotype 3 is the primary contributor. However, the rise of stable lineages associated with non-PCV13 serotypes, particularly 8, 9N, and 22F highlights a shifting epidemiology. The persistence of multidrug-resistant lineages, such as GPSC6-ST156 and GPSC1-ST320, emphasizes the need for continued surveillance. Vaccination of high-risk adults with current and broader coverage PCVs would help to control the burden of pneumonia and IPD among adults., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sara Calvo-Silveria declares that the Project was partially funded by PFIS predoctoral grant number FI22/00279. José Yuste declares grants or contracts paid to his institution from MSD, Pfizer and Meiji. Support for attending meetings and/or travel from MSD and Pfizer. Participation on a Data Safety Monitoring Board or Advisory Board from GSK, MSD and Pfizer. Carmen Ardanuy declares funding to the institution from Instituto de Salud Carlos III through PI18/0339; PI21/1000 and INT22/0096; CB06/06/0037. Grants or contracts paid to her institution from MSD, Pfizer and Menarini. Payment or honoraria for lectures, or educational events from MSD and Pfizer to her o her institution. Support for attending meetings and/or travel from MSD and Pfizer., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

With the advent of whole-genome sequencing, opportunities to investigate the population structure, transmission patterns, antimicrobial resistance profiles, and virulence determinants of Streptococcus pneumoniae at high resolution have been increasingly expanding. Consequently, a user-friendly bioinformatics tool is needed to automate the analysis of Streptococcus pneumoniae whole-genome sequencing data, summarize clinically relevant genomic features, and further guide treatment options. Here, we developed PneusPage, a web-based tool that integrates functions for species prediction, molecular typing, drug resistance determination, and data visualization of Streptococcus pneumoniae. To evaluate the performance of PneusPage, we analyzed 80 pneumococcal genomes with different serotypes from the Global Pneumococcal Sequencing Project and compared the results with those from another platform, PathogenWatch. We observed a high concordance between the two platforms in terms of serotypes (100% concordance rate), multilocus sequence typing (100% concordance rate), penicillin-binding protein typing (88.8% concordance rate), and the Global Pneumococcal Sequencing Clusters (98.8% concordance rate). In addition, PneusPage offers integrated analysis functions for the detection of virulence and mobile genetic elements that are not provided by previous platforms. By automating the analysis pipeline, PneusPage makes whole-genome sequencing data more accessible to non-specialist users, including microbiologists, epidemiologists, and clinicians, thereby enhancing the utility of whole-genome sequencing in both research and clinical settings. PneusPage is available at https://pneuspage.minholee.net/.

Objective: To explore the hypothesis of "pathogen storage pool" by analyzing the local microbial community of adenoids. Methods: Under the guidance of a 70° nasal endoscope, sterile swabs were used to collect secretions from the adenoid crypts of the subjects. The samples were sent to the laboratory for DNA extraction and standard bacterial 16S full-length sequencing analysis. Results: At the species level, the top three microbial communities in adenoid crypts were Bacillus subtilis（18.78%）, Fusobacterium pyogenes（11.42%）, and Streptococcus pneumoniae（9.38%）. Conclusion: The local microbial community of adenoids exhibits a high degree of diversity, including microbial communities from the oral cavity and gastrointestinal tract. Our research results support the hypothesis that adenoids act as a " pathogen reservoir"., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Background: Streptococcus pneumoniae does not produce β-lactamases, and its reduced susceptibility to β-lactam antibiotics is predominantly caused by mutations of penicillin-binding proteins (PBPs). However, mechanisms of non-PBP mutation-related β-lactam antibiotic resistance in pneumococcal strains remain poorly characterized., Methods: Susceptibility of S. pneumoniae ATCC49619 and its ciaR gene knockout, complemented, or overexpression mutant (ΔciaR, CΔciaR, or ciaR OE ) to penicillin, cefotaxime, and imipenem was detected using an E-test. Levels of pneumococcal ciaR-mRNA, 5 ccn-microRNAs, and 6 pbps-mRNAs were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Recombinant CiaR (rCiaR) binding to the promoters of ccn-microRNA genes was confirmed using electrophoresis mobility shift and chromatin immunoprecipitation assays. Sequence matching between the ccn-microRNAs and pbps-mRNAs was analyzed using IntaRNA software., Results: S. pneumoniae ATCC49619 was sensitive to the 3 β-lactam antibiotics, but overexpression of CiaR, a response regulator protein in 2-component system, caused the increase of MICs against these antibiotics. The ciaR OE mutant exhibited the significantly increased transcription of ccn-microRNAs but notably decreased transcription of pbps-mRNAs; conversely, the ΔciaR mutant displayed decreased levels of ccn-microRNAs and increasesed transcription of pbps-mRNAs. rCiaR was able to bind to the promoters of all ccn-microRNA genes in vitro and within cells. The 3 antibiotics at 1/8 minimal inhibitory concentrations caused a significant increase in the ciaR-mRNA and ccn-microRNAs. The mRNA-binding seed sequences in the 5 ccn-microRNAs matched all the promoter-containing sequences of pbps-mRNAs., Conclusions: β-Lactam antibiotics at low concentrations induce non-PBP mutation-related antibiotic resistance conversion of S. pneumoniae by decrease of PBPs through the pathway of CiaR-mediated transcriptional increase of ccn-microRNAs and ccn-microRNA-dependent degradation of pbp-mRNAs., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Streptococcus pneumoniae (S. pneumoniae) employs various metabolic pathways to generate nicotinamide adenine dinucleotide phosphate (NADPH), which is essential for redox balance, fatty acid synthesis, and energy production. GAPN, a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, plays a role in this process by directly reducing NADP + to NADPH, effectively contributing to glucose metabolism. However, its relative importance for S. pneumoniae metabolism and infection has remained unknown. Here, we performed a comprehensive characterization of S. pneumoniae GAPN through kinetic assays, isothermal titration calorimetry (ITC), cryo-EM, mass spectrometry, and infection assays. Despite its structural similarities to its homologues in other species, S. pneumoniae GAPN exhibits negative cooperativity with respect to its substrate, glyceraldehyde-3-phosphate (G3P), suggesting a unique regulatory mechanism. Our results demonstrate that GAPN knockout leads to significant metabolic reprogramming, including increased glycogen storage that leads to enhanced fatty acid metabolism. This collectively reduces the ability of S. pneumoniae to manage oxidative stress and sustain infection. Our findings highlight GAPN as a critical enzyme for S. pneumoniae metabolic balance and suggest that its inhibition could serve as a potential strategy for therapeutic intervention in pneumococcal diseases., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Introduction: To understand the in-vivo dynamics in pneumococci, investigation into the carriage in patients with invasive pneumococcal disease (IPD) is extremely important., Methods: To clarify genomic and morphological differences between pneumococcal strains simultaneously isolated from different sites in a patient with IPD, we conducted comparative analyses of two strains. A capsular strain isolated from the blood and a non-capsular strain isolated from the sputum of a patient with IPD were used., Results: The strain isolated from blood was serotype 24B with capsule. The strain isolated from sputum with capsular type 24 genes was non-encapsulated, and genomic analysis revealed an insertion region in the wcxK gene. Its biofilm-forming capacity was higher than that of the capsular strain, as was that of the pspK-positive true non-encapsulated strain. Furthermore, observing the microbe using transmission electron microscopy revealed that the strain isolated from sputum lacked a capsule, like the pspK-positive true non-encapsulated strain., Conclusions: Our analysis of the two strains isolated from the blood and sputum of a patient with IPD showed one possible in-vivo morphological change in Streptococcus pneumoniae., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Background: Since the introduction of the national routine vaccination program against Streptococcus pneumoniae in Japan from the early 2010s, the incidence of invasive pneumococcal disease (IPD) caused by non-vaccine serotypes has increased. This study focused on non-vaccine serogroup 24 strains derived from IPD and aimed to clarify their genetic characteristics., Methods: Between 2013 and 2022, 121 strains identified as serogroup 24 in patients with IPD were collected and applied to multilocus sequence typing and next-generation sequencing. Whole-genome data were used to delineate phylogenetic relationships and to identify virulence and antimicrobial resistance-associated genes., Results: Recent trends in sequence types (STs) were characterized by an increase in the proportion of ST162 and ST2754 for 24F and 24B, respectively, after 2018. Whole-genome phylogenetic analysis demonstrated that serogroup 24 strains were organized into three clades, closely related to STs but not with serotypes. All ST162 strains were classified as Global Pneumococcal Sequence Cluster (GPSC) 6 and harbored the virulence-associated rlrA islet, with co-trimoxazole-resistance mutations in folA and folP genes. Two ST162 strains with different serotypes 24F and 24B from the same patient were phylogenetically indistinguishable, showing that these strains were derived by serotype conversion during infection., Conclusion: The recent changes in predominant STs were similar to those previously reported throughout Japan, except Tokyo. Little correlation between whole-genome phylogeny and serotypes and the observed serotype conversion in one patient indicate potentially variable immunogenicity of this serogroup., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Streptococcus pneumonia is a Gram-positive and facultative anaerobic bacterium that causes a number of diseases, including otitis media, community-acquired pneumonia, sepsis, and meningitis. With the emergence of antibiotic-resistant strains, there is an urgent need to develop antibiotics with a novel mechanism. The toxin-antitoxin (TA) system, which is primarily found in prokaryotes, consists of a toxin and its equivalent antitoxin genes. The YefM-YoeB module is a Type II TA system, where the YoeB toxin functions as a putative mRNA interferase upon activation, while the YefM antitoxin acts as a transcription repressor by binding to its promoter region along with YoeB. In this study, we determined the crystal structure of the YefM-YoeB complex from S. pneumoniae TIGR4 to comprehend the binding mechanism of the TA system. Furthermore, an in vitro ribonuclease activity assay was conducted to identify the ribonuclease activity of the YoeB toxin. Additionally, furthermore, the oligomeric state of the YefM-YoeB complex in solution was investigated, and a DNA-binding mode was proposed. These structural and functional insights into the YefM-YoeB complex could provide valuable information for the development of novel antibiotics targeting S. pneumonia-associated diseases., (© 2025 Wiley Periodicals LLC.)

Bacterial diseases caused by S. pneumoniae are an important public health problem in Middle Eastern and North African countries. The burden of S. pneumoniae is poorly documented in these regions, local surveillance programs are lacking. This systematic review was conducted in Medline and Embase to obtain a comprehensive overview of the clinical burden of pneumococcal disease and pneumococcal vaccination status in the pediatric population in Middle East and Northern Africa. Overall, 255 articles and gray literature sources were included in the study, with the majority representing Israel ( n  = 82), Türkiye ( n  = 40), Iran ( n  = 27), and Saudi Arabia ( n  = 13). The incidence of pneumococcal disease varied based on time frame and country. The dominant IPD serotypes varied, with serotypes 19F and 14 the most prevalent in the Middle East region and serotypes 19F, 14, and 6B the most prevalent in North Africa. The PCV introduction status was identified in most countries of interest except Egypt, Jordan, and Syria. This review sheds light on the significant impact of PCV on the burden of S. pneumoniae -related diseases. Serotypes 19F, 14, and 6B are still prevalent in the countries of MENA region despite inclusion in licensed PCVs. Genomic analysis could elucidate what the drivers of this persistence are, and future work could be developed around this. Continuous monitoring of serotype epidemiology is important for policy decisions on pediatric pneumococcal vaccination recommendations.

Human pathogen Streptococcus pneumoniae forms multiple epigenetically and phenotypically distinct intra-populations by invertase PsrA-driven inversions of DNA methyltransferase hsdS genes in the colony opacity-determinant (cod) locus. As manifested by phase switch between opaque and transparent colonies, different genome methylation patterns or epigenomes confer pathogenesis-associated traits, but it is unknown how the pathogen controls the hsdS inversion orientations. Here, we report our finding of the SpxA1-TenA toxin-antitoxin (TA) system that regulates the orientations of hsdS inversions, and thereby bacterial epigenome and associated traits (e.g., colony opacity) by targeting pneumococcal protein synthesis. SpxA1 and TenA were found to constitute a highly conserved type II TA system in S. pneumoniae, primarily based on the observation that overexpressing toxin TenA led to growth arrest in E. coli and enhanced autolysis in S. pneumoniae, and the antitoxin SpxA1 repressed the transcription of the spxA1-tenA operon. When the transcription of tenA was de-repressed by a spontaneous AT di-nucleotide insertion/deletion in the promoter region of the spxA1-tenA operon, TenA bound to the ribosome maturation factor RimM, and thereby reduced the cellular level of alternative sigma factor ComX (known for the activation of natural transformation-associated genes). Attenuation of ComX expression in turn enhanced the transcription of the invertase gene psrA, which favored the formation of the transparent colony phase-associated hsdS allelic configurations in the cod locus. Phenotypically, moderate expression of TenA dramatically reshaped pneumococcal epigenome and colony opacity. Because spontaneous variations frequently occur during bacterial growth in the number of the AT di-nucleotides in the promoter region of the spxA1-tenA operon, this locus acts as a programmed genetic switch that generates pneumococcal subpopulations with epigenetic and phenotypic diversity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria and, in doing so, may promote the spread of drug resistance genes in the population.IMPORTANCEThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here, we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface and that this DNA can be imported by the transformation machinery, facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance., Competing Interests: The authors declare no conflict of interest.

Background: While Streptococcus pneumoniae (Spn) is the leading cause of pediatric complicated community-acquired pneumonia (cCAP), it is infrequently recovered by culture-based methods. We studied the real-world clinical impact of an Spn polymerase chain reaction (PCR) assay for pleural fluid., Methods: This pre-post quasi-experimental cohort study compared pathogen detection, antibiotic usage, and outcomes in children hospitalized with cCAP requiring pleural effusion or empyema drainage at Children's Hospital Colorado between 2016 and 2023. Patients were compared across 2 diagnostic periods: pre-Spn PCR and post-Spn PCR. Cox proportional hazard models compared time from admission to pathogen detection, optimal therapy (narrowest pathogen-directed or guideline-recommended empiric therapy), and methicillin-resistant Staphylococcus aureus (MRSA) therapy discontinuation between periods., Results: Compared to the pre-Spn PCR cohort (n = 149), the post-Spn PCR cohort (n = 79) was more likely to have a pathogen detected (73.4% post-PCR vs 38.9% pre-PCR, P < .001), driven by more Spn detections (45.6% vs 14.1%, P < .001). Time to pathogen detection during hospitalization was shorter in the post-Spn PCR period (P < .001). The post-PCR cohort was more likely to receive optimal therapy (84.8% vs 53.0%, P < .001), with shorter median times to optimal antibiotics (4.9 vs 10.0 days, P < .001) and MRSA therapy discontinuation (1.5 vs 2.5 days, P = .03). There were no differences in hospital length of stay or readmissions., Conclusions: Spn molecular testing of pleural fluid in children with cCAP resulted in significantly more microbiologic diagnoses and was associated with the optimization of antibiotics and decreased exposure to MRSA therapy, suggesting its clinical impact for pediatric complicated pneumonia., Competing Interests: Potential conflicts of interest . S. R. D. receives grants from Biofire Diagnostics, Karius, and Pfizer and serves as a consultant for Biofire Diagnostics, Karius, and DelveBio, outside the submitted work. E. J. A. has received research grant support through his institution from Pfizer. M. B. has received conference attendance support from Pfizer. S. J. is a consultant for Roche Diagnostics and Karius, and has received conference attendance support from Diasorin, Karius, and bioMérieux. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: The substantial risk for respiratory and invasive infections with Streptococcus pneumoniae (Spn) among people with HIV-1 (PWH) begins with asymptomatic colonization. The frequency of Spn colonization among US adults with and without HIV-1 infection is not well characterized in the conjugate vaccine era., Methods: We determined Spn colonization frequency by culture and specific lytA gene quantitative polymerase chain reaction (PCR) and microbiota profile by 16S ribosomal RNA gene sequencing in nasopharyngeal (NP) and oropharyngeal (OP) DNA from 138 PWH and 93 control adults and associated clinical characteristics., Results: The frequencies of Spn colonization among PWH and controls did not differ (11.6% vs 8.6%, respectively; P = .46) using combined results of culture and PCR, independent of vaccination or behavioral risks. PWH showed altered microbiota composition (ie, β-diversity; NP: P = .0028, OP: P = .0098), decreased α-diversity (NP: P = .024, OP: P = .0045), and differences in the relative abundance of multiple bacterial taxa. Spn colonization was associated with altered β-diversity in the nasopharynx (P = .011) but not oropharynx (P = .21)., Conclusions: Despite widespread conjugate vaccine and antiretroviral use, frequencies of Spn colonization among PWH and controls are currently consistent with those reported in the preconjugate era. The persistently increased risk of pneumococcal disease despite antiretroviral therapy may relate to behavioral and immunologic variables other than colonization., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The Gram-positive bacteria Streptococcus pneumoniae is part of the Streptococcus mitis group (SMG) and causes life-threatening infections, such as pneumonia, sepsis, and meningitis. The closely related Streptococcus pseudopneumoniae has recently been shown to cause respiratory tract infections, as well as invasive infections, especially in patients with comorbidities. Due to the genetic and phenotypic similarities of species belonging to the SMG, the identification of S. pneumoniae and S. pseudopneumoniae is difficult and unreliable using phenotypic tests, as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and 16S rRNA sequencing. In this study, a loop-mediated isothermal amplification (LAMP)-based assay was developed using molecular markers specific for S. pneumoniae (SPN0001) and S. pseudopneumoniae (SPPN&#95;RS10375). The LAMP assay was evaluated using a collection of SMG clinical isolates, concluding that the method provides a correct, reliable, and fast identification of both S. pneumoniae and S. pseudopneumoniae clinical isolates., Competing Interests: The authors declare no conflict of interest.

Background: Streptococcus pneumoniae is a transmitted respiratory pathogen that causes high morbidity and mortality in children, especially those under 5 years of age. During the implementation of population control measures for COVID-19 in mainland China, the Streptococcus pneumoniae detection rate in pediatric patients decreased. However, with the second wave of the COVID-19 pandemic (2022), the incidence of pneumococcal disease (PD) and even invasive pneumococcal disease (IPD) began to rise again., Methods: This study was conducted from August 2022 to September 2023 at a national regional medical center based mainly in West China Second University Hospital, Sichuan University. The demographic and clinical characteristics of S. pneumoniae -infected pediatric patients were analyzed. All S. pneumoniae isolates were subjected to standardized clinical sample inoculation, culture, subculture, and identification procedures. Next-generation sequencing and analysis were used to determine serotypes and sequence types (STs) and evaluate antibiotic resistance- and virulence-related genes. Antimicrobial susceptibility was determined in AST dishes via the broth microdilution method., Results: The prevalent serotypes in the IPD patients were 14, 6A, and 23F, and the prevalent serotypes in the NIPD patients were 19F and 6A. A significant difference in the proportion of patients with serotype 14 was noted between the two groups. A total of 23 STs were identified and classified into 13 different GPSC lineages, including 4 novel STs (ST18449, ST18451, ST18464 and ST18466) and 1 novel allele ( ddl 1209). According to the interpretation breakpoints for non-meningitis infections, the resistance/nonsusceptibility rates of invasive isolates were as follows: penicillin (0.0%/8.3%), amoxicillin (0.0%/0.0%), cefotaxime (8.3%/16.6%), ceftriaxone (8.3%/8.3%), and cefepime (0.0%/8.3%). The resistance/nonsusceptibility rates of invasive isolates according to the meningitis breakpoints were as follows: penicillin (100.0%), cefotaxime (16.7%/33.4%), ceftriaxone (8.3%/50.0%), and cefepime (8.3%/66.7%). All the isolates were susceptible to rifampicin, levofloxacin, moxifloxacin, linezolid and vancomycin. In addition, the characteristics of the antibiotic resistance-related genes and virulence genes of serotype 19F were significantly different from those of the other serotypes., Conclusion: These data provide valuable information for understanding pediatric pneumococcal disease during the second outbreak of COVID-19 in Southwest China and will contribute to the prevention and treatment of S. pneumoniae infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The handling editor FQ declared a shared parent affiliation with the authors at the time of review., (Copyright © 2024 Yan, Miao, Liu, Fu, Liu, Li, Kuang, Cui and Jiang.)

Using chromosomal barcoding, we observed that >97% of the Streptococcus pneumoniae (Spn) population turns over in the lung within 2 days post-inoculation in a murine model. This marked collapse of diversity and bacterial turnover was associated with acute inflammation (severe pneumococcal pneumonia), high bacterial numbers in the lungs, bacteremia, and mortality. Intra-strain competition mediated by the blp locus, which expresses bacteriocins in a quorum-sensing-dependent manner, was required for each of these effects. Bacterial turnover from the activity of Blp-bacteriocins increased the release of the pneumococcal toxin, pneumolysin (Ply), which was sufficient to account for the lung pathology. The ability of Ply to evade complement, rather than its pore-forming activity, prevented opsonophagocytic clearance of Spn enabling its multiplication in the lung, facilitating the inflammatory response and subsequent invasion into the bloodstream. Thus, our study demonstrates how an appreciation for bacterial population dynamics during infection provides new insight into pathogenesis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Despite growing evidence of reduced invasive and non-invasive pneumococcal disease attributed to public health measures against the COVID-19 pandemic, the effect of these measures on pneumococcal carriage remains unclear. This study aimed to assess pneumococcal nasopharyngeal carriage among children and adults self-confined at home during the COVID-19 national lockdown in Spain while identifying predictors of pneumococcal carriage in children., Methods: Household study conducted across the metropolitan area of Barcelona (Spain) between April-June 2020. Nasopharyngeal samples were collected from young children and adults for real-time PCR pneumococcal lytA and wgz gene detection, quantification, and serotyping, as well as for detection of respiratory viruses., Results: Among 332 children (median age: 3.1 years [IQR: 1.9-4.0 years]; 59% male) and 278 adults (median age: 38.9 years [IQR: 36.1-41.3 years]; 64% female), pneumococcal carriage rates were 28.3% and 2.5%, respectively. Highly invasive serotypes 3, 7F/7A, and 19A were detected in 14.0% of samples from children carriers. Pneumococcal co-infections with respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and influenza virus (IV) were not identified in children. Attendance to kindergarten before the lockdown (aOR: 2.65; IQR: 1.57-4.47; p<0.001) and household crowding (aOR: 1.85; IQR: 1.09-3.15; p = 0.02) were independent risk factors for children's pneumococcal carriage., Conclusions: Pneumococcal carriage rate among quarantined children during a full COVID-19 lockdown was moderate and correlated with limited presence of highly invasive serotypes and absence of pneumococcal co-infections with RSV, hMPV, and IV. Pre-lockdown daycare and household crowding predisposed children to carriage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Brotons et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: A study conducted in the city of Niterói/RJ, four years after the introduction of the pneumococcal conjugate vaccine in Brazil, reported the emergence of non-vaccine serotype 6C Streptococcus pneumoniae associated with carriage in children. The multidrug-resistant (MDR) lineage ST386 was predominant among 6C isolates. A subsequent study, in 2019, reported the continued prevalence of 6C as the main serotype. This study aims to determine the genetic lineages of serotype 6C S. pneumoniae obtained from the 2019 study and evaluate the status of ST386 in this population., Methods: Serotype 6C S. pneumoniae isolates were obtained during the 2019 study. Lineages were determined by MLST and changes in ST386 status between 2014 and 2019 were verified by a two-tailed Fisher's exact test., Results: Of the 16 serotype 6C isolates recovered during 2019, 10 (62.5 %) belonged to ST386, remaining predominant in the population. The second most frequent was ST2777 represented by four (25 %) isolates. Both ST63 and ST3280 only had one (6.25 %) isolate each. Comparison of ST386 proportion between 2014 and 2019 showed no significant changes within the population., Conclusions: This study was able to confirm the stability on the occurrence of the MDR lineage ST386 in children in our setting nine years after the introduction of PCV10 in Brazil., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Streptococcus pneumoniae is a prevalent and virulent global pathogen, with colonization being considered a precondition for pneumococcal disease. Understanding colonization is critical for gaining insights into transmission dynamics and developing effective interventions. This study aimed to determine the prevalence of nasopharyngeal colonization and serotype distribution in the Sicilian population., Methods: Observational study randomly selecting samples belonging to Sicilian individuals whose nasopharyngeal swabs were collected between February 1, 2020, and December 31, 2022. Pneumococcal colonization was determined using PCR for the pneumococcal autolysin (LytA) gene, and positive samples were serotyped., Results: The study sample consisted of 1,196 individuals, with 17.4% testing positive for the LytA gene. Pneumococcal colonization rates fell from birth to 24 years, with a peak in 0-4-year-olds (aOR=6.9; p<0.001). Colonization was higher in colder months, particularly in December (aOR=2.9, p<0.05) and February (aOR=4, p<0.05). Serotypes 22F and 24ABF exhibited strong colonization and an invasive pneumococcal disease (IPD) risk, whereas serotypes 4, 6AB, 9VA, and 13 had high colonization but a low IPD risk. Serotypes 3 and 8 exhibited considerable IPD risk but low colonization., Conclusion: Our findings provide insights into pneumococcal colonization mechanisms, influencing serotype prevalence, colonization risk variables, and serotype comparisons for colonization and pathogenicity propensity., Competing Interests: EA and FV received grants from GlaxoSmithKline SA, Sanofi Pasteur, Moderna, Janssen, MSD and Pfizer for taking part in advisory boards, expert meetings, being a speaker or an organizer of congresses/conferences. FT received grants from MSD for taking part in advisory boards, expert meetings, being a speaker or an organizer of congresses/conferences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Amodio, Tramuto, De Francisci, Genovese, Guzzetta, Pisciotta, Santino, Randazzo, Trapani, Vella and Vitale.)

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9-targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9-targeted sequencing holds immense promise for serotype identification of S. pneumoniae., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: Indonesia commenced the nationwide introduction of pneumococcal conjugate vaccine (PCV) in 2022. Pre-vaccine Streptococcus pneumoniae data from across the country could be critical to enable vaccine impact evaluation in the future. This study evaluates colonization prevalence, factors associated with colonization, serotype distribution, and the antimicrobial susceptibility profile of S. pneumoniae., Methods: Children under 5 years of age were enrolled from Bajau tribe settlements in Wakatobi, southeast Sulawesi, Indonesia, from October 2018 to February 2019. Nasopharyngeal swab specimens were analysed by culture, and isolates were serotyped using sequential multiplex polymerase chain reaction. Antibiotic susceptibility was performed by the disk diffusion method. Multivariable logistic regression was performed for risk factor analysis., Results: A total of 499 NP swab specimens were collected; 61.9% were colonized with S. pneumoniae and 48.9% of the isolates were of PCV13-vaccine type. The most common serotypes were 23F, 6B, 19F, and 6A at 13.2%, 9.8%, 8.9%, and 8.0%, respectively. Exposure to cigarette smoke in the household and runny nose were significant risk factors for colonization, with aORs of 1.6 (95% confidence interval: 1.1-2.3) and 2.1 (95% confidence interval: 1.4-3.3), respectively., Conclusions: The findings of this study may contribute to baseline pre-vaccine data in Indonesia that would be critical for the impact evaluation of vaccines., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Streptococcus pneumoniae is an important human pathogen that can colonize the respiratory tract of healthy individuals. The respiratory tract mucosa is thus the first barrier for this pathogen. In this study, we have tested three models of the respiratory epithelium with immune cells: (i) monolayer of A549 human lung epithelial cells, (ii) A549 + macrophages differentiated from the human monocytic THP-1 cell line (dMφ) and (iii) A549 + dMφ + dendritic cells differentiated from THP-1 (dDC) using a two-chamber system. Pneumococcal strains Rx1 (non-encapsulated) and BHN418 (serotype 6B) were incubated with the cells and secretion of IL-6, IL-8, IL-1β, TNF-α and IL-10 was evaluated. Overall, the models using co-cultures of A549 + dMφ and A549 + dMφ + dDC elicited higher levels of pro-inflammatory cytokines and the non-encapsulated strain elicited an earlier cytokine response. BHN418 pspA (pneumococcal surface protein A) and pspC (pneumococcal surface protein C) knockouts elicited similar cytokine secretion in the co-culture models, whereas BHN18 ply (pneumolysin) knockout induced much lower levels. The results are in accordance with the activation of the inflammasome by Ply. Finally, we evaluated pneumococcal extracellular vesicles (pEVs) in the co-culture models and observed secretion of pro-inflammatory cytokines in the absence of cytotoxicity. Since pEVs are being studied as vaccine candidate against pneumococcal infections, the co-cultures of A549 + dMφ and A549 + dMφ + dDC are simple models that could be used to evaluate pEV vaccine batches., Competing Interests: Declarations. Conflict of interest: ENM is an associate editor of the Brazilian Journal of Microbiology. ENM, TCR and MLSO have deposited patents on pneumococcal vaccines., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

This study aimed to provide data for the clinical features of invasive pneumococcal disease (IPD) and the molecular characteristics of Streptococcus pneumoniae isolates from paediatric patients in China. We conducted a multi-centre prospective study for IPD in 19 hospitals across China from January 2019 to December 2021. Data of demographic characteristics, risk factors for IPD, death, and disability was collected and analysed. Serotypes, antibiotic susceptibility, and multi-locus sequence typing (MLST) of pneumococcal isolates were also detected. A total of 478 IPD cases and 355 pneumococcal isolates were enrolled. Among the patients, 260 were male, and the median age was 35 months (interquartile range, 12-46 months). Septicaemia (37.7%), meningitis (32.4%), and pneumonia (27.8%) were common disease types, and 46 (9.6%) patients died from IPD. Thirty-four serotypes were detected, 19F (24.2%), 14 (17.7%), 23F (14.9%), 6B (10.4%) and 19A (9.6%) were common serotypes. Pneumococcal isolates were highly resistant to macrolides (98.3%), tetracycline (94.1%), and trimethoprim/sulfamethoxazole (70.7%). Non-sensitive rates of penicillin were 6.2% and 83.3% in non-meningitis and meningitis isolates. 19F-ST271, 19A-ST320 and 14-ST876 showed high resistance to antibiotics. This multi-centre study reports the clinical features of IPD and demonstrates serotype distribution and antibiotic resistance of pneumococcal isolates in Chinese children. There exists the potential to reduce IPD by improved uptake of pneumococcal vaccination, and continued surveillance is warranted.

The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in Streptococcus pneumoniae, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed S. pneumoniae EgtU and purified it from E. coli BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization., (Copyright © 2024 Elsevier Inc. All rights reserved.)

StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. In vitro, phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring the proper functioning of the StkP signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The circadian clock is a cell-autonomous process that regulates daily internal rhythms by interacting with environmental signals. Reports across species show that infection can alter the expression of circadian genes; however, in teleosts, these effects are influenced by light exposure. Currently, no reports analyze the direct effects of bacterial exposure on the zebrafish clock. Using zebrafish Z3 cells, we demonstrate that exposure to heat-killed Streptococcus pneumoniae (HK-Spn) augments the expression of core repressive factors in a light- and time-dependent manner. In constant darkness, HK-Spn highly upregulated cry1a, per3, and per1b expression. In the presence of light, HK-Spn exposure rapidly and strongly upregulated per2 and cry1a, and this was proportionally increased with light intensity. The combinatorial effect of light and HK-Spn on per2 and cry1a was not duplicated with H 2 O 2, a known byproduct of light exposure. However, the ROS inhibitor N-acetyl cysteine was sufficient to block HK-Spn augmentation of per2, cry1a, and per3. These findings demonstrate that exposure to an inactive bacteria influences the expression of zebrafish clock genes under different light conditions., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δ fhs and Δ proABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δ fhs and Δ proABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 Δ proABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 Δ proABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δ fhs and Δ proABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δ fhs had an impaired stringent response, and when cultured in sera, BHN418 Δ fhs and Δ proABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae . We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential., Competing Interests: The authors declare no conflict of interest.

In subjects with peculiar susceptibility to severe infections by common pyogenic bacteria, mutations of interleukin-1 receptor-associated kinase proteins (IRAK)1 and IRAK4 had been identified. The IRAK kinases function as downstream signal transductors following the activation of pathogen recognition receptors. In two patients with sequential or repeated invasive infections: herpes simplex virus-triggered hemophagocytic lymphohistiocytosis with tuberculosis, and Streptococcus pneumoniae bacteremia with candidemia respectively, novel mutations of IRAK2 were identified. These mutations compromised the capacity to ubiquinate (or functionally modify) the signal adaptor tumor necrosis factor receptor-associated factor 6. The result is impairment of the cytokine tumor necrosis factor-alpha production. This susceptibility to a varied range of pathogens underlines a potential central role played by IRAK2 in mediating host defense in infectious diseases., Competing Interests: Declarations of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Asthma is the most common chronic medical condition among children ≥5 years of age with invasive pneumococcal disease. How asthma or its management affects pneumococcal colonization is not fully understood. Our objective was to compare pneumococcal colonization rates between children with persistent asthma and children without asthma, and to characterize the pneumococcal serotype distribution., Methods: We used nasal mid-turbinate samples obtained per routine care from 5- to 18-year-old children with upper respiratory symptoms from November to April (respiratory seasons) of 2017 to 2018 and 2018 to 2019 in Kansas City, United States. Pneumococcal immunization status, prior antibiotic use and other clinical data were collected. Samples were tested for pneumococcal colonization by real-time polymerase chain reaction targeting lytA gene. Positive samples underwent multiplex serotype-specific polymerase chain reaction assays to determine the serotype., Results: Of 363 children (120 with persistent asthma and 243 without asthma), 87.6% were 5 to 10 years old, 50.1% were female and 74.1% received ≥3 doses of a pneumococcal conjugate vaccine. The pneumococcal colonization rate was lower in children with persistent asthma than in children without asthma (10% versus 18.9%, P = 0.03). The odds of colonization were lower in children with persistent asthma [OR 0.4 (95% confidence interval: 0.2-0.9)] after adjusting for demographic and clinical data. Pneumococcal serotype was confirmed in 77.6% of positive samples; 35.6% of those samples corresponded to PCV13 serotypes and 64.4% to non-PCV13 serotypes. The most common serotypes were 19F (15%), 3 (13%) and 6C/6D (11%)., Conclusions: Children with persistent asthma had lower rates of pneumococcal colonization than children without asthma when seeking care for respiratory symptoms., Competing Interests: L.O and D.S. were investigators on pneumococcal vaccine clinical trials sponsored by Merck, Pfizer, and Sanofi; research funds for these activities were provided to their institution. C.J.H was an investigator on a SARS-CoV-2 vaccine trial and a pneumococcal vaccine trial both sponsored by Pfizer, a meningococcal vaccine trial sponsored by GSK, and an in vitro antimicrobial study sponsored by Merck. All other authors declared no relevant conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Streptococcus pneumoniae colonization patterns are influenced by host and environmental factors, which may be related to Invasive Pneumococcal Disease (IPD). Interestingly, COVID-19 pandemic witnessed a decline in the incidence of IPDs. Investigations with diligent data collection on the prevalence of nasopharyngeal colonization and associated serotypes during this unique period can yield novel insights. The aim of the current study was to assess the prevalence of S. pneumoniae carriage among children and adults who have sought care at emergency departments with suspected COVID-19., Methods: In this cross-sectional study, adults and children presenting with signs and symptoms likely associated with COVID-19 in two outpatient clinics in Southern Brazil were invited to participate. RT-PCR with a comprehensive molecular panel for pneumococcal identification of the 21 most prevalent serotypes in Latin America was performed on all enrolled subjects. Prevalence of pneumococcal carriage was assessed in the age groups (< 2, ≥ 2-5, ≥ 5-11, ≥ 11-18, ≥ 18-60, ≥ 60)., Results: A total of 1644 subjects were included in the study. Pneumococcal carriage was detected by PCR testing in 14.9% (245/1,644), and serotype identification occurred in 42.0% (103/245) of the participants, with a total frequency of 111. The most frequent serotype identified was 19A (25.2%, n = 28/111), followed by 6C/6D (17.1%, n = 19/111), and 23A (11.7%, n = 13/111), also highlighting the high frequency of non-vaccine serotypes found across all age groups., Discussion: 19A serotype, as well other most frequent serotypes identified are not covered by the PCV-10 in a community setting where PCV-10 is widely available. This finding reinforces the need for continuous surveillance to determine the impact of pneumococcal vaccination and guide public health decision-making. High 19A serotype prevalence is critical in the decision-making process for electing the best options for pneumococcal conjugate vaccines., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier España, S.L.U.)

Aims: To investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization., Methods and Results: Genomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose., Conclusions: Pneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: We aimed to determine the prevalence of and risk factors for nasopharyngeal and oral pneumococcal carriage in adults with community-acquired pneumonia (CAP), and the relationship between carried and disease-causing serotypes., Methods: Between 2016 and 2018, nasopharyngeal swabs, oral-fluid, and urine were collected from hospitalised adults recruited into a prospective cohort study of CAP. Pneumococcal carriage was detected by semi-quantitative real-time PCR of direct and culture-enriched nasopharyngeal swabs and culture-enriched oral-fluid. LytA and piaB positive/indeterminate samples underwent semi-quantitative serotype/serogroup-specific real-time-PCR. Serotypes in urine were identified using a 24-valent serotype-specific urinary-antigen assay., Results: We included 465 CAP patients. Nasopharyngeal carriage was detected in 34/103 (33.0%) swabbed pneumococcal pneumonia patients and oral carriage in 18/155 (12%) of sampled pneumococcal pneumonia patients. Concordance between nasopharyngeal/urine serotypes and oral/urine serotypes was 70.6% and 50% respectively. Serotypes 3 (26%, 22.2%), 8 (19.7%, 19.4%), non-typeable (11.6%, 13.9%) and 19A/F (7.5%, 8.3%) were most prevalent in urine and nasopharyngeal swabs respectively, with non-typeable (35%) and 15A/F (17%) most prevalent in oral-fluid. Pneumococcal carriage was significantly associated with pneumococcal pneumonia (nasopharyngeal adjusted odds ratio [aOR] 8.1, 95% confidence interval [CI] 3.8-17.2; oral aOR 5.5, 95% CI 2.1-13.3). All-cause CAP patients ≥65 years had lower odds of nasopharyngeal carriage (aOR 0.47, 95% CI 0.24-0.91) and current smokers had higher odds of oral carriage (aOR 2.69, 95% CI 1.10-6.60)., Conclusions: The association between nasopharyngeal carriage and pneumococcal CAP was strong. Adult carriage and disease from serotypes 8 and 19A may support direct protection of adults with PCV vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships that may be considered as potential competing interests: Wei Shen Lim reports unrestricted investigator-initiated research funding from Pfizer to WSL’s institution for an unrelated study of pneumonia in which WSL is the Chief Investigator.WSL reports research funding from NIHR for a multi-centre clinical trial of aspirin in community acquired pneumonia in which WSL is a co-applicant. WSL is Deputy Chair of the Joint Committee of Vaccination and Immunisation (JCVI)(unpaid), and unpaid Chair of the NIHR Respiratory-Translational Research Centre’s Acute Respiratory Infection National Strategy Research Group. Caroline Trotte participated in a CMV vaccine advisory board meeting in May 2022, unrelated to the topic of this paper. Seyi Eletu declares participation in a Virtual Advisory Board for a pneumococcal project organised by Sanofi Pasteur SA unrelated to the submitted work. The UK Health Security Agency (UKHSA) received monies from the University of Nottingham from an unrestricted grant from Pfizer for this work. The Vaccine Preventable Bacteria Section of UKHSA has received grants from Pfizer for investigator led research unrelated to the current project. The UKHSA provides vaccine manufacturers (GlaxoSmithKline (GSK), MSD, Pfizer) with post marketing surveillance reports on vaccine-preventable disease, including pneumococcal infections for which a cost recovery charge is made and which is unrelated to the submitted work. No other authors declare competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Genetic evolution of resistance due to mutations and transposon insertions is the primary cause of antimicrobial resistance in Streptococcus pneumoniae. Resistance to macrolide, tetracycline, and chloramphenicol is caused by the insertion of specific genes that carried by transposon (Tn). This study aims to analyze transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of S. pneumoniae serotype 19F in Indonesia. S. pneumoniae serotype 19F isolates were collected from nasopharyngeal swab specimens from different regions in Indonesia. Genomic DNA was extracted from sixteen isolates and whole genome sequencing was performed on Illumina platform. Raw sequence data were analyzed using de novo assembly by ASA 3 P and Microscope server. The presence of transposons was identified with detection of int and xis genes and visualized by pyGenomeViz. The genome size of S. pneumoniae ranges from 2,040,117 bp to 2,437,939 bp, with a GC content of around 39 %. ST1464 (4/16) and ST271 (3/16) were found as the predominant sequence type among isolates. Tn2010 was the most common transposon among S. pneumoniae serotype 19F isolates (7/16) followed by Tn2009 (4/16), and Tn5253 (3/16). We identified two deletion sites within the tetM gene (2 bp and 58 bp) that confer tetracycline susceptibility from one isolate. This study suggests that genomic analysis can be employed for the detection and surveillance of antimicrobial resistance genes among S. pneumoniae strains isolated from various regions in Indonesia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Streptococcus pneumoniae is one of the major pathogens responsible for bacterial meningitis and neurological sequelae. The present study was conducted to identify a non-hematogenous route used by S. pneumoniae to gain access to brain tissue without causing bacteremia or pneumonia, as well as bacterial and host factors involved in this process. To investigate the molecular mechanisms and dissemination pathways of pneumococcal infection in brain tissue, mice were intranasally inoculated with S. pneumoniae strain EF3030, a clinical isolate from a patient with otitis media. Pneumococci were isolated from the frontal olfactory bulb, caudal cerebrum, and cerebellum, with neither bacteremia nor pneumonia observed in the present model. Immunostaining imaging revealed the presence of S. pneumoniae organisms in olfactory nerve fibers. Knockout of the ply gene encoding pneumolysin (PLY) markedly compromised the ability of the bacterial organisms to disseminate into brain tissue, whereas the dissemination efficiency of the complemented strain was restored to nearly the same level as the wild type. Notably, distinct upregulation of Gli1 and Snail1, which are involved in the transcriptional repression of junctional proteins, along with downregulation of E-cadherin, was detected in nasal lavage samples from mice infected with the wild-type or complemented strain, but not in those from mice infected with the ply mutant. Taken together, the present findings indicate that PLY induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, thus allowing pneumococcal dissemination to brain tissue that occurs in a non-hematogenous manner.IMPORTANCEBacterial meningitis, considered to be caused by bacteremia, can lead to blood-brain barrier disruption and bacterial dissemination into the central nervous system. Despite the availability of intravenously administered antibiotics with cerebrospinal fluid transferability, bacterial meningitis remains associated with high rates of morbidity and mortality. Here, we utilized Streptococcus pneumoniae strain EF3030, clinically isolated from otitis media, for the construction of a murine infection model to investigate the molecular mechanisms by which nasally colonized pneumococci disseminate into brain tissue. The obtained findings indicate that pneumolysin (PLY) induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, which facilitates pneumococcal dissemination to brain tissue in a non-hematogenous manner. Our results support the existence of an alternative route by which S. pneumoniae can reach the central nervous system and indicate the need for the development of novel therapeutic strategies, which would be an important contribution to the clinical management of bacterial meningitis., Competing Interests: The authors declare no conflict of interest.

Background: Streptococcus pneumoniae is a major cause of mortality globally. The introduction of pneumococcal conjugate vaccines (PCVs) has reduced the incidence of the targeted serotypes significantly, but expansion of non-targeted serotypes, serotype replacement, and incomplete vaccine-targeting contribute to pneumococcal disease in the vaccine era. Here, we characterize the changing population genetic landscape of S. pneumoniae in Norway over a 41-year period (1982-2022)., Methods: Since 2018, all cases of invasive pneumococcal disease have undergone whole-genome sequencing (WGS) at the Norwegian Institute of Public Health. In order to characterize the changing population over time, historical isolates were re-cultured and sequenced, resulting in a historical WGS dataset. Isolates were assigned to global pneumococcal sequence clusters (GPSCs) using PathogenWatch and assigned to serotypes using in silico (SeroBA) and in vitro methods (Quellung reaction). Temporal phylogenetic analyses were performed on GPSCs of particular interest., Results: The availability of WGS data allowed us to study capsular variation at the level of individual lineages. We detect highly divergent fates for different GPSCs following the introduction of PCVs. For two out of eight major GPSCs, we identified multiple instances of serotype switching from vaccine types to non-vaccine types. Dating analyses suggest that most instances of serotype switching predated the introduction of PCVs, but expansion occurred after their introduction. Furthermore, selection for penicillin non-susceptibility was not a driving force for the changing serotype distribution within the GPSCs over time., Conclusions: PCVs have been major shapers of the Norwegian disease-causing pneumococcal population, both at the level of serotype distributions and the underlying lineage dynamics. Overall, the introduction of PCVs has reduced the incidence of invasive disease. However, some GPSCs initially dominated by vaccine types escaped the effect of vaccination through expansion of non-vaccine serotypes. Close monitoring of circulating lineages and serotypes will be key for ensuring optimal vaccination coverage going forward., (© 2024. The Author(s).)

Chromosome segregation in bacteria is a critical process ensuring that each daughter cell receives an accurate copy of the genetic material during cell division. Active segregation factors, such as the ParABS system or SMC complexes, are usually essential for this process, but they are surprisingly dispensable in Streptococcus pneumoniae . Rather, chromosome segregation in S. pneumoniae relies on the protein Regulator of Chromosome Segregation (RocS), although the molecular mechanisms involved remain elusive. By combining genetics, in vivo imaging, and biochemical approaches, we dissected the molecular features of RocS involved in chromosome segregation. We investigated the respective functions of the three RocS domains, specifically the C-terminal amphipathic helix (AH), the N-terminal DNA-binding domain (DBD), and the coiled-coil domain (CCD) separating the AH and the DBD. Notably, we found that a single AH is not sufficient for membrane binding and that RocS requires prior oligomerization to interact with the membrane. We further demonstrated that this self-interaction was driven by the N-terminal part of the CCD. On the other hand, we revealed that the C-terminal part of the CCD corresponds to a domain of unknown function (DUF 536) and is defined by three conserved glutamines, which play a crucial role in RocS-mediated chromosome segregation. Finally, we showed that the DBD is phosphorylated by the unique serine-threonine kinase of S. pneumoniae StkP and that mimicking this phosphorylation abrogated RocS binding to DNA. Overall, this study offers new insights into chromosome segregation in Streptococci and paves the way for a deeper understanding of RocS-like proteins in other bacteria.IMPORTANCEBacteria have evolved a variety of mechanisms to properly segregate their genetic material during cell division. In this study, we performed a molecular dissection of the chromosome partitioning protein Regulator of Chromosome Segregation (RocS), a pillar element of chromosome segregation in S. pneumoniae that is also generally conserved in the Streptococcaceae family. Our systematic investigation sheds light on the molecular features required for successful pneumococcal chromosome segregation and the regulation of RocS by phosphorylation. In addition, our study also revealed that RocS shares functional domains with the Par protein, involved in an atypical plasmid segregation system. Therefore, we expect that our findings may serve to extend our understanding of RocS and RocS-like proteins while broadening the repertoire of partitioning systems used in bacteria., Competing Interests: The authors declare no conflict of interest.

Objectives: This study aimed to identify the risk factors and construct the diagnostic model associated with pneumocystis pneumonia (PCP) in pediatric patients., Methods: This retrospective observational study analyzed 34 cases of PCP and 51 cases of other types of pneumonia treated at Children's Hospital Affiliated to Shandong University between January 2021 and August 2023. Multivariate binary logistic regression was used to identify the risk factors associated with PCP. Receiver operating characteristic curves and calibration plots were constructed to evaluate the diagnostic model., Results: Twenty clinical variables significantly differed between the PCP and non-PCP groups. Multivariate binary logistic regression analysis revealed that dyspnea, body temperature>36.5°C, and age<1.46 years old were risk factors for PCP. The area under the curve of the diagnostic model was 0.958, the P -value of Hosmer-Lemeshow calibration test was 0.346, the R 2 of the calibration plot for the actual and predicted probability of PCP was 0.9555 ( P <0.001), and the mean Brier score was 0.069. In addition, metagenomic next-generation sequencing revealed 79.41% (27/34) and 52.93% (28/53) mixed infections in the PCP and non-PCP groups, respectively. There was significantly more co-infection with cytomegalovirus and Streptococcus pneumoniae in the PCP group than that in the non-PCP group (p<0.05)., Conclusions: Dyspnea, body temperature>36.5°C, and age<1.46 years old were found to be independent risk factors for PCP in pediatric patients. The probability of co-infection with cytomegalovirus and S. pneumoniae in the PCP group was significantly higher than that in the non-PCP group., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Li, Chen, Wang, Yang, Xu and Wang.)

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated within an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall synthesized by the divisome is split to generate a lateral wall, whose expansion is insured by the insertion of the so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are, thus, crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes, but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy, and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling during the cell cycle that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A penicillin-binding proteins to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein. I MPORTANCEThis study sheds light on fundamental processes governing bacterial growth and division, using integrated click chemistry, advanced microscopy, and computational modeling approaches. It addresses cell wall synthesis mechanisms in the opportunistic human pathogen Streptococcus pneumoniae , responsible for a range of illnesses (otitis, pneumonia, meningitis, septicemia) and for one million deaths every year worldwide. This bacterium belongs to the morphological group of ovococci, which includes many streptococcal and enterococcal pathogens. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, morphogenesis, and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only enhances our understanding of how ovoid bacteria proliferate but also offers the opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, thereby participating in various cell cycle processes., Competing Interests: The authors declare no conflict of interest.