Breastfeeding reduces the risk of severe lower respiratory infections (sLRIs), a leading cause of infant mortality; however, the protective mechanisms remain elusive. Here, we demonstrated that the absence of milk-derived osteopontin (OPN), highly expressed in colostrum, predisposes neonatal mice to viral and bacterial sLRI, consequent to disrupted dendritic cell (DC) hematopoiesis in the developing liver and lung. Amelioration of disease severity by oral OPN supplementation was associated with increased enteric abundance of Lactobacillaceae and elevated levels of serum 3-phenyllactic acid (PLA), a peroxisome proliferator-activated receptor gamma (PPARγ) agonist. Supplementation with PLA or the PPARγ agonist rosiglitazone restored lung DC hematopoiesis via airway epithelium-derived chemokine ligand 25 (CCL25)-mediated recruitment of lymphoid-myeloid primed progenitors and induction of a supportive lung niche. PLA-induced DC hematopoiesis and disease tolerance were attenuated by plasmacytoid DC depletion, immunoneutralization of stem cell factor, or genetic deletion of airway epithelial Flt3L. Our findings elucidate a microbiome-host interaction by which milk OPN confers protection against sLRI.

Background:Streptococcus pneumoniae (the pneumococcus) is one of the main causes of childhood mortality. Understanding pneumococcal serotype and lineage distribution in children is important for vaccine decision-making. We undertook a secondary analysis of nasopharyngeal swabs collected from unvaccinated children as part of pneumococcal vaccine studies to provide a comprehensive picture of pneumococcal carriage epidemiology in Vietnamese children during the first 60 months of life. Methods:We analysed 4375 nasopharyngeal swabs from unvaccinated children to assess overall and vaccine-type pneumococcal carriage at 6, 12, 18, 24, and 60 months of age. For the latter three age groups, serotype distribution and genetic lineages (Global Pneumococcal Sequence Cluster, GPSCs) were described overall and by age. We also evaluated the prevalence of antimicrobial resistance (AMR) genes and multi-drug resistance (MDR), comparing vaccine-type and non-vaccine-type pneumococci. Findings:Overall pneumococcal carriage was 21·7% (952/4375) with a total of 27 serotypes detected. Serotype coverage was similar across products Pneumosil (68·6% [95% CI 65·5-71·7%], 595/867), Prevenar13 (70·0% [95% CI 67·0-73·1%], 607/867), and Vaxneuvance (70·0% [95% CI 67·0-73·1%], 607/867), and lower for Synflorix (41·5% [95% CI 38·2-44·8%], 360/867) p < 0·05 vs Pneumosil, Prevenar13 or Vaxneuvance. In total, 2444 swabs were tested at 18, 24, and 60 months. Thirty distinct GPSCs were identified, with their distribution remaining stable across these ages. AMR genes were highly prevalent, detected in 98·9% (360/364) of samples. Interpretation:Synflorix provided lower serotype coverage than other PCVs, largely driven by the prevalence of serotype 6A, which is not included in Synflorix formulation. Serotype, lineage distribution, and prevalence of AMR genes across the sampled age groups remained consistent, indicating that these distributions are broadly representative of young unvaccinated children, helping to guide optimal approaches for pneumococcal surveillance in low- and middle-income countries. Funding:National Health and Medical Research Council, Bill & Melinda Gates Foundation, Murdoch Children's Research Institute.

Abstract Background After the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. Methods A global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis ( cps ) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. Findings The global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze , wcil , wciJ and fnlA . Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ . However, substantial inter-individual antisera variation limits definitive interpretation. Interpretation Our work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on–off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. Funding Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

Streptococcus pneumoniae vaccine serotypes can persist despite pneumococcal conjugate vaccine (PCV) introduction. To examine serotype persistence, we leverage 6,545 nasopharyngeal swabs collected from children hospitalised with pneumonia before and after PCV13 introduction in Mongolia. DNA microarray was used to identify changes in lineage composition within serotypes 6A, 6B, 14, 19F and 23F over the six-year surveillance period. Children carrying post-PCV lineages are less likely to have severe pneumonia (serotypes 6B, 14 and 19F) and be multi-drug resistant (serotypes 6A, 6B and 23F) than pre-PCV lineages. Of note, the post-PCV lineage of serotype 6B has lower antibody binding to capsule and expression of capsule genes, thinner capsule and is less virulent in mice compared with the pre-PCV lineage. Our data show that although vaccine serotypes can persist in vaccinated populations, vaccination may select for less virulent lineages within vaccine serotypes. Therefore, considering factors beyond serotype is important when evaluating the true value of vaccination.

The unprecedented number of Streptococcus pneumoniae (the pneumococcus) genomes sequenced in recent years has accelerated the discovery of novel serotypes and highlighted the genetic diversity both between and within each serotype. A novel serotype should demonstrate a distinct cps locus, capsular structure and serological profile. In only the past 4years, nine new serotypes have been identified. Accurate and timely serotyping of pneumococcal isolates is key to understanding their global distribution, evolution and the response of the bacterial population to vaccination. However, current bioinformatics serotyping tools are infrequently updated and struggle to accommodate the rapid discovery of new serotypes in a timely manner. To address these limitations, we built a comprehensive and curated library (SeroBAnk) encompassing all known pneumococcal serotypes; this resource is presented as an atlas on a dedicated publicly accessible webpage (https://www.pneumogen.net/ gps/#/serobank). Building upon this resource, we developed SeroBA(v2.0), a tool with an easy-to-update database that can accurately identify 102 of 107 known pneumococcal serotypes (except for serotypes 24B, 24C, 24F, 7D and 6H) and 18 genetic subtypes within serotypes 6A, 6B, 11A, 19A, 19F and 33F. We validated SeroBA(v2.0) on 26,306 genomes from the Global Pneumococcal Sequencing project, reference isolates and simulated reads derived from the reference genetic sequences of capsular polysaccharide biosynthetic (cps) locus. We showed that SeroBA(v2.0) can reliably detect the nine recently discovered serotypes. Additionally, we show that in silico serotypes inferred by SeroBA(v2.0) had high concordance with phenotypic serotypes determined by either Quellung or latex agglutination at the serotype level (88.9%; 15,945/17,933) and at the serogroup level (91.9%; 16,480/17,933). Finally, we propose a community-contribution-based approach to ensure that SeroBA(v2.0) is maintained and updated as novel serotypes continue to be discovered. The global community can submit putative novel serotypes through our public repository on GitHub (https://github.com/GlobalPneumoSeq/seroba/issues). The submitted putative novel serotypes will be curated based on the genetic sequence of the cps region, capsular structure and serological profile by people of relevant expertise in the field. SeroBA(v2.0) can be accessed at https://github.com/GlobalPneumoSeq/seroba.

Background Streptococcus pneumoniae is a leading cause of pneumonia globally. Vaccine serotypes can persist despite pneumococcal conjugate vaccine (PCV) introduction. To examine serotype persistence, we leveraged 6,545 nasopharyngeal swabs collected from children hospitalised with pneumonia before and after PCV13 introduction in Mongolia and undertook molecular, epidemiological and experimental analyses. Methods Serotype, genetic lineage and antimicrobial resistance genes were inferred from DNA microarray. Patients carrying lineages that were predominant pre- and post-PCV introduction were examined for differences in disease severity. We also compared the pre- and post-PCV lineages by bacterial adhesion to hydrocarbon (BATH) and enzyme-linked immunosorbent (ELISA) assays. Capsule gene expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), capsule thickness by transmission electron microscopy, and virulence using an infant mouse model. Findings Changes in lineage composition were observed within serotypes 6A, 6B, 14, 19F and 23F over the six-year surveillance period. Children carrying pre-PCV lineages were more likely to have severe pneumonia than those carrying post-PCV lineages (serotypes 6B, 14 and 19F). Pre-PCV lineages were more likely to be multi-drug resistant than post-PCV lineages (serotypes 6A, 6B and 23F). For serotype 6B the post-PCV lineage had higher cell surface hydrophobicity, lower IgG, lower expression of capsule genes and evidence of thinner capsule than the pre-PCV lineage. Notably, the post-PCV 6B lineage was also less virulent in mice than the pre-PCV 6B lineage. Interpretation Despite persistence of vaccine serotypes in highly vaccinated populations, vaccination may select for lineages with differences in capsule and antibody binding and that are less virulent. When evaluating the true value of vaccination, it is important to consider factors beyond serotype alone. Funding National Health and Medical Research Council, Murdoch Children’s Research Institute, GAVI, the Vaccine Alliance. ### Competing Interest Statement CvM, CS, TM, CDN and EKM were investigators on a Pfizer collaborative research project on the impact of paediatric PCV introduction on adults in Mongolia (2018-2022). CS is an investigator on a pre-study award from Pfizer on pneumonia in Australia outside of this work. CS, CDN and EKM are investigators on a Merck Investigator Study Program grant funded by MSD outside this work. EMD is currently employed by Pfizer. JH is co-founder and shareholder of BUGS Bioscience Ltd., a not-for-profit spin-out company of City St George's, University of London. The other authors have no relevant conflicts of interest to declare. National Health and Medical Research Council, https://ror.org/011kf5r70, GNT1196415, GNT2037205, 1087957 Murdoch Children's Research Institute, https://ror.org/048fyec77 Veski, https://ror.org/04vq43039 Rebecca L. Cooper Medical Research Foundation, https://ror.org/038mte443 Gavi, https://ror.org/0141yg674, PP61690717A2

Viral-bacterial interactions during co-infection are often synergistic and can increase disease severity. However, emerging evidence indicates that some bacteria can antagonise viral infection, although the host responses driving this process remains unclear. Using infant mice co-infected with the nasopharyngeal inhabitant and pathogen Streptococcus pneumoniae and pneumonia virus of mice (PVM) to model antagonistic interactions, we found that prior bacterial colonisation enhances and prolongs anti-viral immune responses during co-infection, compared with viral infection alone. Transcriptomic, immunological, and histological analyses showed that pneumococcal colonisation prior to PVM infection enhanced and prolonged interferon signalling, increased anti-viral cytokine and chemokine protein levels and CD8+ cell responses. Notably, over 50% of differentially expressed host genes during co-infection were not differentially expressed in either infection alone. Our work shows that bacterial colonisation can modulate host immunity, shaping how the immune system responds to incoming viral infections, which has the potential to open novel therapeutic applications. ### Competing Interest Statement SM and CS have received honoraria from Pfizer and MSD for presentations at symposia or attendance at expert advisory meetings unrelated to this study. National Health and Medical Research Council, https://ror.org/011kf5r70, GNT1182442 Jack Brockhoff Foundation, https://ror.org/02ybsyy31, 4212

Streptococcus pneumoniae (the pneumococcus) is a leading cause of community-acquired pneumonia. Pneumococci are categorised into serotypes, based on the type of capsular polysaccharide produced, which has important implications for virulence, vaccine impact and global surveillance. Recently, we identified a novel serotype, which we named 33G, that is comprised of an O-acetylated hexasaccharide repeat unit. In this study, we report and describe variants of 33G, designated 33G-like, which we isolated from the nasopharynx of two adults hospitalised with pneumonia in Mongolia. Serological comparison of 33G and 33G-like pneumococci were conducted by Quellung serotyping. Genetic analysis of the capsular polysaccharide loci was performed using whole genome sequencing. Polysaccharide composition was determined using 1H nuclear magnetic resonance. By Quellung serotyping, 33G pneumococci type as both 10B and 33B whereas 33G-like pneumococci type as both 10B and 33F. Genomic analysis of the capsular polysaccharide locus revealed 33G-like loci are identical to 33G, except for frameshift mutations in the wciG gene which encodes an acetyltransferase responsible for the O-acetylation of beta-galactofuranose (β-Galf) in the capsular polysaccharide repeat unit. We constructed an artificial 33G-like by deleting wciG in a 33G strain and confirmed this gene was responsible for the serological differences between 33G and 33G-like pneumococci. Lastly, 1H nuclear magnetic resonance confirmed the O-acetylation present in the 33G polysaccharide is absent in the 33G-like polysaccharide. Here, we have provided serological, genetic and biochemical evidence that the 33G-like capsule differs to 33G and all other pneumococcal serotypes, meeting the requirements to be designated as a new serotype, which we have named 33H.

SUMMARY Streptococcus pneumoniae (the “pneumococcus”) is a significant human pathogen. The key determinant of pneumococcal fitness and virulence is its ability to produce a protective polysaccharide (PS) capsule, and anti-capsule antibodies mediate serotype-specific opsonophagocytic killing of bacteria. Notably, immunization with pneumococcal conjugate vaccines (PCVs) has effectively reduced the burden of disease caused by serotypes included in vaccines but has also spurred a relative upsurge in the prevalence of non-vaccine serotypes. Recent advancements in serotyping and bioinformatics surveillance tools coupled with high-resolution analytical techniques have enabled the discovery of numerous new capsule types, thereby providing a fresh perspective on the dynamic pneumococcal landscape. This review offers insights into the current pneumococcal seroepidemiology highlighting important serotype shifts in different global regions in the PCV era. It also comprehensively summarizes newly discovered serotypes from 2007 to 2024, alongside updates on revised chemical structures and the de-novo determinations of structures for previously known serotypes. Furthermore, we spotlight emerging evidence on non-pneumococcal Mitis-group strains that express capsular PS that are serologically and biochemically related to the pneumococcal capsule types. We further discuss the implications of these recent findings on capsule nomenclature, pneumococcal carriage detection, and future PCV design. The review maps out the current status and also outlines the course for future research and vaccine strategies, ensuring a continued effective response to the evolving pneumococcal challenge.

ABSTRACTBackgroundData available for RSV and influenza infections among children < 2 years in Mongolia are limited. We present data from four districts of Ulaanbaatar from April 2015 to June 2021.MethodsThis study was nested in an enhanced surveillance project evaluating pneumococcal conjugate vaccine (PCV13) impact on the incidence of hospitalized lower respiratory tract infections (LRTIs). Our study was restricted to children aged < 2 years with arterial O2 saturation < 93% and children with radiological pneumonia. Nasopharyngeal (NP) swabs collected at admission were tested for RSV and influenza using qRT‐PCR. NP swabs of all patients with radiological pneumonia and of a subset of randomly selected NP swabs were tested for S. pneumoniae (S.p.) by qPCR and for serotypes by culture and DNA microarray.ResultsAmong 5705 patients, 2113 (37.0%) and 386 (6.8%) had RSV and influenza infections, respectively. Children aged 2–6 months had a higher percentage of very severe RSV infection compared to those older than 6 months (42.2% versus 31.4%, p‐value Fisher's exact = 0.001). S.p. carriage was detected in 1073/2281 (47.0%) patients. Among S.p. carriage cases, 363/1073 (33.8%) had S.p. and RSV codetection, and 82/1073 (7.6%) had S.p. and influenza codetection. S.p. codetection with RSV/influenza was not associated with more severe LRTIs, compared to only RSV/influenza cases.ConclusionIn Mongolia, RSV is an important pathogen causing more severe LRTI in children under 6 months of age. Codetection of RSV or influenza virus and S.p. was not associated with increased severity.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

The pneumococcus is a major cause of mortality globally. Implementation of NPIs during the COVID-19 pandemic led to reductions in invasive pneumococcal disease in many countries.

ABSTRACT Streptococcus pneumoniae (the pneumococcus) is a human pathogen responsible for a spectrum of diseases such as pneumonia, sepsis, and meningitis. The capsule is the major pneumococcal virulence factor and is encoded by the capsular polysaccharide (cps) locus, a recombination hotspot that has resulted in over 100 distinct capsular polysaccharide types (serotypes) identified to date. Recently, 33X (also known as 10X) was proposed as a putative novel serotype, but the capsule structure had not been elucidated. Here, we provide an in-depth investigation of 33X, demonstrating it is a new pneumococcal capsular serotype. In this study, we screened 12,850 nasopharyngeal swabs from both healthy children and pneumonia patients (adults and children) in Mongolia collected between 2015 and 2022. We identified 20 pneumococcal 33X isolates. Using whole genome sequencing, we found that the 33X cps locus is a chimera of genes from pneumococcal serogroups 35, 10, and 33, as well as other Streptococcal species. Serotyping of 33X pneumococci by the Quellung reaction revealed a unique serological profile, typing as both 10B and 33B. Competitive ELISAs confirmed that antibodies that were generated in mice directed against 33X were inhibited by 33X pneumococci but not 10B or 33B. Lastly, the elucidation of the 33X capsule structure revealed that the polysaccharide is distinct from other serotypes, consisting of an O-acetylated hexasaccharide repeat unit of →5)-β-Galf-(1→3)-β-Glcp-(1→5)-β-Galf 2Ac-(1→3)-β-GalpNAc-(1→3)-α-Galp-(1→4)-Rib-ol-(5→P→. Therefore, 33X meets the requisite genetic, serological, and biochemical criteria to be designated as a new serotype, which we have named 33G. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

Host and microbial factors are critically important for influencing the severity and outcome of infection. Interactions between microbes is an understudied yet important aspect to this process. Such interactions can include interplay of pathogens with members of the microbiome and/or other pathogens, which can play a major role in determining infectious disease severity (Neu and Mainou 2020). These interactions can be synergistic, resulting in enhanced disease severity, or antagonistic, whereby the presence of one microbe might be protective and reduce the risk of infection or disease severity caused by the infecting pathogen. This thematic issue for FEMS Microbes features a collection of papers that explore the microbiology and epidemiology of bacterial-viral coinfections, particularly Streptococcus pneumoniae (pneumococcus), influenza virus, and SARS-CoV-2. One of the best-known examples of bacterial-viral coinfection occurs between pneumococcus and influenza virus. Past influenza pandemics have highlighted the consequences of pneumococcal-influenza co-infection, with severe influenza cases and deaths associated with secondary pneumococcal infection (Siemens et al. 2017). Changes in the host immune system and epithelial structure induced by influenza infection leave individuals more susceptible to pneumococcal infection (Brealey et al. 2015). Recent disruptions to the circulation of respiratory viruses related to the implementation of non-pharmaceutical interventions during the COVID-19 pandemic (e.g. physical distancing, mask wearing, etc) led to a decline in bacterial disease (Brueggemann et al. 2021). In particular, the decline in pneumococcal disease that followed the implementation of these interventions was associated with the decline in co-infecting respiratory viruses, such as influenza and respiratory syncytial virus (RSV), rather than reductions in pneumococcal prevalence (Danino et al. 2021, Dagan, Danino and Weinberger 2022, Nation et al. 2022, Rybak et al. 2022). In this FEMS Microbes thematic issue, we have three research articles that work to unravel the interactions between pneumococci and influenza. Using a ferret model, Mifsud et al. (2022) show that the time ferrets remain susceptible to secondary pneumococcal infection following influenza infection is dependent on viral subtype. Ferrets infected with H3N2 remained susceptible to pneumococcal infection for a longer period compared with ferrets infected with H1N1. In another study using ferrets and the 2009 influenza pandemic strain H1N1pdm09, Mueller Brown et al. (2022) validate previous work showing co-infected animals had enhanced disease severity compared with either infection alone. Additionally, this study explored an asymmetrical relationship between pneumococci and influenza virus. Replication of influenza virus was restricted in ferrets infected with pneumococci, supporting antagonistic observations observed in mouse models of pneumococcal-influenza infection (Diavatopoulos et al. 2010, Ortigoza et al. 2018, Manna et al. 2022). Lastly, Smith et al. (2022) used mathematical and mouse models to examine spatial dynamics in the lung during co-infection. They identified greater influenza dissemination throughout the lung during co-infection with pneumococci. Given historical observations of the key role of secondary bacterial infections during influenza pandemics, there were major concerns that a similar phenomenon would occur with SARSCoV-2 in the COVID-19 pandemic. The concern was so great that many hospitals treated COVID-19 patients with antibiotics as a precaution (Adebisi et al. 2021, Langford et al. 2021). Compared with influenza pandemics, co-infection of SARS-CoV-2 with bacteria appears to be less common, although it does occur (Westblade, Simon and Satlin 2021). In our FEMS Microbes special issue, we have two articles that examine bacterial infection in COVID-19 patients. Stahlfeld et al. (2022) tested a cohort of COVID19 patients from Yale-New Haven Hospital in the United States for pneumococcus. Interestingly, pneumococcus was detected in 11% of patients and might have been more common in patients with severe COVID-19, although the numbers of cases were too small to draw firm conclusions. Another study in our collection by Protonotariou et al. (2022) examined the microbiology of hospitalized COVID-19 patients that had bacteremia in Northern Greece. Of the 1165 COVID-19 patients admitted to hospital, ∼10% developed bacteremia, with the etiologic agents isolated being nosocomial pathogens including Acinetobacter baumannii, Klebsiella pneumoniae and Enterococcus faecium. Co-infected patients had higher disease severity, evidenced by longer hospital stay and higher mortality. The last two articles in our collection are reviews summarising the literature on hot topics in the field of bacterial–viral co-infection. The review by Flynn et al. (2022) concentrates on the upper respiratory tract microbiome including its development over time as well as its contribution to providing protection or susceptibility to infection by other pathogens. This includes a discussion of applications and implications of these interactions for interventions such as vaccines and probiotics. The review by Stein and Bianchini (2022) focuses on our understanding of the heterogeneity that exists in pathogen transmission, including super-spreading events that can be a major driver of outbreaks. They review the literature on how bacterial–viral co-infection is one such factor that can enhance transmission. Examples discussed include enhanced shedding and transmission of bacteria colonizing the upper respiratory tract following respiratory viral infection, as well as increased shedding of HIV in individuals co-infected with sexually transmitted bacterial pathogens. It is evident that microbial interactions are a contributor to enhanced morbidity and mortality caused by infectious diseases. We hope you enjoy this special issue focused on bacterial–viral coinfection, a topic that continues to garner interest in the infectious diseases field.

Non-pharmaceutical interventions (NPIs) implemented to contain SARS-CoV-2 have decreased invasive pneumococcal disease. We undertook an observational study to evaluate the impact of NPIs on pneumococcal carriage and density, drivers of transmission and disease, during the COVID-19 pandemic in Ho Chi Minh City, Vietnam. While NPIs did not significantly impact pneumococcal carriage, mean capsular pneumococcal density decreased by up to 91.5% (1.07 log10genome equivalents/mL, 95% Confidence Interval: 0.74-1.41) after NPI introduction compared with the pre-COVID-19 period. As higher pneumococcal density is a risk factor for disease, the observed decline provides a plausible mechanism for the reductions in invasive pneumococcal disease.### Competing Interest StatementCS is a lead investigator, and KM and CN are co-investigators, on a Merck Investigator Studies Program grant funded by MSD outside of this work. KM is a lead investigator, and CS and CN are co-investigators, on a Pfizer funded study outside of this work. CN is on a Data Safety Monitoring Board outside of this work (no payment). JB prepared a report on pneumococcal serotypes for MSD outside of this work. JH receives project grants from Pfizer that are outside of this work, and is a co-founder and board member of BUGS Bioscience Ltd., a not-for-profit spin-out company (no personal payment). KM is a member of WHO SAGE committee (no payment) and KM and CS are Board members of ISPPD (no payment). None of the other authors have any competing interests to declare.### Funding StatementThe vaccine trial was supported by the Bill & Melinda Gates Foundation (grant number OPP-1116833/INV-008627). We also acknowledge the Victorian Governments Operational Infrastructure Support Program.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethical approval was obtained for the vaccine trial from the Human Research Ethics Committee of the Royal Childrens Hospital Melbourne, the Institutional Review Board at the Pasteur Institute of Ho Chi Minh City, and the Vietnam Ministry of Health Ethical Review Committee for Biomedical Research. The vaccine trial is registered at clinicaltrials.gov ([NCT03098628][1]).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesDeidentified data are available upon reasonable request. Requests must be compliant with the vaccine trial ethical approvals. Requests should be directed to Professor Kim Mulholland (https://orcid.org/0000-0001-7947-680X). [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03098628&atom=%2Fmedrxiv%2Fearly%2F2022%2F05%2F07%2F2022.05.05.22274646.atom

Streptococcus pneumoniae (the pneumococcus) is a leading cause of pneumonia in children under 5 years of age. Coinfection by pneumococci and respiratory viruses enhances disease severity. Little is known about pneumococcal coinfections with respiratory syncytial virus (RSV). Here, we developed a novel infant mouse model of coinfection using pneumonia virus of mice (PVM), a murine analogue of RSV, to examine the dynamics of coinfection in the upper respiratory tract, an anatomical niche that is essential for host-to-host transmission and progression to disease. Coinfection increased damage to the nasal tissue and increased production of the chemokine CCL3. Nasopharyngeal pneumococcal density and shedding in nasal secretions were increased by coinfection. In contrast, coinfection reduced PVM loads in the nasopharynx, an effect that was independent of pneumococcal strain and the order of infection. We showed that this "antagonistic" effect was absent using either ethanol-killed pneumococci or a pneumococcal mutant deficient in capsule production and incapable of nasopharyngeal carriage. Colonization with a pneumococcal strain naturally unable to produce capsule also reduced viral loads. The pneumococcus-mediated reduction in PVM loads was caused by accelerated viral clearance from the nasopharynx. Although these synergistic and antagonistic effects occurred with both wild-type pneumococcal strains used in this study, the magnitude of the effects was strain dependent. Lastly, we showed that pneumococci can also antagonize influenza virus. Taken together, our study has uncovered multiple novel facets of bacterial-viral coinfection. Our findings have important public health implications, including for bacterial and viral vaccination strategies in young children. IMPORTANCE Respiratory bacterial-viral coinfections (such as pneumococci and influenza virus) are often synergistic, resulting in enhanced disease severity. Although colonization of the nasopharynx is the precursor to disease and transmission, little is known about bacterial-viral interactions that occur within this niche. In this study, we developed a novel mouse model to examine pneumococcal-viral interactions in the nasopharynx with pneumonia virus of mice (PVM) and influenza. We found that PVM infection benefits pneumococci by increasing their numbers in the nasopharynx and shedding of these bacteria in respiratory secretions. In contrast, we discovered that pneumococci decrease PVM numbers by accelerating viral clearance. We also report a similar effect of pneumococci on influenza. By showing that coinfections lead to both synergistic and antagonistic outcomes, our findings challenge the existing dogma in the field. Our work has important applications and implications for bacterial and viral vaccines that target these microbes.

Streptococcus pneumoniae (the pneumococcus) is a human pathogen of global importance, classified into serotypes based on the type of capsular polysaccharide produced. Serotyping of pneumococci is essential for disease surveillance and vaccine impact measurement. However, the accuracy of serotyping methods can be affected by previously undiscovered variants. Previous studies have identified variants of serotype 14, a highly invasive serotype included in all licensed vaccine formulations. However, the potential of these variants to influence serotyping accuracy and evade vaccine-induced protection has not been investigated. In this study, we screened 1,386 nasopharyngeal swabs from children hospitalized with acute respiratory infection in Papua New Guinea for pneumococci. Swabs containing pneumococci (n = 1,226) were serotyped by microarray to identify pneumococci with a divergent serotype 14 capsule locus. Three serotype 14 variants ('14-like') were isolated and characterized further. The serotyping results of these isolates using molecular methods varied depending on the method, with 3/3 typing as nontypeable (PneumoCaT), 3/3 typing as serotype 14 (seroBA), and 2/3 typing as serotype 14 (SeroCall and quantitative PCR). All three isolates were nontypeable by phenotypic methods (Quellung and latex agglutination), indicating the absence of capsule. Illumina and nanopore sequencing were employed to examine their capsule loci and revealed unique mutations. Lastly, when incubated with sera from vaccinated individuals, the 14-like isolates evaded serotype-specific opsonophagocytic killing. Our study highlights the need for phenotypic testing to validate serotyping data derived from molecular methods. The convergent evolution of capsule loss underscores the importance of studying pneumococcal population biology to monitor the emergence of pneumococci capable of vaccine escape, globally. IMPORTANCE Pneumococcus is a pathogen of major public health importance. Current vaccines have limited valency, targeting a subset (up to 20) of the more than 100 capsule types (serotypes). Precise serotyping methods are therefore essential to avoid mistyping, which can reduce the accuracy of data used to inform decisions around vaccine introduction and/or maintenance of national vaccination programs. In this study, we examine a variant of serotype 14 (14-like), a virulent serotype present in all currently licensed vaccine formulations. Although these 14-like pneumococci no longer produce a serotype 14 capsule, widely used molecular methods can mistype them as serotype 14. Importantly, we show that 14-like pneumococci can evade opsonophagocytic killing mediated by vaccination. Despite the high accuracy of molecular methods for serotyping, our study reemphasizes their limitations. This is particularly relevant in situations where nonvaccine type pneumococci (e.g., the 14-likes in this study) could potentially be misidentified as a vaccine type (e.g., serotype 14).