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Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3-31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939-1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.

Mass drug administration (MDA) programmes with the macrolide antibiotic azithromycin to reduce childhood mortality are expanding in Africa; however, concerns remain about the long-term effects of these programmes on antimicrobial resistance (AMR). We aimed to evaluate the persistence and spread of Streptococcus pneumoniae AMR following a community-randomised MDA trial. This population-based, cross-sectional, pneumococcal carriage survey was conducted in Mangochi, Malawi, 3·5 years after the MORDOR trial, in which communities received twice-yearly azithromycin or placebo for 2 years. Eligible participants in this carriage survey were children aged 4–9 years who lived in an azithromycin-treated or placebo-treated cluster during the MORDOR trial, and children aged 1–3 years who were resident in a cluster but born after the MORDOR trial ended. Nasopharyngeal swabs were collected from participants and analysed by whole genome sequencing; pneumococcal genomes obtained from a distant site in Malawi, in which MDA had not been conducted, were used as reference genomes. The primary outcome was the prevalence of S pneumoniae macrolide resistance, comparing placebo-treated and azithromycin-treated clusters at baseline, 6 months post-MDA, and 3·5 years post-MDA. Between April 8 and May 14, 2021, 924 children aged 1–9 years were screened, of whom 19 were excluded and 905 were recruited to the follow-up carriage survey: 452 from azithromycin-treated clusters and 453 from placebo-treated clusters of the MORDOR trial. We assessed 426 isolates from these participants (190 from azithromycin-treated clusters and 236 from placebo-treated clusters), as well as samples from the baseline of the MORDOR trial (164 isolates; 83 from azithromycin-treated clusters and 81 from placebo-treated clusters) and from 6 months post-MDA (223 isolates; 119 from azithromycin-treated clusters and 104 from placebo-treated clusters). In azithromycin-treated clusters, macrolide resistance increased from 21·7% (95% CI 14·2–31·7; 18 of 83 isolates) at baseline to 31·9% (24·2–40·8; 38 of 119 isolates) 6 months post-MDA and to 32·1% (25·9–39·0; 61 of 190 isolates) 3·5 years post-MDA. In placebo-treated clusters, resistance increased from 21·0% (13·5–31·1; 17 of 81 isolates) at baseline to 25·0% (17·7–34·1; 26 of 104 isolates) 6 months post-MDA and to 30·9% (25·4–37·1; 73 of 236 isolates) 3·5 years post-MDA. No significant differences were observed in odds ratios between treatment groups across the survey timepoints: 0·97 (95% CI 0·36–2·55) at baseline, 1·46 (0·67–3·17) at 6 months post-MDA, and 1·12 (0·66–1·91) at 3·5 years post-MDA. Macrolide resistance in the non-MDA site remained stable: 16·9% (95% CI 12·8–21·8; 45 of 267 isolates) at baseline, 16·5% (13·3–20·3; 70 of 424 isolates) at 6 months, and 16·5% (12·5–21·4; 44 of 267 isolates) at 2·5 years. Among children born into azithromycin-treated clusters after MDA, macrolide resistance was 36·0% (27·7–45·1; 41 of 114 children). Multidrug resistance to at least three antibiotic classes was significantly higher in azithromycin-treated (p=0·0015) and placebo-treated (p<0·0001) clusters than in the comparator population at 3·5 years post-MDA and was associated with integrative conjugative elements. Azithromycin MDA is associated with macrolide resistance that persists and potentially spreads to untreated populations. The co-existence of multidrug resistance and transmissible resistance on integrative conjugative elements in these populations is a public health concern. Careful monitoring of AMR is essential in areas where MDA is implemented. The Gates Foundation, the National Institute for Health and Care Research, and the Wellcome Trust.

Lack of rapid and comprehensive microbiological diagnosis in patients with community acquired pneumonia (CAP) hampers appropriate antimicrobial therapy. This study evaluates the real-world performance of the BioFire FilmArray Pneumonia panel plus (FAP plus ) and explores the feasibility of evaluation in a randomised controlled trial. Patients presenting to hospital with suspected CAP were recruited in a prospective feasibility study. An induced sputum or an endotracheal aspirate was obtained from all participants. The FAP plus turnaround time (TAT) and microbiological yield were compared with standard diagnostic methods (SDs). 96/104 (92%) enrolled patients had a respiratory tract infection (RTI); 72 CAP and 24 other RTIs. Median TAT was shorter for the FAP plus , compared with in-house PCR (2.6 vs 24.1 h, p < 0.001) and sputum cultures (2.6 vs 57.5 h, p < 0.001). The total microbiological yield by the FAP plus was higher compared to SDs (91% (162/179) vs 55% (99/179), p < 0.0001). Haemophilus influenzae , Streptococcus pneumoniae and influenza A virus were the most frequent pathogens. In conclusion, molecular panel testing in adults with CAP was associated with a significant reduction in time to actionable results and increased microbiological yield. The impact on antibiotic use and patient outcome should be assessed in randomised controlled trials.

Streptococcus pneumoniae can be divided into many strains, each a distinct set of isolates sharing similar core and accessory genomes, which co-circulate within the same hosts. Previous analyses suggested the short-term vaccine-associated dynamics of S. pneumoniae strains may be mediated through multi-locus negative frequency-dependent selection (NFDS), which maintains accessory loci at equilibrium frequencies. Long-term simulations demonstrated NFDS stabilised clonally-evolving multi-strain populations through preventing the loss of variation through drift, based on polymorphism frequencies, pairwise genetic distances and phylogenies. However, allowing symmetrical recombination between isolates evolving under multi-locus NFDS generated unstructured populations of diverse genotypes. Replication of the observed data improved when multi-locus NFDS was combined with recombination that was instead asymmetrical, favouring deletion of accessory loci over insertion. This combination separated populations into strains through outbreeding depression, resulting from recombinants with reduced accessory genomes having lower fitness than their parental genotypes. Although simplistic modelling of recombination likely limited these simulations’ ability to maintain some properties of genomic data as accurately as those lacking recombination, the combination of asymmetrical recombination and multi-locus NFDS could restore multi-strain population structures from randomised initial populations. As many bacteria inhibit insertions into their chromosomes, this combination may commonly underlie the co-existence of strains within a niche.

•This phase I study examined a candidate trivalent pneumococcal protein vaccine.•The vaccine contained recombinant PcpA, PhtD, and PlyD1.•The vaccine was safe and immunogenic in adults, toddlers, and infants.•In infants, aluminum adjuvant improved immunogenicity without changing safety.•The results support and provide guidance for the development of the vaccine. Pneumococcal protein vaccines (PPrVs) may provide improved protection over currently available polysaccharide and conjugated polysaccharide vaccines. Here, we examined the safety and immunogenicity of a trivalent recombinant PPrV containing PcpA, PhtD, and PlyD1. This was a phase I, single-center, randomized, observer-blind study with safety review between cohorts. Adults (18–50 years; n=30) and then toddlers (12–13 months; n=30) were randomized 2:1 to receive aluminum-adjuvanted trivalent PPrV (PPrV+adj) containing 50μg per antigen or placebo. Infants (42–49 days; n=220) were next randomized to be injected at 6, 10, and 14 weeks of age with 10μg PPrV+adj or placebo (n=60; 2:1); 25μg PPrV+adj, 25μg unadjuvanted PPrV, or placebo (n=100; 2:2:1); and 50μg PPrV+adj or placebo (n=60; 2:1). Solicited reactions were recorded for 7 days and unsolicited adverse events for 30 days after each vaccination. Concentrations of antibodies to the three vaccine antigens were measured by enzyme-linked immunosorbent assay. Tenderness/pain was the most frequent injection-site reaction. Abnormal crying and irritability (infants), loss of appetite (toddlers), and headache, malaise, and myalgia (adults) were the most frequent systemic reactions. Reactions were mostly mild or moderate, resolved within 3 days, were not adjuvant- or dose-dependent, and were not increased by repeated vaccination. No immediate adverse events, hypersensitivity reactions, or treatment-related serious adverse events were reported. In all PPrV+adj cohorts, at least 75% of subjects had a ≥2-fold increase in all three antibody concentrations. In infants, antibody concentrations were higher with PPrV+adj than with unadjuvanted PPrV, higher with three than two vaccinations, and similar at the different vaccine doses. The candidate trivalent PPrV was safe and immunogenic in adults, toddlers, and infants. Addition of aluminum adjuvant improved immunogenicity in infants without changing the safety profile.

BackgroundA putative Streptococcus pneumoniae serotype, 6D, resulting from the introduction of wciNβ into serotype 6B has been proposed MethodsWe studied 98 unique serogroup 6 isolates from Fijian children, two-thirds of whom had received at least 1 dose of 7-valent pneumococcal conjugate vaccine, and 51 invasive isolates from Australian children. We used a polymerase chain reaction (PCR) system that targets both wciNβ and the single-nucleotide polymorphism that differentiates serotypes 6A and 6B—wciP584g (6A) and wciP584a (6B) ResultsTwo (9%) of 22 Australian isolates and 24 (38%) of 64 Fijian isolates previously identified as 6A by the Quellung reaction and wciP584g PCR contained wciNβ and were designated as 6C; 14 (41%) of 34 Fijian isolates previously identified as 6B by the Quellung reaction and wciP584a PCR contained wciNβ and were designated as the new putative serotype 6D. A significantly smaller proportion of children from whom serotype 6D was isolated (2/14 [14%]) had not received PCV-7, compared with the proportion of those from whom serotype 6B was isolated (11/20 [55%]) (P<.05) ConclusionThis is the first report of naturally occurring S. pneumoniae serotype 6D

Background There has been increasing interest in the use of newer, culture-independent techniques to study the airway microbiome of COPD patients. We investigated the relationships between the three common potentially pathogenic microorganisms (PPMs) Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis , as detected by quantitative PCR (qPCR), and inflammation and health status in stable patients in the London COPD cohort. Methods We prospectively collected sputum, serum and plasma samples for analysis of airway bacterial presence and load, and airway and systemic inflammation from 99 stable COPD patients between January 2011 and October 2012. Health status was measured with St George’s Respiratory Questionnaire and COPD Assessment Test. Results Airway inflammation and plasma fibrinogen, but not C-reactive protein, were greater in samples with PPM detection (p < 0.001, p = 0.049 and p = 0.261, respectively). Increasing total bacterial load was associated with increasing airway (p < 0.01) but not systemic inflammation (p > 0.05). Samples with high total bacterial loads had significantly higher airway inflammation than both samples without PPM detection and those with lower loads. Haemophilus influenzae presence was associated with significantly higher levels of airway but not systemic inflammation for all given pathogen loads (p < 0.05), and was significantly greater than with other PPMs. No association was observed between inflammation and health status (p > 0.05). Conclusions Airway and systemic inflammation, as measured by fibrinogen, is greater in stable COPD patients with PPMs detected using the culture-independent qPCR technique. The airway, but not systemic inflammatory response, appears to have a total pathogen-load threshold and appears attributable to Haemophilus influenzae . However, discordance between inflammation and health status was observed.

► Pneumococcal PhtD and PcpA vaccine candidates were safe in adults. ► PhtD and PcpA vaccine candidates were immunogenic at 10μg, 25μg, and 50μg. ► PhtD and PcpA provide targets for pneumococcal vaccines. Pneumococcal vaccines based on protein antigens may provide expanded protection against Streptococcus pneumoniae. To evaluate safety and immunogenicity in adults of pneumococcal vaccine candidates comprising S. pneumoniae pneumococcal histidine triad protein D (PhtD) and pneumococcal choline-binding protein A (PcpA) in monovalent and bivalent formulations. This was a phase I, randomized, observer-blinded, placebo-controlled, step-wise dose-escalation study. Following a pilot safety study in which participants received one intramuscular injection of either aluminum hydroxide (AH)–adjuvanted PcpA (25μg) or PhtD–PcpA (10μg each), participants in the main study received AH–adjuvanted PcpA (25μg), AH–adjuvanted PhtD–PcpA (10, 25, or 50μg each), unadjuvanted PhtD–PcpA (25μg each), or placebo as 2 injections 30 days apart. Assignment of successive dose cohorts was made after blinded safety reviews after each dose level. Safety endpoints included rates of solicited injection site and systemic reactions, unsolicited adverse events (AEs), serious AEs (SAEs), and safety laboratory tests. Immunogenicity endpoints included levels of anti-PhtD and anti-PcpA antibodies (ELISA). Six adults 18–50 years of age were included in the pilot study and 125 in the main study. No obvious increases in solicited reactions or unsolicited AEs were reported with escalating doses (adjuvanted vaccine) after either injection, or with repeated administration. Adjuvanted vaccine candidates were associated with a higher incidence of solicited reactions (particularly injection site reactions) than unadjuvanted vaccine candidates. However, no SAE or discontinuation due to an AE occurred. Geometric mean concentrations of anti-PhtD IgG and anti-PcpA IgG increased significantly after injection 2 compared with injection 1 at each dose level. No enhancement of immune responses was shown with adjuvanted vaccine candidates compared with the unadjuvanted vaccine candidate. In the dose-escalating comparison, a plateau effect at the 25μg dose was observed as measured by geometric mean concentrations and by fold increases. Promising safety profiles and immunogenicity of these monovalent and bivalent protein vaccine candidates were demonstrated in an adult population (ClinicalTrials.gov registry no. NCT01444339).

Release of conserved cytoplasmic proteins is widely spread among Gram-positive and Gram-negative bacteria. Because these proteins display additional functions when located at the bacterial surface, they have been qualified as moonlighting proteins. The GAPDH is a glycolytic enzyme which plays an important role in the virulence processes of pathogenic microorganisms like bacterial invasion and host immune system modulation. However, GAPDH, like other moonlighting proteins, cannot be secreted through active secretion systems since they do not contain an N-terminal predicted signal peptide. In this work, we investigated the mechanism of GAPDH export and surface retention in Streptococcus pneumoniae, a major human pathogen. We addressed the role of the major autolysin LytA in the delivery process of GAPDH to the cell surface. Pneumococcal lysis is abolished in the ΔlytA mutant strain or when 1% choline chloride is added in the culture media. We showed that these conditions induce a marked reduction in the amount of surface-associated GAPDH. These data suggest that the presence of GAPDH at the surface of pneumococcal cells depends on the LytA-mediated lysis of a fraction of the cell population. Moreover, we demonstrated that pneumococcal GAPDH binds to the bacterial cell wall independently of the presence of the teichoic acids component, supporting peptidoglycan as a ligand to surface GAPDH. Finally, we showed that peptidoglycan-associated GAPDH recruits C1q from human serum but does not activate the complement pathway.

Pneumococcal conjugate vaccines reduce the prevalence of vaccine serotypes carried in the nasopharynx. Because this could alter carriage of other potential pathogens, we assessed the nasopharyngeal microbiome of children who had been vaccinated with 10-valent pneumococcal non-typeable Haemophilus influenzae protein-D conjugate vaccine (PHiD-CV). Profiles of the nasopharyngeal microbiota of 60 children aged 12-59 months, who had been randomized to receive 2 doses of PHiD-CV (n=30) or Hepatitis A vaccine (n=30) 60 days apart, were constructed by 16S rRNA gene pyrosequencing of swab specimens collected before vaccination and 180 days after dose 1. Prior to vaccination, Moraxella catarrhalis (median of 12.3% of sequences/subject), Streptococcus pneumoniae (4.4%) and Corynebacterium spp. (5.6%) were the most abundant nasopharyngeal bacterial species. Vaccination with PHiD-CV did not significantly alter the species composition, abundance, or prevalence of known pathogens. Distinct microbiomes were identified based on the abundances of Streptococcus, Moraxella, and Haemophilus species. These microbiomes shifted in composition over the study period and were independent of age, sex, school attendance, antibiotic exposure, and vaccination. Vaccination of children with two doses of PHiD-CV did not significantly alter the nasopharyngeal microbiome. This suggests limited replacement carriage with pathogens other than non-vaccine strains of S. pneumoniae. clinicaltrials.gov NCT01028326.