Host and environmental factors shape upper airway microbiota and respiratory health across the human lifespan.

Our understanding of the normal variation in the upper respiratory tract (URT) microbiota across the human lifespan and how these relate to host, environment, and health is limited. We studied the microbiota of 3,104 saliva (<10 year-olds)/oropharynx (≥10 year-olds) and 2,485 nasopharynx samples of 3,160 Dutch individuals 0-87 years of age, participating in a cross-sectional population-wide study (PIENTER-3) using 16S-rRNA sequencing. The microbiota composition was strongly related to age, especially in the nasopharynx, with maturation occurring throughout childhood and adolescence. Clear niche- and age-specific associations were found between the microbiota composition and host/environmental factors and health outcomes. Among others, social interaction, sex, and season were associated with the nasopharyngeal microbial community. By contrast, the oral microbiota was more related to antibiotics, tobacco, and alcohol use. We present an atlas of the URT microbiota across the lifespan in association with environment and health, establishing a baseline for future research.

Serological profiling of pneumococcal proteins reveals unique patterns of acquisition, maintenance and waning of antibodies throughout life.

Streptococcus pneumoniae is a leading cause of morbidity and mortality in children and older adults. Yet knowledge on the development of pneumococcal protein-specific antibody responses throughout life is limited. To investigate this, we measured serum IgG levels to 55 pneumococcal proteins in 11-month old infants (n=73), 24-month old children (n=101), parents (n=99), adults without children <6 years of age (n= 99) and older adults aged >60 years (n=100). Our findings revealed low IgG levels in infancy, with distinct development patterns peaking in adults. A decrease in levels was observed for 27 antigens towards older age. Adults and older adults had increased IgG levels during pneumococcal carriage and at increased exposure risk to S. pneumoniae. Carriage was a stronger predictor than exposure or age for antibody responses. These findings highlight the dynamic nature of naturally acquired humoral immunity to pneumococcal proteins throughout life, offering insights for age-targeted interventions.

Saliva as an alternative sample type for detection of pneumococcal carriage in young children.

For children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillance of pneumococcal colonization and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of paired nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular detection methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic culture. Next, DNA extracted from all plate growth was tested by qPCR for the presence of the pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 161/288 (60 %) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture plates. By qPCR, 155/288 (54 %) culture-enriched saliva samples and 187/288 (65 %) nasopharyngeal swabs tested positive. Altogether, 219/288 (76 %) infants tested positive for pneumococcus, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs detecting significantly more carriers compared to either conventional culture (P<0.001) or qPCR detection of saliva (P=0.002). However, 32/219 (15 %) carriers were only positive in saliva, contributing significantly to the overall number of carriers detected (P=0.002). While testing nasopharyngeal swabs by qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes that may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling of study participants.

Meningococcal carriage in children and young adults: a cross-sectional and longitudinal study, Iceland, 2019 to 2021.

BackgroundNeisseria meningitidis is a commensal bacterium which can cause invasive disease. Colonisation studies are important to guide vaccination strategies.AimThe study's aim was to determine the prevalence of meningococcal colonisation, duration of carriage and distribution of genogroups in Iceland.MethodsWe collected samples from 1 to 6-year-old children, 15-16-year-old adolescents and 18-20-year-old young adults. Carriers were sampled at regular intervals until the first negative swab. Conventional culture methods and qPCR were applied to detect meningococci and determine the genogroup. Whole genome sequencing was done on groupable meningococci.ResultsNo meningococci were detected among 460 children, while one of 197 (0.5%) adolescents and 34 of 525 young adults (6.5 %) carried meningococci. Non-groupable meningococci were most common (62/77 isolates from 26/35 carriers), followed by genogroup B (MenB) (12/77 isolates from 6/35 carriers). Genogroup Y was detected in two individuals and genogroup W in one. None carried genogroup C (MenC). The longest duration of carriage was at least 21 months. Serial samples from persistent carriers were closely related in WGS.ConclusionsCarriage of pathogenic meningococci is rare in young Icelanders. Non-groupable meningococci were the most common colonising meningococci in Iceland, followed by MenB. No MenC were found. Whole genome sequencing suggests prolonged carriage of the same strains in persistent carriers.

High Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Household Transmission Rates Detected by Dense Saliva Sampling.

BACKGROUND: Understanding the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) household transmission is important for adequate infection control measures in this ongoing pandemic. METHODS: Households were enrolled upon a polymerase chain reaction-confirmed index case between October and December 2020, prior to the coronavirus disease 2019 vaccination program. Saliva samples were obtained by self-sampling at days 1, 3, 5, 7, 10, 14, 21, 28, 35, and 42 from study inclusion. Nasopharyngeal swabs (NPS) and oropharyngeal swabs (OPS) were collected by the research team at day 7 and capillary blood samples at day 42. Household secondary attack rate (SAR) and per-person SAR were calculated based on at least 1 positive saliva, NPS, OPS, or serum sample. Whole genome sequencing was performed to investigate the possibility of multiple independent SARS-CoV-2 introductions within a household. RESULTS: Eighty-five households were included consisting of 326 (unvaccinated) individuals. Comparable numbers of secondary cases were identified by saliva (133/241 [55.2%]) and serum (127/213 [59.6%]). The household SAR was 88.2%. The per-person SAR was 64.3%. The majority of the secondary cases tested positive in saliva at day 1 (103/150 [68.7%]). Transmission from index case to household member was not affected by age or the nature of their relationship. Phylogenetic analyses suggested a single introduction for the investigated households. CONCLUSIONS: Households have a pivotal role in SARS-CoV-2 transmission. By repeated saliva self-sampling combined with NPS, OPS, and serology, we found the highest SARS-CoV-2 household transmission rates reported to date. Salivary (self-) sampling of adults and children is suitable and attractive for near real-time monitoring of SARS-CoV-2 transmission in this setting.

Approximate likelihood-based estimation method of multiple-type pathogen interactions: An application to longitudinal pneumococcal carriage data.

While the serotypes of Streptococcus pneumoniae are known to compete during colonization in human hosts, our knowledge of how competition occurs is still incomplete. New insights of pneumococcal between-type competition could be generated from carriage data obtained by molecular-based detection methods, which record more complete sets of serotypes involved in co-carriage than when detection is done by culture. Here, we develop a Bayesian estimation method for inferring between-type interactions from longitudinal data recording the presence/absence of the types at discrete observation times. It allows inference from data containing co-carriage of two or more serotypes, which is often the case when pneumococcal presence is determined by molecular-based methods. The computational burden posed by the increased number of types detected in co-carriage is addressed by approximating the likelihood under a multi-state model with the likelihood of only those trajectories with minimum number of acquisition and clearance events between observation times. The proposed method's performance was validated on simulated data. The estimates of the interaction parameters of acquisition and clearance were unbiased in settings with short sampling intervals between observation times. With less frequent sampling, the estimates of the interaction parameters became more biased, but their ratio, which summarizes the total interaction, remained unbiased. Confounding due to unobserved heterogeneity in exposure could be corrected by including individual-level random effects. In an application to empirical data about pneumococcal carriage in infants, we found new evidence for between-serotype competition in clearance, although the effect size was small.

Influenza-like Illness Exacerbates Pneumococcal Carriage in Older Adults.

BACKGROUND: In older adults, pneumococcal disease is strongly associated with respiratory viral infections, but the impact of viruses on Streptococcus pneumoniae carriage prevalence and load remains poorly understood. Here, we investigated the effects of influenza-like illness (ILI) on pneumococcal carriage in community-dwelling older adults. METHODS: We investigated the presence of pneumococcal DNA in saliva samples collected in the 2014/2015 influenza season from 232 individuals aged ≥60 years at ILI onset, followed by sampling 2-3 weeks and 7-9 weeks after the first sample. We also sampled 194 age-matched controls twice 2-3 weeks apart. Pneumococcal DNA was detected with quantitative polymerase chain reaction assays targeting the piaB and lytA genes in raw and in culture-enriched saliva. Bacterial and pneumococcal abundances were determined in raw saliva with 16S and piaB quantification. RESULTS: The prevalence of pneumococcus-positive samples was highest at onset of ILI (42/232 [18%]) and lowest among controls (26/194 [13%] and 22/194 [11%] at the first and second samplings, respectively), though these differences were not significant. Pneumococcal carriage was associated with exposure to young children (odds ratio [OR], 2.71 [95% confidence interval {CI}, 1.51-5.02]; P < .001), and among asymptomatic controls with presence of rhinovirus infection (OR, 4.23 [95% CI, 1.16-14.22]; P < .05). When compared with carriers among controls, pneumococcal absolute abundances were significantly higher at onset of ILI (P < .01), and remained elevated beyond recovery from ILI (P < .05). Finally, pneumococcal abundances were highest in carriage events newly detected after ILI onset (estimated geometric mean, 1.21 × 10-5 [95% CI, 2.48 × 10-7 to 2.41 × 10-5], compared with preexisting carriage). CONCLUSIONS: ILI exacerbates pneumococcal colonization of the airways in older adults, and this effect persists beyond recovery from ILI.

Distribution of serotypes and patterns of antimicrobial resistance among commensal Streptococcus pneumoniae in nine European countries.

BACKGROUND: Streptococcus pneumoniae is a commensal of the human upper respiratory tract and a major cause of morbidity and mortality worldwide. This paper presents the distribution of serotypes and antimicrobial resistance in commensal S. pneumoniae strains cultured from healthy carriers older than four years of age in nine European countries. METHODS: Nasal swabs from healthy persons (age between 4 and 107 years old) were obtained by general practitioners from each country from November 2010 to August 2011. Swabs were cultured for S. pneumoniae using a standardized protocol. Antibiotic resistance was determined for isolated S. pneumoniae by broth microdilution. Capsular sequencing typing was used to identify serotypes, followed by serotype-specific PCR assays in case of ambiguous results. RESULTS: Thirty-two thousand one hundred sixty-one nasal swabs were collected from which 937 S. pneumoniae were isolated. A large variation in serotype distribution and antimicrobial resistant serotypes across the participating countries was observed. Pneumococcal vaccination was associated with a higher risk of pneumococcal colonization and antimicrobial resistance independently of country and vaccine used, either conjugate vaccine or PPV 23). CONCLUSIONS: Serotype 11A was the most common in carriage followed by serotypes 23A and 19A. The serotypes showing the highest resistance to penicillin were 14 followed by 19A. Serotype 15A showed the highest proportion of multidrug resistance.

Dried Saliva Spots: A Robust Method for Detecting Streptococcus pneumoniae Carriage by PCR.

The earliest studies in the late 19th century on Streptococcus pneumoniae (S. pneumoniae) carriage used saliva as the primary specimen. However, interest in saliva declined after the sensitive mouse inoculation method was replaced by conventional culture, which made isolation of pneumococci from the highly polymicrobial oral cavity virtually impossible. Here, we tested the feasibility of using dried saliva spots (DSS) for studies on pneumococcal carriage. Saliva samples from children and pneumococcus-spiked saliva samples from healthy adults were applied to paper, dried, and stored, with and without desiccant, at temperatures ranging from -20 to 37 °C for up to 35 days. DNA extracted from DSS was tested with quantitative-PCR (qPCR) specifically for S. pneumoniae. When processed immediately after drying, the quantity of pneumococcal DNA detected in spiked DSS from adults matched the levels in freshly spiked raw saliva. Furthermore, pneumococcal DNA was stable in DSS stored with desiccant for up to one month over a broad range of temperatures. There were no differences in the results when spiking saliva with varied pneumococcal strains. The collection of saliva can be a particularly useful in surveillance studies conducted in remote settings, as it does not require trained personnel, and DSS are resilient to various transportation conditions.

Identification of pneumococcal colonization determinants in the stringent response pathway facilitated by genomic diversity.

BACKGROUND: Understanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has rarely been exploited. Here we report use of a diverse collection of 616 carriage isolates of Streptococcus pneumoniae and their genome sequences to help identify a novel determinant of pneumococcal colonization. RESULTS: A spontaneously arising laboratory variant (SpnYL101) of a capsule-switched TIGR4 strain (TIGR4:19F) showed reduced ability to establish mouse nasal colonization and lower resistance to non-opsonic neutrophil-mediated killing in vitro, a phenotype correlated with in vivo success. Whole genome sequencing revealed 5 single nucleotide polymorphisms (SNPs) affecting 4 genes in SpnYL101 relative to its ancestor. To evaluate the effect of variation in each gene, we performed an in silico screen of 616 previously published genome sequences to identify pairs of closely-related, serotype-matched isolates that differ at the gene of interest, and compared their resistance to neutrophil-killing. This method allowed rapid examination of multiple candidate genes and found phenotypic differences apparently associated with variation in SP_1645, a RelA/ SpoT homolog (RSH) involved in the stringent response. To establish causality, the alleles corresponding to SP_1645 were switched between the TIGR4:19F and SpnYL101. The wild-type SP_1645 conferred higher resistance to neutrophil-killing and competitiveness in mouse colonization. Using a similar strategy, variation in another RSH gene (TIGR4 locus tag SP_1097) was found to alter resistance to neutrophil-killing. CONCLUSIONS: These results indicate that analysis of naturally existing genomic diversity complements traditional genetics approaches to accelerate genotype-phenotype analysis.

Viral and bacterial interactions in the upper respiratory tract.

Respiratory infectious diseases are mainly caused by viruses or bacteria that often interact with one another. Although their presence is a prerequisite for subsequent infections, viruses and bacteria may be present in the nasopharynx without causing any respiratory symptoms. The upper respiratory tract hosts a vast range of commensals and potential pathogenic bacteria, which form a complex microbial community. This community is assumed to be constantly subject to synergistic and competitive interspecies interactions. Disturbances in the equilibrium, for instance due to the acquisition of new bacteria or viruses, may lead to overgrowth and invasion. A better understanding of the dynamics between commensals and pathogens in the upper respiratory tract may provide better insight into the pathogenesis of respiratory diseases. Here we review the current knowledge regarding specific bacterial-bacterial and viral-bacterial interactions that occur in the upper respiratory niche, and discuss mechanisms by which these interactions might be mediated. Finally, we propose a theoretical model to summarize and illustrate these mechanisms.

Respiratory microbiota dynamics following Streptococcus pneumoniae acquisition in young and elderly mice.

The upper respiratory tract (URT) is a distinct microbial niche of low-density bacterial communities and, also, a portal of entry for many potential pathogens, including Streptococcus pneumoniae. Thus far, animal models have been used to study the dynamics of and interactions between limited numbers of different species in the URT. Here, we applied a deep sequencing approach to explore, for the first time, the impact of S. pneumoniae acquisition on URT microbiota in a mouse model, as well as potential age-dependent effects. Young-adult and elderly mice were inoculated intranasally with S. pneumoniae, and nasal lavage samples were collected for up to 28 days postcolonization. Bacterial DNA extracted from lavage samples was subjected to barcoded pyrosequencing of the V5-to-V7 hypervariable region of the small-subunit rRNA gene. We observed highly diverse microbial profiles, with the presence overall of 15 phyla and approximately 645 operational taxonomic units (OTUs). We noted differences in the composition of microbiota between young and elderly mice, with a significantly higher abundance of Bacteroidetes in the young mice. The introduction of S. pneumoniae into the URT led to a temporary dominance of pneumococci in the microbiota of all mice, accompanied by a significant decrease in microbial diversity. As mice gradually cleared the colonization, the diversity returned to baseline levels. Diversification was accompanied by an early expansion of Bacteroidetes, Staphylococcus spp., and Lachnospiraceae. Moreover, the Bacteroidetes expansion was significantly greater in young-adult than in elderly mice. In conclusion, we observed differences in URT microbiota composition between naive young-adult and elderly mice that were associated with differences in pneumococcal clearance over time.

Seven-valent pneumococcal conjugate vaccine and nasopharyngeal microbiota in healthy children.

Seven-valent pneumococcal conjugate vaccine (PCV-7) is effective against vaccine serotype disease and carriage. Nevertheless, shifts in colonization and disease toward nonvaccine serotypes and other potential pathogens have been described. To understand the extent of these shifts, we analyzed nasopharyngeal microbial profiles of 97 PCV-7-vaccinated infants and 103 control infants participating in a randomized controlled trial in the Netherlands. PCV-7 immunization resulted in a temporary shift in microbial community composition and increased bacterial diversity. Immunization also resulted in decreased presence of the pneumococcal vaccine serotype and an increase in the relative abundance and presence of nonpneumococcal streptococci and anaerobic bacteria. Furthermore, the abundance of Haemophilus and Staphylococcus bacteria in vaccinees was increased over that in controls. This study illustrates the much broader effect of vaccination with PCV-7 on the microbial community than currently assumed, and highlights the need for careful monitoring when implementing vaccines directed against common colonizers.

Distinct effects on diversifying selection by two mechanisms of immunity against Streptococcus pneumoniae.

Antigenic variation to evade host immunity has long been assumed to be a driving force of diversifying selection in pathogens. Colonization by Streptococcus pneumoniae, which is central to the organism's transmission and therefore evolution, is limited by two arms of the immune system: antibody- and T cell- mediated immunity. In particular, the effector activity of CD4(+) T(H)17 cell mediated immunity has been shown to act in trans, clearing co-colonizing pneumococci that do not bear the relevant antigen. It is thus unclear whether T(H)17 cell immunity allows benefit of antigenic variation and contributes to diversifying selection. Here we show that antigen-specific CD4(+) T(H)17 cell immunity almost equally reduces colonization by both an antigen-positive strain and a co-colonized, antigen-negative strain in a mouse model of pneumococcal carriage, thus potentially minimizing the advantage of escape from this type of immunity. Using a proteomic screening approach, we identified a list of candidate human CD4(+) T(H)17 cell antigens. Using this list and a previously published list of pneumococcal Antibody antigens, we bioinformatically assessed the signals of diversifying selection among the identified antigens compared to non-antigens. We found that Antibody antigen genes were significantly more likely to be under diversifying selection than the T(H)17 cell antigen genes, which were indistinguishable from non-antigens. Within the Antibody antigens, epitopes recognized by human antibodies showed stronger evidence of diversifying selection. Taken together, the data suggest that T(H)17 cell-mediated immunity, one form of T cell immunity that is important to limit carriage of antigen-positive pneumococcus, favors little diversifying selection in the targeted antigen. The results could provide new insight into pneumococcal vaccine design.

Pneumococcal immune evasion: ZmpC inhibits neutrophil influx.

Neutrophil recruitment is essential in clearing pneumococcal infections. The first step in neutrophil extravasation involves the interaction between P-selectin on activated endothelium and P-Selectin Glycoprotein 1 (PSGL-1) on neutrophils. Here, we identify pneumococcal Zinc metalloproteinase C as a potent inhibitor of PSGL-1. ZmpC degrades the N-terminal domain of PSGL-1, thereby disrupting the initial rolling of neutrophils on activated human umbilical vein endothelial cells. Furthermore, mice infected with wild-type strain in the model of pneumococcal pneumonia showed lower lungs neutrophil infiltration compare to animals infected with ZmpC mutant. In addition, we confirmed the association of zmpC with serotype 8 and 11A and found it to be associated with serotype 33F as well. In conclusion, wereport PSGL-1 as a novel target for ZmpC and show that ZmpC inhibits neutrophil extravasation during pneumococcal pneumonia.

Impact of age on pneumococcal colonization of the nasopharynx and oral cavity: an ecological perspective.

Pneumococcal carriage studies have suggested that pneumococcal colonization in adults is largely limited to the oral cavity and oropharynx. In this study, we used total abundance-based ß-diversity (dissimilarity) and ß-diversity components to characterize age-related differences in pneumococcal serotype composition of respiratory samples. quantitative PCR (qPCR) was applied to detect pneumococcal serotypes in nasopharyngeal samples collected from 946 toddlers and 602 adults, saliva samples collected from a subset of 653 toddlers, and saliva and oropharyngeal samples collected from a subset of 318 adults. Bacterial culture rates from nasopharyngeal samples were used to characterize age-related differences in rates of colonizing bacteria. Dissimilarity in pneumococcal serotype composition was low among saliva and nasopharyngeal samples from children. In contrast, respiratory samples from adults exhibited high serotype dissimilarity, which predominantly consisted of abundance gradients and was associated with reduced nasopharyngeal colonization. Age-related serotype dissimilarity was high among nasopharyngeal samples and relatively low for saliva samples. Reduced nasopharyngeal colonization by pneumococcal serotypes coincided with significantly reduced Moraxella catarrhalis and Haemophilus influenzae and increased Staphylococcus aureus nasopharyngeal colonization rates among adults. Findings from this study suggest that within-host environmental conditions, utilized in the upper airways by pneumococcus and other bacteria, undergo age-related changes. It may result in a host-driven ecological succession of bacterial species colonizing the nasopharynx and lead to competitive exclusion of pneumococcus from the nasopharynx but not from the oral habitat. This explains the poor performance of nasopharyngeal samples for pneumococcal carriage among adults and indicates that in adults saliva more accurately represents the epidemiology of pneumococcal carriage than nasopharyngeal samples.

Within-host selection is limited by an effective population of Streptococcus pneumoniae during nasopharyngeal colonization.

Streptococcus pneumoniae (pneumococcus) is a significant pathogen that frequently colonizes the human nasopharynx. Environmental factors, including antimicrobial use and host immunity, exert selection on members of the nasopharyngeal population, and the dynamics of selection are influenced by the effective population size of the selected population, about which little is known. We measured here the variance effective population size (N(e)) of pneumococcus in a mouse colonization model by monitoring the frequency change of two cocolonizing, competitively neutral pneumococcal strains over time. The point estimate of N(e) during nasal carriage in 16 BALB/c mice was 133 (95% confidence interval [CI] = 11 to 203). In contrast, the lower-bound census population exhibited a mean of 5768 (95% CI = 2,515 to 9,021). Therefore, pneumococcal N(e) during nasal carriage is substantially smaller than the census population. The N(e) during day 1 to day 4 of colonization was comparable to the Ne during day 4 to day 8. Similarly, a low Ne was also evident for the colonization of pneumococcus in BALB/c mice exposed to cholera toxin 4 weeks prior to challenge and in another mouse strain (DO11.10 RAG(-/-)). We developed a mathematical model of pneumococcal colonization composed of two subpopulations with differential contribution to future generations. By stochastic simulation, this model can reproduce the pattern of observed pneumococcal N(e) and predicts that the selection coefficients may be difficult to measure in vivo. We hypothesized that such a small N(e) may reduce the effectiveness of within host selection for pneumococcus.

Surface charge of Streptococcus pneumoniae predicts serotype distribution.

Streptococcus pneumoniae (pneumococcus) frequently colonizes the human nasopharynx and is an important cause of pneumonia, meningitis, sinusitis, and otitis media. The outer cell surface of pneumococcus may assume various degrees of negative charge depending on the polysaccharide capsule, of which more than 90 serotypes have been identified. The negative charge of capsular polysaccharides has been proposed to electrostatically repel pneumococci from phagocytic cells, and avoidance of phagocytosis correlates with higher carriage prevalence. We hypothesized that the surface charge of pneumococcus contributes to its success in nasopharyngeal carriage by modulating resistance to phagocyte-mediated killing. Here, we measured the surface charge (zeta potential) of laboratory-constructed strains that share a genetic background but differ in serotype and of clinical strains that differ in serotype and genetic background. A more negative surface charge correlated with higher resistance to nonopsonic killing by human neutrophils in vitro. In addition, a more negative zeta potential was associated with higher carriage prevalence in human populations before and after the widespread use of the pneumococcal conjugate vaccine PCV7. We also confirmed that capsule is the major determinant of net surface charge in clinical isolates with diverse backgrounds. We noted that exceptions exist to the idea that a higher magnitude of negative charge predicts higher prevalence. The results indicated that zeta potential is strongly influenced by pneumococcal capsule type but is unlikely to be the only important mechanism by which capsule interacts with host.

Impaired innate mucosal immunity in aged mice permits prolonged Streptococcus pneumoniae colonization.

Streptococcus pneumoniae is a frequent asymptomatic colonizer of the nasopharyngeal niche and only occasionally progresses toward infection. The burden of pneumococcal disease is particularly high in the elderly, and the mechanisms behind this increased susceptibility are poorly understood. Here we used a mouse model of pneumococcal carriage to study immunosenescence in the upper respiratory tract (URT). Nasal mucosa-associated lymphoid tissue (NALT) showed increased expression of Toll-like receptor 1, interleukin-1ß, NLRp3 inflammasome, and CCL2 in naive elderly compared to young animals. This suggests an increased proinflammatory expression profile in the NALT of aged mice at baseline. Simultaneously, we observed a more tolerogenic profile in respiratory epithelia of naive elderly compared to young adult mice with upregulation of the NF-κß pathway inhibitor peroxisome proliferator-activated receptor gamma (PPARγ). After nasal instillation of pneumococci, pneumococcal colonization was prolonged in elderly mice compared to in young adults. The delay in clearance was associated with absent or delayed upregulation of a proinflammatory mediator(s) in the NALT, diminished influx of macrophages into the URT niche, and absent downregulation of PPARγ in respiratory epithelium, accompanied by diminished expression of cathelicidin (CRAMP) at the site of colonization. These findings suggest that unresponsiveness to pneumococcal challenge due to altered mucosal immune regulation is the key to increased susceptibility to disease in the elderly.

Streptococcus pneumoniae capsular serotype invasiveness correlates with the degree of factor H binding and opsonization with C3b/iC3b.

Different capsular serotypes of Streptococcus pneumoniae vary markedly in their ability to cause invasive infection, but the reasons why are not known. As immunity to S. pneumoniae infection is highly complement dependent, variations in sensitivity to complement between S. pneumoniae capsular serotypes could affect invasiveness. We have used 20 capsule-switched variants of strain TIGR4 to investigate whether differences in the binding of the alternative pathway inhibitor factor H (FH) could be one mechanism causing variations in complement resistance and invasive potential between capsular serotypes. Flow cytometry assays were used to assess complement factor binding and complement-dependent neutrophil association for the TIGR4 capsule-switched strains. FH binding varied with the serotype and inversely correlated with the results of factor B binding, C3b/iC3b deposition, and neutrophil association. Differences between strains in FH binding were lost when assays were repeated with pspC mutant strains, and loss of PspC also reduced differences in C3b/iC3b deposition between strains. Median FH binding was high in capsule-switched mutant strains expressing more invasive serotypes, and a principal component analysis demonstrated a strong correlation between serotype invasiveness, high FH binding, and resistance to complement and neutrophil association. Further data obtained with 33 clinical strains also demonstrated that FH binding negatively correlated with C3b/iC3b deposition and that median FH binding was high in strains expressing more invasive serotypes. These data suggest that variations in complement resistance between S. pneumoniae strains and the association of a serotype with invasiveness could be related to capsular serotype effects on FH binding.