The importance of pneumococcal capsule in inate immunity and biofilm formation

Chapter 1 gives an overview about Streptococcus pneumoniae, its role as a human pathogen and its virulence factors. Additionally, biofilm development and its relevance in clinics are introduced, and the innate immune response to pneumococcus as well as bacterial-viral interactions in the upper respiratory tract are also discussed. Chapter 2 emphasizes the three main topics of this thesis: the role of capsule and pneumolysin in the immune response in the respiratory tract, biofilm formation of S. pneumoniae serotypes and commensal streptococci in vitro, and host innate immune responses to RSV and S. pneumoniae during in vitro co-infections. Aims and hypotheses are provided here. Chapter 3 is divided into two parts: First, the release of the pro-inflammatory cytokines CXCL8 and IL-6 from the human pharyngeal epithelial cell line Detroit 562 and from human bronchial epithelial cells (iHBEC) is described in response to S. pneumoniae. Capsule was shown to suppress the release of both cytokines in both cell lines tested, but release was much less from iHBEC cells. During intranasal colonization of mice, suppression of CXCL8 release by the capsule was also observed in vivo, but the effect was only measured in the absence of pneumolysin. Long term, stable nasopharyngeal carriage in a mouse model resulted in the dissemination of nonencapsulated pneumococci into the lungs, whereas encapsulated strains remained in the nasopharynx. The S. pneumoniae capsule thus plays a role in modulation of the pro-inflammatory immune response in the respiratory tract. Second, results on immunological cells and immune regulation in a long term, stable nasopharyngeal carriage mouse model are presented. Mice were infected with encapsulated or nonencapsulated pneumococcal strains, and after 1, 3, 8 and 15 days, were sacrificed to evaluate the numbers of CD45+ cells, neutrophils, macrophages, FoxP3+ regulatory T-cells and CD3+ T-cells in the nasal mucosa as well as the amount of secreted IL-10 in the nasopharynx. Nasopharyngeal colonization which is effectively silent resulted in the stimulation of FoxP3+ regulatory T-cells and IL-10 release associated with immune homeostasis, whereas lung infiltration was required to increase the number of neutrophils and macrophages resulting in a stronger innate immune response in the nasal mucosa. Chapter 4 contains results of mono- and co-stimulation using RSV and pneumococci or pneumococcal virulence factors on the human bronchial epithelial cell line BEAS-2B. An increase in CXCL8 and IL-6 levels was measured for mixed stimulations of RSV and pneumococcus when encapsulated bacteria were used. Increasing pneumolysin concentrations resulted in enhanced CXCL8 levels. Priming of bronchial epithelial cells with RSV opens the door for more severe pneumococcal infections. Chapter 5 is composed of two parts: The first part describes initial biofilm formation of serotypes 6B and 7F in a static model in vitro. Biofilms of both serotypes contained SCVs, but only serotype 6B increased in SCV formation between 16 and 65h of incubation. SCV stability was tested by passaging clones in complex medium, where SCV production is not associated with advantages in growth. Serotype 6B lost the SCV phenotype indicating a fast adaptation to a changing nutritional environment. Limitations of our in vitro model are discussed. The second part is about initial biofilm formation of mixed culture growth of S. pneumoniae with commensal streptococci. Competition dominates this process. S. oralis and pneumococcus compete for nutrients, whereas mixed species growth of S. mitis or S. pseudopneumoniae with S. pneumoniae is mainly influenced by other factors. In Chapter 6 the findings of chapters 3, 4 and 5 are discussed and an outlook for further studies is provided. Chapters 7, 8, 9, 10 and 11 contain the references, the acknowledgements, the curriculum vitae, the appendix and the declaration of originality.

Serotype epidemiology of invasive pneumococcal disease in Swiss adults: A nationwide population-based study.

BACKGROUND In Switzerland, the heptavalent (PCV7) and 13-valent pneumococcal conjugate vaccine (PCV13) were recommended for all infants aged <2 years in 2007 and 2011, respectively. Due to herd effects, a protective impact on the invasive pneumococcal disease (IPD) rates in adults had been expected. METHODS Within this study, data from the nationwide mandatory surveillance was analyzed for all adult patients ≥16 years with IPD of known serotype/serogroup during 2003-2012. Trend (for IPD cases from 2003 to 2012) and logistic regression analyses (2007-2010) were performed to identify changes in serotype distribution and to identify the association of serotypes with age, clinical manifestations, comorbidities and case fatality, respectively. FINDINGS The proportion of PCV7 serotypes among all IPD cases (n=7678) significantly declined in adults from 44.7% (2003) before to 16.7% (2012) after the recommendation of PCV7 (P<0.001). In contrast, the proportion of non-PCV7 serogroup/serotypes increased for non-PCV13 but also PCV13 serotypes (not included in PCV7) at the same time. Serotype distribution varied significantly across ages, clinical manifestations and comorbidities. Serotype was furthermore associated with case fatality (P=0.001). In a multivariable logistic regression model, analyzing single serotypes showed that case-fatality was increased for the serotypes 3 (P=0.008), 19A (P=0.03) and 19F (P=0.005), compared to serotype 1 and 7F. CONCLUSION There was a significant decline in PCV7 serotypes among adults with IPD in Switzerland after introduction of childhood vaccination with PCV7. Pneumococcal serotypes were associated with case fatality, age, clinical manifestation and comorbidities of IPD in adults. These results may prove useful for future vaccine recommendations for adults in Switzerland.

Decrease in pneumococcal co-colonization following vaccination with the seven-valent pneumococcal conjugate vaccine.

Understanding the epidemiology of pneumococcal co-colonization is important for monitoring vaccine effectiveness and the occurrence of horizontal gene transfer between pneumococcal strains. In this study we aimed to evaluate the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal co-colonization among Portuguese children. Nasopharyngeal samples from children up to 6 years old yielding a pneumococcal culture were clustered into three groups: pre-vaccine era (n = 173), unvaccinated children of the vaccine era (n = 169), and fully vaccinated children (4 doses; n = 150). Co-colonization, serotype identification, and relative serotype abundance were detected by analysis of DNA of the total bacterial growth of the primary culture plate using the plyNCR-RFLP method and a molecular serotyping microarray-based strategy. The plyNCR-RFLP method detected an overall co-colonization rate of 20.1%. Microarray analysis confirmed the plyNCR-RFLP results. Vaccination status was the only factor found to be significantly associated with co-colonization: co-colonization rates were significantly lower (p = 0.004; Fisher's exact test) among fully vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or unvaccinated children of the PCV7 era (18.3%). In the PCV7 era there were significantly less non-vaccine type (NVT) co-colonization events than would be expected based on the NVT distribution observed in the pre-PCV7 era (p = 0.024). In conclusion, vaccination with PCV7 resulted in a lower co-colonization rate due to an asymmetric distribution between NVTs found in single and co-colonized samples. We propose that some NVTs prevalent in the PCV7 era are more competitive than others, hampering their co-existence in the same niche. This result may have important implications since a decrease in co-colonization events is expected to translate in decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution events such as acquisition of antibiotic resistance determinants or capsular switch. This might represent a novel potential benefit of conjugate vaccines.

Inhibition of matrix metalloproteinases attenuates brain damage in experimental meningococcal meningitis.

BackgroundApproximately 7% of survivors from meningococcal meningitis (MM) suffer from neurological sequelae due to brain damage in the course of meningitis. The present study focuses on the role of matrix metalloproteinases (MMPs) in a novel mouse model of MM-induced brain damage.MethodsThe model is based on intracisternal infection of BALB/c mice with a serogroup C Neisseria meningitidis strain. Mice were infected with meningococci and randomised for treatment with the MMP inhibitor batimastat (BB-94) or vehicle. Animal survival, brain injury and host-response biomarkers were assessed 48 h after meningococcal challenge.ResultsMice that received BB-94 presented significantly diminished MMP-9 levels (p¿<¿0.01), intracerebral bleeding (p¿<¿0.01), and blood-brain barrier (BBB) breakdown (p¿<¿0.05) in comparison with untreated animals. In mice suffering from MM, the amount of MMP-9 measured by zymography significantly correlated with both intracerebral haemorrhage (p¿<¿0.01) and BBB disruption (p¿<¿0.05).ConclusionsMMPs significantly contribute to brain damage associated with experimental MM. Inhibition of MMPs reduces intracranial complications in mice suffering from MM, representing a potential adjuvant strategy in MM post-infection sequelae.

The kynurenine pathway is involved in bacterial meningitis.

BackgroundBacterial meningitis (BM) is characterized by an intense host inflammatory reaction, which contributes to the development of brain damage and neuronal sequelae. Activation of the kynurenine (KYN) pathway (KP) has been reported in various neurological diseases as a consequence of inflammation. Previously, the KP was shown to be activated in animal models of BM, and the association of the SNP AADAT¿+¿401C/T (kynurenine aminotransferase II - KAT II) with the host immune response to BM has been described. The aim of this study was to investigate the involvement of the KP during BM in humans by assessing the concentrations of KYN metabolites in the cerebrospinal fluid (CSF) of BM patients and their relationship with the inflammatory response compared to aseptic meningitis (AM) and non-meningitis (NM) groups.MethodsThe concentrations of tryptophan (TRP), KYN, kynurenic acid (KYNA) and anthranilic acid (AA) were assessed by HPLC from CSF samples of patients hospitalized in the Giselda Trigueiro Hospital in Natal (Rio Grande do Norte, Brazil). The KYN/TRP ratio was used as an index of indoleamine 2,3-dioxygenase (IDO) activity, and cytokines were measured using a multiplex cytokine assay. The KYNA level was also analyzed in relation to AADAT¿+¿401C/T genotypes.ResultsIn CSF from patients with BM, elevated levels of KYN, KYNA, AA, IDO activity and cytokines were observed. The cytokines INF-¿ and IL-1Ra showed a positive correlation with IDO activity, and TNF-¿ and IL-10 were positively correlated with KYN and KYNA, respectively. Furthermore, the highest levels of KYNA were associated with the AADAT¿+¿401 C/T variant allele.ConclusionThis study suggests a downward modulatory effect of the KP on CSF inflammation during BM.

Bacterial meningitis: insights into pathogenesis and evaluation of new treatment options: a perspective from experimental studies.

ABSTRACT  Bacterial meningitis is associated with high mortality and morbidity rates. Bacterial components induce an overshooting inflammatory reaction, eventually leading to brain damage. Pathological correlates of neurofunctional deficits include cortical necrosis, damage of the inner ear and hippocampal apoptosis. The hippocampal dentate gyrus is important for memory acquisition and harbors a neuronal stem cell niche, thus being potentially well equipped for regeneration. Adjuvant therapies aimed at decreasing the inflammatory reaction, for example, dexamethasone, and those protecting the brain from injury have been evaluated in animal models of the disease. They include nonbacteriolytic antibiotics (e.g., daptomycin), metalloproteinase inhibitors and modulators of the immunological response, for example, granulocyte colony-stimulating factor. Increasing research interest has recently been focused on interventions aimed at supporting regenerative processes.

Genetic polymorphisms associated with the inflammatory response in bacterial meningitis.

BACKGROUND Bacterial meningitis (BM) is an infectious disease that results in high mortality and morbidity. Despite efficacious antibiotic therapy, neurological sequelae are often observed in patients after disease. Currently, the main challenge in BM treatment is to develop adjuvant therapies that reduce the occurrence of sequelae. In recent papers published by our group, we described the associations between the single nucleotide polymorphisms (SNPs) AADAT +401C > T, APEX1 Asn148Glu, OGG1 Ser326Cys and PARP1 Val762Ala and BM. In this study, we analyzed the associations between the SNPs TNF -308G > A, TNF -857C > T, IL-8 -251A > T and BM and investigated gene-gene interactions, including the SNPs that we published previously. METHODS The study was conducted with 54 BM patients and 110 healthy volunteers (as the control group). The genotypes were investigated via primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) or polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis. Allelic and genotypic frequencies were also associated with cytokine and chemokine levels, as measured with the x-MAP method, and cell counts. We analyzed gene-gene interactions among SNPs using the generalized multifactor dimensionality reduction (GMDR) method. RESULTS We did not find significant association between the SNPs TNF -857C > T and IL-8 -251A > T and the disease. However, a higher frequency of the variant allele TNF -308A was observed in the control group, associated with changes in cytokine levels compared to individuals with wild type genotypes, suggesting a possible protective role. In addition, combined inter-gene interaction analysis indicated a significant association between certain genotypes and BM, mainly involving the alleles APEX1 148Glu, IL8 -251 T and AADAT +401 T. These genotypic combinations were shown to affect cyto/chemokine levels and cell counts in CSF samples from BM patients. CONCLUSIONS In conclusion, this study revealed a significant association between genetic variability and altered inflammatory responses, involving important pathways that are activated during BM. This knowledge may be useful for a better understanding of BM pathogenesis and the development of new therapeutic approaches.

Leptomeningeal contrast enhancement and blood-CSF barrier dysfunction in aseptic meningitis

OBJECTIVE To investigate the blood-CSF barrier (BCSFB) dysfunction in aseptic meningitis. METHODS In our case series of 14 patients with acute aseptic meningitis, we compared MRI characteristics with CSF findings. RESULTS Contrast enhancement in the sulcal space in a leptomeningeal pattern was visualized in 7 patients with BCSFB dysfunction categorized as moderate to severe as evidenced by the CSF/serum albumin ratio (Qalb) but was not present in those with mild or no barrier disturbance (p = 0.001). The Qalb as a marker for the leakiness of the BCSFB and, more indirectly, of the blood-brain barrier (BBB) was positively correlated with the incidence of leptomeningeal contrast enhancement seen on postcontrast fluid-attenuated inversion recovery (FLAIR) MRI (p = 0.003). Patients with a more pronounced brain barrier dysfunction recovered more slowly and stayed longer in the hospital. CONCLUSIONS The severity of meningeal BBB disturbance can be estimated on postcontrast FLAIR MRI, which may be of diagnostic value in patients with aseptic meningitis.

Inhibition and deficiency of the immunoproteasome subunit LMP7 attenuates LCMV-induced meningitis

In addition to antigen processing, immunoproteasomes were recently shown to exert functions influencing cytokine production by monocytes and T cells, T-helper cell differentiation, and T-cell survival. Moreover, selective inhibition of the immunoproteasome subunit LMP7 ameliorated symptoms of autoimmune diseases including CD4(+) T-cell mediated EAE. In this study, we show that LMP7 also plays a crucial role in the pathogenesis of lymphocytic choriomeningitis virus (LCMV)-induced meningitis mediated by CTLs. Mice lacking functional LMP7 display delayed and reduced clinical signs of disease accompanied by a strongly decreased inflammatory infiltration into the brain. Interestingly, we found that selective inhibition and genetic deficiency of LMP7 affect the pathogenesis of LCMV-induced meningitis in a distinct manner. Our findings support the important role of LMP7 in inflammatory disorders and suggest immunoproteasome inhibition as a novel strategy against inflammation-induced neuropathology in the CNS.

Use of pneumococcal vaccine in people with chronic disease in United States

Objective. The risk of complications and deaths related to pneumococcal infections is high among high risk population (i.e. those with chronic diseases such as diabetes or asthma), despite current immunization recommendations. The aim of this study is to evaluate the use of pneumonia vaccine in adults with and without diabetes or asthma by year of age and whether immunization practices conform to policy recommendations. ^ Methods. Data were drawn from 2005 Behavioral Risk Factor Surveillance Study. Age specific estimated counts and proportions of pneumonia vaccination status were computed. The association of socio-demographic factors with vaccination status was estimated from multiple logistic regression and results were presented for adults (18-64yrs) and elderly (65 or older). ^ Results. Overall 12.3% of the adults and 61.5% of elderly reported ever received pneumonia vaccine. 66.8% of diabetics and 72.6% of asthmatics received the vaccine among elderly. 33.4% of diabetics and 21.6% of asthmatics received the vaccine among adults. These numbers are far away from Healthy people 2010 objective coverage rates of 90% for elderly and 60% for high risk adults. Though diabetes was one of the recommendations for the pneumonia vaccine still the status was less than 70% even at older ages. Although asthma was not an indication for pneumonia vaccine, asthmatics still achieved 50% level by an early age of 60 and reached up to 80% at as early as 75 years. In those having both asthma and diabetes, although the curve reaches to 50% level at a very early age of 40yrs, it is not stable until the age of 55 and percentages reached to as high as 90% in older ages. Odds of receiving pneumonia vaccine were high in individuals with diabetes or asthma in both the age groups. But the odds were stronger for diabetics in adults compared to those in the elderly [2.24 CI (2.08-2.42) and 1.32 CI (1.18-1.47)]. The odds were slightly higher in adults than in elderly for asthmatics [1.92 CI (1.80-2.04) and 1.73 CI (1.50-2.00)].The likelihood of vaccination also differed by gender, ethnicity, marital status, income category, having a health insurance, current employment, physician visit in last year, reporting of good to excellent health and flu vaccine status. ^ Conclusion. There is a very high proportion of high risk adults and elderly that remain unvaccinated. Given the proven efficacy and safety of vaccine there is a need for interventions targeting the barriers for under-vaccination with more emphasis on physician knowledge and practice as well as the recipient attitudes.^

Potential role of statins as immunomodulators in pneumococcal pneumonia

Background: Mortality in pneumococcal pneumonia remains as high as 20%, and most deaths occur within the first two weeks of hospitalization despite eradication of the causative organisms by antimicrobials in the first 24 hours. An inflammatory response rather than active infection could be responsible for this early mortality. Statins have been shown to have potent immunomodulatory activity in vitro. We investigated whether there was decreased severity or improved outcome in patients who were receiving statins at the time they were admitted for pneumococcal pneumonia. ^ Methods: Patients seen at the Michael E. DeBakey Veterans Affairs Medical Center in Houston, Texas from January, 2000 to June, 2010 with a diagnosis of pneumococcal pneumonia were included in this retrospective cohort study. Electronic medical records were reviewed to record demographic characteristics, comorbidities, laboratory values and statin use at the time of admission. Severity of pneumonia was determined using the Pneumonia Outcomes Research Team (PORT) classification. Uni- and multivariate Cox regression was used to evaluate survival. We adjusted for all variables in the multivariate model if they were significant in the univariate model at p<0.05. ^ Results: Of 347 patients admitted for pneumococcal pneumonia, 90 (25.9%) were taking statins at the time of presentation. Patients in the statin group were older (age: 68.0±9.7 vs. 62.5±12.3 years, p<0.001) and had higher prevalence of diabetes, coronary artery disease and kidney disease (p<0.05 for each comparison). Liver disease and alcohol consumption were less prevalent among statin users (p<0.05). The PORT scores were normally distributed in both groups with statin users having higher mean scores at admission as compared to patients not on statins (108±32 vs. 96±32, p = 0.002). The Cox proportional hazard analyses, adjusted for age, comorbidities, length of stay and PORT scores, showed a significantly reduced risk of mortality among statin users at 14 days (HR: 0.39; 0.15-0.98, p=0.045), 20 days (0.35; 0.14- 0.88, p=0.03) and 30 days(0.41; 0.17-0.95, p=0.01) after presentation. ^ Conclusion: Statin use is associated with improved clinical outcomes in patients with pneumococcal pneumonia.^

The incidence of necrotizing changes in adults with pneumococcal pneumonia

Background. Necrotizing pneumonia is generally considered a rare complication of pneumococcal pneumonia in adults. We systematically studied the incidence of necrotizing changes in adult patients with pneumococcal pneumonia, and examined the severity of infection, the role of causative serotype and the association with bacteremia. ^ Methods. We used a data base of all pneumococcal infections identified at our medical center between 2000 and 2010. Original readings of chest X-rays (CXR) and computerized tomography (CT) were noted. All images were then reread independently by 2 radiologists. The severity of disease was assessed using the SMART-COP scoring system. ^ Results. There were 351 cases of pneumococcal pneumonia. Necrosis was reported in no original CXR readings and 6 of 136 (4.4%) CTs. With re-reading, 8 of 351 (2.3%) CXR and 15 of 136 (11.0%) CT had necrotizing changes. Overall, these changes were found in 23 of 351 (6.6%, 95% CI 4.0 - 9.1) patients. The incidence of bacteremia and the admitting SMART-COP scores were similar in patients with and without necrosis (P=1.00 and P=0.32, respectively). Type 3 pneumococcus was more commonly isolated from patients with than from patients without necrotizing pneumonia (P=0.05), but a total of 10 serotypes were identified among 16 cases in which the organism was available for typing. ^ Conclusions. Necrotizing changes in the lungs were seen in 6.6% (95% CI 4.0 - 9.1) of a large series of adults with pneumococcal pneumonia. Patients with necrosis were not more likely to have bacteremia or more severe disease. Type 3 pneumococcus was commonly implicated, but 9 other serotypes were also identified.^