Peter W. M. Hermans

Streptococcus pneumoniae colonisation: the key to pneumococcal disease

Streptococcus pneumoniae is an important pathogen causing invasive diseases such as sepsis, meningitis, and pneumonia. The burden of disease is highest in the youngest and oldest sections of the population in both more and less developed countries. The treatment of pneumococcal infections is complicated by the worldwide emergence in pneumococci of resistance to penicillin and other antibiotics. Pneumococcal disease is preceded by asymptomatic colonisation, which is especially high in children. The current seven-valent conjugate vaccine is highly effective against invasive disease caused by the vaccine-type strains. However, vaccine coverage is limited, and replacement by non-vaccine serotypes resulting in disease is a serious threat for the near future. Therefore, the search for new vaccine candidates that elicit protection against a broader range of pneumococcal strains is important. Several surface-associated protein vaccines are currently under investigation. Another important issue is whether the aim should be to prevent pneumococcal disease by eradication of nasopharyngeal colonisation, or to prevent bacterial invasion leaving colonisation relatively unaffected and hence preventing the occurrence of replacement colonisation and disease. To illustrate the importance of pneumococcal colonisation in relation to pneumococcal disease and prevention of disease, we discuss the mechanism and epidemiology of colonisation, the complexity of relations within and between species, and the consequences of the different preventive strategies for pneumococcal colonisation.

Colonisation by Streptococcus pneumoniae and Staphylococcus aureus in healthy children

A trial with a 7-valent pneumococcal-conjugate vaccine in children with recurrent acute otitis media showed a shift in pneumococcal colonisation towards non-vaccine serotypes and an increase in Staphylococcus aureus-related acute otitis media after vaccination. We investigated prevalence and determinants of nasopharyngeal carriage of Streptococcus pneumoniae and S aureus in 3198 healthy children aged 1-19 years. Nasopharyngeal carriage of S pneumoniae was detected in 598 (19%) children, and was affected by age (peak incidence at 3 years) and day-care attendance (odds ratio [OR] 2.14, 95% CI 1.44-3.18). S aureus carriage was affected by age (peak incidence at 10 years) and male sex (OR 1.46, 1.25-1.70). Serotyping showed 42% vaccine type pneumococci. We noted a negative correlation for co-colonisation of S aureus and vaccine-type pneumococci (OR 0.68, 0.48-0.94), but not for S aureus and non-vaccine serotypes. These findings suggest a natural competition between colonisation with vaccine-type pneumococci and S aureus, which might explain the increase in S aureus-related otitis media after vaccination.

4G/5G promoter polymorphism in the plasminogen-activator-inhibitor-1 gene and outcome of meningococcal disease

BACKGROUND Intravascular coagulation with infarction of skin, digits, and limbs is a characteristic feature of meningococcal sepsis. Children with meningococcal sepsis have higher than normal concentrations of plasminogen activator inhibitor 1 (PAI-1) in plasma. Combined with the widespread venous thrombosis, this finding suggests an impairment of fibrinolysis. A common functional insertion/deletion (4G/5G) polymorphism exists in the promoter region of the PAI-1 gene. We tested the hypothesis that children with the 4G/4G genotype produce higher concentrations of PAI-1, develop more severe coagulopathy, and are at greater risk of death during meningococcal sepsis. METHODS The relation between meningococcal disease outcome, PAI-1 concentration, and PAI-1 genotype was investigated in 175 children with meningococcal disease (37 from Rotterdam, the Netherlands, and 138 from London, UK) and 226 controls (137 from Rotterdam, 89 from London). PAI-1 concentrations in plasma were measured by ELISA, and the 4G/5G PAI-1 polymorphism was detected by PCR and hybridisation. FINDINGS Concentrations of PAI-1 on admission correlated with presentation (sepsis or meningitis) and outcome. The median PAI-1 concentration in children who died was substantially higher than that in survivors (2448 [IQR 1115-3191] vs 370 [146-914] ng/mL; p<0.0001). Patients with the 4G/4G genotype had significantly higher PAI-1 concentrations than those with the 4G/5G or 5G/5G genotype (1051 [550-2440] vs 436 [198-1225] ng/mL; p=0.03), and had an increased risk of death (relative risk 2.0 [1.0-3.8] for the two cohorts combined, and 4.8 [1.8-13] for the London cohort). INTERPRETATION A genetic predisposition to produce high concentrations of PAI-1 is associated with poor outcome of meningococcal sepsis. This finding suggests that impaired fibrinolysis is an important factor in the pathophysiology of meningococcal sepsis.

Effect of conjugate pneumococcal vaccine followed by polysaccharide pneumococcal vaccine on recurrent acute otitis media: a randomised study.

BACKGROUND Pneumococcal conjugate vaccine prevents recurrent acute otitis media (AOM) in infants immunised at 2, 4, 6, and 12-15 months of age. We aimed to find out whether this vaccine also prevents AOM in older children who have had previous episodes of AOM. METHODS In this double-blind, randomised study, we enrolled 383 patients aged 1-7 years who had had two or more episodes of AOM in the year before entry. Randomisation was stratified in four groups according to age (12-24 months vs 25-84 months) and the number of previous AOM episodes (two or three episodes vs four or more episodes). Children received either 7-valent pneumococcal conjugate vaccine followed by 23-valent pneumococcal polysaccharide vaccine, or hepatitis A or B vaccines. They were followed up for 18 months for recurrence of AOM. We also cultured samples of middle-ear fluid and nasopharyngeal swabs to assess association of pneumococcal serotypes with AOM after vaccination. FINDINGS We noted no reduction of AOM episodes in the pneumococcal vaccine group compared with controls (intention-to-treat analysis: rate ratio 1.25, 95% CI 0.99-1.57). Although nasopharyngeal carriage of pneumococci of serotypes included in the conjugate-vaccine was greatly reduced after pneumococcal vaccinations, immediate and complete replacement by non-vaccine pneumococcal serotypes took place. INTERPRETATION These data do not lend support to the use of pneumococcal conjugate vaccine to prevent otitis media in previously unvaccinated toddlers and children with a history of recurrent AOM.

Inhibitory and Bactericidal Effects of Hydrogen Peroxide Production by Streptococcus pneumoniae on Other Inhabitants of the Upper Respiratory Tract

ABSTRACT An inverse correlation between colonization of the human nasopharynx by Streptococcus pneumoniae andHaemophilus influenzae, both common upper respiratory pathogens, has been reported. Studies were undertaken to determine if either of these organisms produces substances which inhibit growth of the other. Culture supernatants from S. pneumoniaeinhibited growth of H. influenzae, whereas culture supernatants from H. influenzae had no effect on the growth of S. pneumoniae. Moreover, coculture of S. pneumoniae and H. influenzae led to a rapid decrease in viable counts of H. influenzae. The addition of purified catalase prevented killing of H. influenzae in coculture experiments, suggesting that hydrogen peroxide may be responsible for this bactericidal activity. H. influenzae was killed by concentrations of hydrogen peroxide similar to that produced byS. pneumoniae. Hydrogen peroxide is produced by the pneumococcus through the action of pyruvate oxidase (SpxB) under conditions of aerobic growth. Both an spxB mutant and a naturally occurring variant of S. pneumoniae, which is downregulated in SpxB expression, were unable to kill H. influenzae. A catalase-reversible inhibitory effect of S. pneumoniae on the growth of the respiratory tract pathogens Moraxella catarrhalis andNeisseria meningitidis was also observed. Elevated hydrogen peroxide production, therefore, may be a means by which S. pneumoniae is able to inhibit a variety of competing organisms in the aerobic environment of the upper respiratory tract.

Genome-wide association study identifies variants in the CFH region associated with host susceptibility to meningococcal disease

Meningococcal disease is an infection caused by Neisseria meningitidis. Genetic factors contribute to host susceptibility and progression to disease, but the genes responsible for disease development are largely unknown. We report here a genome-wide association study for host susceptibility to meningococcal disease using 475 individuals with meningococcal disease (cases) and 4,703 population controls from the UK. We performed, in Western European and South European cohorts (consisting of 968 cases and 1,376 controls), two replication studies for the most significant SNPs. A cluster of complement factor SNPs replicated independently in both cohorts, including SNPs within complement factor H (CFH) (rs1065489 (p.936D<E), P = 2.2 × 10−11) and in CFH-related protein 3 (CFHR3)(rs426736, P = 4.6 × 10−13). N. meningitidis is known to evade complement-mediated killing by the binding of host CFH to the meningococcal factor H–binding protein (fHbp). Our study suggests that host genetic variation in these regulators of complement activation plays a role in determining the occurrence of invasive disease versus asymptomatic colonization by this pathogen.

Host genetic determinants of Neisseria meningitidis infections

The clinical presentation of infections caused by Neisseria meningitidis is highly diverse. Some patients develop meningitis, and others present with sepsis or even septic shock. After invasion of the bloodstream by the bacteria, three main cascade pathways are activated. These are the complement system, the inflammatory response, and the coagulation and fibrinolysis pathway. These pathways do not act independently but are able to interact with each other. Genetic polymorphisms among components of these pathways have been shown to be involved in the susceptibility, severity, and outcome of meningococcal disease. We review knowledge of genetic variations associated with susceptibility to and severity of meningococcal infection. Complement deficiencies and defects in sensing or opsonophagocytic pathways, such as the rare Toll-like receptor 4 single nucleotide polymorphisms (SNPs) and combinations of inefficient variants of Fcgamma-receptors, seem to have the most important role in genetically established susceptibility. Effect on severity has repeatedly been reported for FcgammaRIIa and plasminogen activator inhibitor type 1 (PAI1) polymorphisms. Outcome effects have been confirmed for SNPs in properdin deficiencies, PAI1 and combination of the -511C/T SNP in interleukin 1beta, and the +2018C/T SNP in interleukin RN. Conflicting results are reported for the effect of the -308G/A promoter polymorphism in tumour necrosis factor (TNF) alpha. These differences may reflect discrepancies in group definitions between studies or the influence of additional SNPs in the TNFalpha promoter, which can form haplotypes representing different cytokine production capacity. For several SNPs, the potential effect on susceptibility, severity, or outcome has not yet been confirmed in an independent study.

The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms

The Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10) on the surface of lung cells through amino acids 273-341 located in the Basic Region (BR) domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (r)BR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122-166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs) of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae) may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.

Plasminogen-activator-inhibitor-1 4G/5G promoter polymorphism and prognosis of severely injured patients

A single base pair insertion/deletion (4G/SG) promoter polymorphism in the plasminogen-activator-inhibitor-1 (PAI-1) gene is thought to play a part in prognosis after severe trauma. We investigated the relation between outcome of severe trauma, PAI-1 concentrations, and PAP-1 genotype in 61 patients who had been severely injured. 11 (58%) of 19 patients with genotype 4G/4G did not survive, whereas only eight (28%) of 29 patients with heterozygous genotype 4G/SG, and two (15%) of 13 patients with genotype 5G/5G died. The PAI-1 4G allele is associated with high concentrations of PAI-1 in plasma and a poor survival rate after severe trauma.

CodY of Streptococcus pneumoniae: Link between Nutritional Gene Regulation and Colonization

CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.