Dynamics of nasopharyngeal microbial flora; the key to respiratory and invasive disease?

BACKGROUND The nasopharyngeal (NP) microbial flora consists of a wide range of ‘neutral’ bacteria like viridans Streptococci together with potential pathogens like Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus. During health, the microflora seems to represent a balanced ecosystem, preventing overgrowth of single species and subsequent respiratory or invasive disease. During disease, common NP colonizers can become pathogens, although the precise mechanism (dysbalance, overgrowth, viral effect) remains unknown. For example, S. pneumoniae is a major pathogen in both respiratory and invasive disease and therefore currently a target for new vaccination strategies. Target groups for these vaccines are both ends of the age-spectrum, i.e. young children and elderly, who both have high incidence rates of pneumococcal disease. To prevent pneumococcal diseases in children, a 7-valent pneumococcal conjugate vaccine (PCV-7) is currently licensed and implemented in national immunization programs of many countries. Interestingly, however, after implementation of this vaccine a shift in NP colonization and infections from vaccine-type pneumococci towards non-vaccine serotypes was observed in the entire population. Furthermore, other NP species like H. influenzae and S. aureus became more frequently involved in respiratory diseases after implementation of PCV-7. These data suggest that by selective vaccination against certain colonizers, the composition of the NP flora changes, resulting in a subsequent shift in colonizing pathogens involved in infectious diseases. It is therefore clear that there is a strong need for more fundamental knowledge on the detailed composition of the nasopharynx in general, in relation to age, health, disease, and vaccination. RESEARCH PROPOSAL We have formulated several important research questions that altogether could improve the understanding of the complex pathways by which bacterial colonization will lead to disease. A. what is the composition of NP microflora in young children, in healthy adults and in elderly > 65 years of age? Moreover, what is the composition of the NP flora during (pneumococcal) CAP or IPD disease in elderly and how do these profiles relate to health? B. What is the influence of viral presence on the composition of NP microflora in infants and elderly? C. Are invasive pneumococcal strains genetically a direct reflection of colonizing strains in children and elderly ? D. What is the effect of pneumococcal conjugate vaccination on the composition of and dynamics within the NP microflora in infants and elderly in general? What effect does conjugate vaccination have on genotype and serotypes of invasive strains in both age groups? METHODS To study these questions, we have access to a large set of unique NP materials from healthy children 6 weeks to 2 years of age (n=1000), adults (parents) and healthy elderly (n=2000) obtained during placebo-controlled pneumococcal conjugate immunization trials. Furthermore, we have access to invasive pneumococcal strains from children and elderly from the collection of the Netherlands Reference laboratory for Bacterial Meningitis from the pre- and post-PCV-7 implementation era. Lastly, we will obtain NP swabs and blood and sputum cultures from community acquired pneumonia or IPD patients that are vaccinated either with 13-valent pneumococcal conjugate vaccine or placebo-vaccine in the period November 2008-November 2010. To address the first and fourth research question we plan on studying dynamics within NP microflora with and without pneumococcal conjugate vaccination in healthy children, adults and elderly. In addition, samples of elderly presenting with CAP or IPD will be studied. State-of-the-art molecular techniques like massive parallel pyrosequencing and taxonomic micro-array analyses will be applied. To study the effects of viral ‘colonization’ on the dynamics within these NP microflora we will use real-time PCR methods for the presence of a broad spectrum of respiratory viruses in nasopharyngeal samples obtained during the above mentioned trials. To study the third question we will characterize the genotypes of carriage and invasive pneumococcal isolates in children and elderly by means of multi-locus-sequence-typing (MLST). In elderly, strains causing pneumonia will be studied as well. RATIONALE We are confident that worldwide we are the only research group with access to such large numbers of unique and well-defined materials (NP samples and disease-causing isolates from individuals with and without previous pneumococcal vaccinations and during health and disease). In combination with the state-of-the-art techniques proposed, we believe we have the ultimate opportunity to obtain unique data for the understanding of the NP ecology and pathophysiology of disease by NP colonizing species, which will ultimately support decision-making around future preventive strategies.