Background
Chapters 2 5 have contributed to our understanding of dynamics in commensal
nasopharyngeal pneumococcal carriage in healthy adults. Although the number of microbes administered in the experimental model may differ from natural exposure, its appropriateness to study the course and microbial consequences of experimental carriage was confirmed by its resemblance to naturally acquired carriage. Acquisition of pneumococcal carriage upon exposure could be seen as the resultant of a battle between adherence mechanisms by the pneumococcus and clearing mechanisms by the host and other microbes resident to the nasopharynx. If so, how do we know when this battle is settled? Our observation that pneumococcal density is highly variable during the first five weeks of a carriage episode suggests that the battle is ongoing during a major part of a carriage episode. Therefore, we should realize that single measurements may be subject to dips or peaks in carriage density that distort our interpretation of pneumococcal acquisition. The balance between sampling frequency and disturbance of natural homeostasis in the nasopharynx hampers the level of detail we can reach in studying pneumococcal carriage episodes. However, the nasal wash sampling method applied in the present model allowed for relatively frequent sample collection, compared to conventional nasopharyngeal swab technique.6 While in vitro culture of human samples is the gold standard for detection of pneumococcal carriage, one can argue whether this method is representative for dynamics in the nasopharyngeal niche. Recently it was described that genetically homogeneous bacterial populations, contain subgroups of members with different metabolic strategies.7 Therefore, a change in metabolic environment (i.e. from nasopharynx to culture medium) can result in outgrowth of a different portion of the population8,9, which could theoretically skew pneumococcal density assessment by culture. However, our observation that estimating pneumococcal density by culture highly correlates with qPCR, indicates that the relative distances between sample densities are not affected by this phenomenon when cultured on a rich medium.
CHAPTER 14
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In healthy adults, pneumococcal acquisition and carriage were associated with high microbial diversity and heterogeneity and little interaction between specific members. These results suggest that reduction of diversity in the nasopharyngeal microbiome would limit pneumococcal acquisition. However, in general such a diverse microbiome with low interaction strength is thought to be highly stable and little receptive to imbalances in health.10 Perhaps, for this adult population we should actually reconsider our thought of pneumococcal carriage as a detrimental phenomenon that should be eliminated. Whereas a 'poor' microbiome triggers inflammation, a diversified and balanced microbiome induces regulatory T‐cells and a broad IgA repertoire which fosters its diversified composition.6 As such, for their preservation in carriage (their existence) pneumococci would benefit from keeping their density low. And in fact, in adult natural carriers we observed that only a small fraction of the microbiome was occupied by Streptococcal reads. Pneumoccocci are indeed capable of assessing their own population density via quorum sensing and, in case of crowding, produce proteinaceous toxins that lyse their equals in the same niche.11 Whereas Gram positive pneumococci are insensitive to lysis via the membrane attack complex (MAC), they do secrete peptides inhibiting MAC‐formation12 possibly enhancing the presence of other bacteria in their nasopharyngeal niche. Furthermore, in mice it was observed that pneumococci elicit TGF‐β1 production by nasopharyngeal epithelial cells, thereby inducing regulatory T cells which facilitate prolonged, yet contained pneumococcal carriage. While inhibition of TGF‐β1 led to fierce inflammation and rapid clearance of carriage, it was accompanied by tissue damage and spread of pneumococci to the lungs.13 This low abundant pneumococcal carriage may actually be beneficial to the host as it has been demonstrated to elicit a systemic immune response important for protection from invasive disease, and should therefore be intermittently tolerated.14 Consequently, instead of eliminating pneumococcal carriage, it may be more appropriate to control the density of pneumococcal carriage. The interest for pneumococcal carriage density as a determinant of pneumococcal disease is supported by a study in South African HIV positive patients with CAP. The nasopharyngeal pneumococcal density was higher in pneumococcal CAP compared with CAP caused by other pathogens and compared with asymptomatic carriers, and was positively associated with the severity of pneumococcal CAP.15‐17 The basis of this relationship between carriage density and disease is yet unclear. It may be purely distributional, or otherwise might rely on low carriage density limiting the odds for natural selection of (to men unfavorable) pneumococcal variants.18 While in adults high density pneumococcal carriage is associated with disease, tolerance of low density pneumococcal carriage on the other hand seems to be protective. Therefore, while containing the density of pneumococcal carriage, its elimination should be avoided.
For future pneumococcal carriage studies we recommend the use of qPCR as it is more sensitive than culture and molecular determination of the serotype and antibiotic susceptibility are within reach. If the present viability of (not just exposure to) the pneumococcus is crucial to the research question considered, it is recommended to complement qPCR with culture. In the experimental human model pneumococcal acquisition
SUMMARIZING DISCUSSION
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was determined by the capacity of the pneumococcus to remain viable in the nasopharynx for at least 48 hours after inoculation, avoiding the risk of early wash out of the inoculated bacteria. Recently, sampling of the oropharynx and saliva was demonstrated to be more sensitive than nasopharyngeal sampling for detection of pneumococcal carriage in adolescents and elderly.19,20 If these alternative sampling methods would also provide reliable data on pneumococcal concentration in adults, they could allow for more frequent mapping of pneumococcal density over time. Longitudinal sampling revealed that in some volunteers carriage of a specific strain can be ongoing for many months, yet rapidly cleared in others. Should these carriage episodes be considered to be equally successful, or do density and duration affect the likelihood of pneumococcal spread or invasion? Whereas carriage is a known prerequisite for disease, we now developed accurate tools to study which aspects of pneumococcal carriage and the nasopharyngeal microbiome influence the risk of pneumococcal transmission and disease, and how these variables can be modified by interventions like vaccination.