influenzae population (Figure 6A). Figure 6 Neutrophil infiltration: comparison of strains and species at 48 hours and dynamics over 96 hours. A) Neutrophils in the nasal epithelium from rats inoculated 48 hours earlier CB-839 order with 104 cfu of bacteria from a single species (Rm154, TIGR4 and Poland(6b)-20) or from rats inoculated 96 hours earlier with 106 cfu of H. influenzae and 48 hours earlier with 104 cfu of Poland(6b)-20

were quantified using the MPO assay. Lines indicate median MPO values. P-value is calculated by the Wilcoxon rank sum test. B) Dynamics of neutrophil infiltration in response to nasal colonization by S. pneumoniae (TIGR4) or H. influenzae. Following inoculation groups of 5-8 rats were sacrificed and neutrophil infiltration was measured by MPO assay. Median MPO Units are plotted. Error bars represent SE. Dashed line represents median MPO of uninoculated rats. No difference in neutrophil infiltration is observed Screening Library between rats colonized by the two different S. pneumoniae strains (TIGR4 and Poland(6b)-20). The neutrophil infiltration observed 48 hours after Poland(6b)-20 STA-9090 chemical structure invaded on an established H. influenzae population (when immune-mediated competition was observed in the nasal wash)

was significantly higher than rats with just Poland(6b)-20 colonizing alone. However, neutrophil infiltration was not significantly higher than in rats with only H. influenzae. While these results suggest that H. influenzae is primarily responsible for the neutrophil infiltration that reduces the nasal lumen populations of some strains of S. pneumoniae, S. pneumoniae may still have a role in eliciting the immune response (perhaps with slower dynamics than H. influenzae). We observed that the neutrophil infiltration in response to S. pneumoniae colonizing alone increases from 48-96 hours after inoculation, compared to the constant

neutrophil presence with H. influenzae (Figure 6B). Discussion Population Dynamics All three species that we studied (S. aureus, S. pneumoniae and Adenosine H. influenzae) can colonize the nasal passages of neonatal rats and each reaches a bacterial load that is independent of the initial inoculum size; they increase in density when initially below this level and decline when initially above it. This indicates that the steady-state density is tightly controlled – perhaps by a limiting resource or the host’s immune response. The total density of each of these colonizing species is relatively low and there is wide-spread variation in the densities of individuals, similar to what has been observed in colonized humans [27].