- Background: Co-colonization by Streptococcus pneumoniae and Haemophilus influenzae among children has been noted in numerous studies, as has an inverse relationship involving colonization with these species and Staphylococcus aureus. Interactions among these pathogens could mediate unanticipated outcomes of clinical interventions, including changes in H. influenzae and S. aureus disease incidence following pneumococcal vaccine introduction. However, it remains unclear whether co-colonization patterns represent true interspecies interactions or owe to confounding factors. Methods: We investigated polymicrobial carriage using longitudinal data from 369 Bedouin children and 400 Jewish children in Israel enrolled in a 7-valent pneumococcal conjugate vaccine (PCV7) trial. Children were swabbed ten times between two and 30 months of age. Results: The pathogens followed distinct age and seasonal distributions, however polymicrobial carriage associations persisted after controlling for these and other confounding factors. Receipt of PCV7 resulted in pneumococcal serotype replacement but did not impact total carriage of S. pneumoniae, H. influenzae, or S. aureus. Conclusions: The fact that S. pneumoniae, H. influenzae, and S. aureus polymicrobial carriage patterns do not owe to confounding by age and season supports the idea of active interspecies interactions. However, pneumococcal serotype replacement may prevent changes in H. influenzae and S. aureus carriage among PCV7 recipients.