The three most common bacterial species found colonizing the nasopharynx of children in the study were Streptococcus pneumoniae (31%), Haemophilus influenzae (19%), and alpha-hemolytic streptococci (6%). Over 20% of samples showed co-colonization, most often S. pneumoniae and H. influenzae. Of S. pneumoniae-positive samples, 25% were co-colonized with H. influenzae, and 43% of H. influenzae-positive samples were co-colonized with S. pneumoniae, demonstrating significant co-occurrence of these species.

1. Introduction Polysaccharide conjugate vaccines against the respiratory pathogens: Streptococcus pneumoniae , Haemophilus influenzae serogroup B and Neisseria meningitidis serogroup C, have successfully reduced childhood morbidity and mortality (Finn, 2004). However, targeting only specific subgroups may result in changes in species and serotype prevalence in carriage and disease (Pletz et al. , 2008). Such changes are also observed in the absence of vaccine pressure (Jefferies et al. , 2010). Bacterial populations, interact both positively and negatively within a niche, these interactions can determine the presence and persistence of particular species (Pettigrew et al. , 2008). These niches are therefore sensitive to any changes within the respiratory bacterial community. We analysed co-colonisation by common bacterial upper respiratory tract pathogens in the nasopharynx of children as part of a longitudinal carriage study. Methods Nasopharyngeal swabs were collected from 328 children ≤4 years old, attending the paediatric outpatient department of a UK hospital during winter 2008-2009. The swabs were plated onto both chocolate blood agar and blood agar plates on the same day the sample was taken. Conventional culture techniques were then used to screen for and positively identify isolates of S. pneumoniae , other α haemolytic streptococci (α-HS), H. influenzae , Staphylococcus aureus and N. meningitidis . References Finn, A. (2004). Bacterial polysaccharide-protein conjugate vaccines. Br Med Bull 70, 1-14. Jefferies, J. M., Smith, A. J., Edwards, G. F. S., McMenamin, J., Mitchell, T. J. & Clarke, S. C. (2010). Temporal analysis of invasive pneumococcal clones from Scotland illustrates fluctuations in diversity of serotype and genotype in the absence of pneumococcal conjugate vaccine. J Clin Microbiol 48, 87-96. Pettigrew, M. M., Gent, J. F., Revai, K., Patel, J. A. & Chonmaitree, T. (2008). Microbial interactions during upper respiratory tract infections. Emerg Infect Dis 14, 1584-1591. Pletz, M. W., Maus, U., Krug, N., Welte, T. & Lode, H. (2008). Pneumococcal vaccines: mechanism of action, impact on epidemiology and adaption of the species. International Journal of Antimicrobial Agents 32, 199-206. Results The two most commonly carried bacteria were S. pneumoniae , 31% carriage (95% CI, 26-36%), and H. influenzae , 19% carriage (95% CI, 14-23%) (figure 1). α-HS and S. aureus were isolated from 6% and 4% of swabs respectively (figure 1), all S. aureus strains that were isolated were found to be meticillin sensitive. N. meningitidis was not isolated from any sample. More than one species was isolated from 22% of swabs, predominantly due to co-colonisation of S. pneumoniae and H. Influenzae . 2 species were isolated from the same swab on 32 occasions, while co-colonisation by 3 species occurred only 3 times. Of the 102 swabs from which S. pneumoniae was identified, 25% (95% CI, 17-34%) were co-colonized with H. influenzae . Of the 61 swabs from which H. influenzae was isolated 43% (95% CI, 30-55%) were co-colonised with S. pneumoniae (figure 2). Polymicrobial colonisation of the nasopharynx in UK children R.A. Gladstone 1 , A.S. Tocheva 1 , G. Afimeke 1, 2 , J. Garland 2 , J.M. Jefferies 1, 3 , S.N. Faust 1,2 , S.C. Clarke 1, 3 1. Division of Infection, Inflammation and Immunity, University of Southampton School of Medicine, UK. 2. Wellcome Trust Clinical Research Facility, University of Southampton, Southampton, UK. 3. Health Protection Agency, Southampton, UK [email_address] Conclusions We have demonstrated the presence of co-colonising bacteria in the nasopharynx of young children. We continue to monitor molecular epidemiological fluctuations over time in a longitudinal study. Bacterial populations in this niche are likely to be dynamic following widespread conjugate vaccine use. Replacement of colonising vaccine types by non-vaccine types and species may impact on invasive disease. Further studies to characterise the relationships between co-colonising bacteria are being undertaken. Haemophilus influenzae (Hi) Staphylococcus aureus (Sa) Streptococcus pneumoniae (Spn) Alpha haemolytic streptococci ( α -HS) 11 72 23 23 9 35 35 77 5 1 1 1 2 1 96 13 10 1 1 Spn (n=102) Spn (n=102) Spn (n=102) Hi (n=61) Hi (n=61) Sa (n=12) α -HS (n=19) α -HS (n=19) Sa (n=12) 5 Figure 2. Bacterial Co-colonisation of the nasopharynx Figure 1. Number of each organism isolated 13 36