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1 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
2 Department of Pediatrics, Erasmus MC, University Medical Center Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands
3 Department of Microbial Genomics, Keygene NV, Agro Business Park 90, 6708 PW Wageningen, The Netherlands
4 Department of Bioinformatics, Erasmus MC, University Medical Center Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands
5 PathoFinder BV, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands
6 Laboratory of Pediatric Infectious Diseases, University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands
Correspondence
Damian C. Melles
d.melles{at}erasmusmc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Steinberg et al., 1996). The anterior nares are the primary ecological reservoir of Staph. aureus in humans (Moss et al., 1948), and it has been determined that most infections result from endogenous nasal carriage (Luzar et al., 1990; Nguyen et al., 1999; von Eiff et al., 2001; Wertheim et al., 2005; Yu et al., 1986). The molecular basis of nasal colonization with Staph. aureus is not properly understood, but environmental factors (Bogaert et al., 2004; Peacock et al., 2003) as well as host factors (Cole et al., 1999, 2001) play an important role (Peacock et al., 2001; Wertheim et al., 2005). Age, sex, fasting glucose levels, diabetes mellitus and smoking were recently demonstrated to be independent determinants of Staph. aureus nasal carriage in adults (Nouwen et al., 2004). Furthermore, two recent large-scale population studies revealed an inverse relationship between nasopharyngeal Staph. aureus colonization and vaccine-type strains of Streptococcus pneumoniae (Bogaert et al., 2004; Regev-Yochay et al., 2004), suggesting natural competition between these bacterial species in the nasopharyngeal niche. This could not be confirmed in HIV-infected children (McNally et al., 2006). A recent trial with the 7-valent pneumococcal-conjugate vaccine (PCV7) in children with recurrent acute otitis media (AOM) showed a shift in pneumococcal colonization towards non-vaccine serotypes and an increase in Staph. aureus-related AOM after vaccination (Veenhoven et al., 2003). A similar trend was recently observed for bacteraemia in young children (Herz et al., 2006). Hence, bacterial co-colonization is considered to have important clinical implications for the widespread use of PCV7 in young children. It has become obvious that the nasopharyngeal microbial ecology is complex, and microbial inter-species interactions, as well as host–pathogen interactions, may define whether or not potential infectious pathogens can persist locally. We aimed to determine whether the capacity of Staph. aureus to compete with Strep. pneumoniae is dependent on bacterial genotype, the main question being whether certain successful staphylococcal clones are better equipped to compete with pneumococci. We also studied demographic and bacteriological determinants of carriage of specific genotypes of Staph. aureus in children.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Informed consent was obtained from the child or the accompanying parent. A single nasopharyngeal sample was obtained per child with rayon-tipped pernasal swabs (Copan, Brescia, Italy). Swabs were transported in Amies medium and plated within 6 h on gentamicin blood agar and Thayer–Martin medium, and finally submerged in phenyl mannitol broth for selective isolation of Strep. pneumoniae, Neisseria meningitidis and Staph. aureus, respectively. Bacterial identification was performed according to standard procedures (Bannerman, 2003). Staph. aureus and Strep. pneumoniae strains were isolated from 1117 and 598 children, respectively. In this study cohort a negative correlation for co-colonization of Staph. aureus and vaccine-type pneumococci (odds ratio 0.68, condfidence interval 0.48–0.94) was identified (Bogaert et al., 2004). All isolates were stored at –80 °C in broth containing glycerol. A random sample of 400 Staph. aureus isolates was drawn for the present study.
Demographics.
Demographic information was obtained from each child through a standardized questionnaire which was completed under supervision of an instructed interviewer. Questions addressed gender, date of birth, zip code, and active and passive smoking habits. In our analysis we grouped zip codes into four categories (north, south, centre and suburbs of Rotterdam).
DNA isolation and bacterial genotyping.
The 400 Staph. aureus isolates were grown on Columbia III agar (Becton Dickinson) supplemented with 5 % sheep blood. Three to five colonies were suspended in 25 mM Tris, 10 mM EDTA, 50 mM glucose containing lysostaphin (50 µg ml–1) and incubated at 37 °C for 1 h. DNA was extracted with the MagNA Pure LC DNA Isolation Kit III using the MagNA Pure LC robot (Roche Diagnostics) and stored at –20 °C. High-throughput amplified fragment length polymorphism (ht-AFLP) analysis was performed as described by Melles et al. (2004). AFLP is a whole-genome typing method that scans for polymorphism in actual restriction sites but also among the nucleotides bordering these sites. As such it documents nucleotide sequence variation, insertions and deletions across genomes. Briefly, using the predictive software package Recomb, the optimal enzyme and primer combinations were selected. Bacterial DNA was digested with the enzymes MboI and Csp6I, and the linker oligonucleotide pairs for MboI (5'-CTCGTAGACTGCGTACC-3' and 5'-GATCGGTACGCAGTCTAC-3') and for Csp6I (5'-GACGATGAGTCCTGAC-3' and 5'-TAGTCAGGACTCAT-3') were ligated. Subsequently, a non-selective pre-amplification was performed using the MboI primer (5'-GTAGACTGCGTACCGATC-3') and Csp6I primer (5'-GACGATGAGTCCTGACTAC-3'). In the final amplification, a 33P-labelled MboI primer containing one selective nucleotide (either +C or +G) and a Csp6I primer containing two selective nucleotides (+TA) were used. Amplified material was analysed using standard polyacrylamide slab gels and subsequent autoradiography. Marker fragments were scored, and a binary table, scoring marker fragment absence (0) or presence (1), was compiled.
Pneumococcal DNA was extracted (from 578 of the 598 pneumococcal isolates) and analysed by restriction fragment end labelling (RFEL) as described before (van Steenbergen et al., 1995; Bogaert et al., 2006). Briefly, purified pneumococcal DNA was digested by the restriction enzyme EcoRI. The DNA restriction fragments were end-labelled at 72 °C with [
-32P]dATP using DNA polymerase (Goldstar; Eurogentec). After the radiolabelled fragments had been denatured and separated electrophoretically on a 6 % polyacrylamide sequencing gel containing 8 M urea, the gel was transferred onto filter paper, vacuum dried (HBI, Saddlebrook, NY, USA), and exposed for variable times at room temperature to ECL hyperfilm (Amersham Laboratories).
Multilocus sequence typing (MLST).
MLST was carried out for a selection of 45 of the Staph. aureus strains using DNA arrays (van Leeuwen et al., 2003). The selected strains were equally distributed across an AFLP dendrogram by selecting approximately 1 out of 10 carriage strains isolated from healthy children, going from top to bottom through the AFLP dendrogram (Melles et al., 2004).
Data analysis.
Analysis of the AFLP data was performed as described by Melles et al. (2004). The method used for 2D clustering of the AFLP data was agglomerative (successive) hierarchical. This was performed using the unweighted pair group method with arithmetic means (UPGMA). The similarity metric used was Tanimoto, which defines similarity for binary data (0 and 1) based on the number of positive attributes that two records have in common. The resulting dendrogram was ordered by mean value.
Principal component analysis (PCA) is a standard multivariate method used to reduce the dimensional space of the data to its principal components (PCs). PCA aims to reduce a large number of variables to a smaller set that explain most of the variation in the data. It is, basically, a rotation of axes after centering data to the means of the variables, the rotated axes being the PCs, which are linear combinations of the original variables. The PC computation is displayed as a 3D scatter plot in which the position along the axes shows the PCA score of the strain. The distribution of the strains in the four phylogenetic branches was defined on the basis of PCA. Hierarchical and PCA cluster analysis was performed using Spotfire DecisionSite 7.2 software (http://www.spotfire.com).
To compare the distribution of strain categories in different genetic clusters, chi-squared analysis was used. Logistic regression analysis was used to adjust for possible confounding factors. A two-sided P-value of 0.05 or less was considered significant.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Melles et al., 2004). This earlier study also revealed that the AFLP-clustering matched strongly with the major clonal complexes as defined by MLST (Melles et al., 2004). Moreover, computer algorithms used to solve the Staph. aureus population structure based on MLST data (eBURST) generated clustering identical to the AFLP Spotfire analyses. In the present study, using the subset of 400 Staph. aureus carriage strains obtained from children, a total of 147 different genetic markers per strain were generated, covering 58 800 AFLP fragments (Fig. 1). The AFLP data revealed two distinct, homogeneous clusters (II and III) and several other smaller subclusters within the two main ht-AFLP clusters I and IV (Fig. 1, Fig. 2a). MLST analysis of the Staph. aureus isolates identified different clonal complexes in AFLP group I (including CC5, CC7, CC8, CC15, CC20 and CC25), indicating its heterogeneity. In contrast, AFLP-clusters II and III harbour single clonal complexes, CC30 and CC45, respectively. The fact that these two clonal complexes account for 47.3 % of all carriage isolates in our study population suggests that they have evolved to be very successful in colonizing humans (Melles et al., 2004). Clusters IVa and IVb are associated with CC22 and CC121, respectively (see Fig. 1).
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summarizes the main characteristics of the study population. Age, gender, zip code, active smoking and co-colonization with N. menigitidis or Strep. pneumoniae were not significantly associated with carriage of a particular Staph. aureus genotype as defined by AFLP. Although age is an important determinant of Staph. aureus colonization in children, with highest incidences at the age of 11 years (Bogaert et al., 2004), we found no association between age distribution and the various genotypes of Staph. aureus. This suggests that unknown host or environmental factors are more likely to be prime determinants of the parabolic age-versus-carriage distribution in children (Bogaert et al., 2004). Seventy-four (18.5 %) of the 400 children with Staph. aureus carriage were co-colonized with Strep. pneumoniae. Twenty-four (32.4 %) of the 74 pneumococcal isolates expressed capsular serotypes included in PCV7 (vaccine-type pneumococci) (Table 1). We found no association between co-colonization with vaccine- or non-vaccine-type pneumococci and the different genotypes of Staph. aureus (Table 2, Fig. 2a
). In addition, when the RFEL types obtained for the pneumococci were included in the comparison, no additional overlaps in co-colonizing types were observed (Fig. 2b). Notably, genetic heterogeneity among the pneumococci was much higher (showing no genetic clusters; Fig. 2b) (see also Bogaert et al., 2006), than in Staph. aureus (showing four major genetic clusters; Fig. 2a).
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). This suggested that AFLP clusters were evenly distributed geographically; this contrasts with Staph. aureus infection in specific disease categories, where local spread of certain clones is well established (Koning et al., 2003).
However, passive smoking was significantly associated with carriage of Staph. aureus AFLP-cluster IV (P=0.003; see also footnote to Table 2). An earlier study had already revealed that passive smoking is associated with an increased risk of Staph. aureus colonization, with active smoking being protective against colonization (Bogaert et al., 2004). This suggests that passive smoking excites colonization opportunities for specific genotypes of Staph. aureus that have been shown to be possibly hypervirulent before (AFLP cluster IV) (Melles et al., 2004). We additionally performed logistic regression analysis with AFLP cluster IV as outcome, and active smoking, passive smoking, age, gender, co-colonization and zip code as possible confounders in the model. This analysis revealed that passive smoking was independently associated with carriage of AFLP cluster IV (odds ratio 2.8; confidence interval 1.5–5.5; P=0.002). Additional statistical analysis of the different subclusters from major AFLP clusters I and IV (dotted lines in Fig. 1) did not reveal novel associations with carriage of specific Staph. aureus genotypes (data not shown).
In our search for factors co-determining success of colonization with certain Staph. aureus genotypes, we found no evidence for involvement of age, gender, zip code, active smoking and co-colonization with N. menigitidis and vaccine or non-vaccine serotypes of Strep. pneumoniae. This suggests that with respect to Staph. aureus, bacterial inter-species competition in the nasopharynx probably depends on host characteristics or currently unspecified microbial features (e.g. receptor and ligands), rather than AFLP-defined overall Staph. aureus genotypes. Further studies involving molecular typing or gene expression testing at a more detailed level may still generate data that could help identify strain- rather than clone-specific factors involved in baterial interference. Maturation of the nasopharyngeal niche, including its diverse innate immunity factors during ageing, might be an important driving force, but this hypothesis should be substantiated by further investigations. Recently, it was suggested that hydrogen peroxide production by Strep. pneumoniae is important in the bacterial interference process, a lead that warrants further investigation (Regev-Yochay et al., 2006). In conclusion, neither of the staphylococcal clones identified by AFLP has a better competitive edge over the pneumococcus. Thus, success of Staph. aureus clones is not explained on the basis of improved competition with co-colonizing pneumococci. Furthermore, we found an association between passive smoking and carriage of a specific Staph. aureus cluster in children.
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ACKNOWLEDGEMENTS |
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AFLP is a registered trademark of Keygene NV and the AFLP® technology is covered by patents (US 6 045 994A, EP0534858B1) and patent applications owned by Keygene NV.
Edited by: J. M. van Dijl
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Received 7 September 2006;
revised 15 November 2006;
accepted 27 November 2006.
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