Rapid Increase of Oral Bacteria in Nasopharyngeal Microbiota After Antibiotic Treatment in Children With Invasive Pneumococcal Disease

Abstract

Introduction: Antibiotics are commonly prescribed to young children for treating bacterial infections such as invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae. Despite the obvious benefits of antibiotics, little is known about their possible side effects on children's nasopharyngeal microbiota. In other ecological niches, antibiotics have been described to perturb the balanced microbiota with short- and long-term effects on children's health. The present study aims to evaluate and compare the nasopharyngeal microbiota of children with IPD and different degree of antibiotic exposure.

Methods: We investigated differences in nasopharyngeal microbiota of two groups of children <18 years with IPD: children not exposed to antibiotics before sample collection (n=27) compared to children previously exposed (n=54). Epidemiological/clinical data were collected from subjects, and microbiota was characterized by Illumina sequencing of V3-V4 amplicons of the 16S rRNA gene.

Results: Main epidemiological/clinical factors were similar across groups. Antibiotic-exposed patients were treated during a median of 4 days (IQR: 3-6) with at least one beta-lactam (100.0%). Higher bacterial richness and diversity were found in the group exposed to antibiotics. Different streptococcal amplicon sequence variants (ASVs) were differentially abundant across groups: antibiotic use was associated to lower relative abundances of Streptococcus ASV2 and Streptococcus ASV11 (phylogenetically close to S. pneumoniae), and higher relative abundances of Streptococcus ASV3 and Streptococcus ASV12 (phylogenetically close to viridans group streptococci). ASVs assigned to typical bacteria from the oral cavity, including Veillonella, Alloprevotella, Porphyromonas, Granulicatella, or Capnocytophaga, were associated to the antibiotic-exposed group. Common nosocomial genera such as Staphylococcus, Acinetobacter, and Pseudomonas were also enriched in the group exposed to antibiotics.

Conclusion: Our results point toward a reduction of S. pneumoniae abundance on the nasopharynx of children with IPD after antibiotic treatment and a short-term repopulation of this altered niche by oral and nosocomial bacteria. Future research studies will have to evaluate the clinical implications of these findings and if these populations would benefit from the probiotic/prebiotic administration or even from the improvement on oral hygiene practices frequently neglected among hospitalized children.

Keywords: antibiotics; children; invasive pneumococcal disease (IPD); nasopharyngeal microbiota; nosocomial bacteria; oral bacteria.

Figure 1

Alpha- and beta-diversity comparisons between patients with IPD not exposed to antibiotics before sample collection (yellow) and IPD patients with previous antibiotic exposure (blue). Boxplots showing the Chao1 richness (A) and Shannon diversity indexes (B) according to antibiotic-exposure groups at the ASV level. Differences by group were assessed with simple linear regression analyses including confounding variables as copredictors (age, gender, seasonality, vaccination, ICU admission, and length of hospital stay). Three observations from the exposed group were deleted due to missing values in vaccination variable. (C) Nonmetric multidimensional scaling (NMDS) plot based on Bray–Curtis dissimilarities of nasopharyngeal microbiota composition of samples from all patients included in the study. Samples of each group are connected with their corresponding centroids using the function “ordispider” (Vegan R package). P-value corresponds to Adonis PERMANOVA test on the antibiotic- exposure group variable and including confounding variables as covariates. Significance codes: *** ≤0.001; ** ≤0.01; * ≤0.05. MDS, nonmetric multidimensional scaling.

Figure 2

Main ASVs assigned to Streptococcus in the nasopharynx of children with IPD and their relative abundance according to antibiotic-exposure groups. (A) Bar plot showing the ASVs with a relative contribution >0.1% to the total number of streptococcal reads. (B) Boxplot showing the relative abundance of main streptococcal ASVs differentially represented in antibiotic-exposure groups. Significance codes: *** ≤0.001; ** ≤0.01; * ≤0.05.

Figure 3

Classification of children with IPD according to antibiotic-exposure using a Random Forest model based on nasopharyngeal microbiota composition. All ASVs as well as confounding factors (age, gender, seasonality, vaccination, ICU admission, and length of hospital stay) were included in the model. (A) ROC curve showing the performance of the RF model at the ASV level. (B) Bar plot showing top 50 most important features to class separation according to the Mean Decrease Accuracy score (confounding factors were not found among the top important features). Color-coding shows directionality of the association for each of the 50 top features to either the not exposed (yellow) or antibiotic-exposed (blue) group based on a post hoc analyses with Cliff’s delta estimation of the effect size. In addition, a heatmap displaying relative abundance (%) of these top 50 features across samples is shown on the right. Each column represents a sample, while each row represents a different feature. In the x-axis, samples are split by group and ordered according to hierarchical clustering using a Bray–Curtis dissimilarity measure.

Figure 4

Bacterial genera associated to nosocomial infections in the nasopharynx of patients with IPD according to antibiotic-exposure and length of hospitalization prior to sample collection. Heatmap displaying relative abundances (%) of bacterial genera with characteristic species implicated in healthcare-associated infections. Each column represents a sample, while each row a bacterial genus. Samples are split by the antibiotic-exposure group and ordered by days of antibiotic intake. Rows are sorted by total relative abundance in decreasing order. Bottom bars indicate the number of days of antibiotic exposure and days of hospitalization prior to sample collection for each patient with distinct color scales. Differences in relative abundances of these bacteria by group were assessed with Wilcoxon tests and FDR adjusted p-values are shown. Significance codes: *** ≤0.001; ** ≤0.01; * ≤0.05.