Changes in the intestinal microbiota following the administration of azithromycin in a randomised placebo-controlled trial among infants in south India

Abstract

Macrolides are among the most widely prescribed antibiotics worldwide. However, their impact on the gut's bacterial microbiota remains uncertain. We characterised the intestinal microbiota in 6-11 month-old infants in India who received a 3-day course of azithromycin or placebo during a randomised trial of oral poliovirus vaccine immunogenicity (CTRI/2014/05/004588). In 60 infants per study arm, we sequenced the V4 region of the bacterial 16S rRNA gene in stool samples collected before and 12 days after finishing treatment. We also tested for the presence of common bacterial, viral, and eukaryotic enteropathogens in the same samples using real-time PCR in a Taqman array card (TAC) format. Azithromycin induced a modest decline in microbiota richness and a shift in taxonomic composition driven by a reduction in the relative abundance of Proteobacteria and Verrucomicrobia (specifically Akkermansia muciniphila). The former phylum includes pathogenic strains of Escherichia coli and Campylobacter spp. that declined in prevalence based on the TAC assay. These findings differ from previous observations among older children and adults in Europe and North America, suggesting that the effects of azithromycin on the bacterial microbiota may be specific to the age and geographic setting of its recipients.

Figure 1

Composition of the bacterial microbiota at enrolment. (a) Mean relative taxon abundance at phylum and genus level among day-0 infant samples (n = 120) and adult samples (n = 40). Genera with a mean relative abundance of >1% in infants are included. For categories labeled as ‘other’ we include genera below this abundance threshold, including 11 taxa with a mean abundance of >1% in adults. Overall, we observed 125 genera, of which 89 were present in infants and 85 were present in adults. (b) Number of OTUs by age. (c) Unweighted Unifrac distances, visualised via principal coordinates analysis, for day-0 infant samples and adult samples. Abbreviations: m, months; OTU, 97%-identity operational taxonomic unit; PC, principal coordinate.

Figure 2

Impact of azithromycin on the bacterial microbiota. (a) OTU count and Shannon index (mean ± standard error) according to study arm. (b) Unweighted and (c) weighted Unifrac distances between day-14 infant samples, visualised via principal coordinates analysis. Mean values for each principal coordinate are indicated by dotted lines. (d) OTU-level differences in relative taxon abundance at day 14 according to study arm. Bars display p values on a negative log₁₀ scale. Comparisons with a p value of <0.05 prior to FDR correction are indicated. The tree was constructed from de novo OTU sequences spanning the V4 region of the 16S rRNA gene. Owing to the use of a relatively short (and hypervariable) segment of the 16S rRNA gene for tree construction, phyla do not always separate into discrete lineages. (e) Class-level differences in taxon abundance according to study arm. Mean relative abundance values for each study arm are indicated by horizontal lines. Values beyond the scale of the y-axis are indicated in red. *P < 0.05 (after FDR correction for abundance comparisons); **FDR-corrected P < 0.005. Abbreviations: AZ, azithromycin; OTU, 97%-identity operational taxonomic unit; Rel. abund., relative abundance; PC, principal coordinate; PL, placebo.

Figure 3

Highest ranking taxa by Random Forest importance score for prediction of study arm after treatment with azithromycin. Importance scores were calculated based on the decrease in Gini impurity index associated with inclusion of each variable in a tree. Mean importance scores (±standard deviation) were calculated across 200 cross-validation iterations of the Random Forests algorithm, with 5,000 trees per iteration.