Investigation of the Impact of Antibiotic Administration on the Preterm Infants' Gut Microbiome Using Next-Generation Sequencing-Based 16S rRNA Gene Analysis

Abstract

Background: The human gut microbiota is an extensive population of microorganisms, and it shows significant variations between periods of optimal health and periods of illness. Vancomycin-resistant Enterococcus (VRE) and carbapenem-resistant Klebsiella pneumoniae (CRKP) are both pathogenic agents (BPAs) that can colonize in the gut after dysbiosis of microbiotal composition following antibiotic treatment. Methods: This study aimed to investigate the impact of antibiotics on the microbiotal composition of the gut. For this purpose, the first pass meconiums of 20 patients and the first rectal swabs containing BPAs of the same patients after antibiotic treatment were studied using next-generation sequencing-based 16S rRNA gene analysis. The V1-V9 region of 16S rRNA was sequenced with Oxford Nanopore. Results: Twenty-five phyla were detected in the meconiums, and 12 of them were absent after antibiotic treatment. The four most prevalent phyla in meconiums were Bacillota, Pseudomonadota, Bacteroidota, and Actinomycetota. Only the relative abundance of Pseudomonadota was increased, while a significant decrease was observed in the other three phyla (p < 0.05). A significant decrease was observed in alpha-diversity in rectal swabs containing BPAs versus meconiums (p = 0.00408), whereas an increased variance was observed in beta-diversity in all samples (p < 0.05). As a result of a LEfSe analysis, Pseudomonadota was found to have a higher relative abundance in rectal swabs, and Bacillota was significantly higher in the meconiums of the twins. Conclusions: Our study strongly verified the relationship between the administration of antibiotics, dysbiosis, and colonization of BPAs in the infants' gut microbiota. Further research would be beneficial and needed, comprising the natural development process of the infants' gut microbiota.

Keywords: 16S rRNA; antibiotic; carbapenem-resistant Klebsiella pneumoniae; meconium; microbiome; microbiota; preterm infants; vancomycin-resistant Enterococcus.

Figure 1

The results of the alpha and beta diversity calculations at the phylum level are presented in a visual format. The observed, Shannon, and InvSimpson calculations are displayed in the (A) alpha diversity visualization. The presentation employs a combination of violin and box plots, with the middle thick line in the box plot representing the median. In (B,C), Jaccard and Bray–Curtis dissimilarity distances, which are beta diversity metrics, are presented on the PCoA graph. The ellipses were drawn using the 95% confidence interval. In (A–C) the blue color indicates meconiums, and the red color indicates BPAs.

Figure 2

The stacked graphs of phyla. In the figure, “A” represents meconium and “B” represents BPAs. (The chart classifies branches below 1% as others. For interpretation of the references to color in this figure legend, the reader is referred to Supplement File S3).

Figure 3

The results obtained in the alpha-diversity (A), Bray–Curtis (B), and Jaccard (C) PCoA graph. In (A–C) the blue color indicates meconiums, and the red color indicates BPAs.

Figure 3

The results obtained in the alpha-diversity (A), Bray–Curtis (B), and Jaccard (C) PCoA graph. In (A–C) the blue color indicates meconiums, and the red color indicates BPAs.

Figure 4

Alpha diversity plots (A), PCoA plots of Bray–Curtis dissimilarity (B) and Jaccard distance (C). In images (A–C), the blue color indicates meconiums, and the red color indicates rectal swabs containing BPAs.

Figure 4

Alpha diversity plots (A), PCoA plots of Bray–Curtis dissimilarity (B) and Jaccard distance (C). In images (A–C), the blue color indicates meconiums, and the red color indicates rectal swabs containing BPAs.

Figure 5

LEfSe at Phylum level. LEfSe analysis of the meconiums (A) vs. BPAs (B) at phylum level. While the red color represents bacteria found in meconiums, green represents the phyla in BPAs, separating the groups. As the image (a) represents the analysis performed on only the twins’ samples, (b) shows the LEfSe conducted for all of the samples.

Figure 6

LEfSe analyses of twins’ (A) and all (B) samples at species-level. While the red color represents species-level bacteria found in meconiums, green represents the species in BPAs discriminating between the meconiums and BPAs. As the image (a) represents the analysis performed on only the twins’ samples, (b) shows the LEfSe conducted for all of the samples.