Metagenomic and Antibiotic Resistance Analysis of the Gut Microbiota in Larus relictus and Anatidae Species Inhabiting the Honghaizi Wetland of Ordos, Inner Mongolia, from 2021 to 2023

Abstract

Gut microbes thrive by utilising host energy and, in return, provide valuable benefits, akin to a symbiotic relationship. Here, metagenomic sequencing was performed to characterise and compare the community composition, diversity and antibiotic resistance of the gut microbiota of Relict gull (Larus relictus) and Anatidae species. Alpha diversity analysis revealed that the intestinal microbial richness of L. relictus was significantly lower than that of Anatidae, with distinct differences observed in microbial composition. Notably, the intestines of L. relictus harboured more pathogenic bacteria such as clostridium, which may contribute to the decline in their population and endangered status. A total of 117 strains of Escherichia coli were isolated, with 90.60% exhibiting full susceptibility to 21 antibiotics, while 25.3% exhibited significant biofilm formation. Comprehensive Antibiotic Resistance Database data indicated that glycopeptide resistance genes were the most prevalent type carried by migratory birds, alongside quinolone, tetracycline and lincosamide resistance genes. The abundance of resistance genes carried by migratory birds decreased over time. This metagenomic analysis provides valuable insights into the intestinal microbial composition of these wild bird species, offering important guidance for their conservation efforts, particularly for L. relictus, and contributing to our understanding of pathogen spread and antibiotic-resistant bacteria.

Keywords: Anatidae; Escherichia coli; Larus relictus; biofilm; gut microbiota; metagenomic.

Figure 1

Gut bacterial alpha diversity. (A) Chao1 diversity. (B) ACE index of bacteria genera in each sample. YO: L. relictus; NH: Anatidae. ** p  < 0.01.

Figure 2

Gut bacterial beta diversity. (A) Bray–Curtis distance nonmetric multidimensional scaling plots. (B) Analysis of similarities (ANOSIM) of bacterial genera in each sample. (C) Bray–Curtis Distance Cluster Tree Analysis of relative abundance at the genus level in each sample. YO: L. relictus; NH: Anatidae; ES: environmental samples.

Figure 3

Bar graph of the relative bacterial abundance at phylum and genus levels. The bacterial composition of each sample was examined, and the top 10 phyla/genera were used to plot histograms. (A,C) Bar graphs of the relative bacterial abundance at the phylum level. (B,D) Bar graphs of the relative bacterial abundance at the genus level. YO: L. relictus; NH: Anatidae.

Figure 4

Metastat analysis of species’ differences between groups. (A) Metastat analysis of species’ differences at the phylum level. (B) Metastat analysis of species’ differences at the genus level. YO: L. relictus; NH: Anatidae. * p  < 0.05; ** p  < 0.01.

Figure 5

Application of antibiotic resistance genes in intestinal microbes of migratory birds. (A) Wayne plots of three groups of migratory bird faecal samples versus environmental samples carrying resistance genes from 2021 to 2023, grouped by time. (B) Box plots of differences in carrying resistance genes between three groups of migratory bird faecal samples and environmental samples from 2021 to 2023, using time as a subgroup. (C) Clustering heatmap of the top 30 resistance genes for each sample. (D) Clustering heatmap for L. relictus, Anatidae and environmental carriage of the top 30 resistance genes. (E) Plotting Chord diagram illustrating the antibiotic resistance ontologies with maximum abundance of the top 30 resistance genes. (F) Chord diagram showing the distribution of resistance mechanisms based on the mechanism of action of resistance genes relative to species. It is worth noting that the outermost ring features the nomenclature of resistance genes, with each gene distinctly labelled by a unique colour. The length of the band on this ring represents the relative abundance or expression level of the resistance genes across various samples or groups. The interconnecting lines bridging the layers of rings serve to delineate the association relationships among the resistance genes. The thickness and colouration of these lines provide insights into the strength and nature of these associations, respectively, enabling a deeper understanding of the intricate interplay among the genes. YO: L. relictus; NH: Anatidae. ES: environmental samples.

Figure 6

Mobile genetic element prediction for L. relictus and Anatidae (faecal) and environmental samples. (A) Relative abundance of the top 20 Insertion Sequences (ISs) among samples. (B) Relative abundance of the top 20 integrons (integrases and gene cassettes) across samples. (C) Relative abundance of the top 20 plasmids among samples.

Figure 7

Bacterial community functional prediction using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. (A) Class 1 level. (B) Class 2 level. (C) Class 3 level.