Metagenomic Analysis of the Gut Microbiota of Wild Mice, a Newly Identified Reservoir of Campylobacter

Abstract

Campylobacter, the most common etiologic agent of zoonotic gastroenteritis in humans, is present in many reservoirs including livestock animals, wildlife, soil, and water. Previously, we reported a novel Campylobacter jejuni strain SCJK02 (MLST ST-8388) from the gut of wild mice (Micromys minutus) using culture-dependent methods. However, due to fastidious growth conditions and the presence of viable but non-culturable Campylobacter spp., it is unclear whether M. minutus is a Campylobacter reservoir. This study aimed to: 1) determine the distribution and proportion of Campylobacter spp. in the gut microbiota of wild mice using culture-independent methods and 2) investigate the gut microbiota of wild mice and the relationship of Campylobacter spp. with other gut microbes. The gut microbiota of 38 wild mice captured from perilla fields in Korea and without any clinical symptoms (18 M. minutus and 20 Mus musculus) were analyzed. Metagenomic analysis showed that 77.8% (14 of 18) of the captured M. minutus harbored Campylobacter spp. (0.24-32.92%) in the gut metagenome, whereas none of the captured M. musculus carried Campylobacter spp. in their guts. Notably, 75% (6 of 8) of M. minutus determined to be Campylobacter-negative using culture-dependent methods showed a high proportion of Campylobacter through metagenome analysis. The results of metagenome analysis and the absence of clinical symptoms suggest that Campylobacter may be a component of the normal gut flora of wild M. minutus. Furthermore, linear discriminant analysis (LDA) showed that Campylobacter was the most enriched genus in the gut microbiota of M. minutus (LDA score, 5.37), whereas Lactobacillus was the most enriched genus in M. musculus (LDA score, -5.96). The differences in the presence of Campylobacter between the two species of wild mice may be attributed to the differential abundance of Campylobacter and Lactobacillus in their respective gut microbiota. In conclusion, the results indicate that wild M. minutus may serve as a potential Campylobacter reservoir. This study presents the first metagenomics analysis of the M. minutus gut microbiota to explore its possible role as an environmental Campylobacter reservoir and provides a basis for future studies using culture-independent methods to determine the role of environmental reservoirs in Campylobacter transmission.

Keywords: Campylobacter; Lactobacillus; Micromys minutus; environmental reservoir; gut microbiota; metagenomics; transmission cycle; wild mouse.

Figure 1

Taxonomic composition of the gut microbiota of wild mice. Taxonomy bar plot of the gut microbiota of Micromys minutus at the (A) phylum and (B) genus levels. (C) The relative abundance of Campylobacter in the gut microbiota of Micromys minutus and Mus musculus. The blue and orange boxes represent the relative abundance of Campylobacter in the Campylobacter culture-positive and culture-negative M. minutus groups. Circle (●) and square (▪) represent the maximum point of relative abundance of Campylobacter, respectively. (D) Taxonomic composition of gut microbiota of two species of wild mice (Micromys minutus and Mus musculus) at the genus level.

Figure 2

Core gut microbiota of Micromys minutus. Box plots showing the relative abundance of the members of the core microbiota at the (A) phylum and (B) genus levels. Plus sign (+) represents the mean value. Heatmaps showing the relative abundance of core microbiota (C) at the phylum and (D) genus levels in individual M. minutus samples. The X-axis represents the individual samples of M. minutus. The Y-axis represents the core taxa. The color scale represents the relative abundance of core taxa in individual samples.

Figure 3

Differences in the gut microbiota of Micromys minutus according to Campylobacter culture status. (A) Alpha diversity of the gut microbiota of two groups of Micromys minutus. The distribution of the number of observed amplicon sequence variants, the Simpson’s index and the Shannon’s index of each group is shown in the box plot. The blue box denotes the Campylobacter culture-positive group, and the red box denotes the Campylobacter culture-negative group. (B) Principle coordinate analysis plot of Bray-Curtis dissimilarity between the gut microbiota of the Campylobacter culture-negative and -positive groups of M. minutus. Ellipses indicate 95% confidence intervals. (C) Histograms of the linear discriminant analysis scores for genera with differential abundance identified using linear discriminant analysis effect size in a culture-positive (blue) and culture-negative (red) group of Micromys minutus.

Figure 4

Differences in the gut microbiota of two species of wild mice. (A) Alpha diversity of the gut microbiota of two species of wild mice. The distribution of the number of observed amplicon sequence variants, the Simpson’s index and the Shannon’s index of each group is shown in the box plot. (B) Principle coordinate analysis plot of Bray-Curtis dissimilarity between the gut microbiota of Micromys minutus (orange) and Mus musculus (blue). Ellipses indicate 95% confidence intervals. (C) Histograms of the linear discriminant analysis scores for genera with differential abundance identified using linear discriminant analysis effect size in M. minutus (orange) and M. musculus (blue).