Results

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two species shared the same field, C. jejuni was isolated only from Micromys minutus (Table 13). No difference was observed in the prevalence of C. jejuni in Micromys minutus based on the gender/age of the wild mice or the region/year in which wild mice were captured.

MLST analysis for all isolates revealed that the sequences of seven housekeeping genes (aspA, glnA, gltA, glyA, pgm, tkt, and uncA) were identical, regardless of various factors (gender, age, region, and year). In other words, only one ST was found among all 42 C. jejuni isolates from Micromys minutus. All sequences were submitted to PubMLST and a new ST, ST-8388 (aspA: 7, glnA: 618, gltA: 303, glyA: 688, pgm: 823, tkt: 645, uncA: 6), was determined.

Virulence/survival factors profile and antibiotic-resistance patterns of C. jejuni in Micromys minutus

Among 10 virulence- and survival-related genes, flhB, cadF, pldA, iamA, cdtB, crsA, perR, and htrA were present in all (100%) C. jejuni isolates (Figures 27A and B). However, hcp was not detected in any isolates (0%). In addition, virB11 was detected in 20/42 (47.6%) of isolates with a similar geographical distribution. Specifically, all 20 isolates found in fields 02 (3 isolates), 13 (5 isolates), 17 (10 isolates), and 22 (2 isolates) were positive for virB11, whereas

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22 isolates from other fields (fields 03, 05, 09, 14, 15, 16, 18, 19, 20, and 21) were negative for this gene. Wild mice were caught in different two years from fields 03 and 13, but an identical pattern was observed. In an antibiotic- resistance test, all C. jejuni isolates were susceptible to all tested antibiotics, regardless of testing method used (MIC, disk diffusion, or a sequence-based method).

Complete genome sequence and genomic characterization of Micromys minutus-derived C. jejuni

One representative C. jejuni strain (SCJK2) was selected for sequencing, since all 42 isolates from Micromys minutus were presumed to be genetically identical, according to the MLST results. Based on the whole-genome sequence of Micromys minutus-derived C. jejuni (strain SCJK2), the complete genome has a total length of 1,859,287 bp and consists of three contigs (one chromosome and two plasmids) with an average GC content of 30.15%. The genome harbors 1,781 coding genes (chromosome: 1,642 genes; plasmid 1: 93 genes; plasmid 2: 46 genes), 44 tRNA, 9 rRNA, and 3 ncRNA (Table 14).

Among these, 1,278 coding genes are assigned to EggNog/COG categories (Figure 28). The complete genome sequence of Micromys minutus-derived C.

jejuni (strain SCJK2) was deposited in GenBank (accession numbers CP038862–CP038864). In virulence-factor analysis based on VFDB, the

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Micromys minutus-derived C. jejuni genome (strain SCJK2) contains most virulence-related genes associated with adherence (cadF, jlpA, porA, and pebA), colonization (capsule biosynthesis and transport-related genes), glycosylation (N-linked protein and O-linked flagella glycosylation-related genes), immune evasion (LOS-related genes), invasion (ciaB), motility (flagella-related genes), the secretion system (type IV secretion system-related genes), and toxins (cdtA/B/C). Antibiotic-resistance analysis indicated that the genome did not contain antibiotic-resistance genes which correspond to genes registered in CARD.

Genetic relatedness between Micromys minutus-derived C.

jejuni and C. jejuni derived from other sources, based on MLST and comparative-genomic analysis

A minimum-spanning tree was constructed based on MLST and was used to assess allelic differences between sequences of Micromys minutus-derived C.

jejuni and previously reported sequences (Figure 29). In the case of C. jejuni from Micromys minutus, the allelic difference to the nearest ST was 5.0, indicating a low level of genetic relatedness with C. jejuni from other sources, such as humans, livestock, and the environment. The complete genome of Micromys minutus-derived C. jejuni (strain SCJK2) was compared with 174 other C. jejuni genome sequences from different sources (Figure 30). Among

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1,203 core genes of 175 genomes, Micromys minutus-derived C. jejuni (strain SCJK2) did not harbor 12 genes, including aer_1 (aerotaxis receptor), glnM (putative glutamine ABC transporter permease protein), cheY (chemotaxis protein), tyrS (tyrosine-tRNA ligase), and peb1A (major cell-binding factor). In the approximately-maximum-likelihood phylogenetic tree, this genome sequence formed clades with other sequences. However, unlike most other sequences that were clustered by isolated country or source, this genome was not deeply clustered with other sequences, including GCF 002214785.1 and GCF 004328905.1, which were isolated from South Korea. In addition, the branch length from the closet bootstrap (0.009) in the tree of this strain was much longer than that of most other strains. The complete genome of Micromys minutus-derived C. jejuni (strain SCJK2) was compared with 19 representative C. jejuni genome sequences from different sources. For most sequences, except for Micromys minutus-derived C. jejuni (strain SCJK2), the highest orthoANI values and tetra-nucleotide values were 98.84–99.98 and 99.92–100, respectively, when compared to other sequences. However, the highest orthoANI value and tetra-nucleotide value of Micromys minutus-derived C.

jejuni (strain SCJK2) was only 98.37 (with GCA_001865435.1, C. jejuni subsp.

jejuni, chicken, Tulsa, US) and 99.87 (with GCA_002101355.1, C. jejuni subsp.

jejuni, calf, Philadelphia, US), respectively. In addition, the Micromys minutus- derived C. jejuni (strain SCJK2) genome did not cluster with other sequences in the orthoANI value-derived and tetra-nucleotide value-derived UPGMA

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dendrogram. In POG analysis, 499 genes were present in the Micromys minutus-derived C. jejuni genome (strain SCJK2), but not in the other 19 genome sequences; however, most of the 499 genes had unknown names and functions. In the UPGMA dendrogram and heat map based on the presence/absence of POGs, Micromys minutus-derived C. jejuni (strain SCJK2) did not cluster with C. jejuni derived from other sources.

Diversity in the gut microbiota of each wild mouse species (Mus musculus and Micromys minutus)

In alpha diversity, the number of OTUs in the gut of Mus musculus was significantly higher than those in the gut of Micromys minutus (Mus musculus:

347.6 ± 27.2, Micromys minutus: 248.3 ± 38.2, Kruskal-Wallis test, p = 0.04).

However, there was no significant difference in microbial richness and evenness between the two wild mice species. The Shannon diversity indices of Mus musculus and Micromys minutus were 6.84 ± 0.27 and 6.19 ± 0.36, respectively; the Pielou evenness indices were 0.82 ± 0.02 and 0.79 ± 0.03, respectively. In beta diversity using the principal coordinates analysis (PCoA) plot based on ‘unweighted UniFrac distance metric, all 20 Mus musculus clustered together and all 6 Micromys minutus also clustered together (regardless of the isolation result of C. jejuni) (Figure 31).

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Taxonomic composition of gut microbiota of each wild mice species (Mus musculus and Micromys minutus)

The taxonomic composition of the gut microbiota of each wild mouse species at the genus level (with an average of ≥ 1% in at least one species) is shown in Figure 32. A total of 251 genera were identified; among them, 18 genera comprised the core gut microbiota of wild mice regardless of the wild mice species and constituted ca. 85.0% of the total gut microbiota. In Mus musculus, 15 genera comprised the core gut microbiota and constituted ca. 84.0%

of the total gut microbiota. The predominant core gut microbiota of Mus musculus included Lactobacillus (32.9%), followed by Lachnospiraceae;

uncultured (11.5%), Bacteroides (10.7%), and Muribaculaceae; uncultured (10.7%). In Micromys minutus, 10 genera comprised the core gut microbiota and constituted ca. 80.3 % of the total gut microbiota. The predominant core gut microbiota of Micromys minutus included Lactobacillus (26.2%), followed by Bacteroides (14.8%), Campylobacter (13.9%), and Lachnospiraceae;

uncultured (11.4%).

Particularly, the relative abundance of Campylobacter in the gut of Micromys minutus was significantly higher than that in Mus musculus (unpaired t test, p < 0.01) (Figure 33A). Campylobacter was not present in the gut of all Mus musculus, while the relative abundance of Campylobacter in the gut of

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Micromys minutus was 13.9 ± 7.4% (mean ± SEM). In Micromys minutus, there was no significant difference in the relative abundance of Campylobacter according to the culture-based isolation result of C. jejuni (Micromys minutus negative for C. jejuni: 9.88 ± 8.83%, Micromys minutus were positive for C.

jejuni: 17.90 ± 13.33%) (Figure 33B).

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