ABSTRACT Despite its central role in host fitness, the gut microbiota may differ greatly between individuals. This variability is often mediated by environmental or host factors such as diet, genetics, and infections. Recently, a particular attention has been given to the interactions between gut bacteriome and helminths, as these latter could affect host susceptibility to other infections. Further studies are still required to better understand the three-way interactions between gut bacteriome, helminths and other parasites, especially because previous findings have been very variable, even for comparable host-parasite systems. In our study, we used the V4 region of the 16S rRNA gene to assess the variability of gut bacteriome diversity and composition in wild populations of a small mammal, the bank vole Myodes glareolus. Four sites were sampled at a fine geographical scale (100 km) along a North-South transect in Eastern France. We applied analyses and concepts of community and microbial ecology to evaluate the interactions between the gut bacteriome, the gastro-intestinal helminths and the pathogenic bacteria detected in the spleen. Regarding the gut bacteriome composition and diversity among bank voles, we identified important variations that were mainly explained by sampling localities and reflected the North/South sampling transect. In addition, we detected two main enterotypes, that might correspond to contrasted diets. We found geographic variations of the gut bacteriome richness and Firmicutes/Bacteroidetes ratio, both correlating positively with body mass index. We found positive correlations between the specific richness of the gut bacteriome and of the helminth community, as well as between the composition of these two communities, even when accounting for the influence of geographical distance. For the pathogenic bacteria community, no broad patterns have been detected. The helminths Aonchothecha murissylvatici, Heligmosomum mixtum and the bacteria Bartonella sp were the main taxa associated with the whole gut bacteriome composition. Besides, changes in relative abundance of particular gut bacteriome taxa were specifically associated with other helminths (Mastophorus muris, Catenotaenia henttoneni, Paranoplocephala omphaloides and Trichuris arvicolae) or pathogenic bacteria. Especially, infections with Neoehrlichia mikurensis, Orientia sp or Rickettsia sp were associated with lower relative abundance of the family Erysipelotrichaceae (Firmicutes), while coinfections with higher number of bacterial infections were associated with lower relative abundance of a Bacteroidales family (Bacteroidetes). These results emphasize complex interlinkages between gut bacteriome and infections in wild animal populations. They remain difficult to generalize due to the strong impact of environment, even at fine geographical scales, on these interactions. Abiotic features, as well as small mammal community composition and within host parasite coinfections, should now be considered to better understand the spatial variations observed in the relationships between gut bacteriome, gastro-intestinal helminths and bacterial infections. FILE DESCRIPTION: Individual information on rodent samples whose colons have been sequenced This CSV file contains 161 rodent individuals for which the DNA extracted from the colon organ was successfully sequenced (16S rRNA V4 gene). The table is organized with the sequencing code in row and the different individual variables in column: the unique identifier of each individual "Code_ind", the gender (F=female; M=male), the sampling locations "Locality" and the corresponding abbreviations "Loc"; Mont-sous-Vaudrey=”Mont”, Chaux-des-Crotenay=”Chau”, Chatillon=”Chat”, Cormaranche=”Corm”, the body mass index "Body_index" and the age class "Age" (juvenil, adult). File name: Gut_samples_data.csv Individual information on rodent samples whose spleens have been sequenced CSV file contains 138 rodent individuals for which the DNA extracted from the splee was successfully sequenced (16S rRNA V4 gene). The table is organized with the sequencing code in row and the different individual variables in column: the unique identifier of each individual "Code_ind", the gender (F=female; M=male), the sampling locations "Locality" and the corresponding abbreviations "Loc"; Mont-sous-Vaudrey=”Mont”, Chaux-des-Crotenay=”Chau”, Chatillon=”Chat”, Cormaranche=”Corm”, the body mass index "Body_index" and the age class "Age" (juvenil, adult). File name: PathBact_samples_data.csv Individual information on rodent samples whose gastrointestinal helminths have been studied This CSV file contains 153 rodent individuals for which the gastrointestinal helminths have been analysed (morphological identification). The table is organized with the sequencing code in row and the different individual variables in column: the unique identifier of each individual "Code_ind", the gender (F=female; M=male), the sampling locations "Locality" and the corresponding abbreviations "Loc"; Mont-sous-Vaudrey=”Mont”, Chaux-des-Crotenay=”Chau”, Chatillon=”Chat”, Cormaranche=”Corm”, the body mass index "Body_index" and the age class "Age" (juvenil, adult). File name: GIHel_samples_data.csv Information concerning the Myodes glareolus samples and the positive and negative controls multiplexed in the 16Sv4 MiSeq sequencing runs 1 and 4 This CSV file contains the Sequencing_run_ID, PCR_ID, PCR_replicate, Sequence file name (read 1 fastq file), Sequence file name (read 2 fastq file), Rodent_ID, Sex, Site, Site_ID, Body_index, Age, Helminth_analysis, Sample_ID, Sample_type, Sample_included_in_this_study, and Extraction Kit for the 1165 PCR products multiplexed in two different Illumina MiSeq runs. Only the sequences from 647 PCR products were used in this study (see “Sample_included_in_this_study”), corresponding to 168 individuals showing results on spleen pathobiome and/or colon bacteriome and/or helminth detection, and excluding the caecum samples. File name: Sequencing_informations.csv MiSeq raw sequences of the 16Sv4 rRNA gene from Myodes glareolus samples (Runs 1 and 4) This ZIP file contains the Runs 1 and 4 FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for each Myodes glareolus sample using the MiSeq platform. The 1165 multiplexed PCR products were indexed using both forward and reverse indices. The list of the multiplexed samples and positive & negative controls are provided in the following CSV file titled: Sequencing_informations.csv File name: MiSeq_Reads_16S_Myodes_glareolus_Run1and4.zip Table of abundance of gut bacteriome OTUs for rodent samples whose colons have been sequenced This CSV file contains the number of reads obtained after data filtering for each OTU of the MiSeq runs and each rodent sample whose colon has been sequenced. The results of the two PCR replicates from the same sample have been summed and filtered using negative and positive controls. File name: Gut_otu_table.csv Table of presence/absence of potentially pathogenic OTUs for rodent samples whose spleens have been sequenced This CSV file contains the presence/absence of each OTU obtained after data filtering from the MiSeq runs and for each sample whose spleen has been sequenced. The abundance of reads of the two PCR replicates from the same sample were summed and corrected by negative and positive controls. Only OTUs considered to be potentially pathogenic following the Gideon database, and after removal of possible contaminations (<500 reads and non-bacterial cluster), were retained. A transformation of abundance into presence/absence was further performed. File name: PathBact_otu_table.csv Table of presence/absence of gastrointestinal helminths This CSV file contains the presence/absence of each gastrointestinal helminth, for each sample analysed. File name: GIHel_otu_table.csv Affiliation table of gut bacteriome OTUs This CSV file contains the 578 clusters (=OTUs) corresponding to the filtered OTUs found in the table provided in the file Gut_otu_table.csv. Each cluster has been affiliated to the SILVA database. Phylum, Class, Order, Family, Genus and Species correspond to the different affiliation levels. File name: Gut_tax_table.csv Affiliation table of pathogenic bacteria OTUs This CSV file contains the 10 clusters (=OTUs) corresponding to the filtered OTUs found in the table provided in the file PathBact_otu_table.csv. Each cluster has been affiliated to the SILVA database. Phylum, Class, Order, Family, Genus and Species correspond to the different affiliation levels. File name: PathBact_tax_table.csv Affiliation table of gastro-intestinal helminth OTUs This CSV file contains the 8 clusters (=gastro-intestinal helminth species) found to the table provided in the file GIHel_otu_table.csv. Phylum, Class, Order, Family, Genus and Species correspond to the different affiliation levels. File name: GIHel_tax_table.csv Matrix of geographical distance between samples This CSV file contains a geographic distance dissimilarity matrix between each sample, in km. File name: Geographical_distance_samples.csv R scripts This R file contains the scripts used for the statistical analyses and for the figure generation. File name: Script_Ms2014Mglareolus20221213_v2.R