A 16S rRNA Gene and Draft Genome Database for the Murine Oral Bacterial Community

To date, 325 16S rRNA gene sequences from murine oral bacterial isolates have been analyzed and found to constitute 103 mouse oral taxa (MOT) (Table 1 and Fig. 1). Twelve of the assigned MOTs (12.36%) represent novel, previously unidentified species that need further characterization in order to be assigned a formal species name. Representative 16S rRNA gene sequences for these novel taxa have been submitted to the National Center for Biotechnology Information (NCBI) database and are available under accession numbers MN095260 to MN095271. The 16S rRNA gene sequences for all the isolates analyzed in this study are available to download under accession numbers MW175535 to MW175859.

The mouse oral taxa are distributed across four bacterial phyla (Fig. 1): Firmicutes (54 taxa), Proteobacteria (27 taxa), Actinobacteria (14 taxa), and Bacteroidetes (8 taxa). The Firmicutes phylum, having the greatest number of taxa, is represented by species of the Streptococcus, Staphylococcus, Lactobacillus, Gemella, Enterococcus, Aerococcus, Jeotgalicoccus, Granulicatella, Facklamia, Dubosiella, and Ileibacterium genera. Proteobacteria are comprised of members of the Enterobacter, Klebsiella, Shigella, Escherichia, Pasteurella, Providencia, Proteus, Actinobacillus, Acinetobacter, Neisseria, Rodentibacter, Rhodanobacter, and Devosia genera. The Bacteroidetes phylum comprises the Bacteroides, Parabacteroides, Porphyromonas, Helicobacter, Muribaculum, and Dysgonomonas genera, while Actinobacteria are represented by the Micrococcus, Sanguibacter, Microbacterium, Rothia, Corynebacterium, Actinomyces, Cutibacterium, and Turicella genera. At the species level, the species Streptococcus danieliae MOT10 was most frequently observed among the samples tested. The periodontal pathogen Porphyromonas gingivalis, albeit never isolated in the laboratory from the murine swab samples, was identified among the amplicon sequencing data of the 16S rRNA gene. The species has been observed in the wild mouse species and occasionally in experimental SPF laboratory mice, but never in healthy, untreated SPF mice.

The SPF laboratory mice included in this study represented diverse background strains (C57BL/6J, C3H/Orl, CD-1, BALB/c) and were also obtained from a range of diverse sources (commercially purchased, in-house colonies, conventionalized from germfree mice, and genetic knockouts). In addition, samples were also collected from the formerly wild wood mouse Apodemus sylvaticus and the wild house mouse Mus musculus domesticus. Beta-diversity analyses of the oral microbial populations of the sampled mice (Fig. 2) showed distinct separation in the clustering of all three groups of mice. However, a number of shared species have also been observed between them, including Lactobacillus murinus (MOT93) and Streptococcus danieliae (MOT10) (Table 1).

Custom reference data sets for community profiling analysis were constructed using the current version of the database and used for data analysis. Analysis of the Illumina MiSeq amplicon sequencing data (the V1-V2 region of the 16S rRNA gene) in SPF mouse samples using the custom reference data set revealed that 98% of the total number of reads could be assigned to species level. Further comparative analysis of culturing data with this amplicon sequencing data set revealed a significant consensus in the SPF mouse samples, with all of the cultured species being represented at comparable levels with the next-generation sequencing (NGS) data (Fig. 3). Among the wild Mus musculus domesticus samples, 95% of the reads were identified to the species level using the custom reference data set, even though very little consensus has been observed with the culturing data in terms of abundance of individual species (Fig. 4). This could be explained by the fact that the species that show significantly higher abundance in the NGS data set (Muribacter muris, Neisseria species 1, and Porphyromonas gingivalis) are predominantly slow growers and therefore could have been missed being detected in the 48-h culturing protocol followed in this study.

In the samples from the formerly wild wood mouse Apodemus sylvaticus, the proportion of reads positively identified using the custom data set fell to 83%, even though a higher consensus between culturing and NGS was observed compared to the wild Mus musculus domesticus, despite the increased diversity (Fig. 5).

Current versions of the reference data sets have been constructed for both the mothur and DADA2 pipelines and are available to download at https://figshare.com/s/2470f05ab77cdf40b2f8.

Draft genomes of 55 murine oral bacterial isolates have been generated, sourced from all the mouse groups included in this study (Table 2). Representative genome sequences have been uploaded to the NCBI bacterial genome database and are publicly available at NCBI BioProject PRJNA671681. After assembly, the genomes had a mean contig number of 135 (±134) and GC% ratios ranging from 28 to 73%.

The following three genera were found to be of particular interest due to their presence in a wide range of samples tested.

(i) Streptococcus. S. danieliae MOT10 was the most commonly detected species in all SPF laboratory mouse groups from multiple sources and mouse strains (Fig. 6A). This species was also isolated from the wild Mus musculus domesticus. Streptococcus species 5 MOT45 was isolated from the wood mouse A. sylvaticus (Fig. 5) and was found to be very closely related to, but distinct from, S. danieliae (16S rRNA gene sequence similarity, 98.4%) forming a murine S. danieliae cluster.

Some isolates of S. acidominimus MOT11 were observed in a group of SPF mice belonging to the BALB/c strain, whereas a few novel streptococcal species were also isolated in single occurrences (Streptococcus species 1, 2, and 3; MOTs 12 to 14).

(ii) Lactobacillus. With only two exceptions, all of the lactobacilli in mice were identified as L. murinus MOT93 (Fig. 6B). The two exceptions belonged to the L. taiwanensis-gasseri-johnsonii cluster at >99% identity and could not be distinguished based on the 16S rRNA gene sequence analysis. In silico DNA-DNA hybridization gene sequence analysis identified them as L. johnsonii MOT51 at 72.1% genome identity.

(iii) Gemella. All Gemella isolates from laboratory mice were found to belong to a novel, as yet unnamed, species, Gemella species 2 MOT43 (Fig. 6C). The nearest phylogenetic neighbor is the canine oral species, Gemella palaticanis at 97.4% 16S rRNA gene sequence identity. Another single, novel Gemella isolate was observed in A. sylvaticus wood mice and designated Gemella species 1 MOT33.

All animal experiments were conducted in accredited facilities in accordance with the UK Animals (Scientific Procedures) Act 1986 (Home Office license number 7006844). Fieldwork for the wild Mus musculus domesticus work was approved by the Animal Welfare Ethical Review Body (AWERB) at the Department of Zoology, University of Oxford.

Conventional SPF C3H/Orl and BALB/c mice were maintained in individually ventilated cages (IVCs) at the animal care facilities of Queen Mary University of London. Conventional SPF C57BL/6J mice were maintained in IVCs at the animal care facilities of King’s College London. SPF C57BL/6J and CD-1 mice were also purchased commercially from Charles River Laboratories UK. In all, 191 SPF mice have been sampled over the course of 6 years for various experimental studies, and isolates obtained from the swabbing of these mice have been used for the analysis and development of this database.

One set (set 1) of formerly wild, now laboratory-bred, A. sylvaticus wood mice sampled in 2014 (n = 20) was originally established from wild wood mice that were live trapped from the UK woodlands and are now housed at the University of Edinburgh. Wild wood mice sampled in January 2017 (n = 39) were housed at the facilities at Fera Science (Sand Hutton, York), and the colony began with wild-caught wood mice from the grounds of the Institute; these animals had been laboratory-bred for 20 years and are referred to as set 2.

Wild house mice (Mus musculus domesticus [n = 21]) were sampled on the island of Skokholm in Wales, UK. These mice were captured temporarily in traps, sampled, and then released back into the wild.

The murine oral cavity was swabbed for 30 s, using sterile fine-tip rayon swabs (VWR International), while the animal was held in a scruff. The swab was then placed in a tube containing 100 μl of reduced John’s transport medium (see Text S1 in the supplemental material). Swabs from wild mice were stored at −20°C before being transported on ice to the laboratory. Serial dilutions of the suspension were spread onto blood agar plates containing 5% defibrinated horse blood (TCS Biosciences, UK) and incubated for aerobic and anaerobic (80% N₂, 10% H₂, and 10% CO₂) growth at 37°C for 48 h in a Don Whitley anaerobic chamber. The CFUs of predominant cultivable bacteria on each plate were counted. On an average, four to six different colony types could be identified on each blood agar plate. Every different colony morphology type observed was isolated and purified by subculture by restreaking the samples twice on fresh blood agar plates. Once the purity was established, the cultured isolate was cryopreserved for storage in Microbank bead tubes (Prolab Diagnostics) in duplicate. Briefly, a loopful of pure culture was aseptically transferred into the manufacturer’s cryopreservative liquid containing beads, the tube was inverted four to five times for emulsification and allowed to stand for 2 min. Any excess liquid was aseptically removed, and the bead tube was then frozen at −70°C.

Genomic DNA for each isolated bacterial strain was extracted by using a GenElute bacterial DNA kit (Sigma-Aldrich), following the Gram-positive protocol according to manufacturer’s instructions, and used as a template for PCR. The 16S rRNA gene in the bacterial strains was amplified using modified versions of the universal 27FYM and 1492R 16S rRNA gene primers with built-in redundancies (see Text S2), using Phusion Green Hot Start II High Fidelity PCR Master Mix (Thermo Fisher Scientific). PCR conditions were as follows: initial denaturation at 98°C for 30 s; followed by 25 cycles of 98°C for 10 s, 47°C for 45 s, and 72°C for 30 s; followed in turn by a final extension at 72°C for 10 min. The amplified products were purified using Macherey-Nagel NucleoSpin gel and PCR Clean-Up (Fisher Scientific), followed by Sanger sequencing using the universal M13 primers M13 uni(-21) and M13 rev(-29) (Eurofins Genomics). For certain samples, internal primers for the 16S rRNA gene (342R, 357F, 519R, 907R, 926F, 1100R, 1114F, and 1392R) were also used for sequencing to improve the sequence coverage accuracy. All primer sequences have been provided in Text S2.

The forward and reverse sequences for each sample were assembled using the CAP3 assembly tool (37). Sequences were identified by BLAST interrogation of the NCBI nucleotide database. A sequence identity threshold of 98.5% was used for assignation to species, which is consistent with current recommendations (38) and the value used previously for the related human and canine oral databases (17, 18). Sequences were aligned by means of the CLUSTALW algorithm in Bioedit (39), and maximum-likelihood phylogenetic trees for each genus were constructed using MEGA7 (40) with 100 bootstrap replicates. Each species-level taxon was assigned an MOT number. For taxa that were not identified as validly proposed species at 98.5% identity, a novel species-level designation was assigned as “Genusname_species1,” and a new MOT number was allocated. For isolates that could not be definitively distinguished by 16S rRNA gene sequence analysis, each of the possible matching species was assigned a unique MOT ID in order to ensure maximum diversity capture. Assignment of taxa was also carried out using 16S rRNA gene amplicons selected from the MiSeq sequencing data that were not assigned an ID based on the existing reference database and then following the same BLAST protocol as described above. Visualization and annotation of the final phylogenetic tree of the identified 103 MOTs was performed using the web interface of iTOL v4 (41).

Whole genomic DNA was extracted from the above swabs using the DNeasy PowerSoil kit (Qiagen) according to the manufacturer’s instructions. Cell lysis was performed in the PowerBead tubes provided with the kit by bead beating on a vortex at maximum speed for 20 min. PCRs were performed with Phusion Green Hot Start II High Fidelity PCR Master Mix (Thermo Scientific) targeting the V1-V2 variable regions of the 16S rRNA gene using fusion primers 27F-YM (AGAGTTTGATYMTGGCTCA) and 338R-R (TGCTGCCTCCCGTAGRAG) combined with MiSeq adaptors and barcodes to achieve a double indexing system. The PCR conditions were as follows: initial denaturation for 5 min at 95°C; followed by 25 cycles of 95°C for 45 s, 53°C for 45 s, and 72°C for 45 s; followed in turn by a final extension of 72°C for 5 min. The amplified PCR products were cleaned and normalized in equimolar amounts by using a Sequal Prep normalization plate kit (Thermo Fisher Scientific). Extraction kit controls and PCR negative controls were included in the amplification plates, as well as sequencing pools. Pooled amplicons were sequenced at the Barts and the London Genome Centre using an Illumina MiSeq 2 × 250 flow cell for paired-end sequencing. The generated reads were quality checked, filtered, trimmed, denoised, dereplicated, and assembled into amplicon sequence variants (ASVs) using the DADA2 v1.8 pipeline (42). The assembled ASVs were then assigned taxonomy at the genus and species level using a custom-formatted reference database constructed using the taxa included in this database. Compared to the mean of total reads in the murine swab samples, the negative controls generated a very low percentage of reads (<0.1% in the PCR control and <1% in the kit control). Once this was confirmed, the negative samples were eliminated from further analyses. The generated ASV counts were normalized for sequencing depth by using the median of ratios method in the DeSeq2 (43) package in R, followed by beta-diversity and relative abundance analyses of the microbial population. Graphical analysis and plots were created using the R packages phyloseq (44) and ggplot2 (45). The raw sequencing reads have been uploaded to the NCBI SRA database (accession no. PRJNA642845).

A selection of bacterial isolates from the database were cultured, purity checked, and genomic DNA extracted using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich). These isolates are representatives of the cultured microbial population observed in various murine studies and include multiple candidates of the more commonly observed Streptococcus, Lactobacillus, and Staphylococcus species-level taxa. Genomic DNA libraries were prepared by MicrobesNG UK using Nextera XT Library Prep kit (Illumina, San Diego, CA) and sequenced on the Illumina HiSeq using a 250-bp paired-end protocol. Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 (46). De novo assembly was performed on samples using SPAdes version 3.7 (47), and contigs were annotated using Prokka 1.11 (48). Further genome analysis and annotation was also performed using the RAST server (https://rast.nmpdr.org/) (49). Phylogenetic distance and relatedness of certain isolates were determined using the genome-to-genome distance calculator, in the form of an in silico DNA-DNA hybridization (50).

The 16S rRNA gene V1-V2 region amplicon sequencing data from this study are available from the NCBI SRA database under accession no. PRJNA642845. Draft genome sequences of representative murine oral bacterial isolates are available to download from NCBI BioProject no. PRJNA671681. 16S rRNA gene sequences of the novel, unnamed bacterial isolates from this study are available under NCBI accession numbers MN095260 to MN095271. The 16S rRNA gene sequences for all the isolates analyzed in this study are available under NCBI accession numbers MW175535 to MW175859. Custom taxonomy reference data sets of the database for NGS analysis using mothur and DADA2 pipelines are available to download from https://figshare.com/s/2470f05ab77cdf40b2f8.