Draft genome sequence of Ruoffia tabacinasalis isolated from a bovine nasal swab: a novel member of the bovine nasal microbiota

TL;DR

Scientists discovered a new bacteria in cow noses and sequenced its genome.

Contribution

The isolation and draft genome sequencing of Ruoffia tabacinasalis, a new bovine nasal microbiota member.

Findings

The draft genome is 90.5% complete with one contig of 2,363,349 bp.

The GC content of the genome is 36.66%.

Abstract

We report the isolation and draft genome sequence of Ruoffia tabacinasalis, a novel member of the bovine nasal microbiota. The genome, which is estimated to be 90.5% complete, is composed of one contig comprising 2,363,349 bp with a GC content of 36.66%.

Figures

SNP-based unrooted phylogenetic tree of jzb001 and select members of the Aerococcaceae family. A consensus tree was constructed based on 100 bootstraps. Node labels indicate bootstrap values. Other sequences were acquired from NCBI RefSeq. Light purple indicates Ruoffia clade. Purple tip point indicates jzb001 and its closest match according to GTDBTk. Best fit model: GTR+F+I+G4; consensus tree log-likelihood: −15,106.811.

Tables

TABLE 1. jzb001 draft genome sequence metrics and results

Metric	Result	Tool
Number of contigs	1	QUAST
GC content	36.66%	QUAST
Total size	2,363,349 bp	QUAST
N 50	2,363,349 bp	QUAST
Coverage/depth	89×	Flye
Estimated completeness	90.5%	Busco
Predicted genes	2,307 CDS (total), 49 tRNA, 15 rRNA, 4 ncRNA,and 124 pseudogenes	PGAP
Virulence factors	None detected	VFDB
Antimicrobial resistance (AMR) genes	tet(M), ANT (6)-Ia	CARD

Full text

ANNOUNCEMENT

Bovine respiratory disease (BRD) is the most devastating disease affecting American cattle producers, and research has illustrated a strong connection between the respiratory microbiota and BRD. Regardless, historically, most research regarding BRD and the respiratory microbiota has focused on the four major opportunistic pathogens (1). Commensal members of the bovine respiratory microbiota were isolated from nasal swabs. jzb001 was isolated from the nasal swab of a healthy steer from a research feedlot in West Texas (34.966086647966726,–101.80246082318041). A 1:10 dilution of swab buffer was plated onto Mycoplasma base agar (Criterion, Hardy Diagnostics, Santa Maria, CA, USA) + Mycoplasma supplement (Difco, BD, Sparks, MD, USA) inside a BSL-2 certified BioSafety Cabinet, and jzb001 was isolated after 72 hours of aerobic incubation at 37°C. Ruoffia tabacinasalis (formerly Facklamia tabacinasalis) was initially isolated and identified as a tobacco powder contaminant (2) and was recently reclassified as R. tabacinasalis (3). To our knowledge, this is the first time R. tabacinasalis has been isolated from the bovine nasal cavity.

For whole-genome sequencing, jzb001 was cultured overnight at 37°C on brain heart infusion agar. Three loops of overnight culture (originating from a single colony) were suspended in phosphate-buffered saline and vortexed in a bead beater at 3,500 rpm for 2 minutes for genomic DNA isolation using the Qiagen QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Next, the Rapid Barcoding Library Preparation Kit and AMPure XP beads were used to prepare the sequencing library (Oxford Nanopore Technologies). Sequencing was performed using the Oxford Nanopore MinION Mk1C sequencer with the flow cell R9.4.1 (FLO-MIN106D) for 30 hours. Guppy (version 6.1.2) was used for basecalling and trimming of DNA sequencing data obtained from the sequencer (4). The circular genome was assembled with Flye (version 2.9) (5) and polished with Racon (version 1.5.0) (6). Quality was assessed using QUAST (version 5.0.2) (7). The estimated N50 for jzb001 was 2.36 Mb (Table 1), and one contig was generated. Default parameters were used unless otherwise noted.

jzb001’s genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (version 6.6) (8) (Table 1), and GTDBtk (version 2.1.0), along with its database (R207_v2), was used for taxonomic classification (9). jzb001 was identified as R. tabacinasalis [fastani_reference: GCF_005864045.1; average nucleotide identity (ANI): 96.5; alignment fraction: 0.82] using only ANI. As the above genome was not the R. tabacinasalis type strain, taxonomic assignment was confirmed by comparing jzb001 to the R. tabacinasalis type strain (GCF_015863285.1) using fastANI (version 1.33) (10) (ANI: 95.7). Busco (version 5.5) (11) (lineage: Lactobacillales_odb10) was used to estimate genome completeness (Table 1). PhaME (version 1.0.5) (12) was used to calculate the core genome SNPs of jzb001 and members of the Aerococcaceae family [reference: Globicatella sanguinis (GCF_002847845.2)]. The SNP alignment was used to reconstruct an unrooted phylogenetic tree using IQ-TREE (version 2.2.5) (13, 14), which was visualized using the ggtree R package (version 3.6.2) (15) (Fig. 1). Virulence factors were detected using the Virulence Factor Database (accessed 13 December 2023) (16, 17). AMR genes were detected using the Comprehensive Antibiotic Resistance Database (version 3.2.8) (18, 19) (Table 1).

SNP-based unrooted phylogenetic tree of jzb001 and select members of the Aerococcaceae family. A consensus tree was constructed based on 100 bootstraps. Node labels indicate bootstrap values. Other sequences were acquired from NCBI RefSeq. Light purple indicates Ruoffia clade. Purple tip point indicates jzb001 and its closest match according to GTDBTk. Best fit model: GTR+F+I+G4; consensus tree log-likelihood: −15,106.811.