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Microbiol Resour Announc
Microbiol Resour Announc
mra
Microbiology Resource Announcements
2576-098X
American Society for Microbiology 1752 N St., N.W., Washington, DC

39082814
mra00087-24
10.1128/mra.00087-24
mra.00087-24
Genome Sequences
bacteriologyBacteriologyDraft genome sequence of Ruoffia tabacinasalis isolated from a bovine nasal swab: a novel member of the bovine nasal microbiota
https://orcid.org/0000-0003-0632-8106
Howe Samantha 1 Data curation Formal analysis Writing – original draft Writing – review and editing
Wei Xiaoyuan 2 Data curation Writing – review and editing
https://orcid.org/0000-0002-9465-4552
Kovac Jasna 2 Data curation Writing – review and editing
https://orcid.org/0000-0002-2894-8560
Zhao Jiangchao 1 Conceptualization Writing – review and editing jzhao77@uark.edu

1 Department of Animal Science, University of Arkansas , Fayetteville, Arkansas, USA
2 Department of Food Science, Pennsylvania State University , State College, Pennsylvania, USA
Editor Klepac-Ceraj Vanja Wellesley College Department of Biological Sciences , Wellesley, Massachusetts, USA

Address correspondence to Jiangchao Zhao, jzhao77@uark.edu
The authors declare no conflict of interest.

9 2024
31 7 2024
31 7 2024
13 9 e00087-2425 2 2024
08 6 2024
Copyright © 2024 Howe et al.
2024
Howe et al.
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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%.

KEYWORDS

whole-genome sequencing
nasal microbiota
bovine
U.S. Department of Agriculture (USDA) https://doi.org/10.13039/100000199 20196701629869 Zhao Jiangchao cover-dateSeptember 2024
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pmcANNOUNCEMENT

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.

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	

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).

Fig 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.

ACKNOWLEDGMENTS

This study was supported by the USDA National Institute of Food and Agriculture (NIFA) Agriculture and Food Research Initiative (AFRI) Competitive Grant no. 20196701629869 and funds from the Arkansas Biosciences Institute.

Part of the data was also included in an author’s dissertation.

DATA AVAILABILITY

The assembled and annotated genome sequence was deposited at NCBI under the RefSeq accession number GCF_036866465.1, the BioProject accession number PRJNA1063262, and the BioSample accession number SAMN39331681. The raw reads were deposited into the NCBI SRA database under the run number SRR27966697. The other genome sequences utilized for SNP alignment and phylogenetic reconstruction include the following NCBI RefSeq accessions: GCF_013267415.1, GCF_002847845.2, GCF_023195815.2, GCF_003546865.1, GCF_002871685.1, GCF_000183205.1, GCF_000301035.1, GCF_000245795.1, GCF_012396555.1, GCF_002847645.1, GCF_000518205.1, GCF_014049385.1, GCF_005864045.1, and GCF_015863285.1.
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