Complete Genome Sequence of Brucella abortus 68, Isolated from Aborted Fetal Sheep in Ukraine

The complete genome sequence of Brucella abortus 68, isolated from an aborted sheep fetus in Luhansk, Ukraine, was assembled using Nanopore sequences. Two circular chromosomes totaling 3,281,317 bp (N50, 2,124,943 bp) comprised the complete genome sequence. The strain encodes the fosfomycin antibiotic resistance gene FosX, highlighting the risk of cross-species livestock and human infection.

A s part of a scientific initiative to enhance veterinary diagnostic capacity in Ukraine, we sequenced the genome of a Brucella sp. strain isolated in 1974 from the Luhansk region in Ukraine. This isolate is part of a historical collection of bacterial livestock pathogens archived at the National Scientific Center Institute of Experimental and Clinical Veterinary Medicine in Ukraine. The sequencing of this strain using the Oxford Nanopore Technologies (ONT) MinION platform in veterinary laboratories in Ukraine represents a genomic exploration of this collection and will provide insight into circulating livestock pathogens.
The abomasum content of an aborted sheep's fetus was added to meat-peptoneliver-glucose-glycerol (MPLGG) broth at 37°C overnight and then plated onto MPLGG agar. All media contained 20% bovine serum. A single colony was taken for pure culture. The isolated strain was lyophilized and stored until revival for sequencing in 2019. To revive the lyophilized strain, we added MPLGG broth without serum and then plated it onto MPLGG agar. For DNA extraction, we isolated a single colony by loop and added it to lysing buffer as input for the DNeasy UltraClean microbial kit (Qiagen).
We used 1 mg of DNA as input for a rapid sequencing library (SQK-RAD004; ONT) and sequenced it on an R9.4.1 flow cell (FLO-MIN106; flow cell ID FAK90503) for 48 h using a MinION Mk1B device. We base called the raw data using Guppy v4.2.2 (ONT) using the high accuracy model (-c dna_r9.4.1_450bps_hac.cfg) and default parameters. This run generated 11,677,814,462 bp in 3,163,635 reads with an average read length of 3,691 bp. We used Filtlong v0.2.0 (https://github.com/rrwick/Filtlong) to filter reads #50 bp long (-min_length 50) and with a Q score of #10 (-min_mean_q 90). After filtering, we had 8,786,405,255 bp in 2,101,589 reads with a read length N 50 of 7,747 bp and a median Q score of 13.0.
Data availability. This whole-genome project has been deposited in GenBank under accession no. CP066175 and CP066176. The versions described in this paper are the first versions, CP066175.1 and CP066176.1. The raw data for this project can be found in the SRA under accession no. PRJNA685163.

ACKNOWLEDGMENTS
Many thanks go to Xiao Bai, Elaina Milton, Cora Lyon, and Oleksander Tarasov for technical assistance. We thank Ihor Halka, Nataliya Mykhaylovska, and Olga Fedorenko for project support.
This work was funded by the U.S. Defense Threat Reduction Agency (DTRA) through the Biological Threat Reduction Program in Ukraine (Ukraine Project-9). The contents of this publication are the responsibility of the authors and do not necessarily reflect the views of the DoD, NIH, or the United States Government. The research reported in this publication was in part supported by an NIH NIGMS Institutional Development Award (IDeA), grant no. 2P20GM103395 (Alaska INBRE). We also acknowledge the Antimicrobial resistance mechanism Gene(s) Antibiotic inactivation enzyme fosX Antibiotic target in susceptible species alr, ddl, EF-G, EF-Tu, folA, dfr, folP, gyrA, gyrB, inhA, fabI, Iso-tRNA, kasA, murA, rho, rpoB, rpoC, S10p, S12p Efflux pump conferring antibiotic resistance macA, macB, triABC-opmH Gene conferring resistance via absence gidB Protein altering cell wall charge conferring antibiotic resistance gdpD, pgsA Regulator modulating expression of antibiotic resistance genes oxyR generous support of the Institute of Arctic Biology at UAF and the UAA College of Arts and Sciences.