Draft genome sequences of six Brucella melitensis isolates collected from humans and livestock in Tanzania

TL;DR

This paper reports the genome sequences of six Brucella melitensis strains from Tanzania, offering insights into the spread of this disease in humans and livestock.

Contribution

The study provides new genome sequences of Brucella melitensis from Tanzania, enhancing understanding of local strain diversity.

Findings

Six Brucella melitensis isolates were sequenced from Tanzania’s Kagera region.

The genomes will aid in comparative genomics and brucellosis control strategies in East Africa.

Abstract

We present genome assemblies of six Brucella melitensis strains isolated from goats and humans in Tanzania’s Kagera region. These sequences provide insight into circulating Brucella strains in Tanzania and East Africa. These data will support future comparative genomics, epidemiological investigations, and regional brucellosis control strategies.

Figures

Phylogenomic tree of newly sequenced Brucella strains from Tanzania along with closely related sequences from NCBI assemblies. Leaves are labeled with NCBI’s accession and country of origin, and the symbol color at leaf tips indicates the broader region of origin, while the new sequences are indicated by pink symbols. Branch lengths are proportional to the inferred number of mutations/site. All splits are supported with 100% of aLRT 1000 replicates. Tree was inferred from all available B. melitensis assemblies in RefSeq/Genbank (accessed 3 Oct 2024) with the OrthoPhyl pipeline (v2.0) (14). OrthoPhyl was run with defaults except only strict single-copy orthologs were concatenated into the final codon supermatrix, and iqtree[] options “--lmap 2000 --symtest -B 1000 -t PARS --ninit 2 -m MFP -p partions.txt” were invoked. Tree was visualized with ggtree in R (15).

Tables

TABLE 1. Genome assembly statistics, annotation results, and accession numbers

	Sample
	TZ_Kagera_H01	TZ_Kagera_H02	TZ_Kagera_H03	TZ_Kagera_G01	TZ_Kagera_G02	TZ_Kagera_G03
Genome size	3,280,749	3,281,053	3,281,099	3,277,679	3,283,154	3,283,061
Contigs	38	38	37	236	37	37
Longest contig	609,536	609,552	609,536	80,579	609,570	609,586
N50	143,366	174,766	174,766	23,443	174,791	174,791
Mean contig length	86,335.50	86,343.50	88,678.35	13,888.47	88,733.89	88,731.37
GC_percent	0.57	0.57	0.57	0.57	0.57	0.57
CDSa	3,125	3,128	3,126	3,195	3,129	3,129
rRNAa	3	3	3	4	3	3
tRNAa	49	51	50	50	51	51
Coding density	0.87	0.87	0.87	0.87	0.87	0.87
Completeness	99.45	99.45	99.45	99.06	99.45	99.45
Contamination	0.67	0.67	0.67	1.79	0.67	0.67
NCBI BioSample	SAMN44407420	SAMN44407421	SAMN44407422	SAMN44407423	SAMN44407424	SAMN44407425
NCBI accessions	JBIQWR000000000	JBIQWQ000000000	JBIQWP000000000	JBIQWU000000000	JBIQWT000000000	JBIQWS000000000
SRA accession	SRR31174980	SRR31174979	SRR31174978	SRR31174977	SRR31174976	SRR31174975
Raw reads	3,302,222	3,294,052	3,219,745	13,196,957	3,211,131	3,081,903
Collection GPS	−1.603449831.141049	−1.603449831.141049	−1.534632431.1533194	−1.31258531.20451	−1.31258531.20451	−1.31258531.20451

Full text

ANNOUNCEMENT

The molecular epidemiology of brucellosis in Tanzania is critical for effective disease control. The country’s heterogeneous agricultural practices create complex multi-host transmission networks for Brucella species. Whole genome sequencing and phylogenetic analyses comparing Tanzanian isolates with global data sets provide insights into strain diversity and regional pathogen dissemination patterns across East Africa (1).

Brucella melitensis, the predominant cause of human brucellosis, poses significant public health challenges on the African continent (2, 3). Here, we present six Brucella melitensis isolates from the Kagera region of Tanzania, providing molecular insights into strain diversity from this important livestock-producing area. Three isolates were obtained from goat milk (TZ_Kagera_G0-) and three from human patients in Karagwe district (TZ_Kagera_H0-). The animals and humans sampled were from a region where goats intermingled with cattle and sheep during grazing. Goat milk was collected (~5 mL) into sterile Falcon tubes, pooled from all four quarters after cleaning the udder and discarding the first few drops. Collected milk was immediately stored at 18°C. For human subjects, 5 mL of blood was aseptically taken by registered nurses from the brachial vein using heparinized tubes and immediately stored at −20°C. Both blood and milk were cultured on Brucella selective medium (Farrell’s medium) under both aerobic and anaerobic conditions (5% CO_2_) at 37°C for about 4 days (4, 5). Suspected Brucella colonies were subcultured to obtain pure isolates. Cultures were incubated at 37°C in 5%–10% CO_2_ and observed for 10 days. Suspected Brucella-positive cultures were confirmed by subculture on Farrell’s medium and MacConkey agar (6).

Isolates were plated on blood agar and incubated at 37°C in 5%–10% CO_2_ for up to 3 days. Two to three colonies were selected, heat-inactivated at 100°C for 10 minutes (7), and DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). DNA quality was confirmed via qPCR targeting the Brucella-specific IS711 sequence (8), NanoDrop spectrophotometer, and Qubit 4.0 fluorometer. Genomic libraries were prepared using the Illumina DNA Prep kit. One sample was sequenced with the P1 600-cycle kit (Kagera_G01), and the others with the P1 300-cycle kit (2 × 151 bp reads), all on an Illumina NextSeq 2000 platform.

Sequence analysis was performed within the EDGE bioinformatics UI platform (v2.4.0) (9). Reads were trimmed and filtered using faQC (v2.08) (10) with three bases clipped from each end and removing reads below 20 average quality and/or 50 bp length. Reads were assembled using IDBA (v1.1.1) (11) with options "--pre_correction --mink 31 --maxk 121 --step 20 --min_contig 200.” CheckM (v1.2.2) (12) was used to estimate completeness and contamination, while Prokka (v1.14.5) (13) was used to predict coding sequences (CDSs), tRNAs, and rRNAs (Table 1). Contigs less than 700 bp long were filtered out of the final assemblies.

Phylogenetic relationship between the six new sequences and publicly available Brucella sequences shows the new sequences cluster tightly with the closest relatives being from Belgium, Kuwait, Somalia, and Norway (Fig. 1). These six new sequences increase our understanding of local B. melitensis strain diversity, supporting future comparative genomics, epidemiological studies, and regional brucellosis control efforts.

Phylogenomic tree of newly sequenced Brucella strains from Tanzania along with closely related sequences from NCBI assemblies. Leaves are labeled with NCBI’s accession and country of origin, and the symbol color at leaf tips indicates the broader region of origin, while the new sequences are indicated by pink symbols. Branch lengths are proportional to the inferred number of mutations/site. All splits are supported with 100% of aLRT 1000 replicates. Tree was inferred from all available B. melitensis assemblies in RefSeq/Genbank (accessed 3 Oct 2024) with the OrthoPhyl pipeline (v2.0) (14). OrthoPhyl was run with defaults except only strict single-copy orthologs were concatenated into the final codon supermatrix, and iqtree[] options “--lmap 2000 --symtest -B 1000 -t PARS --ninit 2 -m MFP -p partions.txt” were invoked. Tree was visualized with ggtree in R (15).