Introduction

Brucellosis is a neglected zoonotic disease caused by Brucella bacteria, characterized by its complex genomic adaptability that enables the pathogen to evade the host’s immune system and persist intracellularly. Brucella, a Gram-negative bacterium, affects both humans and various animals, primarily ruminants such as cattle and sheep. In these animals, the immune system produces specific responses to counteract the pathogen’s invasion. However, humans lack immunity to Brucellosis, which leads to persistent infection with minimal or no immediate immune response. The disease is mainly transmitted through the digestive and respiratory tracts, but can also spread via contact with the skin, mucous membranes, or contaminated environments and objects from infected animals. Once infected, humans may exhibit clinical symptoms such as fever, excessive sweating, joint pain, fatigue, and swollen lymph nodes. The Brucella species most significant to human health include Brucella suis, Brucella melitensis, and Brucella abortus1,2,3. Brucellosis primarily spreads through direct contact within the same animal species but can also infect secondary hosts, such as humans. Human exposure typically occurs through contact with infected sheep or their products, as well as through activities such as slaughtering, handling raw meat, selling, and transferring live animals. Additional routes of transmission include person-to-person spread, such as through blood transfusions or organ transplants, and exposure to contaminated environments4,5.

In developed countries, the incidence of brucellosis has steadily declined, with some regions even managing to eradicate it. However, in low- and middle-income countries, Brucellosis remains a significant public health and animal health challenge6. The disease causes substantial reproductive losses in livestock and debilitating illness in humans7. Furthermore, its management remains problematic due to emerging complications, demographic changes, persistence in pastoral communities, and increasing prevalence in regions with high numbers of small ruminants8,9. In China, Brucellosis is a key zoonotic disease for prevention and control. It remains prevalent in cattle and sheep populations, severely impacting the economy through decreased fertility, higher abortion rates, and reduced milk and meat production10.

Recent bioinformatics advancements have significantly improved our understanding of Brucella pathogenesis and evolution. Whole-genome sequencing and comparative genomics have identified key virulence factors, such as lipopolysaccharide (LPS) and O-antigen biosynthesis genes, which play crucial roles in immune evasion and intracellular survival. Liu et al.11 designed CRISPR RNA (crRNA) and RAA primers based on the conserved sequence of the Brucella Bcsp31 gene. In addition to this work, which developed integrated RAA-CRISPR/Cas13a detection methods (readable by both fluorescence and paper strips), machine learning models have identified novel antigenic targets12, aiding the design of higher-specificity subunit vaccines. These computational strategies, combined with phylogenetic networks, emphasize the importance of horizontal gene transfer in Brucella diversification, particularly in zoonotic strains13. Bioinformatics analysis of Brucella genomes has revealed conserved virulence-associated loci14, such as ialB homologs in Rhizobiales, and identified novel type IV secretion system (T4SS) effectors through comparative proteomics15. These findings underscore evolutionary convergence in pathogenic mechanisms across zoonotic pathogens.

Tettelin et al.16 established the pan-genome as a foundational framework for genomics, characterizing it as the full complement of non-redundant genes within a species, which includes universal core genes and strain-specific variable genes. Unlike bacteria, eukaryotes are not prone to frequent horizontal gene transfer and thus display relatively limited gene presence polymorphisms17. The application of this pan-genome concept now enables comprehensive studies of intraspecific variation, interspecies gene exchange, and the genomic basis of domestication and improvement across plants, fungi, and animals.Many bacterial species are characterized by a pangenome, a collective gene pool that exceeds the genome of any individual strain due to variation in accessory genes18.The growth of public genomic repositories, driven by progress in sequencing, now enables the in-depth dissection of these extensive pangenomes19.

This study conducted a comprehensive analysis of 40 Brucella strains with well-documented origins. After retrieving their complete genomes from NCBI, we performed Average Nucleotide Identity (ANI) analysis, constructed a phylogenetic tree, and characterized their pan-genomic architecture. Furthermore, secondary metabolite biosynthetic gene clusters were identified using AntiSMASH, and the resistome and virulome were profiled using the Fabric platform. These discoveries elucidate key aspects of Brucella biology and pave the way for developing effective prevention and treatment strategies.

Materials and methods

Ethical statement

As no animal subjects were involved in this study, ethical approval or written consent was not required.

Data collection

The genomes of 40 Brucella strains were retrieved from the NCBI database to investigate Brucellosis at the genomic level.The genome sizes of all strains range from 4.88 MB to 6.00 MB, with the largest genomes found in the Brucella suis NPDC055919 and IPA7.2 strains, and the smallest genome in the BU72 strain derived from pituitary tumors. The whole-genome G+C content of these strains varies between 53.00% and 60.50%. A common characteristic shared by all strains is GC content and the number of ribosomal clusters. G+C content correlates with various intrinsic and extrinsic factors; a high G+C content indicates greater stability in genetic material, providing a more robust genome that is less susceptible to environmental changes20.As detailed in Table 1.

Table 1 Basic genomic information of 40 strains of Brucella.
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Construction of genomic phylogenetic trees

Genomic sequences from 40 Brucella strains were analyzed using FastTree v2.1.11 and fastANI v1.33. Phylogenetic relationships inferred from the phylogenetic tree were compared with those derived from Average Nucleotide Identity (ANI) analysis.

Pan-genome and core genome analysis

The pan-genome and core genome dynamics across the 40 Brucella strains were assessed using PGAP v1.2.1 and an upset plot generated using R software.Gene family (GF) evolution was modeled based on alignments generated with Diamond BLASTP v2.0.14, under the following parameter thresholds: –matrix BLOSUM45, –evalue 1e-5, score ≥ 40, identity ≥ 0.5, and coverage ≥ 0.5.

Analysis of secondary metabolites

Putative secondary metabolite biosynthetic gene clusters (BGCs) in the 40 Brucella genomes were predicted using antiSMASH v7.1.0. The analysis was conducted with the following parameters: -t bacteria, enabling comprehensive detection of –cb-general, –cb-subclusters, and –cb-knownclusters.

Analysis of antibiotic resistance genes and pathogenicity genes

Using Blastp, we conducted an analysis of antibiotic resistance genes and pathogenicity genes in the genomic sequences of 40 Brucella strains.The threshold parameters for the identification of resistance genes were set as follows: E-value ≤ 1 × 10⁻5, maximum target sequences = 1, and sequence identity ≥ 45%. Additionally, the alignment length of the query sequence was required to cover no less than 70% of the corresponding reference resistance gene sequence. For virulence gene detection, the following criteria were applied: E-value ≤ 1 × 10⁻5, maximum target sequences = 1, and sequence identity ≥ 60%. Moreover, the aligned region of the query sequence must span at least 70% of the length of the respective virulence factor-associated gene.

Results

Genomic phylogenetic tree analysis of 40 Brucella strains

The whole genome phylogenetic tree (Figs. 1 and 2) illustrates that the 40 Brucella strains are categorized into three distinct branches: the first branch contains the Brucella anthropi CCUG 34461 strain, the second branch consists of the Brucella sp. NBRC 13694 strain, and the remaining strains are grouped into the third branch, represented by Brucella anthropi MAG47. Phylogenetic analysis results align with those from the ANI analysis, with most strains from distantly related genetic backgrounds originating from Brucella tritici, Brucella intermedia, and similar species. The likelihood of strains from the same source clustering within smaller branches is higher, indicating closer evolutionary relationships between Brucella strains from human and animal sources.

Fig. 1
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Whole genome phylogenetic tree of 40 Brucella strains.

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Fig. 2
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Build phylogenetic tree based on ANI data.

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Pan-genome and core genome analysis of 40 strains of Brucella

Pan-genome and core-genome analysis was performed on 40 representative Brucella strains from various isolated sources. The analysis revealed a total of 187,623 functional genes encoding proteins, which were grouped into 21,800 gene families. Among these, the core genome consists of 198 gene families, representing 0.91% of the Brucella pan-genome. The number of unique genes was 10,371, accounting for 47.6% of the pan-genome. The strain with the highest number of unique genes is B. endophytica CGMCC1.15082, which contains 1464 unique genes.

To assess the relationship between the pan-genome, core genome, and genome number, PanGP was used to fit the data, resulting in the data box plot shown in Fig. 3. In this plot, red represents the pan-genome size, while green represents the core genome size. Figure 3 illustrates that as the number of genomes increases, the size of the pan-genome expands. The fitting equation for the relationship between the pan-genome (y) and genome size (x) is y = 3281.2x0.496 + 1327.0. Meanwhile, the core genome size decreases with an increasing number of genomes, with the fitting equation for the relationship between the core genome (M) and genome size (N) being M = 3975.6e−0.142N + 308.5. This indicates that Brucella has an open pan-genome.The results were validated using an upset plot generated with R software, confirming no discrepancies (Fig. 4) .

Fig. 3
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Characteristic curves of pan-genome and core-genome.

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Fig. 4
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An upset plot created using R software.

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Analysis of secondary metabolites of 40 Brucella strains

Using antiSMASH online software for secondary metabolite prediction, a total of 18 categories and 350 gene clusters related to secondary metabolism were identified across the 40 Brucella strains. These gene clusters were found to produce 298 secondary metabolites, including 39 arylpolyene, 38 acyl-amino acids, 38 betalactones, 38 terpenes, 36 hydrogen cyanide, 33 NAGGN, 18 NI-siderophores, 16 hserlactones, 15 ectoines, and 13 NAPAA, among others (Fig. 5).

Fig. 5
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Statistics of secondary metabolites of 40 Brucella strains.

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Analysis of resistance genes and virulence genes in 40 strains of Brucella

Genome sequences of the 40 Brucella strains were uploaded in FASTA format to the CARD resistance gene database for alignment, resulting in the identification of seven resistance genes: rpsE, rpsL, rosA, golS, fabG, fabI, and uL3. Among these, the B. anthropi SBA01 and B. media Q1108 strains exhibited the highest number of resistance genes (Fig. 6). Information regarding each gene was retrieved from the database: rpsE encodes a product that confers resistance to spectinomycin; rpsL encodes resistance to aminoglycoside antibiotics; rosA encodes an antimicrobial peptide efflux pump, providing resistance to antimicrobial peptides; golS encodes an antibiotic efflux pump, which facilitates resistance to carbapenems, cephalosporins, monobactams, and benicol antibiotics; fabG and fabI are involved in resistance to isoniazid drugs; and uL3 encodes an efflux pump that provides resistance to pleurodesin antibiotics.

Fig. 6
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Venn diagram of resistance genes in 40 Brucella strains.

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Virulence gene analysis via the VFDB database identified eight virulence genes: lpxC, acpXL, fliY, bspJ, lpxA, fliI, fliQ, and bvrR. The lpxC gene immunoregulates N-acetylglucosamine deacetylase, while acpXL encodes an immunomodulatory acyl carrier protein. BspJ is a T4SS effector, and lpxA encodes an immunomodulatory UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase. FliY, fliI, and fliQ are flagellar-specific ATP synthases, and bvrR is a transcription factor that regulates responses. All 40 Brucella strains were found to contain the virulence genes lpxC, acpXL, fliY, and bspJ. (Fig. 7).

Fig. 7
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Venn diagram of virulence genes in 40 Brucella strains.

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Discussion

Brucella is a Gram-negative, non-motile bacterium that lacks a capsule (presenting as a smooth type with microcapsules). It is positive for urease and oxidase, strictly aerobic, capable of reducing nitrate, and exhibits intracellular parasitism, enabling it to survive in a wide range of livestock species. This study selected 40 representative Brucella strains from various isolated sources obtained from the NCBI database. Analysis of their pan-genome, core genome, and genome number revealed that Brucella possesses an open pan-genome with high genetic diversity. Similar findings have been observed in other Gram-negative bacteria, such as Hansen’s disease21, Francisella tularensis22, and Chlamydia trachomatis23, indicating a genetic similarity between Brucella and other Gram-negative bacterial strains.

This study further characterized the secondary metabolites produced by the 40 Brucella strains, identifying 350 metabolites distributed across 18 distinct categories. Among these, arylpolyene was found to possess antibacterial activity, suppressing the growth of pathogenic bacteria while helping maintain intestinal microbiota homeostasis. It also contributes to the regulation of host metabolism, enhancement of immune responses, and mitigation of host health disorders. A structurally related compound, referred to as the “yellow-affinity substance” (YAS) in the anaerobic thermophilic archaeon Thermococcus, has been identified as a specific aryl-polyene alkaloid complex known as fibroflavone. Such compounds have demonstrated antibiotic effects against Gram-positive bacteria, including clinically relevant strains24. In addition, research by Zai et al.25 on Brucella metabolic pathways revealed that a coordinated set of processes—including oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle (TCA cycle), thiamine metabolism, and nitrogen metabolism—plays a central role in restoring cellular homeostasis and metabolic equilibrium under stress conditions. Dutta S26 found that clusters of arylpolyene and an unidentified small linear peptide exhibit antifungal and biocontrol activity, demonstrating their potential in biocontrol. Acyl-amino acids, which are formed by the amide bond between an amino acid and a fatty acid, play pivotal roles in energy metabolism, neuroprotection, and anti-inflammatory processes. Research by Larrick JW27 indicates that N-acyl amino acids, at physiological concentrations, act as endogenous uncoupling agents for mitochondrial respiration. In cell cycle regulation, Clasto Lactacystin beta-lactone significantly impacts the progression of osteosarcoma cell lines, inhibiting the cell cycle. This has potential therapeutic applications for controlling the growth and proliferation of tumor cells, offering new insights and targets for osteosarcoma treatment. From an enzyme inhibition perspective, Clasto Lactacystin beta-lactone serves as an irreversible 20S proteasome inhibitor. The 20S proteasome is pivotal in the degradation and turnover of cellular proteins, and its inhibition can affect various cellular processes, including cell signaling and gene expression regulation. Additionally, beta-lactone acts as a partial inhibitor of branched-chain amino acid-preferring peptidase (BrAAP), inactivating approximately 60% of BrAAP activity, with the remaining 40% inactivated at a slower rate. This suggests that its inhibition of BrAAP is stage-specific, contributing to the precise regulation of intracellular metabolic pathways28.

The FASTA sequences of 40 Brucella strains were uploaded to the CARD resistance gene database for comparison, which identified a total of seven resistance genes in Brucella. The strains B. anthropi SBA01 and B. media Q1108 were found to carry the highest number of antibiotic resistance genes, indicating that these two strains are more difficult to eliminate with antibiotics. These resistance genes were also identified in other bacterial strains. For example, Erin E. Killeavy29 isolated spontaneous tiamulin-resistant mutants of the thermophilic bacterium Thermus thermophilus, containing either single amino acid substitutions in ribosomal protein uL3 or single base substitutions in the peptidyltransferase active site of 23S rRNA30. Rui-ru Shi31 investigated the rpsL and rrs gene mutations in Mycobacterium tuberculosis.Antimicrobial resistance in Brucella is generally mediated through five principal mechanisms facilitated by resistance genes: (1) enzymatic inactivation of antibiotics via protease degradation or functional group modification; (2) alteration of drug targets to circumvent antibacterial activity; (3) reduction in membrane permeability to limit intracellular antibiotic accumulation; (4) active efflux of antimicrobial agents through efflux pump systems; and (5) metabolic bypass mechanisms that diminish drug efficacy. Notably, the effectiveness of several conventional antibiotics—including vancomycin, carbapenems, and macrolides, which target cell wall integrity, membrane function, RNA, and protein synthesis—is substantially compromised by these resistance traits. These findings underscore growing concerns regarding the clinical utility of such drugs in treating brucellosis and emphasize the need to develop alternative therapeutic strategies.Wang32 revealed that the YejABEF ABC transporter contributes to Brucella virulence by mediating resistance to host antimicrobial agents, a process that underpins the pathogen’s ability to maintain long-term infection.

Additionally, the FASTA sequences of the 40 Brucella strains were uploaded to the VFDB virulence gene database for comparison, revealing a large number of virulence genes. A total of 8 virulence gene types were identified in Brucella, many of which are also present in other bacterial strains, posing significant risks to human safety. For example, Brevundimonas species harbor virulence-associated genes such as icl, tufA, kdsA, htpB, and acpXL, which encode isocitrate lyase, elongation factor, 2-dehydro-3-deoxyphosphooctonate aldolase, heat shock protein, and acyl carrier protein, respectively33.The study by Zhang et al.34 indicates that while BtpA and VjbR proteins potentially contribute to the residual virulence of Brucella melitensis M5-90, ABC transporters and thiamine metabolic proteins are also implicated as newly identified virulence factors.

Brucellosis remains a major zoonotic infectious disease in China, particularly prevalent in cattle and sheep populations. It exerts a significant economic impact, manifesting as reduced fertility, increased miscarriage rates, and declines in milk and meat production, all of which severely affect the agricultural sector and directly threaten human health2,10. Human Brucellosis infections primarily occur through contact with infected animals or contaminated livestock products, highlighting the need for enhanced public awareness and protective measures. Nationwide reports indicate an increasing incidence of Brucellosis, particularly in Xinjiang, where the rise in cases has become a pressing public health concern. Human exposure to Brucella-infected sheep, their byproducts, and contaminated items, as well as activities like slaughtering, processing raw lamb, sales, and animal transfers, are common transmission routes4,5. These factors not only lead to economic hardship for herders but also present a serious health risk to their communities, as emphasized in China’s “Brucellosis Diagnosis and Treatment” guidelines.

A daily inspection system has been implemented to strengthen the prevention and control of Brucellosis in livestock, reduce its incidence and transmission risks, promote the sustainable development of animal husbandry, and safeguard public health. This system ensures timely inspection, quarantine, sampling, testing, and reporting on the movement of cattle and other livestock, addressing human brucellosis infections, high miscarriage rates, and other suspicious conditions. Additionally, a mandatory immunization plan for Brucellosis has been developed and improved, targeting cattle and sheep farms. This includes strengthening file management, enhancing immunization records, conducting regular immunization assessments, and ensuring both immunization coverage and efficacy. These measures aim to bolster population immunity and lay a strong foundation for Brucellosis prevention and control. Special public awareness campaigns have been conducted for key groups, such as those involved in breeding, transportation, slaughtering, and processing, to continuously improve their understanding of prevention and guide them on proper disinfection, isolation, and other protective measures35.

Conclusion

In conclusion, this study systematically analyzed the genomic and biological characteristics of Brucella strains from various sources. Brucella possessed an open pan-genome, with 12 gene clusters associated with the synthesis of secondary metabolites, several of which exhibit potential antibacterial properties. Seven types of resistance genes were identified (rpsE, rpsL, rosA, golS, fabG, fabI, and uL3), with strains B. anthropi_SBA01 and B. media_Q1108 harboring the highest number of resistance genes. Additionally, eight virulence genes (lpxC, acpXL, fliY, bspJ, lpxA, fliI, fliQ, and bvrR) were identified, with B. cytisi_IPA7.2 carrying the most virulence genes. Notably, lpxC and acpXL may serve as unique virulence factors in Brucella compared to other strains. These findings provide a comprehensive understanding of Brucella and offer a solid foundation for its prevention and treatment.