Crocins, the main active ingredients in Crocus sativus, have anti-inflammatory, antioxidant, hypolipidemic, hypoglycemic, and antitumor properties. Due to the harsh growing conditions and the limited yield of C. sativus, sustainable alternative methods for crocin production need to be explored. In this study, a new rice germplasm resource, "crocin rice", was developed by expressing eight genes of the crocin synthesis pathway in rice endosperm. The content of crocins in rice seeds was up to 9.25 μg/g dry weight, and it was also rich in a variety of carotenoids, including α-carotene, lutein, and violaxanthin. The expression of exogenous genes did not affect normal growth or the major nutritional structure of rice. Feeding experiments on mice showed that crocin rice could effectively reduce lipopolysaccharide-induced liver injury. With the adjuvant therapeutic benefits, crocin rice could be used to improve human nutrition and health. In addition, rice callus also showed the ability to synthesize crocins, which could serve as a potential substitute for C. sativus to alleviate resource shortage.

Over two decades after its initial detection in Klebsiella pneumoniae, the aminoglycoside resistance gene armA is now widely disseminated, having been reported in 16 genera from eight families, predominantly in Klebsiella spp. (Enterobacteriaceae) and Acinetobacter spp. (Moraxellaceae). Here, we identified a 243-bp remnant of the insertion sequence common region (ISCR) 28 containing a 2-bp truncation at the origin of insertion (oriIS) end (∆ISCR28) in the intergenic region between insertion sequence Escherichia coli 28 (ISEc28) and armA within the original armA dissemination vehicle, transposon Tn1548, extending the inferred mobilization history of armA. Comparative analysis of genetic contexts in 17 representative sequences from different hosts revealed conservation of ∆ISCR28 in all sequences, implicating ISCR28 in the initial capture of armA, and suggesting, under a parsimonious evolutionary model, that this event preceded its association with ISEc28 and ISEc29. A rapid amplification of cDNA ends (5'-RACE) experiment confirmed a transcription start site of armA located 146 bp upstream of the start codon, and showed that the promoter region PCR28-1 lies within the right arm of ISCR28 and is required for armA expression. The tandem array ISCR1-ISEc28-∆ISCR28 extending to the downstream insertion sequence (IS)26 was more conserved during transmission than the IS26-ISCR1 region. Analysis of 8-bp target site duplications flanking IS26 indicated that downstream IS26 in Tn1548 co-transferred with armA, whereas the upstream IS26 did not. Collectively, these findings clarify the role of ISCR28 in mediating armA expression and initiating its mobilization.

INTRODUCTION:Brucellosis remains a severe zoonotic threat in the Inner Mongolia Autonomous Region of China. METHODOLOGY:This study integrates a comprehensive epidemiological trend analysis with a novel methodological comparison of forecasting techniques to inform control strategies. RESULTS:Using reported human brucellosis surveillance data from Inner Mongolia for 2004-2024, joinpoint regression analysis revealed a persistently increasing yet fluctuating long-term trend (AAPC = 5.13%, P < 0.001), characterized by significant epidemic surges in 2004-2010 (APC = 22.43%, P < 0.001) and 2016-2021 (APC = 29.83%, P < 0.001), interrupted by a decline phase in 2010-2016 (APC = -17.17%, P < 0.001) and 2021-2024 (APC = -12.15). The disease demonstrated strong seasonality with June-August peaks, and predominance among farmers and herdsmen aged 30-60 years. Building on this epidemiological foundation, we rigorously compared the predictive performance of the standard Seasonal Autoregressive Integrated Moving Average (SARIMA) model against its Bootstrap-enhanced version for 24-month-ahead forecasting (2023-2024 validation). This finding offers a novel perspective on enhancing the predictive performance of brucellosis models. While the Bootstrap approach achieved superior point forecast accuracy by reducing Mean Absolute Error by 39.95% and Median Absolute Percentage Error by 33.55% compared to SARIMA, it produced severely overconfident prediction intervals, with only 33.33% empirical coverage compared to SARIMA's 91.67%. This study validates the SARIMA model as a robust baseline for brucellosis forecasting and introduces a Bootstrap ensemble method as a powerful tool for significantly enhancing point prediction accuracy. CONCLUSION:The findings provide novel epidemiological insights that offer a scientific basis for disease control measures and decision-making. Future work should aim to develop hybrid models that bridge this gap, and delivering high accuracy in both point and interval forecasts.

Floral organs typically function as heterotrophic tissues, heavily reliant on imported photoassimilates for respiration and development. However, under specific genetic or environmental conditions, they can reacquire photosynthetic capabilities. Here, we identified a rare green-flower (GF) mutant in Narcissus tazetta var. chinensis that exhibits stable green coloration and leaf-like characteristics. Physiological assays demonstrated that this mutant undergoes a fundamental metabolic transition from a heterotrophic state to a photoautotrophic state, characterized by functional Photosystem II activity and positive net photosynthetic rates. Integrated transcriptomic and metabolomic analyses identified the ectopic accumulation of Cytokinins (specifically Zeatin) as the primary key promoter of this transition. High Cytokinin levels correlated strongly with the activation of GOLDEN2-LIKE (NcGLK) transcription factors, which coordinated chloroplast biogenesis, stomatal development, and cell cycle re-entry. We propose a “Cytokinin-GLK” regulatory module governing floral greening, providing new insights into floral organ plasticity and potential strategies for breeding long-lasting ornamental varieties.

Abstract Subgingival biofilms in periodontitis exhibit spatial heterogeneity, yet the organization of microbial communities across periodontal niches remains incompletely defined. Using paired sampling and 16S rRNA gene sequencing, we characterized non-attached and attached subgingival plaque from patients with periodontitis, together with non-attached plaque from periodontally healthy individuals. Across diversity metrics and ordination analyses, non-attached plaque from periodontitis patients occupied positions between healthy-associated and attached-plaque communities. Taxonomically, these communities contained both health-associated commensals and anaerobic genera commonly enriched in periodontitis. Network analysis identified differences in association-network topology among niches, with the non-attached periodontitis network containing more retained associations than the healthy network. These cross-sectional results describe niche-associated patterns of subgingival community composition and association structure. They do not establish temporal progression, direct microbial interactions, or clinical utility.

Southwest China is a global biodiversity hotspot, and its complex and diverse ecosystems harbor vast amounts of “microbial dark matter.” This paper systematically examines the distribution characteristics of microbial dark matter in hosts such as arthropods, mammals, and birds, as well as in environments including soil, hot springs, and high-altitude lakes, with a particular focus on the cross-species transmissibility and pathogenic potential of emerging pathogens. Research indicates that new microbial species in the Southwest exhibit significant geographic concentration and host specificity: Yunnan Province is a core hotspot, while the Tibet Autonomous Region contributes a wealth of microbial resources due to its extreme environments, with arthropods and mammals accounting for the highest proportion of novel species. Regarding public health risks, eight novel pathogens with evidence of human infection have been identified, spanning the three major groups of viruses, bacteria, and parasites. The cross-species transmission potential of some pathogens (such as DPRV rhabdovirus, PPV arenaviridae, Luxi hantavirus, Banna virus and a novel Babesia species) has been confirmed through serological surveys or molecular testing. Deepening the exploration of microbial dark matter and risk early warning in this region will provide critical scientific support for public health safety monitoring.

The northeastern region of China is characterized by complex ecosystems, including forests and wetlands, and borders North Korea, Russia, and Mongolia. It serves not only as a natural reservoir for various microorganisms but also as a critical geographical and ecological hub for cross-border exchanges in Northeast Asia. Based on metagenomics and meta-transcriptomics investigations, this study systematically reviews the current research status of novel pathogens in the northeastern border region of China. It systematically organizes the newly discovered species, their classifications, and geographical distributions, with a focus on analyzing novel viruses that have potential pathogenicity to humans. The novel viruses identified in the northeastern border region belong to 11 viral families, including 9 from the Nairoviridae, 4 from the Rhabdoviridae, 3 each from the Astroviridae, Picornaviridae, and Parvoviridae, and 1-2 from other viral families, indicating a broad diversity of newly discovered viruses. These novel viruses are found in a wide range of hosts, including humans, ticks, minks, Marmota sibirica, and Myodes rufocanus, underscoring the significant public health risks these viruses pose. Geographically, the novel viruses discovered in the northeastern border region show a clustering pattern, with new species primarily concentrated in areas bordering Russia and North Korea. This highlights the unique role of the region as a hotspot for cross-border pathogen transmission and risk. The findings provide a systematic scientific reference for understanding the spectrum of unknown novel pathogens and their geographical distribution in the northeastern border region, assessing the risk of emerging infectious diseases, and optimizing active surveillance systems.

Abstract Small open reading frames (sORFs) are a potentially rich, yet error-prone, source of antimicrobial-peptide (AMP) candidates: short sequences are readily prioritized by AMP classifiers but may derive from incomplete gene calls. We developed a genome-context-aware discovery workflow that separates AMP-like sequence properties from evidence for a complete, recurrent coding locus. From 649,653 RefSeq assemblies representing 327 clinically relevant bacterial species, species-aware clustering and length filtering yielded 4,442,548 representative 10–100-aa sequences. AmpScanner v2, Macrel and AMPlify identified 585 non-haemolytic records supported by all three models. However, genome-context auditing of 11,918 mapped candidates showed that 529 of 536 mapped consensus candidates were supported exclusively by partial ORFs near contig termini. By contrast, 3,382 candidates had at least one complete non-edge occurrence; 1,069 recurred in ≥2 assemblies and 251 in ≥10 assemblies. We therefore assembled a 20-peptide panel through two explicitly labelled routes: sequence/structure-led selection (n=8) and genome-supported selection (n=12). Broth microdilution against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 identified low-micromolar activity in both routes. CAND_04141, a recurrent complete non-edge candidate, had the strongest combined profile (MICs of 4 and 2 μM, respectively), while CAND_07825 and CAND_04265 were also active at low micromolar concentrations. In plate-count MBC assays, all three advanced peptides achieved ≥3-log10 reductions at 128 μM. These findings show that high classifier agreement is not a substitute for genomic evidence and provide an auditable framework for prioritizing both synthetic AMP-like sequences and candidate genome-encoded peptides.

Influenza A virus antigenic drift forces annual vaccine reformulation, motivating the search for conserved epitope candidates that could support broadly protective vaccines. We systematically screened influenza A virus sequences (H1N1, H3N2, H5N1; nine viral proteins) to define 98 conserved candidate regions, 38 of which were identical across the H1N1, H3N2, and H5N1 consensus sequences, all in the polymerase complex and nucleoprotein (PB2, PB1, PA, NP), whereas the ten surface-glycoprotein (HA/NA) candidates were subtype-specific. We then benchmarked two protein-language-model (ESM-2) features against alignment conservation. Group-masked log-probability correlated moderately with MSA conservation (Spearman rho = 0.25 to 0.39 for HA) but provided no incremental value for T-cell epitope discrimination (change in AUROC +0.004, p = 0.46); attention-derived contact-density was not a valid solvent-accessibility proxy. A curated antibody-epitope benchmark (22 clusters, 5 neutralization-supported) was underpowered for a high-confidence B-cell test. We document data-quality and reproducibility pitfalls (length heterogeneity, coordinate mapping, and pseudoreplication) and release the auditable benchmark. These results provide an auditable candidate resource and show that, in the evaluated benchmarks, ESM-2 sequence scores did not improve epitope prioritization beyond alignment-derived conservation.

Human brucellosis is a re-emerging zoonotic disease in Yunnan Province, China; however, the molecular epidemiological profile of circulating strains remains unclear. In this study, bacteriology, multilocus sequence typing (MLST), and core genome MLST (cgMLST) were applied to investigate the epidemiological relationships among strains. Bacteriological analysis revealed that among the 127 strains, nine were identified as Brucella melitensis (B. melitensis) biovar. 1 and 118 as B. melitensis bv. 3, with these strains distributed across 13 cities and autonomous prefectures. Among the patients, 70.9% were male, 87.4% were livestock-exposed farmers, and 95.3% were aged 41–80 years, with infection peaking in the summer–autumn agricultural season (70.1% of cases). The 127B. melitensis strains were classified into two sequence types (STs): ST8 (n = 116) and ST39 (n = 11). ST8, is widely distributed in Northern China, whereas ST39 has mainly been reported in Southwestern China (Guizhou and Sichuan Provinces). These data indicate that B. melitensis sequence types ST8 and ST39 are jointly associated with the brucellosis epidemic in the human population of Yunnan Province. cgMLST analysis classified 127 strains into eight clusters (CI–VIII) and 21 STs, with 19 STs comprising strains from at least two cities isolated across two years. These findings suggest that the brucellosis epidemic in this region is driven by strains sharing a high degree of genetic homology. Global cgMLST analysis revealed high genetic similarity between the majority of strains in this study and isolates from Northern China, implying latent epidemiological links among these populations. Prioritizing dominant ST8 lineages is essential for effective brucellosis control and surveillance.

Nocardiosis is an uncommon infection that can cause severe damage or be life-threatening. However, the epidemiology and strain distribution of nocardiosis in China remain poorly characterized. In this study, a comprehensive literature search was conducted using the keyword “(‘Nocardia’ OR ‘Nocardiosis’) AND (‘China’ OR ‘Chinese’)” in PubMed and Web of Science, and “(Nuokajun) OR (Nukajun)” in the China National Knowledge Infrastructure (CNKI) database. All published cases of nocardiosis between April 1, 1990, and September 13, 2024, were included. Geographic analysis identified a concentration of cases in eastern coastal China. The mean patient age was 52.6 ± 17.4 years, with a male predominance (61.0%) and the highest burden in the 50–59 age group (24.4%). The primary infection sites were the lungs (69.9%), skin (30.3%), and brain/central nervous system (CNS) (19.3%). Disseminated infections accounted for 29.0% of all cases and were associated with a markedly higher mortality rate (16.1%) compared with the overall rate (9.0%), underscoring the importance of early diagnosis and treatment. The predominant species were Nocardia farcinica, Nocardia cyriacigeorgica, and Nocardia brasiliensis. Most isolates remained highly susceptible to trimethoprim-sulfamethoxazole, amikacin, and linezolid. This study has established a national epidemiological pattern of Nocardia infections and strain distribution in China. The elevated risk and mortality associated with disseminated infections warrant vigilance. This underscores the need for heightened clinical awareness of Nocardia infections and ensure timely screening and treatment.

A strain exhibiting high nitrate-removal efficiency under anaerobic conditions was isolated and taxonomically identified as Pseudomonas sp. DE6. Subsequent investigations revealed that supplementation with specific amino acids, including L-asparagine and glycine, substantially enhanced its nitrate-removal capacity under aerobic conditions. The results indicated that when sodium succinate was used as the carbon source at a carbon-to-nitrogen ratio of 4 (mass basis), and the culture was maintained at 30 degrees C, pH 7.0, and 160 rpm under anaerobic tube-incubation conditions, strain DE6 removed 620 mg/L KNO3 (equivalent to 85.87 mg/L nitrate nitrogen) within 12 h, achieving a removal rate of 1.37 mg N/(g DCWh) and nearly complete removal of total inorganic-nitrogen within 24 h. However, under aerobic conditions, the nitrate-nitrogen removal rate was substantially lower. Complete removal was achieved only after 24 h with the addition of 0.4 g/L amino acids, yielding a nitrate-nitrogen removal rate of 0.87 mg N/(g DCWh) and a final total inorganic-nitrogen removal efficiency of 80.94 %. RT-qPCR analysis indicated that nitrate-nitrogen removal was mainly achieved by denitrification and nitrogen assimilation. Following supplementation with amino acids, the expression levels of genes involved in the nitrate assimilation and partial denitrification pathways were significantly upregulated under aerobic conditions. This suggests that amino acid metabolism enhances both denitrification and assimilation by strain DE6 under aerobic conditions, thereby improving the removal efficiency of nitrate nitrogen. These findings highlight the potential for the flexible application of the nitrate-removal mechanism of the amino-acid-coordinated strain DE6 across diverse wastewater-treatment contexts.

Pollution caused by polycyclic aromatic hydrocarbons (PAHs) seriously threatens ecosystem sustainability. Persistent PAHs pollutants such as naphthalene are extremely difficult to completely degrade once emitted. Microorganisms have natural advantages in remediating pollutants, but the scarcity of efficient and complete degrading bacteria hinders their application in naphthalene remediation. In response to these challenges, this study focuses on the development of engineered strain scavengers for naphthalene bioremediation. We redesigned and assembled a new pathway containing 17 genes for complete degradation of naphthalene and validated it in E. coli. Briefly, these modified strains can quickly and completely degrade 1mM naphthalene within 4h. The transformant shows increased tolerance to naphthalene and attenuated the impact of naphthalene stress by four-dimensional label-free quantitative proteomic analysis. Stable isotope tracing analysis showed that the engineered bacteria could incorporate naphthalene, salicylate and catechol into the tricarboxylic acid cycle. Naphthalene in polluted water and soil could be rapidly remediated by engineered strains under laboratory conditions. In addition, the engineered bacteria can alleviate the negative effects of naphthalene on soil microbial community and eliminated its biotoxicity to zebrafish. In conclusion, the engineered bacterial scavenger developed in this study demonstrates significant potential, offering a viable strategy for the rapid remediation of naphthalene-contaminated sites.

Abstract Dental caries is a biofilm-mediated disease associated with ecological changes in the oral microbiome. How microbial community organization differs among healthy plaque, caries-associated plaque, and carious dentin remains incompletely defined. We used 16S rRNA gene sequencing to profile paired supragingival plaque and carious dentin samples from patients with caries, together with supragingival plaque from healthy controls. Caries-associated plaque showed higher diversity than healthy plaque, whereas diversity was lower in carious dentin. Ecological ordering placed the three sample types along a health–plaque–dentin continuum. Association-network analysis showed distinct network structures in caries-associated plaque and carious dentin, with the dentin network displaying greater density and lower modularity. By integrating differential-abundance and network-centrality results, we identified taxa associated with the dentin niche. A sparse logistic-regression model using three genera distinguished plaque from dentin in patient-grouped cross-validation (AUROC, 0.780; AUPRC, 0.718). These cross-sectional findings describe niche-associated microbiome organization in dental caries and provide candidate features for future validation in independent, clinically relevant cohorts.

Background:Cutaneous nocardiosis differs from pulmonary and disseminated nocardiosis in that it can occur in immunocompetent individuals and is usually localized with a favorable prognosis. However, the phylogenetic relationships, genomic characteristics, and virulence and antimicrobial resistance profiles of human cutaneous Nocardia isolates remain poorly understood. Purpose:This study aimed to characterize the phylogenetic relationships and genomic features of human cutaneous Nocardia isolates and to investigate their virulence-associated and antimicrobial resistance determinants, as well as their phenotypic antimicrobial susceptibility profiles. Materials and Methods:A total of 31 human cutaneous Nocardia genomes were included for comparative genomic analysis, comprising nine clinical isolates sequenced in this study and 22 publicly available genomes retrieved from the NCBI database. Phylogenetic reconstruction, average nucleotide identity (ANI) analysis, and pan-genome analysis were performed to investigate the genomic relationships and diversity of the isolates. Putative virulence-associated and antimicrobial resistance genes were identified through genome-based annotation. In vitro antimicrobial susceptibility testing was performed for 13 isolates against 15 antimicrobial agents. Results:Phylogenetic analysis demonstrated clear species-level clustering and identified a potentially novel cutaneous pathogen, Nocardia sp. NK_136. Pan-genome analysis revealed an open pan-genome with substantial genomic plasticity. Virulence profiling identified a broadly conserved core framework mainly associated with stress response, immune evasion, iron acquisition, secretion systems, and host interaction, together with variable virulence-associated modules distributed in a species- or strain-specific manner. A total of 40 antimicrobial resistance genes were identified, showing marked species-dependent distribution. Phenotypic antimicrobial susceptibility testing demonstrated that all tested isolates were susceptible to trimethoprim-sulfamethoxazole (TMP-SMX) and linezolid, whereas variable resistance was observed to cefepime, cefoxitin, ciprofloxacin, tobramycin, and clarithromycin. Conclusion:Human cutaneous Nocardia isolates possess a conserved core virulence-associated genomic framework accompanied by variable virulence modules, while their antimicrobial resistance determinants and phenotypic resistance profiles show substantial species-dependent variation. These findings provide genomic insights into the diversity, potential pathogenicity, and antimicrobial resistance of cutaneous Nocardia isolates and provide a basis for further investigation of the pathogenesis and clinical management of cutaneous nocardiosis.

Klebsiella pneumoniae (K. pneumoniae) is a leading cause of hospital- and community-acquired lower respiratory infections (LRI). The emergence of antimicrobial resistance (AMR), especially carbapenem-resistant strains (CRKP), poses a serious global health threat. Using Global Burden of Disease (GBD) 2021 data, we estimated AMR-related mortality for K. pneumoniae-associated LRI across regions from 1990 to 2021. Joinpoint regression analysis identified temporal trends. Global mortality declined overall, with a 70% reduction in under-5 deaths but a doubling among adults over 70 years. Deaths associated with and attributable to AMR decreased by 18.9% and 4.5%, respectively. The age-standardized mortality rate (ASMR) fell by nearly 50%. In contrast, the mortality burden associated with carbapenem resistance increased markedly, with AMR-associated deaths rising by 157% and disproportionately affecting high-burden regions such as Sub-Saharan Africa and South Asia. Despite overall progress, the increasing mortality burden associated with carbapenem resistance in K. pneumoniae-associated LRI poses a critical and persistent global health threat.

Basic helix-loop-helix proteins (bHLHs) are one of the largest classes of transcriptional regulators in plants. They have been shown to be involved in various biological responses to external stimuli. Relatively limited information exists concerning biotic stress-associated bHLH genes in tomato, highlighting a gap in current knowledge that warrants further exploration. In this study, a MeJA-induced bHLH gene, SlJAIB14, was isolated from tomato and functionally characterized. Localization studies with SlJAIB14-sGFP fusion protein revealed its nuclear localization in Arabidopsis roots. Real-time PCR analysis revealed that the expression of SlJAIB14 could be induced by methyl jasmonic acid, wounding, but not by ABA, ACC and SA. In an attempt to investigate the in vivo functions of SlJAIB14, we generated overexpression (OE) and SlJAIB14-silenced (AS) transgenic tomato plants. The SlJAIB14 OE plants showed increased resistance to cotton bollworm attacks. By contrast, the silencing of SlJAIB14 resulted in an opposite effect. Moreover, the transcript levels of some defense-related marker genes were differentially regulated in SlJAIB14 OE and AS plants. A transient transactivation assay in tobacco leaves and the in vitro gel-shift assay indicated that SlJAIB14 may have the potential to directly activate the expression of one stress responsive gene encoding threonine deaminase. Our results indicate that SlJAIB14 functions as a transcriptional activator in response to biotic stresses in plants, and may have potential applications in crop protection against insect attacks.

IntroductionHuman brucellosis is re-emerging in the Chinese Southwest, and its epidemic characteristics remain unclear.MethodologyDescriptive epidemiology, Joinpoint regression, spatial autocorrelation, serological surveillance and pathogen analysis were adopted to clarify the epidemiological evolution of the disease.ResultsBetween 2004 and 2024, 9,822 brucellosis cases occurred, dominated by 7,270 cases in Yunnan. The epidemic showed a persistent upward trend (AAPC ≥5.28, P < 0.05), and 51 cities/prefectures were identified with statistically significant increse (AAPC ≥ 9.30, P < 0.05). Serological surveillance of 36,459 individuals revealed a seropositivity rate of 2.13% (95%CI, 2.00%, 2.26%), identifying 1,831 new cases. Sheep/goats seropositivity at 1.02% (95%CI, 0.97%, 1.06%) and cattle at 0.45% (95%CI, 0.42%, 0.48%). Sustained elevated human incidence was validated by serological evidence, and seroprevalence trends in sheep/goats aligned closely with human epidemic dynamics. Human brucellosis has evolved from random to significantly geographically and spatial aggregated, spreading continuously with intensified clustering in central-eastern Yunnan, the Sichuan basin and Tibet's Ngari-Qamdo axis, highlighting these as priority control zones. Human brucellosis has transitioned from sporadic to locally endemic, with phased progression and increasing geographical clustering recently. Human brucellosis in the Chinese Southwest is characterized by the persistent dominance of B. melitensis bv. 3, primarily represented by the co-circulating MLVA-11 genotypes GT116 and GT125, which form a stable, widely disseminated lineage across the region.ConclusionFrom the re-emergence to persistent endemicity of human brucellosis in study area, the extensive distribution of B. melitensis with unique genotyping reveals a stable zoonotic cycle that requires coordinated regional control.

The COVID-19 pandemic has exposed vulnerabilities in global health systems while accelerating the adoption of metagenomic next-generation sequencing (mNGS) as a transformative tool for culture-independent, unbiased microbial detection. In clinical diagnostics, mNGS enables simultaneous detection of diverse pathogens without prior hypothesis, though its yield depends heavily on specimen type and clinical context. In public health, mNGS has demonstrated remarkable utility in outbreak tracing, novel pathogen discovery, antimicrobial resistance (AMR) surveillance, and One Health initiatives. However, massive data volumes pose persistent challenges in bioinformatics, standardization, and computational demands. Future integration of artificial intelligence, automated platforms, and multi-omics approaches will enhance the conversion of raw data into actionable insights. Collectively, mNGS is poised to drive a paradigm shift from reactive responses to proactive, system-level microbial surveillance across human, animal, and environmental health.

Although brucellosis is endemic to the Qinghai-Tibet Plateau, the molecular epidemiology of circulating Brucella abortus is still poorly characterized. Thus, in this study, we adopted an integrated approach of bacteriology and whole-genome sequencing (WGS) to genetically characterize B. abortus isolates from sheep, yak, and cattle in Qinghai, China. Conventional biotyping assays and multilocus sequence typing (MLST) show that the three strains were conclusively identified as B. abortus biovar 1, sequence type 2 (ST2). Furthermore, average nucleotide identity (ANI) analysis indicated that the three strains (BA0611, BAHYS, and BAQHM) showed ANI values above 99.99% and over 99.91% identity with the B. abortus reference strain, confirming their species assignment. The three B. abortus isolates BA0611, BAHYS, and BAQHM exhibited identical virulence gene profiles, harboring 69 virulence-related genes altogether and uniformly lacking three crucial virulence-associated genes, namely bmaA, btpB, and virB10. Core-genome SNP phylogenetic analysis revealed close genetic relatedness to Tibetan strain XZ19-1 and Russian isolates, distinguishing them from previously identified local human and marmot strains. The high genetic similarity observed among the three strains indicates a common source of infection, supporting the classification of these cases as a cluster infection. These data together confirm the prevalence of genetically divergent B. abortus lineages within the Qinghai-Tibet Plateau, which enriches current insights into the host spectrum and genomic diversity of this zoonotic pathogen in the region. These findings provide robust support for optimizing local genomic surveillance and formulating precise prevention and control strategies to reduce brucellosis prevalence in both animal and human populations.