Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication.

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum-derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system-mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans-Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase-activating Protein (GAP) ACAP1 to dysregulate Arf6-/Rab8a-dependent transport within the recycling endosome, which resulted in accretion of TGN-associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.

A 14th century CE Brucella melitensis genome and the recent expansion of the Western Mediterranean clade.

Brucellosis is a disease caused by the bacterium Brucella and typically transmitted through contact with infected ruminants. It is one of the most common chronic zoonotic diseases and of particular interest to public health agencies. Despite its well-known transmission history and characteristic symptoms, we lack a more complete understanding of the evolutionary history of its best-known species-Brucella melitensis. To address this knowledge gap we fortuitously found, sequenced and assembled a high-quality ancient B. melitensis draft genome from the kidney stone of a 14th-century Italian friar. The ancient strain contained fewer core genes than modern B. melitensis isolates, carried a complete complement of virulence genes, and did not contain any indication of significant antimicrobial resistances. The ancient B. melitensis genome fell as a basal sister lineage to a subgroup of B. melitensis strains within the Western Mediterranean phylogenetic group, with a short branch length indicative of its earlier sampling time, along with a similar gene content. By calibrating the molecular clock we suggest that the speciation event between B. melitensis and B. abortus is contemporaneous with the estimated time frame for the domestication of both sheep and goats. These results confirm the existence of the Western Mediterranean clade as a separate group in the 14th CE and suggest that its divergence was due to human and ruminant co-migration.

Intracellular growth of Brucella is mediated by Dps-dependent activation of ferritinophagy.

Bacteria of the genus Brucella cause brucellosis, one of the world's most common zoonotic diseases. A major contributor to Brucella's virulence is the ability to circumvent host immune defense mechanisms. Here, we find that the DNA-binding protein Dps from Brucella is secreted within the macrophage cytosol, modulating host iron homeostasis and mediating intracellular growth of Brucella. In addition to dampening iron-dependent production of reactive oxygen species (ROS), a key immune effector required for immediate bacterial clearance, cytosolic Dps mediates ferritinophagy activation to elevate intracellular free-iron levels, thereby promoting Brucella growth and inducing host cell necrosis. Inactivation of the ferritinophagy pathway by Ncoa4 gene knockout significantly inhibits intracellular growth of Brucella and host cell death. Our study uncovers an unconventional role of bacterial Dps, identifying a crucial virulence mechanism used by Brucella to adapt to the harsh environment inside macrophages.

Bacillus Calmette-Guérin-Trained Macrophages Elicit a Protective Inflammatory Response against the Pathogenic Bacteria Brucella abortus.

The bacillus Calmette-Guérin (BCG) can elicit enhanced innate immune responses against a wide range of infections, known as trained immunity. Brucella abortus is the causative agent of brucellosis, a debilitating disease that affects humans and animals. In this study, we demonstrate that C57BL/6 mouse bone marrow-derived macrophages under BCG training enhance inflammatory responses against B. abortus. BCG-trained macrophages showed increased MHC class II and CD40 expression on the cell surface and higher IL-6, IL-12, and IL-1ß production. The increase in IL-1ß secretion was accompanied by enhanced activation of canonical and noncanonical inflammasome platforms. We observed elevated caspase-11 expression and caspase-1 processing in BCG-trained macrophages in response to B. abortus compared with untrained cells. In addition, these BCG-trained cells showed higher NLRP3 expression after B. abortus infection. From a metabolic point of view, signaling through the Akt/mammalian target of rapamycin/S6 kinase pathway was also enhanced. In addition, BCG training resulted in higher inducible NO synthase expression and nitrite production, culminating in an improved macrophage-killing capacity against intracellular B. abortus. In vivo, we monitored a significant reduction in the bacterial burden in organs from BCG-trained C57BL/6 mice when compared with the untrained group. In addition, previous BCG immunization of RAG-1-deficient mice partially protects against Brucella infection, suggesting the important role of the innate immune compartment in this scenario. Furthermore, naive recipient mice that received BM transfer from BCG-trained donors showed greater resistance to B. abortus when compared with their untrained counterparts. These results demonstrate that BCG-induced trained immunity in mice results in better control of intracellular B. abortus in vivo and in vitro.

A Single Nasal Dose Vaccination with a Brucella abortus Mutant Potently Protects against Pulmonary Infection.

The Brucella abortus double-mutant (ΔznuA ΔnorD Brucella abortus-lacZ [znBAZ]) was assessed for its protective efficacy after vaccination with a single nasal dose. Superior protection was achieved in znBAZ-vaccinated mice against pulmonary, wild-type B. abortus 2308 challenge when compared with conventional livestock Brucella abortus vaccines, the smooth S19 (smooth B. abortus strain 19 vaccine) and rough RB51 (rough mutant vaccine strain of B. abortus) strains. Nasal znBAZ vaccination reduced splenic and lung colonization by wild-type brucellae by >3-4 logs. In contrast, S19 reduced lung colonization by only 32-fold, and RB51 failed to reduce colonization. One profound attribute of znBAZ vaccination was the >3-fold increase in pulmonary CD8+ T cells when compared with other vaccinated groups. S19 vaccination increased only CD4+ T cells. All vaccines induced IFN-γ and TNF-α production by CD4+ T cells, but only znBAZ vaccination enhanced the recruitment of polyfunctional CD8+ T cells, by >100-fold. IL-17 by both CD4+ and CD8+ T cells was also induced by subsequent znBAZ vaccination. These results demonstrate that, in addition to achieving protective immunity by CD4+ T cells, CD8+ T cells, specifically resident memory T cells, also confer protection against brucellosis. The protection obtained by znBAZ vaccination was attributed to IFN-γ-producing CD8+ T cells, because depletion of CD8+ T cells throughout vaccination and challenge phases abrogated protection. The stimulation of only CD4+ T cells by RB51- and S19-vaccinated mice proved insufficient in protecting against pulmonary B. abortus 2308 challenge. Thus, nasal znBAZ vaccination offers an alternative means to elicit protection against brucellosis.

Outer membrane protein 25 of Brucella suppresses TLR-mediated expression of proinflammatory cytokines through degradation of TLRs and adaptor proteins.

Toll-like receptors (TLRs) are essential components of innate immunity that serves as the first line of defense against the invaded microorganisms. However, successful infectious pathogens subvert TLR signaling to suppress the activation of innate and adaptive responses. Brucella species are infectious intracellular bacterial pathogens causing the worldwide zoonotic disease, brucellosis, that impacts economic growth of many countries. Brucella species are considered as stealthy bacterial pathogens as they efficiently evade or suppress host innate and adaptive immune responses for their chronic persistence. However, the bacterial effectors and their host targets for modulating the immune responses remain obscure. Brucella encodes various outer membrane proteins (Omps) that facilitate their invasion, intracellular replication, and immunomodulation. Outer membrane protein 25 (Omp25) of Brucella plays an important role in the immune modulation through suppression of proinflammatory cytokines. However, the mechanism and the signaling pathways that are targeted by Omp25 to attenuate the production of proinflammatory cytokines remain obscure. Here, we report that Omp25 and its variants, viz. Omp25b, Omp25c, and Omp25d, suppress production of proinflammatory cytokines that are mediated by various TLRs. Furthermore, we demonstrate that Omp25 and its variants promote enhanced ubiquitination and degradation of TLRs and their adaptor proteins to attenuate the expression of proinflammatory cytokines. Targeting multiple TLRs and adaptor proteins enables Omp25 to effectively suppress the expression of proinflammatory cytokines that are induced by diverse pathogen-associated molecular patterns. This can contribute to the defective adaptive immune response and the chronic persistence of Brucella in the host.

Brucella targets the host ubiquitin-specific protease, Usp8, through the effector protein, TcpB, for facilitating infection of macrophages.

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host ubiquitin-specific protease, USP8, negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Upon silencing or chemical inhibition of USP8, the membrane localization of the CXCR4 receptor was enriched, which augmented the invasion of Brucella into macrophages. Activation of USP8 through chemical inhibition of 14-3-3 protein affected the invasion of Brucella into macrophages. Brucella suppressed the expression of Usp8 at its early stage of infection in the infected macrophages. Furthermore, we found that only live Brucella could negatively regulate the expression of Usp8, suggesting the role of secreted effector protein of Brucella in modulating the gene expression. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB, plays a significant role in downregulating the expression of Usp8 by targeting the cyclic-AMP response element-binding protein pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonists showed a diminished bacterial load. Our experimental data demonstrate a novel role of Usp8 in the host defense against microbial intrusion. The present study provides insights into the microbial subversion of host defenses, and this information may ultimately help to develop novel therapeutic interventions for infectious diseases.

Genomic Diversity and Zoonotic Potential of Brucella neotomae.

After reports in 2017 of Brucella neotomae infections among humans in Costa Rica, we sequenced 12 strains isolated from rodents during 1955-1964 from Utah, USA. We observed an exact strain match between the human isolates and 1 Utah isolate. Independent confirmation is required to clarify B. neotomae zoonotic potential.

Phylogenetic Analysis and Comparative Genomics of Brucella abortus and Brucella melitensis Strains in Egypt.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice.

Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B. melitensis. Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B. melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B. melitensis in RAW 264.7 macrophages in vitro. As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B. melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and ß oxidation of fatty acids are no longer required in Interleukin (IL)-17RA-/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B. melitensis in vivo.

Innate Lymphoid Cells and Interferons Limit Neurologic and Articular Complications of Brucellosis.

Brucellosis is a globally significant zoonotic disease. Human patients with brucellosis develop recurrent fever and focal complications, including arthritis and neurobrucellosis. The current study investigated the role of innate lymphoid cells (ILCs) in the pathogenesis of focal brucellosis caused by Brucella melitensis. After footpad infection, natural killer cells and ILC1 cells both limited joint colonization by Brucella. Mice lacking natural killer cells, and in particular mice lacking all ILCs, also developed marked arthritis after footpad infection. Following pulmonary infection, mice lacking adaptive immune cells and ILCs developed arthritis, neurologic complications, and meningitis. Adaptive immune cells and ILCs both limited colonization of the brain by Brucella following pulmonary infection. Transcriptional analysis of Brucella-infected brains revealed marked up-regulation of genes associated with inflammation and interferon responses, as well as down-regulation of genes associated with neurologic function. Type II interferon deficiency resulted in colonization of the brain by Brucella, but mice lacking both type I and type II interferon signaling more rapidly developed clinical signs of neurobrucellosis, exhibited hippocampal neuronal loss, and had higher levels of Brucella in their brains than mice lacking type II interferon signaling alone. Collectively, these findings indicate ILCs and interferons play an important role in prevention of focal complications during Brucella infection, and that mice with deficiencies in ILCs or interferons can be used to study pathogenesis of neurobrucellosis.

Efficacy of antibiotic prophylaxis to preventing brucellosis in accidental exposure: A systematic review.

BACKGROUND: Antibiotic prophylaxis to prevent brucellosis after accidental exposure to Brucella is an important topic in public health. This study aimed to systematically review the efficacy of antibiotic prophylaxis following accidental exposure to Brucella in preventing human brucellosis disease. METHODS: The study protocol was registered in PROSPERO (CRD42023456812). The outcomes included the incidence of brucellosis disease, adverse events rate, and antibiotic prophylaxis adherence. A comprehensive literature search, conducted until 20 November, 2023, involved Medline, Embase, Cochrane Library, and LILACS databases. Descriptive analysis and meta-analysis using R software were performed, risk of bias was assessed using JBI Critical appraisal tools, and certainty of evidence was assessed using the GRADE tool. RESULTS: Among 3102 initially identified records, eight studies involving 97 individuals accidentally exposed, all focused on high-risk accidental exposure to Brucella in laboratory settings, were included in the review. All studies reported the prophylactic treatment comprising doxycycline at a dosage of 100 mg twice daily, combined with rifampicin at 600 mg, both administered over 21 days. Prophylaxis adherence was reported in 86% of cases, and incidence of brucellosis post-treatment was 0.01. Adverse events, mainly gastrointestinal, occurred in 26% of cases. Critical appraisal revealed limitations in reporting demographics and clinical information. The certainty of evidence was rated as 'very low,' emphasising the need for caution in interpreting the observed outcomes due to study design constraints and the absence of comparative groups. CONCLUSIONS: PEP is an alternative practice reported in the literature, used in accidents with high-risk exposure to Brucella. The currently available evidence of the efficacy of antibiotic prophylaxis is insufficient to support a recommendation for or against the widespread use of antibiotic prophylaxis, so caution is needed in interpreting results due to the very low certainty of evidence, primarily stemming from case series and lack of comparative groups.

Pleurisy due to brucellosis.

Any system or organ involvement can be seen in brucellosis, which is still a significant public health problem in developing countries. The rate of respiratory system involvement is lower than that of other systems and which is also difficult to document. Brucellosis-associated pleurisy is a rare complication even in endemic regions. In this case report, a 78-year-old male patient who was assessed for pleural effusion etiology is presented. Brucella spp. were isolated on the 14th day of the pleural fluid incubation in the blood culture set and the patienthas been treated successfully for brucellosis. Based on our experience we think that it is important to use blood culture media for sterile body fluids, particularly for microorganisms that are difficult to isolate such as Brucella spp.

A comparison of clinical features between neurobrucellosis and tuberculous meningitis.

BACKGROUD: This study aims to compare the clinical manifestations, imaging findings, routine tests, biochemistry indicators and cerebrospinal fluid cytology between neurobrucellosis and tuberculous meningitis. The objective is to evaluate the similarities and differences of these two diseases and improve early diagnosis. METHODS: A comprehensive evaluation was conducted by comparing clinical data, imaging results, routine tests findings, biochemistry indicators and cerebrospinal fluid cytology of patients admitted to the Department of Neurology, the Second Hospital of Hebei Medical University from 2019 to 2021. Statistical analysis was applied to identify significant differences and similarities between the two diseases. RESULTS: Preliminary analysis demonstrated both diseases commonly present with symptoms such as fever, headache. However, there were no statistical differences between neurobrucellosis and tuberculous meningitis in early clinical data, imaging results, routine tests findings, biochemistry indicators. Further analysis indicates there is a statistically significantly difference in the lymphocyte ratio and neutrophil ratio in the cerebrospinal fluid between the two groups. CONCLUSIONS: Neurobrucellosis and tuberculous meningitis share similarities in early clinical manifestations, imaging findings and initial cerebrospinal fluid parametes, making early-stage differentiation challenging. The ratio of lymphocytes and neutrophil in the cerebrospinal fluid and a detailed medical history investigation can provide clues for early clinical diagnosis. So the examination of CSF cytology might be a potential to distinguish these two diseases and become a powerful tool in the future.

Two cases of MPO-ANCA-positive hypertrophic pachymeningitis mimicking as intracranial infection.

Hypertrophic pachymeningitis (HP) is a rare disorder marked by thickening of the dura mater due to diverse etiologies. MPO-ANCA-positive HP represents a variant of AAV confined to the central nervous system, distinguished by the presence of serum MPO antibodies. Distinguishing HP triggered by MPO-ANCA from other causes can be challenging.In this study, we present two cases of MPO-ANCA-positive HP initially misdiagnosed as intracranial infections. Case 1 underwent surgery for chronic suppurative otitis media, with histopathological findings revealing inflammatory changes without definitive suppuration. He was presumed to have a secondary intracranial infection resulting from the surgery. However, his condition deteriorated despite two weeks of antibiotic and antiviral treatment. Case 2 presented with headache and was initially suspected of having intracranial Brucellosis given his serum Brucella positivity. Despite treatment for brucellosis, his symptoms persisted, and he developed visual and hearing impairments. Both patients were ultimately diagnosed with MPO-ANCA-positive HP, exhibiting serum MPO antibody positivity. Their symptoms showed improvement with glucocorticoid and immunosuppressive therapy.Based on these observations, we propose that MPO-ANCA-positive HP may initially present as intracranial infection. For HP patients presenting with headache, mastoiditis, otitis media, and visual loss, it is imperative to conduct ANCA antibody-related tests to enhance diagnostic precision.

Construction of recombinant Omp25 or EipB protein loaded PLGA nanovaccines for Brucellosis protection.

Safe and effective vaccine candidates are needed to address the limitations of existing vaccines against Brucellosis, a disease responsible for substantial economic losses in livestock. The present study aimed to encapsulate recombinant Omp25 and EipB proteins, knowledged antigen properties, into PLGA nanoparticles, characterize synthesized nanoparticles with different methods, and assessed theirin vitro/in vivoimmunostimulatory activities to develop new vaccine candidates. The recombinant Omp25 and EipB proteins produced with recombinant DNA technology were encapsulated into PLGA nanoparticles by double emulsion solvent evaporation technique. The nanoparticles were characterized using FE-SEM, Zeta-sizer, and FT-IR instruments to determine size, morphology, zeta potentials, and polydispersity index values, as well as to analyze functional groups chemically. Additionally, the release profiles and encapsulation efficiencies were assessed using UV-Vis spectroscopy. After loading with recombinant proteins, O-NPs reached sizes of 221.2 ± 5.21 nm, while E-NPs reached sizes of 274.4 ± 9.51 nm. The cumulative release rates of the antigens, monitored until the end of day 14, were determined to be 90.39% for O-NPs and 56.1% for E-NPs. Following the assessment of thein vitrocytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells,in vivoimmunization experiments were conducted using concentrations of 16µg ml-1for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing producedBrucella-specific IgG antibody levels for 3 times in contrast to control. Furthermore, it was also demonstrated that vaccine candidates stimulated Th1-mediated cellular immunity as well since they significantly raised IFN-gamma and IL-12 cytokine levels in murine splenocytes rather than IL-4 following to immunization. Additionally, the vaccine candidates conferred higher than 90% protection from the infection according to challenge results. Our findings reveal that PLGA nanoparticles constructed with the encapsulation of recombinant Omp25 or EipB proteins possess great potential to triggerBrucella-specific humoral and cellular immune response.

Motor polyradiculoneuropathy as an unusual presentation of neurobrucellosis: a case report and literature review.

Brucellosis, a zoonotic disease caused by Brucella species, poses a significant global health concern. Among its diverse clinical manifestations, neurobrucellosis remains an infrequent yet debilitating complication. Here, we present a rare case of neurobrucellosis with unusual presentations in a 45-year-old woman. The patient's clinical course included progressive lower extremity weakness, muscle wasting, and double vision, prompting a comprehensive diagnostic evaluation. Notable findings included polyneuropathy, elevated brucella agglutination titers in both cerebrospinal fluid and blood, abnormal EMG-NCV tests, and resolving symptoms with antibiotic therapy. The clinical presentation, diagnostic challenges, and differentiation from other neurological conditions are discussed. This case underscores the importance of considering neurobrucellosis in regions where brucellosis is prevalent and highlights this rare neurological complication's distinctive clinical and radiological features. Early recognition and appropriate treatment are crucial to mitigate the significant morbidity associated with neurobrucellosis.

Neurobrucellosis: laboratory features, clinical characteristics, antibiotic treatment, and clinical outcomes of 21 patients.

BACKGROUND: Neurobrucellosis (NB) is a rare and serious complication of brucellosis. Its clinical manifestations vary, with no obvious specificity. At present, there is no clear clinical diagnosis or treatment for reference. In this study, we retrospectively analyzed the clinical data for 21 patients with NB to provide reference data for its further study. METHODS: We analyzed the epidemiological and clinical manifestations, laboratory tests, imaging examinations, cerebrospinal fluid, and treatment plans of 21 patients diagnosed with NB in the Department of Neurology, Xuanwu Hospital, Capital Medical University Beijing, China. RESULTS: The ages of the patients ranged from 15 to 60 years old (mean age 40.1 ± 13.33 years), the male: female ratio was 4.25:1. Thirteen patients had a history of animal (sheep, cattle) contact, three had no history of animal contact, and the contact status of four was unknown. Brucella can invade various systems of the body and show multi-system symptoms, the main general manifestations were fever (66.67%), fatigue (57.14%) and functional urination or defecation disturbance (42.86%). The main nervous system manifestations were limb weakness (52.38%) and hearing loss (47.62%).The main positive signs of the nervous system included positive pathological signs (71.43%), sensory abnormalities (52.38%), limb paralysis (42.86%). Nervous system lesions mainly included spinal cord damage (66.67%), cranial nerve involvement (61.90%), central demyelination (28.57%) and meningitis (28.57%). In patients with cranial nerve involvement, 69.23% of auditory nerve, 15.38% of optic nerve and 15.38% of oculomotor nerve were involved. The blood of eight patients was cultured for Brucella, and three (37.5%) cultures were positive and five (63.5%) negative. The cerebrospinal fluid (CSF) of eight patients was cultured for Brucella, and two (25.00%) cultures were positive and six (75.00%) negative. Nineteen of the patients underwent a serum agglutination test (SAT), 18 (94.74%) of whom were positive and one (5.26%) of whom were negative. A biochemical analysis of the CSF was performed in 21 patients, and the results were all abnormal. Nineteen patients underwent magnetic resonance imaging (MRI). Twenty-one patients were treated with doxycycline and/or rifampicin, combined with ceftriaxone, quinolone, aminoglycoside, or minocycline. After hospitalization, 15 patients improved (71.43%), two patients did not recover, and the status of four patients was unknown. CONCLUSIONS: The clinical manifestations, CSF parameters, and neurological imaging data for patients with NB show no significant specificity or correlations. When patients with unexplained neurological symptoms accompanied by fever, fatigue, and other systemic manifestations in a brucellosis epidemic area or with a history of contact with cattle, sheep, animals, or raw food are encountered in clinical practice, the possibility of NB should be considered. Treatment is based on the principles of an early, combined, and long course of treatment, and the general prognosis is good.

Delay in the diagnosis of Brucella abortus bacteremia in a nonendemic country: a case report.

BACKGROUND: It is challenging to diagnose brucellosis in nonendemic regions because it is a nonspecific febrile disease. The accurate identification of Brucella spp. in clinical microbiology laboratories (CMLs) continues to pose difficulties. Most reports of misidentification are for B. melitensis, and we report a rare case of misidentified B. abortus. CASE PRESENTATION: A 67-year-old man visited an outpatient clinic complaining of fatigue, fever, and weight loss. The patient had a history of slaughtering cows with brucellosis one year prior, and his Brucella antibody tests were negative twice. After blood culture, the administration of doxycycline and rifampin was initiated. The patient was hospitalized due to a positive blood culture. Gram-negative coccobacilli were detected in aerobic blood culture bottles, but the CML's lack of experience with Brucella prevented appropriate further testing. Inaccurate identification results were obtained for a GN ID card of VITEK 2 (bioMérieux, USA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a MALDI Biotyper (Bruker, Germany). The strain showed 100.0% identity with Brucella spp. according to 16S rRNA sequencing. MALDI-TOF MS peaks were reanalyzed using the CDC MicrobeNet database to determine Brucella spp. (score value: 2.023). The patient was discharged after nine days of hospitalization and improved after maintaining only doxycycline for six weeks. The isolate was also identified as Brucella abortus by genomic evidence. CONCLUSION: Automated identification instruments and MALDI-TOF MS are widely used to identify bacteria in CMLs, but there are limitations in accurately identifying Brucella spp. It is important for CMLs to be aware of the possibility of brucellosis through communication with clinicians. Performing an analysis with an additional well-curated MALDI-TOF MS database such as Bruker security-relevant (SR) database or CDC MicrobeNet database is helpful for quickly identifying the genus Brucella.