Misidentification of Brucella melitensis as Ochrobactrum species: potential pitfalls in the diagnosis of brucellosis.

We describe a case of brucellosis in a man in his 20s, who presented to the emergency department with a 1-month history of fevers, dry cough and knee pain. Blood cultures were positive after 55 hours and Ochrobactrum daejeonense was identified on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. Ochrobactrum spp are Gram-negative organisms that are phylogenetically related to Brucella spp but commercially available MALDI-TOF libraries cannot distinguish between the two genera. Further positive blood cultures for O. daejeonense combined with characteristic growth patterns for Brucella spp led to targeted questioning of the patient regarding potential exposure risks, which revealed a history of consumption of unpasteurised camel milk in the Middle East 3 months earlier. Treatment of brucellosis was initiated and subsequent whole genome sequencing identified the blood culture isolate as Brucella melitensis confirming the diagnosis of brucellosis. This case highlights the challenges in the diagnosis of brucellosis in low-incidence settings.

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.

Brucella melitensis Rev1Δwzm: Placental pathogenesis studies and safety in pregnant ewes.

One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16MΔwzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study.

Molecular Characterization and Analysis of Risk Factors Associated with Brucellosis in Central Indian and Meghalaya Population.

Objective: In this study we evaluated the utility of Abortus Melitensis Ovis Suis Brucella PCR (AMOS PCR) for the molecular characterization of Brucella species and analyzed the associated risk factors for brucellosis in Central Indian and Meghalayan population. Methods: AMOS PCR was carried out in a total of 160 BSCP-31 PCR-positive DNA samples isolated previously from the blood of Central Indian (n = 90) and Meghalayan cohorts (n = 70). Clinical and associated risk factors recorded earlier were used to establish strain-specific disease outcomes in study cohorts. Results: Brucella melitensis was found to be the dominant strain in both Central Indian and Meghalayan cohorts (57.7% and 54.28%, respectively) followed by Brucella abortus (42.22% and 38.57%). Although rare, brucellosis cases in the Meghalayan population also showed the presence of Brucella suis (7.14%) and Brucella ovis (2.85%). Febrile illness was a major clinical risk factor in both study cohorts, while occupational risk factors like exposure to animals and raw milk consumption were major mediating factors for brucellosis in Central Indian cohorts. On the contrary, meat consumption was found to be significant predisposing factor for brucellosis in Meghalaya. Conclusion: Molecular characterization of Brucella species provides important public health data for mitigation, advocacy, and antimicrobial stewardship.

Phylogenetic Analysis of Brucella melitensis Strains Isolated from Humans Using 16S rRNA Sequencing and Multiple Locus Variable Number of Tandem Repeats Analysis-16.

Background: Brucellosis is the most important public health problem worldwide, and the annual incidence of the disease in humans is 2.1 million. The Brucella genome is highly conserved, with over 90% similarity among species. The aim of this study was to perform species-level identification of Brucella spp. strains isolated from humans diagnosed with brucellosis and to further investigate the phylogenetic relationships using multiple locus variable number of tandem repeats analysis (MLVA)-16 and 16S rRNA sequencing analysis. Materials and Methods: Brucella spp. was isolated from the blood cultures of 54 patients who tested positive for brucellosis through serological examinations. Real-time PCR was used to identify the isolates in species, and the genus level of Brucella was confirmed with 16S rRNA. All isolates were subjected to phylogenetic analysis using variable number of tandem repeat analysis with multiple loci. Results: Subsequent analysis via real-time PCR confirmed these isolates to be of the Brucella melitensis species. The 16S rRNA sequence analysis showed 100% homogeneity among the isolates. MLVA revealed the formation of five different genotypic groups. While two groups were formed based on the 16S rRNA sequence analysis, five groups were formed in the MLVA. Conclusions: The study concluded that 16S rRNA sequence analysis alone did not provide sufficient discrimination for phylogenetic analysis but served as a supportive method for identification. MLVA exhibited higher phylogenetic power. The widespread isolation of B. melitensis from human brucellosis cases highlights the importance of controlling brucellosis in small ruminants to prevent human infections.

Hemophagocytic lymphohistiocytosis associated with brucellosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder categorized as familial HLH or secondary HLH. Our case report describes a 63-year-old woman with epilepsy whose clinical signs were unremitting fever and altered consciousness. Primary abnormalities consisted of fever, splenomegaly, cytopenia, hypertriglyceridemia, hyperferritinemia and hemophagocytosis in the bone marrow. Results of blood next generation sequencing and blood culture confirmed Brucella infection. This report illustrates a sHLH case caused by Brucella melitensis infection. Here, we review the classification, clinical features, diagnostic methods, treatment regimens, differential diagnosis, and prognosis of HLH and brucellosis.

Molecular epidemiological characteristics of osteoarthritis-associated Brucella melitensis in China: evidence from whole-genome sequencing-based analysis.

BACKGROUND: Brucellosis, developing complications including arthritis, spondylitis, sacroiliitis, and osteomyelitis, is one of the most common zoonotic diseases in the current world which causes economic losses to the livestock industry and is a great public health concern. Brucella melitensis are the main pathogen of brucellosis epidemics in China, most of which are located in northern China. However, there is limited knowledge about the epidemiology of osteoarthritis-associated brucellosis. This study was aimed to reveal the prevalence of osteoarthritis-associated brucellosis in Inner Mongolia and also to investigate the molecular characteristics of B. melitensis isolates. METHODS AND RESULTS: In 2018, the osteoarthritis symptoms of brucellosis in the Brucellosis department of a hospital in Inner Mongolia were investigated. Twenty osteoarthritis-associated B. melitensis strains, isolated from the inpatients in Inner Mongolia during 2013-2017, were subjected to whole genome sequencing. The multilocus sequence type (MLST) and core genome SNP (cgSNP) analysis were conducted to detect molecular epidemiological characteristics. The incidence of brucellosis osteoarthritis symptoms in males (85/120, 70.8%) was significantly higher than that in females (35/120, 29.2%), and the age of patients was concentrated between 41 and 60 years old. In silico analyses indicated ST8 was the prevalent sequence type and the transmission of osteoarthritis-associated B. melitensis among different geographical areas. All strains carry virulence genes, including cgs, lpsA, manCoAg, pgm, pmm, virB4, wbdA and wboA. CONCLUSION: Our study showed the close epidemiologically connection of osteoarthritis-associated B. melitensis strains in northern China. And ST8 was the prevalent sequence type which need our attention.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains / La genómica comparada de Brucella abortus y Brucella melitensis revela el intercambio de genes, los factores de virulencia y la diversidad de SNP entre las cepas estándar, vacunales y de campo

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.(AU)

Molecular examination for Coxiella burnetii and Brucella spp. infections in Iranian women experiencing spontaneous miscarriage.

BACKGROUND: Spontaneous miscarriage, a leading health concern globally, often occurs due to various factors, including infections. Among these, Coxiella burnetii and Brucella spp. may have adverse effects on pregnancy outcomes. While previous research has established a link between infections and spontaneous miscarriage, our study aimed specifically to investigate the presence of these two pathogens in abortion samples from women who experienced spontaneous miscarriages in Iran. Our study can add to the existing knowledge by focusing on Iran, a region with a high prevalence of C. burnetii and Brucella spp. As a result, it could provide a better understanding and unique insights into the relationship of these pathogens with spontaneous miscarriages in endemic regions. METHODS: From March 2021 to March 2022, a total of 728 abortion samples (including placenta and cotyledon) were collected from 409 women who had experienced spontaneous miscarriages in the provinces of Tehran, Fars, and West Azerbaijan in Iran. The specimens included 467 Formalin-Fixed Paraffin-Embedded (FFPE) and 261 fresh frozen samples. After DNA extraction from abortion samples, the quantitative real-time PCR (qPCR) assay targeted a specific fragment of the IS1111 and IS711 elements for molecular identification of C. burnetii and Brucella spp., respectively. Furthermore, the qPCR assay employing specific primers for different species was used to determine the species of Brucella. RESULTS: Among the studied women, 1 out of 409 (0.24%) samples tested positive for Brucella spp., specifically Brucella melitensis. There were no positive specimens for C. burnetii. CONCLUSIONS: Our study contributes to understanding the potential involvement of Brucella species in spontaneous infectious abortion within endemic regions. The identification of B. melitensis in this study highlights the need for further research in this area. However, while our results suggest a relatively low or zero identification of these pathogens in our sample population, this does not rule out the possibility of undetected infections. Therefore, it is critical to acknowledge the limitations of the molecular techniques used (qPCR), which may have potential limitations such as sensitivity and specificity. Moreover, because 64.15% of our samples were FFPE, the sensitivity of the qPCR test may be reduced. These raise concerns about the accuracy of the reported prevalence rates and the potential for false positives or negatives.

Prevalence of Brucella melitensis and Brucella abortus aminoglycoside-resistant isolates: a systematic review and meta-analysis.

INTRODUCTION: Aminoglycosides are vital antibiotics for treating Brucella infections, because they interfere with bacterial protein production and are often combined with other antibiotics. They are cost-effective, have fewer side effects, and can penetrate biofilms. The prevalence of brucellosis has increased in recent years, increasing the need for effective treatments. In addition, the emergence of multidrug-resistant Brucella strains has highlighted the need for an updated and comprehensive understanding of aminoglycoside resistance. This systematic review aimed to provide a comprehensive overview of the global prevalence of aminoglycoside resistance in B. melitensis and B. abortus. METHODS: A systematic search of online databases was conducted and eligible studies met certain criteria and were published in English. Quality assessment was performed using the JBI Checklist. A random-effects model was fitted to the data, and meta-regression, subgroup, and outlier/influential analyses were performed. The analysis was performed using R and the metafor package. RESULTS: The results of this systematic review and meta-analysis suggested that the average prevalence rates of streptomycin, gentamicin, and amikacin resistance were 0.027 (95% confidence interval [CI], 0.015-0.049), 0.023 (95% CI, 0.017-0.032), and 0.008 (95% CI, 0.002-0.039), respectively. The prevalence of streptomycin resistance was higher in the unidentified Brucella group than in the B. abortus and B. melitensis groups (0.234, 0.046, and 0.017, respectively; p < 0.02). The prevalence of gentamicin resistance increased over time (r = 0.064; 95% CI, 0.018 to 0.111; p = 0.007). The prevalence of resistance did not correlate with the quality score for any antibiotic. Funnel plots showed a potential asymmetry for streptomycin and gentamicin. These results suggest a low prevalence of antibiotic resistance in the studied populations. CONCLUSION: The prevalence of aminoglycoside resistance in B. melitensis and B. abortus was low. However, gentamicin resistance has increased in recent years. This review provides a comprehensive and updated understanding of aminoglycoside resistance in B. melitensis and B. abortus.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains.

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.

Molecular detection of Brucella species among aborted small ruminants in southeast Iran.

Brucellosis, caused by Brucella bacteria, is a common zoonotic infectious disease with various clinical manifestations in humans and animals. The disease is endemic in human and ruminant populations in Iran, with a particular prevalence in areas where humans have close interactions with livestock. Since domestic animals serve as the primary reservoir for brucellosis, this study aimed to identify the presence of Brucella spp. among aborted small ruminants in southeast Iran. Between 2021 and 2022, aborted fetuses of small ruminants (46 sheep and 4 goats) were collected from Zarand County in the Kerman province. Swab samples from the abomasum contents of these fetuses were obtained and subjected to DNA extraction. The samples were then tested for Brucella spp. detection using the polymerase chain reaction (PCR) method. Out of the 50 aborted fetuses examined, Brucella spp. was detected in 15 (30%) specimens, comprising 13 (28%) sheep and 2 (50%) goats. Species typing revealed the presence of Brucella ovis (6 sheep and 1 goat), Brucella melitensis (6 sheep), and Brucella abortus (1 sheep) among the positive specimens. This cross-sectional study highlights the high prevalence of various Brucella species in samples from small ruminant abortions in southeast Iran. Additionally, the identified Brucella species were not limited to their primary host livestock. These indicated potential cross-species transmission among small ruminants.

Genotypic peculiarities of a human brucellosis case caused by Brucella suis biovar 5.

Human brucellosis cases are rare in non-endemic countries, such as Germany, where infections are predominantly caused by Brucella melitensis. The German National Reference Laboratory for Bovine, Porcine, Ovine and Caprine Brucellosis received a suspected Brucella sp. isolate from a patient for identification. Bacteriological tests and PCR-based diagnostics showed the isolate to be B. suis, but did not yield cohesive results regarding the biovar. Whole genome sequencing and subsequent genotyping was employed for a detailed characterization of the isolate and elucidating the reason for failure of the diagnostic PCR to correctly identify the biovar. The isolate was found to be B. suis bv. 5, a rare biovar with limited geographical distribution primarily found in the Northern Caucasus. Due to a deletion in one of the target regions of the diagnostic PCR, the isolate could not be correctly typed. Based on in silico genotyping it could be excluded that the isolate was identical to one of the B. suis bv. 5 reference strains. Here, we report a rare case of a B. suis bv. 5 field isolate. Furthermore, by reporting this finding, we want to make practitioners aware of possible misinterpretation of PCR results, as it cannot be excluded that the detected deletion is common among the B. suis bv. 5 community, as there is currently a lack of field isolates.

Prevalence of Brucella melitensis and Brucella abortus tetracyclines resistance: A systematic review and meta-analysis.

INTRODUCTION: Brucellosis is a zoonotic disease that can be transmitted from animals to humans. Brucellosis is caused by bacteria of the genus Brucella, which are typically transmitted through contact with infected animals, unpasteurized dairy products, or airborne pathogens. Tetracyclines (tetracycline and doxycycline) are antibiotics commonly used to treat brucellosis; however, antibiotic resistance has become a major concern. This study assessed the worldwide prevalence of tetracycline-resistant Brucella isolates. METHODS: A systematic search was conducted in Scopus, PubMed, Web of Science, and EMBASE using relevant keywords and Medical Subject Headings (MeSH) terms until August 13, 2022, to identify relevant studies for meta-analysis. A random effects model was used to estimate the proportion of resistance. Meta-regression analysis, subgroup analysis, and examination of outliers and influential studies were also performed. RESULTS: The prevalence rates of resistance to tetracycline and doxycycline were estimated to be 0.017 (95% confidence interval [CI], 0.009-0.035) and 0.017 (95%CI, 0.011-0.026), respectively, based on 51 studies conducted from 1983 to 2020. Both drugs showed increasing resistance over time (tetracycline: r = 0.077, P = 0.012; doxycycline: r = 0.059, P = 0.026). CONCLUSION: The prevalence of tetracycline and doxycycline resistance in Brucella was low (1.7%) but increased over time. This increase in tetracycline and doxycycline resistance highlights the need for further research to understand resistance mechanisms and develop more effective treatments.

Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes was identified using qRT-PCR. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.

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.

The canonical Brucella species-host dependency is changing, however, the antibiotic susceptibility profiles remain unchanged.

Brucellosis is a chronic disease caused by Brucella species with a wide range of hosts, from marine mammals to terrestrial species, but with strict host preferences. With the zoonotic character, the prevalence of human brucellosis cases is a reflection of animal infections. This study aimed to identify 192 Brucella isolates obtained from various sources by Bruce-ladder PCR and to determine their antibiotic susceptibilities by gradient diffusion method (E-test). As a result of the PCR, all human isolates (n = 57) were identified as B. melitensis. While 58 (82.9%) of the cattle isolates were identified as B. abortus, 59 (90.8%) of the sheep isolates were identified as B. melitensis. In addition, 12 (17.1%) of the cattle isolates and 6 (9.2%) of the sheep isolates were determined as B. melitensis and B. abortus, respectively. The primary host change behavior of B. melitensis was 1.9 times higher than that of B. abortus. While gentamicin and ciprofloxacin susceptibilities of Brucella isolates were 100%, tetracycline, doxycycline, streptomycin, trimethoprim/sulfamethoxazole and rifampicin susceptibilities were 99%, 99%, 97.4%, 91.7% and 83.9%, respectively. The lowest sensitivity of the isolates was determined against to cefoperazone as 26%. A triple-drug resistance was detected in 1 B. abortus isolate that included simultaneous resistance to cefoperazone, rifampicin, and trimethoprim/sulfamethoxazole. The high susceptibility profiles we found against to antibiotics such as tetracycline, doxycycline gentamicin and ciprofloxacin, used widely in treatment, are encouraging. However, the change in the canonical Brucella species-primary host preference suggests the need to reconsider eradication program, including updating vaccine formulations.

A rapid and sensitive triplex-recombinase polymerase amplification for simultaneous differentiation of Brucella abortus, Brucella melitensi s, and Brucella suis in sera and foods.

Brucella is the causative agent of brucellosis and can be transmitted to humans through aerosolized particles or contaminated food. Brucella abortus (B. abortus), Brucella melitensis (B. melitensis), and Brucella suis (B. suis) are the most virulent of the brucellae, but the traditional detection methods to distinguish them are time-consuming and require high instrumentation. To obtain epidemiological information on Brucella during livestock slaughter and food contamination, we developed a rapid and sensitive triplex recombinant polymerase amplification (triplex-RPA) assay that can simultaneously detect and differentiate between B. abortus, B. melitensis, and B. suis. Three pairs of primers (B1O7F/B1O7R, B192F/B192R, and B285F/B285R) were designed and screened for the establishment of the triplex-RPA assay. After optimization, the assay can be completed within 20 min at 39°C with good specificity and no cross-reactivity with five common pathogens. The triplex-RPA assay has a DNA sensitivity of 1-10 pg and a minimum detection limit of 2.14 × 104-2.14 × 105 CFU g-1 in B. suis spiked samples. It is a potential tool for the detection of Brucella and can effectively differentiate between B. abortus, B. melitensis, and B. suis S2, making it a useful tool for epidemiological investigations.

Genomic analysis of Brucella melitensis reveals new insights into phylogeny and evolutionary divergence.

PURPOSE: Brucellosis is a bacterial zoonotic disease caused by genus Brucella. The disease is often transmitted to humans by direct or indirect contact with infected livestock or from laboratory exposure. In this study two clinical isolates of Brucella melitensis were subjected to whole genome sequencing (WGS) using Ion Torrent PGM and Oxford Nanopore MinIon platform. METHODS: The two hybrid complete genomes were subjected to core gene SNP analysis to identify the relative evolutionary position. To distinguish between the various lineages of B. melitensis, Pangenome analysis was carried out. RESULTS: Phylogenetic analysis revealed that both the study isolates (ST8) clustered along the other Asian isolates that formed genotype II. Genome wide analyses of 326 B melitensis isolates suggests 2171 gene clusters were shared across all the genomes while 3552 gene clusters were considered as accessory genes. CONCLUSION: Here we attempted to provide the gain and loss of six unique genes that defined the phylogenetic lineages and complex evolutionary process. As the severity and prevalence of human brucellosis is increasing a better understanding of Brucella genomics and transmission dynamics is needed.