Analysis of the Brucella melitensis epidemic in Xinjiang: genotyping, polymorphism, antibiotic resistance and tracing.

Brucella spp. are facultative intracellular pathogens that cause zoonosis- brucellosis worldwide. There has been a trend of the re-emergence of brucellosis worldwide in recent years. The epidemic situation of brucellosis is serious in Xinjiang. To analyze the epidemic situation of Brucella spp. in Xinjiang among humans and animals, this study identified 144 Brucella isolates from Xinjiang using classical identification and 16 S rRNA sequencing. MLVA, drug resistance testing, and wgSNP detection were also performed. At the same time, analysis was conducted based on the published data of Brucella isolates worldwide. The results showed that the dominant species was B. melitensis biovar 3, which belonged to GT42 (MLVA-8 typing) and the East Mediterranean lineage. The correlation among isolates was high both in humans or animals. The isolates in Xinjiang exhibited higher polymorphism compared to other locations in China, with polymorphism increasing each year since 2010. No amikacin/kanamycin-resistant strains were detected, but six rifampicin-intermediate isolates were identified without rpoB gene variation. The NJ tree of the wgSNP results indicated that there were three main complexes of the B. melitensis epidemic in Xinjiang. Based on the results of this study, the prevention and control of brucellosis in Xinjiang should focus on B. melitensis, particularly strains belonging to B. melitensis bv.3 GT42 (MLVA-8 typing) and East Mediterranean lineage. Additionally, the rifampicin- and trimethoprim-sulfamethoxazole- resistance of isolates in Xinjiang should be closely monitored to avoid compromising the therapeutic efficacy and causing greater losses. These results provide essential data for the prevention and control of brucellosis in Xinjiang and China. Although the isolates from Xinjiang have significant characteristics among Chinese isolates and can reflect the epidemiological situation of brucellosis in China to some extent, this study cannot represent the characteristics of isolates from other regions.

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.

Brucella species-induced brucellosis: Antimicrobial effects, potential resistance and toxicity of silver and gold nanosized particles.

Brucellosis is an endemic zoonotic disease caused by Brucella species, which are intramacrophage pathogens that make treating this disease challenging. The negative effects of the treatment regime have prompted the development of new antimicrobials against brucellosis. A new treatment modality for antibiotic-resistant microorganisms is the use of nanoparticles (NPs). In this study, we examined the antibacterial activities of silver and gold NPs (SNPs and GNPs, respectively), the resistance developed by Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) strains and the toxicity of both of these NPs in experimental rats. To test the bactericidal effects of the SNPs and GNPs, we used 22 multidrug-resistant Brucella isolates (10 B. melitensis and 12 B. abortus). The minimal inhibitory concentrations (MICs) of both types of NPs were determined utilizing the microdilution technique. To test the stability of resistance, 7 B. melitensis and 6 B. abortus isolates were passaged ten times in culture with subinhibitory concentrations of NPs and another ten times without NPs. Histopathological analysis was completed after rats were given 0.25, 0.5, 1, and 2 mg/kg NPs orally for 28 consecutive days. The MIC values (µg/ml) of the 10-nm SNPs and 20-nm GNPs against B. melitensis were 22.43 ± 2.32 and 13.56 ± 1.22, while these values were 18.77 ± 1.33 and 12.45 ± 1.59 for B. abortus, respectively. After extensive in vitro exposure, most strains showed no resistance to the 10-nm SNPs or 20-nm GNPs. The NPs and antibiotics did not cross-react in any of the evolved Brucella strains. SNPs and GNPs at doses below 2 mg/kg were not harmful to rat tissue according to organ histopathological examinations. However, a greater dose of NPs (2 mg/kg) harmed all of the tissues studied. The bactericidal properties of NPs are demonstrated in this work. Brucella strains develop similar resistance to SNPs and GNPs, and at low dosages, neither SNPs nor GNPs were hazardous to rats.

Identifying potential natural inhibitors of Brucella melitensis Methionyl-tRNA synthetase through an in-silico approach.

BACKGROUND: Brucellosis is an infectious disease caused by bacteria of the genus Brucella. Although it is the most common zoonosis worldwide, there are increasing reports of drug resistance and cases of relapse after long term treatment with the existing drugs of choice. This study therefore aims at identifying possible natural inhibitors of Brucella melitensis Methionyl-tRNA synthetase through an in-silico approach. METHODS: Using PyRx 0.8 virtual screening software, the target was docked against a library of natural compounds obtained from edible African plants. The compound, 2-({3-[(3,5-dichlorobenzyl) amino] propyl} amino) quinolin-4(1H)-one (OOU) which is a co-crystallized ligand with the target was used as the reference compound. Screening of the molecular descriptors of the compounds for bioavailability, pharmacokinetic properties, and bioactivity was performed using the SWISSADME, pkCSM, and Molinspiration web servers respectively. The Fpocket and PLIP webservers were used to perform the analyses of the binding pockets and the protein ligand interactions. Analysis of the time-resolved trajectories of the Apo and Holo forms of the target was performed using the Galaxy and MDWeb servers. RESULTS: The lead compounds, Strophanthidin and Isopteropodin are present in Corchorus olitorius and Uncaria tomentosa (Cat's-claw) plants respectively. Isopteropodin had a binding affinity score of -8.9 kcal / ml with the target and had 17 anti-correlating residues in Pocket 1 after molecular dynamics simulation. The complex formed by Isopteropodin and the target had a total RMSD of 4.408 and a total RMSF of 9.8067. However, Strophanthidin formed 3 hydrogen bonds with the target at ILE21, GLY262 and LEU294, and induced a total RMSF of 5.4541 at Pocket 1. CONCLUSION: Overall, Isopteropodin and Strophanthidin were found to be better drug candidates than OOU and they showed potentials to inhibit the Brucella melitensis Methionyl-tRNA synthetase at Pocket 1, hence abilities to treat brucellosis. In-vivo and in-vitro investigations are needed to further evaluate the efficacy and toxicity of the lead compounds.

Dexamethasone-induced stress exacerbates shedding, tissue antigen distribution, and pathology of caprine Brucellosis.

This study investigated dexamethasone-treatment, shedding routes, tissue antigen distribution, and pathology of caprine Brucellosis. Eighteen non-pregnant goats were randomly grouped into A, B, and C. Group A was administered dexamethasone for 7 days at 2 mg/kg before inoculating 0.5 mL B. melitensis at 107 CFU ocularly while group B was inoculated 0.5 mL B. melitensis only, and C as control negative. Blood samples, ocular, nasal, and vaginal swabs were obtained for evaluation. Three goats were sacrificed from each group at days 21 and 42 post-inoculation (pi) and selected tissues collected for PCR, histopathology, and immunohistochemistry. Brucella melitensis was detected in the ocular swabs of group A significantly higher than group B. Shedding was prolonged in group A compared to B. The overall shedding was 22.2% in group A and 9.4% in group B. The uterus of both groups A and B revealed mild inflammation and microgranuloma, extensive necrotic lesions in lymph nodes. Liver showed multifocal necrosis predominantly in group A. Lesion scoring showed significantly higher scores in A compared to B. Strong immunostaining was observed in the liver, lungs, and spleen, predominantly at day 21 pi. This study demonstrated dexamethasone prolonged shedding, tissue antigen distribution, and pathology in dexamethasone-treated goats.

Genome-wide Core Proteome Analysis of Brucella melitensis Strains for Potential Drug Target Prediction.

INTRODUCTION: Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. In humans, chronic infection occurs through contact with infected animals or their waste products. METHODS: The subtractive genomic approach is considered as a powerful and useful method for the identification of potential drug and vaccine targets. In this study, an attempt has been made through a subtractive proteomic strategy to identify novel drug targets in Brucella melitensis strains. Total 2604 core proteins of 56 strains of B. melitensis were taken, of which 545 non-human homologs were found to be essential for pathogen growth. Metabolic pathway analysis of these essential proteins revealed that 129 proteins are exclusively involved in 21 unique metabolic pathways in B. melitensis reference strain. RESULTS: Of these, 31 proteins were found to be involved in 10 metabolic pathways that are unique to the pathogen. We selected Nitrate reductase subunit-ß, Urease subunit α-2, Pantoate-ß-alanine ligase, Isochorismatase, 2-dehydro-3-deoxyphosphooctonate aldolase and Serine O-acetyltransferase as drug targets in Brucella melitensis strains. Among these druggable targets, we selected only Pantoate-ß- alanine ligase as high confidence target based on intensive literature curation, which is nonhomologous to the human gut metagenome involved in biosynthesis of secondary metabolites pathway. Pantothenate synthetase is the best chemotherapeutic target to combat Brucellulosis. CONCLUSION: Furthermore, in vitro and in vivo validation is needed for the evaluation of lead compounds against Brucella melitensis strains.

Cell membrane components of Brucella melitensis play important roles in the resistance of low-level rifampicin.

Brucella spp. are facultative intracellular pathogens that can persistently colonize host cells and cause the zoonosis- brucellosis. The WHO recommended a treatment for brucellosis that involves a combination of doxycycline, rifampicin, or streptomycin. The aim of this study was to screen rifampicin-resistance related genes by transcriptomic analysis and gene recombination method at low rifampicin concentrations and to predict the major rifampicin- resistance pathways in Brucella spp. The results showed that the MIC value of rifampicin for B. melitensis bv.3 Ether was 0.5 µg / mL. Meanwhile, B. melitensis had an adaptive response to the resistance of low rifampicin in the early stages of growth, while the SNPs changed in the rpoB gene in the late stages of growth when incubated at 37°C with shaking. The transcriptome results of rifampicin induction showed that the functions of significant differentially expressed genes were focused on metabolic process, catalytic activity and membrane and membrane part. The VirB operon, ß-resistance genes, ABC transporters, quorum-sensing genes, DNA repair- and replication -related genes were associated with rifampicin resistance when no variations of the in rpoB were detected. Among the VirB operons, VirB7-11 may play a central role in rifampicin resistance. This study provided new insights for screening rifampicin resistance-related genes and also provided basic data for the prevention and control of rifampicin-resistant Brucella isolates.

Treatment of Brucellosis in Guinea Pigs via a Combination of Engineered Novel pH-Responsive Curcumin Niosome Hydrogel and Doxycycline-Loaded Chitosan-Sodium Alginate Nanoparticles: an In Vitro and In Vivo Study.

Brucellosis is a common zoonotic infection, particularly in the developing world. The recommended treatment regimens for brucellosis involve the use of two medications such as doxycycline and curcumin in order to avoid relapses and prolonged use of these drugs. Doxycycline has excellent activity in the acidic phagolysosomal environment, while curcumin modulates the immune system function and macrophage activity. Due to the intracellular existence of Brucellae and the different anti-immune mechanisms of Brucella, the treatment of Brucella infection faces many limitations. The design of nanosystems is a promising treatment approach for brucellosis. The objective of this study was to design and evaluate the efficacy of in situ pH-responsive curcumin-loaded niosome hydrogel and doxycycline-loaded chitosan-sodium alginate nanoparticles as chemotherapeutic agents against brucellosis. The prepared formulae showed a spherical nano shape with a slow drug release pattern and small particle size. The prepared formulae were evaluated in vivo using Guinea pigs experimentally infected with Brucella melitensis biovar3. The prepared formula combination gave a significant high reduction rate of Brucella spleen viable count compared with that of untreated controls at p < 0.05. The results showed that the treatment schemes were not fully successful in eliminating Brucella infection in Guinea pigs; however, they significantly (p < 0.05) reduced the viable Brucella count in a shorter time and sub-therapeutic doses. Collectively the novel prepared formulae could be a successful therapy for the effective treatment of brucellosis infection at the recommended therapeutic doses. Graphical abstract.

Whole-genome sequencing of rough Brucella melitensis in China provides insights into its genetic features.

Brucella spp. can cause the zoonosis brucellosis, which affects public health and safety and even economic development. B. melitensis has a smooth phenotype, while 28 B. melitensis isolates had a rough phenotype in 2018. In this study, rough phenotype detection and whole genome sequencing methods were used to analyze the genetic features of rough B. melitensis. A drug susceptibility test was also performed. The results showed that the rough B. melitensis strains originated from strains isolated in China rather than from foreign strains. Furthermore, an MS tree showed that 9 complexes to be epidemic in China. For the rough B. melitensis strains, expression of the metabolic function genes varied in the earlier stages of evolution compared to the cellular process and signalling function genes. Expression of some transcriptional regulatory factors also varied in the later stages of evolution, and compared to MFS transporter genes, ABC transporter genes varied in the earlier stages. Moreover, as there was no significant difference in rifampicin, doxycycline and streptomycin susceptibility between the smooth and rough B. melitensis strains, treatment of brucellosis was not affected by strain type. This study provided important information for understanding the genetics and evolution of rough B. melitensis in China.

Epidemiologically characteristics of human brucellosis and antimicrobial susceptibility pattern of Brucella melitensis in Hinggan League of the Inner Mongolia Autonomous Region, China.

BACKGROUND: Hinggan League is located in the Northeast of the Inner Mongolia Autonomous Region, the historically endemic area of animal and human brucellosis. In this study, the epidemiological characteristics of human brucellosis were analyzed, and the genotypic profile and antimicrobial susceptibilities of Brucella melitensis strains isolated from humans in Hinggan League were investigated. METHODS: The epidemic characteristics were described using case number, constituent ratio, and rate. The 418 human blood samples were collected and tested by bacteriology, and suspect colonies were isolated and identified by conventional biotyping assays, the VITEK 2.0 microbial identification system, and AMOS (Brucella abortus, B. melitensis, B. ovis, and B. suis)-PCR. Subsequently, all strains were genotyped using multiple-locus variable-number tandem repeat analysis (MLVA) assays, and the antimicrobial susceptibility pattern of Brucella strains against the 10 most commonly used antibiotics was determined by microdilution method. RESULTS: A total of 22 848 cases of human brucellosis were reported from 2004 to 2019, with an annual average incidence of 87.2/100 000. The incidence rates in developed areas of animal husbandry (Horqin Youyi Qianqi [161.2/100 000] and Horqin Youyi Zhongqi [112.1/100 000]) were significantly higher than those in forest areas (Arxan [19.2/100 000]) (χ2 = 32.561, P < 0.001). In addition, peak morbidity occurred during May-August, accounting for 72.6% (16582/22 848) of cases. The highest number of cases occurred in the 40+ age group, accounting for 44.4% (10 137/22484) of cases, and morbidity in males was significantly higher than that in females in all age groups (χ2 = 299.97, P < 0.001), the most common occupation was farmers. A total of 54 B. melitensis strains were divided into 37 genotypes (GT1-37) with 80-100% genetic similarity. All 25 strains were sensitive to seven tested antibiotics, phenotypic resistance to cotrimoxazole and azithromycin was observed in 5 (20%) and 25 (100%) of the isolates, respectively. CONCLUSIONS: Human brucellosis exhibited a significant increasing trend and B. melitensis is the main pathogen responsible for human brucellosis in this region. Improved surveillance of infected animals (sheep) and limiting their transfer and trade are optional strategies for decreasing the incidence of this disease.

Evaluation of Poly(I:C) and combination of CpG ODN plus Montanide ISA adjuvants to enhance the efficacy of outer membrane vesicles as an acellular vaccine against Brucella melitensis infection in mice.

Brucellosis is the most common zoonotic disease worldwide and still there is no vaccine for human use. The commercial animal vaccines also have major problems that limit their use. Therefore, there is a need for an effective Brucella vaccine which is multivalent and produces a good protective immunity with minimal disadvantages. Due to their heterogeneous composition and diverse functions, OMVs are promising acellular vaccine candidates against brucellosis. In the present study, the potential of Poly(I:C) or CpG ODN 1826+ Montanide ISA 70 VG adjuvant formulations were evaluated to enhance the immunity and protection levels conferred by OMVs against Brucella challenge in mice. The results indicated that both vaccine regimens were able to induce strong Th1-biased responses and confer protective levels significantly higher than REV.1 live vaccine. With regard to the results, it is concluded that OMVs in either adjuvant can be introduced as a new vaccine candidate against B. melitensis infection.

Tulathromycin treatment does not affect bacterial dissemination or clearance of Brucella melitensis 16M following experimental infection of goats.

Brucellosis in sheep and goats, a zoonotic disease primarily associated with Brucella melitensis infections, causes significant economic losses and public health concerns worldwide. Although control measures are effective, economic limitations and nomadic lifestyles may limit vaccination coverage, and test and removal policies may not be feasible. In this study, we evaluated the effects of therapy with a long acting antimicrobial tulathromycin on the pathogenesis of brucellosis. Thirty-five goats were randomly assigned for experimental infection with B. melitensis strain 16M while open or during mid-gestation. Approximately half of the animals in each group were then treated with tulathromycin and subsequently assessed for the development of humoral responses to infection, clinical presentation, and bacterial dissemination and colonization. All animals, regardless of treatment group were successfully challenged with B. melitensis 16M demonstrated by bacterial recovery from conjunctival swabs and development of positive antibody titers. In goats infected while open, no animals aborted and Brucella was recovered from only one animal in tulathromycin-treated and one animal from the untreated group. Tulathromycin treatment of pregnant goats did not prevent abortion nor did it reduce bacterial dissemination, colonization, or shedding. Our data suggests that treatment of goats in mid-gestation with tulathromycin at the labeled dose does not influence disease pathogenesis or tissue colonization after experimental B. melitensis challenge.

Doxycycline-encapsulated solid lipid nanoparticles for the enhanced antibacterial potential to treat the chronic brucellosis and preventing its relapse: in vivo study.

BACKGROUND: Brucellosis is one of the most important infection of diseases. Due to its large period of treatment and survival ability of bacteria inside the macrophages, relapse of this disease is the main challenge, especially, after the treatment. OBJECTIVE: The current study was carried out to evaluate the antibacterial effect of solid lipid nanoparticles loaded with doxycycline on the Brucella melitensis in in vivo conditions. METHODS: The double emulsion synthesized doxycycline-encapsulated solid lipid nanoparticles (DOX-SLN) was characterized using DLS and FE-SEM. The efficacy of the DOX-SLN on the acute and chronic Wistar rat infected brucellosis was investigated. The pathological assessments were made on the spleen and liver in the treated rates. RESULTS: The in vivo experimental results demonstrated that the treated rats with DOX-SLN had significantly decreased the B. melitensis CFUs in their spleen and liver compared to that of the treated rates with free doxycycline and untreated ones. The pathologic results indicate that the improvement trend of spleen and liver tissues in rats treated by DOX-SLN was satisfactory. CONCLUSION: According to in vivo results, the DOX-SLN has better effects on the treatment of chronic brucellosis. Therefore, DOX-SLN is recommended to treat the brucellosis and avoid its relapse.

Effects of resveratrol on shedding and pathological dynamics in experimental B. melitensis infection in dexamethasone-treated nonpregnant Boer goats.

Brucellosis constitutes an infectious re-emerging zoonosis. Spread of diseases could be exacerbated by stress-induced immunosuppression. This study evaluated relationship between Brucella melitensis infection, shedding dynamics, dexamethasone-induced stress, pathological alterations and resveratrol ameliorative effects in goats. Twelve nonpregnant goats were divided into four groups A, B, C, and D of three animals each. Groups A and B were administered 107 CFU/mL of B. melitensis ocularly, 21 days prior to 7 days consecutive administration of dexamethasone (2 mg/kg). Group A was further administered resveratrol (5 mg/kg) intravenously for 5 consecutive days from day 31 post B. melitensis inoculation. Group C was administered similar dose of B. melitensis while group D was inoculated normal saline. Blood, nasal, ocular, and vaginal swabs were collected at intervals for analysis. The does were sacrificed at day 42 post inoculation (pi). Tissues were collected for tissue bacterial load determination, histopathology, and immunohistochemistry. Dexamethasone administration from day 21 pi increased the frequency in the shedding dynamics, tissue bacterial load, pathological alterations (frequency of microgranuloma and intensity of immunostaining) in group B while 5 days treatment with resveratrol following dexamethasone administration significantly reduced tissue bacterial load, decline in shedding dynamics, and ameliorate damage by dexamethasone administration/B. melitensis infection.

Doxycycline-encapsulated solid lipid nanoparticles as promising tool against Brucella melitensis enclosed in macrophage: a pharmacodynamics study on J774A.1 cell line.

Background: Brucellosis is a zoonotic disease caused by Brucella species. It has been estimated that more than 500,000 new cases of Brucellosis occur annually all around the world. Relapse of the disease is one of the most important challenges. The most important reason for the relapse of brucellosis is the survival of the bacteria inside the macrophages, which makes them safe from the immune system and disrupts drug delivery mechanism. Objectives: The present study was performed to assess the effects of Doxycycline-loaded Solid Lipid Nanoparticles (DOX-SLN) on the Brucella melitensis inside macrophages. Methods: DOX-SLN was prepared using double emulsion method. The technological characterization of DOX-SLN, including particle size, zeta potential, polydispersity index (PDI), drug loading and encapsulation efficiency were used. Fourier-transform infrared spectroscopy (FTIR) and Differential scanning calorimetry (DSC) were used to assess the interactions between Nanoparticles (NPs) components and crystalline form of doxycycline. Moreover, the effect of DOX-SLN on the bacteria were compared with that of the doxycycline using various methods, including well diffusion, Minimum Inhibitory Concentration (MIC), and investigation of their effects on murine macrophage-like cells cell line J774A.1. Results: The means of particle size, zeta potential, PDI, drug loading and encapsulation efficiency were 299 ± 34 nm, - 28.7 ± 3.2 mV, 0.29 ± 0.027, 11.2 ± 1.3%, and 94.9 ± 3.2%, respectively. The morphology of NPs were spherical with a smooth surface. No chemical reaction was occurred between the components. Doxycycline was located within NP matrix in its molecular form. The DOX-SLN significantly decreased the microbial loading within macrophages (3.5 Log) in comparison with the free doxycycline. Conclusions: Since the DOX-SLN showed better effects on B. melitensis enclosed in macrophages than the free doxycycline, it is recommended to use it for treating brucellosis and preventing relapse.

Antimicrobial susceptibility profile, Adherence and invasion to mammalian cells of Brucellamelitensis isolates.

Susceptibilities of 66 Brucella isolates were tested in vitro. All isolates were susceptible to doxycycline, gentamic in and streptomycin. In addition, propyl paraben, cresol and benzalkoniumchloride were found to be the most powerful tested preservative, disinfectant and antiseptic, respectively. All isolates adhered to and invaded into Vero cells by variable degrees. Adherence and invasion of most isolates were significantly reduced by: (1) pretreatment of test isolates with trypsin and sodium metaperiodate; (2) pretreatment of Vero cells with lipase, neuraminidase and sodium metaperiodate; (3) Presence of Ca++, Mg++ and 200mM mannose in the assay medium and (4) growth of test isolates in half MICs of different antimicrobial agents. On the other hand, pretreatment of Vero cells with trypsin increased the adherence and invasion of most test isolates. No significant change in adhesion and invasion by changing the temperature from 27°C to 42°Cor the pH from 6 to 8. Log phase cultures showed higher adherence and invasion than stationary phase cultures.

Case Report: Visceral Leishmaniasis with Salmonella Paratyphi and Brucella melitensis Coinfection as a Cause of Persistent Fever in a Patient from Sudan.

We describe the case of a 12-year-old boy from Sudan who presented with fever of 1-week duration, headache, cough, and vomiting. A set of diagnostic tests led to the diagnosis of three infectious diseases: visceral leishmaniasis (probable diagnosis based on positive direct agglutination test), enteric fever (blood culture grown with Salmonella Paratyphi), and brucellosis (blood culture grown with Brucella melitensis). The patient received specific treatment of the three infections and recovered. This case illustrates the occurrence and possible implications of coinfections in patients with persistent fever, including conditions that are hard to diagnose in field settings, such as brucellosis and enteric fever.

Whole-genome sequencing and antimicrobial resistance in Brucella melitensis from a Norwegian perspective.

Brucellosis is a rarely encountered infection in Norway. The aim of this study was to explore all Brucella melitensis isolates collected in Norway from 1999 to 2016 in relation to origin of infection and antimicrobial resistance patterns. A total of 23 isolates were analysed by whole-genome sequencing and compared with selected sequences of B. melitensis available from NCBI. Additionally, SNP analysis in antibiotic resistance determining genes was performed. The majority belonged to the East Mediterranean clade (genotype II), while the remaining isolates belonged to the African clade (genotype III). These results indicate that human brucellosis in Norway is related to travels or migration from the Middle East, Asia or Africa, in accordance with results from Germany, Denmark and Sweden. Antibiotic susceptibility patterns were determined by broth microdilution method and/or gradient strip method. All isolates were susceptible for all tested antibiotics, except for rifampicin where phenotypical results indicated resistance or intermediate resistance in all isolates based on broth microdilution method, and in four isolates based on gradient strip testing. In contrast, screening of the rpoB gene did not reveal any mutations in the previously described rpoB "hot spot" regions related to rifampicin resistance, indicating overestimation of resistance based on phenotypical results.

Anti-Brucella activity of Caryopteris mongolica Bunge root extract against Brucella melitensis infection in mice.

BACKGROUND: The current treatment for human brucellosis requires a combination of antibiotics for long periods of time, and the reported incidence and prevalence of the disease vary widely in nomadic livestock of Mongolia. The objective of the present study was to evaluate the in vivo antibacterial activity of the C. mongolica root extract against B. melitensis. METHODS: In this study, we used of 6 groups of mice (n = 5). Five groups of BALB/c mice were inoculated intraperitoneally with the M16 strain of B. melintensis, as follows: (i) one group was used for pretreatment monitoring; (ii) the control group was administered 2% Tween 80 and was used as the non-treatment group; and the other three groups were treated with one oral gavage per day for 21 days with (iii) doxycycline (2 mg/day), (iv) doxycycline (1 mg/day) with root extract (20 mg/day), and (v) C. mongolica root extract (20 mg/day). The one group that was kept non-infected was used as a healthy control group. RESULTS: This study demonstrated that daily treatment with doxycycline alone and in combination with C. mongolica root extract significantly reduced splenic infection at the end of treatment. However, the spleen index of both the doxycycline-treated and the combination-treated groups of mice decreased by approximately 50% compared to that of the healthy control mouse group. Treatment with the C. mongolica root extract resulted in a 1.47log reduction in splenic infection compared to the non-treatment group, and the spleen index of the C. mongolica-treated group of mice was the same as that of the normal mouse group. In all treatment groups, neutrophil phagocytic activity significantly decreased, and all treatment groups demonstrated splenic regeneration. CONCLUSIONS: The present study showed that the C. mongolica root extract may be useful in the treatment of brucellosis patients, in combination with doxycycline or other antibiotics, to reduce the toxicity of high-dosage antibiotics, to prevent the development of antibiotic resistance and to prevent Brucella infection.

Pigs, pooches and pasteurisation: The changing face of brucellosis in Australia

BACKGROUND: Brucellosis, also known as undulant, Mediterranean or Malta fever, is a systemic infection that causes fever, sweats, arthralgias and myalgias. A globally important disease, brucellosis is re-emerging in Australia in association with feral pig hunting activities. OBJECTIVE: This article aims to provide clinicians with an overview of brucellosis, covering epidemiology, clinical features, diagnosis, management and prevention. DISCUSSION: Brucellosis should be suspected in all patients with non-specific, flu-like illness who fall into one of the major risk groups (feral pig hunters, overseas travellers and migrants). Depression is common and often severe, relative to other symptoms. Early diagnosis and treatment are important for preventing complications, which include osteoarticular, genitourinary or, more rarely, neurological or cardiovascular diseases. Diagnosing acute infections is based on serology and blood cultures; imaging and biopsy may be required for diagnosis of focal infections. Dual therapy with doxycycline and gentamicin is the recommended treatment. Relapse occurs in up to 10% of patients. Prevention is achieved through the use of protective gear during hunting and avoidance of unpasteurised dairy products in countries where occur in animals.