Real-time PCR for detection of Brucella spp. DNA in human serum samples

Presented here are the results of an evaluation of an in-house real-time PCR assay for the rapid and specific diagnosis of human brucellosis. The assay was based on direct amplification from serum samples of a 169-bp portion of bcsp31, a gene found in all Brucella species and biovars. Species specificity and selectivity of this real-time PCR assay were evaluated using genomic DNA from 15 Brucella strains and 42 non-Brucella strains, and the results were 100%. Among 17 culture-proven brucellosis patients, sera from 11 gave a positive amplification signal, corresponding to a sensitivity of 64.7%. In contrast, negative results were obtained for all sera from 60 control patients, corresponding to a specificity of 100%. The results indicate this test is well adapted for definite confirmation of brucellosis cases, when Brucella cultures remain sterile and serological tests demonstrate the presence of cross-reacting antibodies against Brucella sp. and Yersinia enterocolitica O:9 antigens.     

Human brucellosis in a nonendemic country: a report from Germany, 2002 and 2003

Human brucellosis has become a rare disease in Germany since the eradication of bovine and ovine/caprine brucellosis in this country. Therefore, most physicians are unfamiliar with the illnesses clinical presentation, diagnostic tools, and therapeutic strategies. This retrospective study was carried out to evaluate the epidemiological, clinical, and laboratory features of human brucellosis in Germany in the years 2002 and 2003. Thirty-one bacterial isolates from 30 patients sent to the German national reference laboratory were characterized using the genus-specific bcsp31 real-time PCR, the species-specific AMOS-PCR, and standard microbiological methods for the detection and identification of Brucella spp. The medical records of all patients with bacteriologically confirmed brucellosis were evaluated. All 31 isolates proved to be Brucella (30 Brucella melitensis and 1 Brucella suis). Most of the brucellosis patients were infected in endemic countries while visiting friends and relatives during their summer holidays. One case of laboratory-acquired infection was identified. Brucellosis was transmitted mainly by the consumption of contaminated unpasteurized milk or cheese from goats and sheep. The patients presented primarily with flu-like symptoms, i.e. fever, chills, sweating, headaches, arthralgia, and myalgia. In most cases, however, symptoms and signs of focal complications, e.g. spondylitis, endocarditis, and meningoencephalitis, predominated. The rate of complications was much higher than that in endemic countries, presumably as a result of diagnostic delay due to a low index of suspicion. In summary, physicians in nonendemic countries such as Germany must be aware of brucellosis being a possible cause of fever of unknown origin in immigrants and tourists travelling from endemic countries.     

Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis.     

Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.     

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.     

Diagnostic Usefulness of Antibodies against Ribosome Recycling Factor from Brucella melitensis in Human or Canine Brucellosis

The diagnostic usefulness of an enzyme-linked immunosorbent assay (ELISA) using a purified recombinant ribosome recycling factor from Brucella melitensis (CP24 antigen) was tested in human and canine infections caused by smooth and rough Brucella species, respectively. Anti-CP24 antibodies were detected in 9 (43%) of 21 consecutive cases of canine brucellosis and in 8 (53%) of 15 dogs followed for 60 days after the diagnosis of acute brucellosis. Among eight patients with acute brucellosis, anti-CP24 antibodies were detected in four in the 10 weeks following diagnosis, but the remaining four were negative during the whole follow-up (22 weeks). The frequency of anti-CP24 antibodies was also low among 24 patients with subacute brucellosis and 23 patients with chronic illness (29 and 26%, respectively). While all patients positive for anti-CP24 antibodies were also positive for antibodies to total cytoplasmic proteins of Brucella (CP), five were negative for antibodies to another cytoplasmic protein, the Brucella lumazine synthase (BLS). When a larger sample of 35 human sera negative for anti-BLS antibodies was assayed, 85.7% were positive for anti-CP24 antibodies, suggesting that the combined measurement of both reactivities could yield a higher sensitivity than any test alone. To test this hypothesis, an ELISA combining both antigens was designed. The percentage of positive results among chronic cases was higher for this assay than for the individual measurement of anti-CP24 or anti-BLS antibodies (83 versus 26 and 65%, respectively) and was closer to the value obtained for anti-CP antibodies (91%). The frequency of anti-CP24 antibodies is low in both canine and human brucellosis. In the latter case, however, an ELISA combining CP24 and BLS is more sensitive than assays measuring anti-CP24 or anti-BLS antibodies separately and almost as sensitive as the ELISA using CP.     

Asymptomatic brucellosis infection in humans: implications for diagnosis and prevention

Human brucellosis is mainly caused by contact with Brucella-infected animals and their secretions and carcasses. Individuals who are continuously in contact with animals are considered to be at a high risk but only some show symptoms and are diagnosed as cases of brucellosis. Here, we showed that asymptomatic brucellosis infections occur among humans. Asymptomatic infections mainly result from less frequent contact with Brucella and/or contact with low-virulence Brucella. In our study, patients with asymptomatic infection had low antibody titres and different contact patterns. Awareness of asymptomatic infection is important for early diagnosis of brucellosis and prevention of chronic infection.     

Online-Only Abstract

Human brucellosis is mainly caused by contact with Brucella-infected animals and their secretions and carcasses. Individuals who are continuously in contact with animals are considered to be at a high risk but only some show symptoms and are diagnosed as cases of brucellosis. Here, we showed that asymptomatic brucellosis infections occur among humans. Asymptomatic infections mainly result from less frequent contact with Brucella and/or contact with low-virulence Brucella. In our study, patients with asymptomatic infection had low antibody titres and different contact patterns. Awareness of asymptomatic infection is important for early diagnosis of brucellosis and prevention of chronic infection.     

Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.     

Evaluation of the PANBIO Brucella Immunoglobulin G (IgG) and IgM Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Brucellosis

PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis.     

Brucellosis in patients infected with the human immunodeficiency virus

Brucellosis has been described rarely in patients infected with HIV, despite the fact that eradication of intracellular brucellae is largely dependent on cell-mediated immunity. The characteristics of all patients with HIV infection and brucellosis seen in seven Spanish hospitals are reported. Since the beginning of the AIDS epidemic, 12 HIV-infected patients were diagnosed with brucellosis (8 with cultures positive forBrucella spp., 4 with high anti-Brucella antibody titers). Most patients were male and intravenous drug users. Eleven patients had no symptoms of HIV infection when first diagnosed with brucellosis and had relatively preserved cellular immunity (median CD4+ cell count 588, range 136–1006). There was a clear epidemiologic antecedent for acquisition of brucellosis in 11 patients. Clinical symptoms included fever, arthromyalgia, and sweating in all patients; four patients presented with focal disease. All patients had high agglutinin titers, and eight of nine had cultures positive forBrucella. Therapy with doxycycline and streptomycin was curative in all cases. Two patients experienced a recurrence of symptoms after initial treatment, although no microbiological relapses were documented after a median follow-up period of 18 months. HIV infection does not seem to increase the incidence of brucellosis. Since most cases occur in asymptomatic patients with relatively preserved immunity, the epidemiology, clinical presentation, diagnosis, response to therapy, and outcome are similar to those observed in non-HIV infected patients.     

Chronic Brucellosis and Persistence of Brucella melitensis DNA

After acute brucellosis infection, symptoms persist in a minority of patients for more than 1 year. Such patients are defined as having chronic brucellosis. Since no objective laboratory methods exist to confirm the presence of chronic disease, these patients suffer delays in both diagnosis and treatment. The aim of the current study was to evaluate the usefulness of quantitative real-time PCR (Q-PCR) in the diagnosis and follow-up of these patients. Thirty-five subjects with a well-documented history of brucellosis that had been diagnosed between 2 and 33 years previously were screened by Q-PCR for the presence of Brucella melitensis DNA and by serological tests and blood culture. Subjects were divided into three groups: 8 (23%) focal-disease subjects, 9 (26%) nonfocal-disease subjects with subjective complaints, such as fatigue, malaise, arthralgia, and/or myalgia, and 18 (51%) asymptomatic subjects. All (100%) focal-disease patients and symptomatic nonfocal-disease patients had at least one positive Q-PCR sample. Only six (33%) of the asymptomatic subjects had Q-PCR-positive samples (P < 0.05). Eleven patients (five focal-disease patients and six nonfocal-disease patients with subjective complaints) received therapy during the study. For those patients who completed treatment, six (60%) still had Q-PCR-positive samples at the posttreatment follow-up. The proportion of individuals with B. melitensis DNA was significantly higher for symptomatic nonfocal-disease patients than for asymptomatic subjects. Therefore, Q-PCR appears to be a useful method for identifying chronic brucellosis patients.Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations. The causative organisms, Brucella spp., are facultatively intracellular bacteria that are capable of evading a number of host defense mechanisms and can survive within phagocytic cells for long periods. These properties may account for focal complications, relapses, and chronic disease (21).The chronic course of the disease was initially explored during the 1930s by Evans (6a) and was further explored in the 1950s by Davies (5). In 1951, Spink and associates stated, with respect to the duration of the illness, that the majority of patients with brucellosis recover within a year after the administration of an antibiotic, while a small but significant number of patients continue to have clinical manifestations despite such therapy (22). These patients can be divided into two groups: those with a focal disease, such as spondylitis, and those without a focal disease who complain, nevertheless, of poor health and have symptoms such as chronic fatigue syndrome (CFS), musculoskeletal pain, depression, or anxiety.The diagnosis of chronic brucellosis is often based on clinical complaints together with the presence of high immunoglobulin G titers (2). However, the specificity of current serological assays is considered to be low, since titers may remain positive for years after the successful resolution of symptoms. In 1980, Buchanan and Faber described a serological test for evaluation of the effectiveness of treatment and for excluding a diagnosis of chronic brucellosis (3). The authors observed that, in assays with 2-mercapthoethanol, results remained positive for 9% of patients 1 year after the initiation of treatment. Among these patients, 50% still had signs and symptoms of brucellosis and required further treatment. Recently, PCR has been used to detect Brucella spp. in the diagnosis of primary infections, relapse, and focal complications of the disease (12, 13, 20). Initially, the persistence of Brucella sp. DNA after therapy was linked to relapse (9, 12, 17). However, our group and others have demonstrated the persistence of Brucella sp. DNA for long periods of time after the conclusion of therapy in asymptomatic patients (10, 15, 24). The practical role of quantitative real-time PCR (Q-PCR) in the laboratory diagnosis of chronic brucellosis and the assessment of clinical manifestations remains to be demonstrated. In the present study, we examined 35 subjects with a well-documented history of brucellosis and followed them to evaluate the usefulness of Q-PCR and the relationship between a positive result by the Q-PCR assay and clinical course.     

Identification of Brucella by Ribosomal-Spacer-Region PCR and Differentiation of Brucella canis from Other Brucella spp. Pathogenic for Humans by Carbohydrate Profiles

Molecular and chemical characteristics often provide complementary information in the differentiation of closely related organisms. The genus Brucella consists of a highly conserved group of organisms. Identification of the four species pathogenic in humans (Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis) is problematic for many clinical laboratories that depend primarily on serology and phenotypic characteristics to differentiate species. PCR amplification of the 16S-23S ribosomal DNA interspace region was evaluated for species-specific polymorphism. B. abortus, B. melitensis, B. suis, and B. canis produced identical PCR interspace profiles. However, these PCR products were unique to brucellae, allowing them to be readily distinguished from other gram-negative bacteria (including Bartonella spp. and Agrobacterium spp.). Carbohydrate profiles differentiated B. canis from the other three Brucella species due to the absence of the rare amino sugar quinovosamine in the three other species. PCR of the rRNA interspace region is useful in identification of the genus Brucella, while carbohydrate profiling is capable of differentiating B. canis from the other Brucella species.     

Review of Detection of Brucella sp. by Polymerase Chain Reaction

Here we present a review of most of the currently used polymerase chain reaction (PCR)-based methods for identification of Brucella bacteria in biological samples. We focused in particular on methods using single-pair primers, multiplex primers, real-time PCRs, PCRs for marine Brucella, and PCRs for molecular biotyping. These methods are becoming very important tools for the identification of Brucella, at the species level and recently also at the biovar level. These techniques require minimum biological containment and can provide results in a very short time. In addition, genetic fingerprinting of isolates aid in epidemiological studies of the disease and its control. PCR-based methods are more useful and practical than conventional methods used to identify Brucella spp., and new methods for Brucella spp identification and typing are still being developed. However, the sensitivity, specificity, and issues of quality control and quality assurance using these methods must be fully validated on clinical samples before PCR can be used in routine laboratory testing for brucellosis.Brucellosis is caused by Brucella spp which is composed of seven terrestrial species and at least two marine species. Terrestrial Brucella spp. include B. abortus, B. melitensis, B. suis. B. ovis, B. canis, B. Neotomae, and a new species, B. microti. Brucella isolated from marine mammals are B. ceti and B. pinnipedialis (1). The first 3 terrestrial species include several biovars. The terrestrial Brucella species display a high degree of DNA homology based on DNA-DNA hybridization studies. Nevertheless, DNA polymorphism sufficient to differentiate the first 6 Brucella species and some of their biovars has been shown to exist (2). Brucella isolated from marine mammalian species is still under investigation. According to the classical criteria of host preference and DNA polymorphism at their outer membrane protein 2 (omp2) locus, at least 2 species that infect marine mammals exist (3).The gold standard for the diagnosis of brucellosis is isolation of Brucella bacteria. However, to isolate Brucella bacteria is time- and resource-intensive; it requires level 3 biocontainment facilities and highly skilled technical personnel to handle samples and live bacteria for eventual identification and biotyping. Handling all live Brucella involves risk of laboratory infection and very strict biosafety rules must be observed. In order to avoid these disadvantages, methods based on the polymerase chain reaction (PCR) are becoming very useful and considerable progress has been made recently to improve their sensitivity, specificity, and technical ease and to lower costs. To date, at least 400 reports have been published dealing with various PCR-based methods for Brucellosis detection. In this review, we discuss extraction of DNA and various PCR methods using different primers and reaction conditions.     

Immunochromatographic Brucella-Specific Immunoglobulin M and G Lateral Flow Assays for Rapid Serodiagnosis of Human Brucellosis

To fulfill the need for a simple and rapid diagnostic test for human brucellosis, we used the immunochromatographic lateral flow assay format to develop two assays, one for the detection of Brucella-specific immunoglobulin M (IgM) antibodies and one for the detection of Brucella-specific IgG antibodies. The diagnostic values of these tests were examined. The tests are shown to detect acute, persistent, and relapsing disease and can be used to monitor treatment. The sensitivity of Brucella IgM and IgG flow assays calculated for the combined assay results is 96%, and specificity amounts to 99%. The flow assay requires neither specialized training nor equipment, the assay is very easy to perform and to read, and the components are stable without a requirement for refrigeration and well standardized. Together these characteristics indicate that the Brucella IgM and IgG flow assays are ideal for use in clinical settings in rural and suburban areas in which brucellosis is endemic.     

Application of a Polymerase Chain Reaction Enzyme Immunoassay in Peripheral Whole Blood and Serum Specimens for Diagnosis of Acute Human Brucellosis

A simple polymerase chain reaction-enzyme immunoassay (PCR-EIA) was employed for the rapid laboratory diagnosis of human brucellosis directly from peripheral blood. Whole blood and serum specimens were collected from 243 patients with acute brucellosis as determined by blood culture, serological tests, and the patients clinical characteristics and from a control group of 50 healthy individuals. Diagnosis of brucellosis was established in 179 cases by isolation of Brucella spp. in blood culture and in 64 cases by clinical signs and serological investigation. Following the amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenic membrane protein specific for the Brucella genus, the amplified product was detected in a microtiter plate by hybridization. Two hundred forty-one of the 243 patients tested had detectable Brucella DNA in either whole blood or serum specimens: 149 (61.3%) patients were positive in both whole blood and serum specimens, 43 (17.7%) were positive in serum specimens only, and 49 (20.2%) were positive in whole blood specimens only. The diagnostic specificity of the PCR-EIA assay for both specimen categories was 100%, while the sensitivity was 81.5% for whole blood specimens, 79% for serum specimens, and 99.2% for whole blood and serum specimens combined. The results suggest that the detection of Brucella DNA in whole blood and serum specimens by PCR-EIA assay is a sensitive and specific method that could assist the rapid and accurate diagnosis of acute human brucellosis.     

Specificity of a Polymerase Chain Reaction Assay of a Target Sequence on the 31-Kilodalton Brucella Antigen DNA Used to Diagnose Human Brucellosis

 The aim of this study was to evaluate the specificity of a polymerase chain reaction assay for detecting Brucella DNA using primers specific for the amplification of a 223 bp region of the sequence encoding a 31 kDa immunogenic Brucella abortus protein (BCSP31). DNA from all Brucella strains, including type, reference, vaccine and field strains, were correctly amplified. With the exception of Ochrobactrum spp., no other amplification was detected with a broad panel of microorganisms serologically or phylogenetically related to Brucella spp. This very good degree of specificity, together with its high yield demonstrated in previous clinical studies, confirms that this polymerase chain reaction assay could be a useful tool for the diagnosis of human brucellosis.     

Evaluation of the Efficacy of Outer Membrane Protein 31 Vaccine Formulations for Protection against Brucella canis in BALB/c Mice

Canine brucellosis is an infectious disease caused by the Gram-negative bacterium Brucella canis. Unlike conventional control programs for other species of the genus Brucella, currently there is no vaccine available against canine brucellosis, and preventive measures are simply diagnosis and isolation of infected dogs. New approaches are therefore needed to develop an effective and safe immunization strategy against this zoonotic pathogen. In this study, BALB/c mice were subcutaneously immunized with the following: (i) the recombinant Brucella Omp31 antigen formulated in different adjuvants (incomplete Freund adjuvant, aluminum hydroxide, Quil A, and Montanide IMS 3012 VGPR), (ii) plasmid pCIOmp31, or (iii) pCIOmp31 plasmid followed by boosting with recombinant Omp31 (rOmp31). The immune response and the protective efficacy against B. canis infection were characterized. The different strategies induced a strong immunoglobulin G (IgG) response. Furthermore, spleen cells from rOmp31-immunized mice produced gamma interferon and interleukin-4 (IL-4) after in vitro stimulation with rOmp31, indicating the induction of a mixed Th1-Th2 response. Recombinant Omp31 administered with different adjuvants as well as the prime-boost strategy conferred protection against B. canis. In conclusion, our results suggest that Omp31 could be a useful candidate for the development of a subcellular vaccine against B. canis infection.     

Antimicrobial susceptibility testing of Brucella melitensis isolated from patients with acute brucellosis in a centre of Iran

Structured to Purpose: Human brucellosis is one of the most common zoonotic infections worldwide, which remains one of the major problems for public health. Despite the World Health Organization’s recommendation for human brucellosis treatment, sporadic cases of relapse have been reported. The aim of this study was to assess the susceptibility of Brucella isolates to common antibiotics that are prescribed by the physician for the treatment of brucellosis and also to determine the minimum inhibitory concentration 50% (MIC₅₀) and MIC₉₀ for these antibiotics. Materials and Methods: Forty-eight Brucella strains were collected from patients with acute brucellosis. Species identification was made based on the conventional methods. MIC of rifampin, doxycycline, ciprofloxacin, trimethoprim-sulfamethoxazole, streptomycin, azithromycin and ceftriaxone was determined by E-test. Results: All the 48 Brucella isolates (47 blood samples and one synovial fluid) were identified as Brucella melitensis. No antimicrobial-resistant strains were recognised. Trimethoprim-sulfamethoxazole had the lowest MIC₅₀ (0.016 μg/ml) and MIC₉₀ (0.064 μg/ml), whereas MIC₅₀ and MIC₉₀ of streptomycin and azithromycin had the highest level at 0.625, 1.5 µg/ml and 0.25, 1 µg/ml, respectively. All the isolates were susceptible to rifampin, and only one of the isolates had a reduced sensitivity to rifampin (1 μg/ml). Conclusions: Although all the Brucella isolates were susceptible, antimicrobial susceptibility test should be recommended in patients with recurrent brucellosis or life-threatening organ involvement.