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.

Identification and characteristics of a novel aminoglycoside phosphotransferase, APH(3')-IId, from an MDR clinical isolate of Brucella intermedia.

OBJECTIVES: To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3')-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. METHODS: Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. RESULTS: APH(3')-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3')-IIb. Expression of aph(3')-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3')-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3')-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3')-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M-1 s-1. Genetic environment and comparative genomic analyses suggested that aph(3')-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. CONCLUSIONS: APH(3')-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3')-IId represents the fourth characterized example of an APH(3')-II enzyme.

Investigation of antibiotic susceptibilities of Brucella Strains isolated from various clinical samples in eastern Turkey.

BACKGROUND: Brucellosis is a worldwide zoonotic disease that causes serious public health problems. This study aimed to identify Brucella strains isolated from various clinical samples by conventional and molecular methods and to determine antimicrobial susceptibilities against doxycycline (DOX), streptomycin (STR), ciprofloxacin (CIP) and rifampicin (RIF) by the gradient strip (E test) test method. METHODS: A total of 87 Brucella strains isolated from various clinical specimens between 2004 and 2018 were included in this study. While four of the 87 strains included in the study were identified only at the genus level, the remaining 83 strains were identified at the species level by the Real-Time Multiplex PCR (M-RT-PCR) method and conventional methods were used for biotyping. RESULTS: According to molecular identification results, 83 strains were identified as B. melitensis by the M-RT-PCR method, with 82 strains identified as Brucella melitensis biovar (bv) 3 and one as B. melitensis bv 1 according to the conventional biotyping method. Among the antibiotics studied, CIP was found to be the most active agent according to the minimum inhibitory concentrations (MIC)90 values. This was followed by DOX and STR, respectively. While all of the isolates were sensitive to CIP, DOX and STR, 18 (20.7%) strains were found to be moderately susceptible to RIF, with the highest values of MIC50 and MIC90. CONCLUSIONS: In our study, all strains were identified as B. melitensis. DOX, STR, CIP and RIF used in the treatment of brucellosis were found to be effective.

Dual versus triple therapy for uncomplicated brucellosis: A retrospective cohort study.

INTRODUCTION: Brucellosis is a zoonotic disease caused by Brucella spp. affecting multiple body systems and may lead to complications. Saudi Arabia is a country where brucellosis is endemic. This study aimed to describe the epidemiological characteristics of uncomplicated brucellosis and to assess outcomes of different antibiotic regimens. METHODOLOGY: A retrospective cohort study in a Saudi tertiary academic medical center. Adults with confirmed uncomplicated brucellosis between January 2008 and December 2018 who received antibiotics were included. The primary endpoint was clinical cure. Secondary endpoints included all-cause mortality and length of stay. RESULTS: Fifty-four patients met the inclusion criteria and were included in the study. Twenty five patients received a combination of doxycycline, rifampin, and aminoglycoside (group 1), whereas 29 patients received doxycycline and rifampin (group 2). There was no significant difference between the two groups in clinical cure, all-cause mortality, length of stay, and end of therapy parameters, including temperature, white blood cells count, C-reactive protein levels, and erythrocyte sedimentation rates. CONCLUSIONS: Due to lack of differences in clinical outcomes, mortality, length of stay, and end of therapy parameters between the two groups, a regimen comprising two, rather than three, agents can be sufficient for uncomplicated brucellosis. This finding conforms to previous studies. Therefore, replacing rifampin with an aminoglycoside for its presumed superior efficacy as per the World Health Organization's guidelines is not substantiated by our study. Further studies with a larger sample size are required to confirm these findings.

The expression of type II TA system genes following exposure to the sub-inhibitory concentration of gentamicin and acid stress in Brucella spp.

BACKGROUND: Brucellosis is one of the most common diseases that afflicts both humans and animals. Bacteria react to stress conditions using different mechanisms one of which is Toxin-Antitoxin (TA) systems. It is believed that the Toxin-Antitoxin (TA) systems have a key role in the chronicity of the disease. This study investigated the expression of TA system genes under acid and antibiotic stresses in Brucella spp. METHODS: Fifty Brucella isolates (17 isolated from animals and 31 isolated from human specimens, and two standard strains) were analyzed using PCR (using two pairs of primers). Then, to determine the effects of sub-MIC of gentamicin on bacterial survival and growth, colony forming unit was quantitated and turbidity was assessed following the treatment of Brucella spp, with ½ MIC of gentamicin at different time intervals. Furthermore, the colony forming unit of Brucella spp, was assessed under acid stress (pH = 5.5) compared to the control (pH = 7.6). Moreover, the expression of TA system genes in Brucella spp, was evaluated 1 h after treatment using qRT-PCR method. RESULTS: A total of 50 isolates, including 41 (82%) Brucella melitensis and 7 (14%) Brucella abortus with two standard strains Brucella melitensis (16 M) and Brucella abortus (B19) were investigated. Our results revealed the reduced growth of Brucella spp. in the presence of sub-MIC of gentamicin compared to the control. Furthermore, according to the results of qRT-PCR assay, gentamicin could increase the expression of TA system genes. Also, results of qRT-PCR showed that under acid stress, the expression of TA system gene COGT/COGAT decreased compared to the control. CONCLUSION: Although the exact role of the TA systems in response to stress is still unclear, our study provided information on the effect of the type II TA systems under the acid and antibiotic stress conditions. However, further studies are still required.

Nanoniosome-encapsulated levoflaxicin as an antibacterial agent against Brucella.

A study was conducted to examine the prevalence of brucellosis (in animal farms) in the vicinity of Islamabad and Rawalpindi. A total of 170 milk samples were collected randomly from several farmhouses. The collected milk samples were initially screened by a Brucella selective medium. The bacterial isolates grown on the selective medium were subjected to biochemical identification for further confirmation of Brucella species. Among the tested samples, 28 (16.4%) were found positive for selective medium and 14 (8.2%) were found positive after biochemical confirmation. The antimicrobial susceptibility of several antibiotics performed by the disc-diffusion method did not yield any significant findings. Encapsulating antimicrobial drugs in unilamellar niosomes is an effective approach to treat the endemic infection. In this study, the antimicrobial activity of niosome-encapsulated levofloxacin is compared with free drug. The drug-encapsulating and empty niosomes were synthesized by using two surfactants Tween 80 and Span 40. Niosomal characterization included electron microscopy, dynamic light scattering, and zeta potential. The encapsulation efficiency was found to be 78% and 74% for Span 40 and Tween 80 niosomes, respectively. The antibacterial activity of niosomal levofloxacin was evaluated against the identified Brucella species and the antimicrobial activity of the free drug was increased many folds after encapsulation. In this study, levofloxacin niosomes were successfully synthesized against Brucellosis.

Monitoring the course of Brucella infection with qPCR-based detection.

OBJECTIVES: To determine blood Brucella DNA loads between brucellosis patients and those without brucellosis. METHODS: The patient group included 350 brucellosis patients. The control was composed of 200 subjects without brucellosis. The extracted DNA from blood was tested by quantitative polymerase chain reaction (qPCR). The cutoff value was determined by receiver operating characteristic curve analysis. A portion of the brucellosis patients were monitored by qPCR during therapy. RESULTS: The detection limit of qPCR was between 1E+01cfu/µL and 1E+08cfu/µL. The standard curve R2 reached 0.998. The cutoff value was 4E+01cfu/µL, which was determined by comparison of the patient group and the control. The qPCR assay had a specificity of 100% and a sensitivity of 93.14%. The monitoring results showed that the Brucella DNA load decreased in most patients during the first 4 weeks of treatment. One patient with bad treatment compliance showed a rebound. CONCLUSIONS: The qPCR results were in accordance with the course of brucellosis in the clinic. The DNA load often reflects the situation of the Brucella-infected patient. The cutoff value provides an important reference of infection. This qPCR-based method can be used to assist in the diagnosis of brucellosis and to adjust the therapy.

Brucellosis in pregnancy: results of multicenter ID-IRI study.

Brucellosis in pregnant women is reported to be associated with obstetric complications (OCs), and adequate data for human brucellosis during pregnancy are largely lacking. We performed this multicenter retrospective cross-sectional study to evaluate the epidemiology, clinical course, treatment responses, and outcomes of brucellosis among pregnant women. The study period comprised a 14-year period from January 2002 to December 2015. All consecutive pregnant women diagnosed with brucellosis in 23 participating hospitals were included. Epidemiological, clinical, laboratory, therapeutic, and outcome data along with the assessment data of the neonate were collected using a standardized questionnaire. Data of 242 patients were analyzed. The OC rate was 14.0% (34/242) in the cohort. Of the 242 women, 219 (90.5%) delivered at term, 3 (1.2%) had preterm delivery, 15 (6.2%) aborted, and 5 (2.1%) had intrauterine fetal demise. Seventeen (7.0%) of the newborns were considered as low birth weight. Spontaneous abortion (6.1%) was the commonest complication. There were no maternal or neonatal deaths and pertinent sequelae or complications were not detected in the newborns. Splenomegaly (p = 0.019), nausea and/or vomiting (p < 0.001), vaginal bleeding (p < 0.001), anemia (blood hemoglobin < 11 g/dL; p < 0.001), high level of serum aspartate aminotransferase (> 41 IU/L; p = 0.025), oligohydramnios on ultrasonography (p = 0.0002), history of taking medication other than Brucella treatment during pregnancy (p = 0.027), and Brucella bacteremia (p = 0.029) were the significant factors associated with OCs. We recommend that pregnant women with OC or with fever should be investigated for brucellosis if they live in or have traveled to an endemic area.

Research Progress on Brucellosis.

Brucellosis is a debilitating febrile illness caused by an intracellular Brucella. The disease is distributed in humans and animals widely, especially in developing countries. Ten species are included in the genus Brucella nowadays; four species of them are pathogenic to humans, which make brucellosis a zoonosis with more than 500,000 new cases reported annually. For human brucellosis, the most pathogenic species is B. melitensis followed by B. suis, while B. abortus is the mildest type of brucellosis. The infection mechanism of Brucella is complicated and mostly relies on its virulence factors. The therapy of the disease contains vaccination and antibiotic. However, there are some defects in currently available vaccines such as the lower protective level and safety. Thus, safe and efficient vaccines for brucellosis are still awaited. The dual therapy of antibacterial is effective in the treatment of brucellosis if a rapid and exact detection method is found.

Antimicrobial Resistance in Leptospira, Brucella, and Other Rarely Investigated Veterinary and Zoonotic Pathogens.

Leptospira, Brucella, and Borrelia are major agents of zoonotic disease, causing high morbidity and, in some cases, significant mortality in humans. For all three genera, prompt diagnosis and appropriate antimicrobial therapy are required to prevent the development of chronic, debilitating illness. Leptospira spp. are intrinsically resistant to several antimicrobial classes; however, there is little evidence in the literature for development of acquired resistance to antimicrobial agents used for clinical treatment of acute leptospirosis. For Brucella infections, there are numerous reports of relapses following therapy, but it is unclear whether this is due to sequestration within infected sites (e.g., bone) or the development of acquired resistance. Brucella have maintained their susceptibility to doxycycline and rifampicin, which in combination remain the most common treatments of brucellosis in humans. In vitro induced point mutations are described as imparting resistance to rifampicin (rpoB) and fluoroquinolones (gyrA). The clinical significance of these mutations is unclear. For Borrelia burgdorferi, although acquired resistance to some antimicrobial agents has been described, resistance due to bacterial persister cells surviving in the presence of antimicrobial, with no apparent increase in the MIC of the organism, have been recently described. Of the remaining veterinary fastidious pathogens, Lawsonia intracellularis is the most interesting from an antimicrobial resistance perspective because it can only be grown in cell culture, making in vitro susceptibility testing challenging. MIC testing has been undertaken on a small number of isolates, and some differences in susceptibility to macrolides have been demonstrated between isolates obtained from different regions.

Case Report: Neurobrucellosis with Plastered Spinal Arachnoiditis: A Magnetic Resonance Imaging-Based Report.

Diffuse spinal arachnoiditis in neurobrucellosis is a rare manifestation. We report a boy aged 17, presenting with hearing impairment and recurrent vomiting for 18 months, weight loss for 12 months, dysphagia, dysarthria, hypophonia for 6 months, and gait unsteadiness for 5 months. He had bilateral 5th (motor) to 12th cranial nerve palsy, wasting and weakness of limbs, fasciculations, absent tendon reflexes, and positive Babinski's sign. Cerebrospinal fluid (CSF) showed raised protein and pleocytosis. Magnetic resonance imaging (MRI) showed extensive enhancing exudates in cisterns and post-contrast enhancement of bilateral 5th, 6th, 7th, and 8th nerves. Spine showed clumping with contrast enhancement of the cauda equina roots and encasement of the cord with exudates. Serum and CSF were positive for anti-Brucella antibodies. He showed significant improvement with antibiotics. At 4 months follow-up, MRI demonstrated near complete resolution of cranial and spinal arachnoiditis. It is important to recognize such rare atypical presentations of neurobrucellosis.

[Determination of in vitro susceptibilities of Brucella spp. strains against 11 different antibacterial gents isolated from blood cultures]. / Kan kültürlerinden izole edilen Brucella spp. suslarinin 11 farkli antibakteriyel ilaca karsi in vitro duyarliliklarinin belirlenmesi.

Brucellosis is a worldwide zoonotic disease and still continuous to be a major public health problem. In this study, it was aimed to identify the Brucella strains to the species level isolated from blood cultures, and to determine the rate of antimicrobial susceptibility against eleven antibacterial agents. A total of 106 Brucella spp. strains were included in the study, which were isolated from blood cultures in University of Health Sciences, Konya Training and Research Hospital, Medical Microbiology Laboratory between January 2011 and June 2013. Identification of the isolated strains were mainly based on conventional methods. In vitro antibacterial susceptibilities of azithromycin, ciprofloxacin, doxycycline, gentamicin, levofloxacin, moxifloxacin, rifampicin, streptomycin, tetracycline, tigecycline, and trimethoprim/sulfamethoxazole, were evaluated by using the gradient (E-test, bioMerieux, France) strip method. The bacterial suspensions adjusted to 0.5 McFarland turbidity was inoculated to Mueller Hinton agar plates, supplemented with 5% sheep blood, and E-test strips of selected antibacterial were applied. The plates were incubated in ambient air 48 hours at 37ºC and Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as quality control strains for antimicrobial susceptibility testing. Minimum inhibitors concentration (MIC) values were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines for slow-growing bacteria such as Haemophilus spp. Of the 106 Brucella spp. strains included in to the study, 90 were identified as Brucella melitensis, and 16 were Brucella abortus. MIC90 values of azithromycin, ciprofloxacin, doxycycline, gentamicin, levofloxacin, moxifloxacin, rifampicin, streptomycin, tetracycline, tigecycline, and trimethoprim/sulfamethoxazole were determined as 1 µg/ml, 0.25 µg/ml, 0.19 µg/ml, 0.25 µg/ml, 0.19 µg/ml, 0.75 µg/ml, 0.25 µg/ml, 0.75 µg/ml, 0.38 µg/ml, 0.64 µg/ml, and 0.19 µg/ml respectively. According to MIC90 values, gentamicin, moxifloxacin, and trimethoprim/sulfamethoxazole, were the most effective antibacterial agents. All the Brucella strains were sensitive to all the tested antibacterial agents except rifampicin. Only six isolates showed intermediate susceptibility to rifampicin. With regard to fluoroquinolones, the most active antibacterial agent was moxifloxacin, followed by ciprofloxacin and levofloxacin. In our study, no resistance was found for the classically recommended antibacterial agents used in the treatment of Brucella species in our hospital but antibiotic susceptibility patterns of Brucella spp. may vary geographically. As a result it was concluded that, the antimicrobial susceptibilities of Brucella species should be determined and controlled periodically to avoid the possible development of resistance problems in the future.

Central Nervous System Brucellosis Granuloma and White Matter Disease in Immunocompromised Patient.

Brucellosis is a multisystem zoonotic disease. We report an unusual case of neurobrucellosis with seizures in an immunocompromised patient in Saudi Arabia who underwent renal transplantation. Magnetic resonance imaging of the brain showed diffuse white matter lesions. Serum and cerebrospinal fluid were positive for Brucella sp. Granuloma was detected in a brain biopsy specimen.

[Study on antimicrobial susceptibility of Brucella in a city].

Objective: To investigate the antimicrobial susceptibility of Brucella and to provide a scientific basis for rational drug use and effective treatment of patients with brucellosis. Methods: A total of 41 Brucella strains were isolated from the blood of patients with brucellosis in 5 counties and 2 districts in Yuxi City, China from 2014 to 2016. The susceptibility to 23 antimicrobial drugs was tested using Kirby-Bauer (K-B) disk diffusion method and the sizes of antimicrobial rings were recorded. The susceptibility testing results were interpreted according to the Drug Susceptibility Testing Guideline (2009 version) . Results: The susceptibility rate of Brucella was 100.00% to ofloxacin, ciprofloxacin, levofloxacin, and amikacin and >90% to cefotaxime, cefepime, imipenem, doxycycline, cefoperazone, minocycline, tobramycin, rifampicin, cefoperazone/sulbactam, and chloramphenicol. The high resistance to aztreonam and ampicillin was observed (87.80% and 41.46%). Doxycycline-intermediate strains, rifampicin-intermediate strains, and rifampicin-resistant strains were identified. Conclusion: Doxycycline and rifampicin are commonly used in the treatment of brucellosis, but doxycycline/rifampicin-intermediate and-resistant strains have been identified. The susceptibility of Brucella to fluoroquinolones and cephalosporins was high, so the two drugs can be considered in the treatment of brucellosis.

The role of 'atypical' Brucella in amphibians: are we facing novel emerging pathogens?

AIMS: To discuss together the novel cases of Brucella infections in frogs with the results of published reports to extend our current knowledge on 'atypical' brucellae isolated from amphibians and to discuss the challenges we face on this extraordinary emerging group of pathogens. METHODS AND RESULTS: Since our first description, an additional 14 isolates from four different frog species were collected. Novel isolates and a subset of Brucella isolates previously cultured from African bullfrogs were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), Fourier transform-infrared (FT-IR) spectroscopy and broth microdilution susceptibility testing. MALDI-TOF MS worked very efficiently for an accurate bacterial identification to the genus level. Within the cluster analysis, 'atypical' brucellae grouped distant from Brucella melitensis and were even more separated by FT-IR spectroscopy with respect to their geographical origin. Minimum inhibitory concentrations of 14 antimicrobial substances are provided as baseline data on antimicrobial susceptibility. CONCLUSIONS: The case history of Brucella infections in amphibians reveals a variety of pathologies ranging from localized manifestations to systemic infections. Some isolates seem to be capable of causing high mortality in zoological exhibitions putting higher demands on the management of endangered frog species. There is considerable risk in overlooking and misidentifying 'atypical' Brucella in routine diagnostics. SIGNIFICANCE AND IMPACT OF THE STUDY: Brucella have only recently been described in cold-blooded vertebrates. Their presence in frog species native to Africa, America and Australia indicates a more common occurrence in amphibians than previously thought. This study provides an extensive overview of amphibian brucellae by highlighting the main features of their clinical significance, diagnosis and zoonotic potential.

[THE PERSPECTIVES OF STUDYING OF POLYMORPHISM OF GENES OF GAMMA-INTERFERON UNDER CHRONIC BRUCELLOSIS].

The brucellosis is an actual zoonotic disease in many countries, Russia included. The complexity of individual prognosis of disease and choice of tactics of maintenance of patients is explained by heterogeneity of clinical manifestations of brucellosis and different rate of progression of organs pathology. Despite of low mortality, this pathology quite often results in disability of patient. The frequent transition of acute process into chronic one (40-60%), probability of development of primary chronic brucellosis determines interest of researchers to issues of immunopathogenesis of this disease. The article presents review of achievements in studies of polymorphism of genes of gamma-interferon in the given area.

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Analysis.

We describe a simple protocol to inactivate the biosafety level 3 (BSL3) pathogens Brucella prior to their analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. This method is also effective for several other bacterial pathogens and allows storage, and eventually shipping, of inactivated samples; therefore, it might be routinely applied to unidentified bacteria, for the safety of laboratory workers.

Cellular Internalization Mechanisms of Polyanhydride Particles: Implications for Rational Design of Drug Delivery Vehicles.

Polyanhydride nanoparticles have emerged as a versatile delivery platform, due to their ability to encapsulate diverse drugs, immunogens, antibodies, and proteins. However, mechanistic studies on the effects of particle chemistry interactions with immune cells have yet to be described. Understanding the mechanism by which these particles are internalized by immune cells will enable rational selection of delivery vehicles for specific applications. In the present study, the internalization, mechanism(s) of uptake by monocytes, and intracellular fate of polyanhydride nanoparticles were evaluated using copolymers based on 1,6-bis(p-carboxyphenoxy)hexane (CPH), sebacic acid (SA), and 1,8-bis(p-carboxyphenoxy)3,6-dioxaoctane (CPTEG). The results showed that 20:80 CPH:SA and 20:80 CPTEG:CPH nanoparticles were internalized to a greater extent by monocytes as compared to the 50:50 CPH:SA and 50:50 CPTEH:CPH nanoparticles. Further, cytochalasin-D treatment of cells inhibited uptake of all the particles, regardless of chemistry, indicating that actinmediated uptake is the primary mechanism of cellular entry for these particles. The insights gained from these studies were used to identify lead nanoparticle formulations to enhance treatment of intracellular bacterial infections. The use of doxycycline-loaded nanoparticles exhibited enhanced therapeutic efficacy compared to soluble drug in treating monocyte monolayers infected with the virulent intracellular pathogen Brucella abortus. Altogether, these studies demonstrate how rational design and selection of nanoscale delivery platforms can be used for a wide spectrum of biomedical applications.

Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.

Evaluation of three formulations of culture media for isolation of Brucella spp. regarding their ability to inhibit the growth of contaminating organisms.

Three culture media (Brucella agar, Farrell medium, and CITA) were compared for their effectiveness in inhibiting contamination and for isolating Brucella spp. One hundred lymph nodes from pigs (n = 50) and wild boars (n = 50) with lymphadenitis were collected in slaughterhouses in the State of São Paulo and were assessed on these three selective media for Brucella spp. All of the samples were negative for Brucella spp. on the three culture media. On the agar medium, fungal (70 plates) and Gram-positive bacterial (59 plates) contaminants were observed; in the CITA medium, the absence of fungal and Gram-positive bacteria on 15 plates was observed; no bacterial or fungal growth was observed on the Farrell media. The results demonstrated that the CITA and Farrell media inhibited the growth of contaminants better than the Brucella agar.