RESUMO
The evolutionary "success" of the genus Brucella depends on the ability to persist both in the environment as well as inside of even activated macrophages of the animal host. For that, the Brucellae produce catalase and superoxide dismutase to defend against oxidative stress. Since the deletion of the mglA gene in the B. abortus S19 vaccine strain resulted not only in an increased tolerance to H2O2 but also in the induction of cytokines in macrophages, we here investigated the effect of oxidative stress (Fe2+ and H2O2) on the survival of B. abortus S19 and the isogenic B. abortus S 19 ∆mglA 3.14 deletion mutant in comparison with B. neotomae 5K33, Brucella strain 83/13, and B. microti CCM4915. These Brucellae belong to different phylogenetic clades and show characteristic differences in the mgl-operon. From the various Brucellae tested, B. abortus S19 showed the highest susceptibility to oxidative stress and the lowest ability to survive inside of murine macrophages. B. abortus S19 ∆mglA 3.14 as well as B. neotomae, which also belongs to the classical core clade of Brucella and lacks the regulators of the mgl-operon, presented the highest degree of tolerance to H2O2 but not in the survival in macrophages. The latter was most pronounced in case of an infection with B. 83/13 and B. microti CCM4915. The various Brucellae investigated here demonstrate significant differences in tolerance against oxidative stress and different survival in murine macrophages, which, however, do not correlate directly.
Assuntos
Brucella abortus/fisiologia , Macrófagos/microbiologia , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Animais , Brucella/classificação , Brucella/fisiologia , Linhagem Celular , Contagem de Colônia Microbiana , Citocinas/metabolismo , Genes Bacterianos , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Macrófagos/imunologia , Camundongos , Viabilidade Microbiana , Mutação , Especificidade da EspécieRESUMO
Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.
Assuntos
Anuros/microbiologia , Brucella/classificação , Brucella/isolamento & purificação , Brucelose/microbiologia , Filogenia , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Evolução Biológica , Brucella/genética , Brucella/metabolismo , Brucelose/mortalidade , Carbono/metabolismo , Linhagem Celular/patologia , Criança , DNA Bacteriano/genética , Células Epiteliais/microbiologia , Feminino , Genes Bacterianos , Genoma Bacteriano , Células HeLa/patologia , Humanos , Lipopolissacarídeos/classificação , Lipopolissacarídeos/genética , Macrófagos/microbiologia , Camundongos , Família Multigênica , Antígenos O/genética , Fenótipo , Ramnose/metabolismo , Texas , Virulência , Zoonoses/microbiologiaRESUMO
Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3â236â779 and 3â237â765âbp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2â%. A genome-to-genome distance of >80â%, an average nucleotide identity (ANI) of 97â% and an average amino acid identity (AAI) of 98â% compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5â% of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).
Assuntos
Brucella/classificação , Raposas/microbiologia , Linfonodos/microbiologia , Filogenia , Animais , Áustria , Técnicas de Tipagem Bacteriana , Tipagem de Bacteriófagos , Composição de Bases , Brucella/genética , Brucella/isolamento & purificação , DNA Bacteriano/genética , Análise de Sequência de DNARESUMO
Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) (â=âNCTC 13660(T)â=âCIRMBP 0958(T)).
Assuntos
Brucella/classificação , Papio/microbiologia , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Brucella/genética , Brucella/isolamento & purificação , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
We aimed to describe clinical and diagnostic features of vertebral osteomyelitis for differential diagnosis and treatment. This is a prospective observational study performed between 2002 and 2012 in Ankara Numune Education and Research Hospital in Ankara, Turkey. All the patients with vertebral osteomyelitis were followed for from 6 months to 3 years. In total, 214 patients were included in the study, 113 out of 214 (53%) were female. Out of 214 patients, 96 (45%) had brucellar vertebral osteomyelitis (BVO), 63 (29%) had tuberculous vertebral osteomyelitis (TVO), and 55 (26%) had pyogenic vertebral osteomyelitis (PVO). Mean number of days between onset of symptoms and establishment of diagnosis was greater with the patients with TVO (266 days) than BVO (115 days) or PVO (151 days, p <0.001). In blood cultures, Brucella spp. were isolated from 35 of 96 BVO patients (35%). Among 55 PVO patients, the aetiological agent was isolated in 11 (20%) patients. For tuberculin skin test >15 mm, sensitivity was 0.66, specificity was 0.97, positive predictive value was 0.89, negative predictive value was 0.88, and receiver operating characteristics area was 0.8. Tuberculous and brucellar vertebral osteomyelitis remained the leading causes of vertebral osteomyelitis with delayed diagnosis. In differential diagnosis of vertebral osteomyelitis, consumption of unpasteurized cheese, dealing with husbandry, sweating, arthralgia, hepatomegaly, elevated alanine transaminase, and lumbar involvement in magnetic resonance imaging were found to be predictors of BVO, thoracic involvement in magnetic resonance imaging and tuberculin skin test > 15 mm were found to be predictors of TVO, and history of spinal surgery and leucocytosis were found to be predictors of PVO.
Assuntos
Osteomielite/diagnóstico , Osteomielite/patologia , Coluna Vertebral/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brucella/classificação , Brucella/isolamento & purificação , Brucelose/sangue , Brucelose/diagnóstico , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Osteomielite/sangue , Osteomielite/microbiologia , Estudos Prospectivos , Fatores de Risco , Coluna Vertebral/microbiologia , Tuberculose Osteoarticular/sangue , Tuberculose Osteoarticular/diagnóstico , Tuberculose Osteoarticular/microbiologia , Tuberculose Osteoarticular/patologia , Turquia , Adulto JovemRESUMO
BACKGROUND: Recently, novel atypical Brucella strains isolated from humans and wild rodents have been reported. They are phenotypically close to Ochrobactrum species but belong to the genus Brucella, based on genetic relatedness, although genetic diversity is higher among the atypical Brucella strains than between the classic species. They were classified within or close to the novel species Brucella inopinata. However, with the exception of Brucella microti, the virulence of these novel strains has not been investigated in experimental models of infection. METHODS: The type species B. inopinata strain BO1 (isolated from a human) and Brucella species strain 83-210 (isolated from a wild Australian rodent) were investigated. A classic infectious Brucella reference strain, B. suis 1330, was also used. BALB/c, C57BL/6, and CD1 mice models and C57BL/6 mouse bone-marrow-derived macrophages (BMDMs) were used as infection models. RESULTS: Strains BO1 and 83-210 behaved similarly to reference strain 1330 in all mouse infection models: there were similar growth curves in spleens and livers of mice and similar intracellular replication rates in BMDMs. However, unlike strain 1330, strains BO1 and 83-210 showed lethality in the 3 mouse models. CONCLUSIONS: The novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species.
Assuntos
Brucella/classificação , Brucella/patogenicidade , Brucelose/microbiologia , Brucelose/mortalidade , Animais , Células Cultivadas , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos , VirulênciaRESUMO
Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk, leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a, and omp2b gene sequences and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb, whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by phage R/C. B. microti exhibited high metabolic activities similar to those of closely related soil organisms, such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be a matter of gene regulation rather than a matter of gene configuration.
Assuntos
Biodiversidade , Brucella/classificação , Animais , Técnicas de Tipagem Bacteriana , Bacteriólise , Bacteriófagos/crescimento & desenvolvimento , Brucella/genética , Brucella/isolamento & purificação , Brucella/fisiologia , Brucelose/microbiologia , Brucelose/veterinária , Genes Bacterianos , Genótipo , Mamíferos/microbiologia , Tipagem de Sequências Multilocus , Fenótipo , Análise de Sequência de DNA , Microbiologia do SoloRESUMO
Brucella spp. were isolated from marine mammals for the first time in 1994. Two novel species were later included in the genus; Brucella ceti and Brucella pinnipedialis, with cetaceans and seals as their preferred hosts, respectively. Brucella spp. have since been isolated from a variety of marine mammals. Pathological changes, including lesions of the reproductive organs and associated abortions, have only been registered in cetaceans. The zoonotic potential differs among the marine mammal Brucella strains. Many techniques, both classical typing and molecular microbiology, have been utilised for characterisation of the marine mammal Brucella spp. and the change from the band-based approaches to the sequence-based approaches has greatly increased our knowledge about these strains. Several clusters have been identified within the B. ceti and B. pinnipedialis species, and multiple studies have shown that the hooded seal isolates differ from other pinniped isolates. We describe how different molecular methods have contributed to species identification and differentiation of B. ceti and B. pinnipedialis, with special emphasis on the hooded seal isolates. We further discuss the potential role of B. pinnipedialis for the declining Northwest Atlantic hooded seal population.
Assuntos
Brucella/fisiologia , Brucelose/epidemiologia , Brucelose/microbiologia , Caniformia , Cetáceos , Animais , Brucella/classificação , Brucella/genética , Brucella/isolamento & purificação , Brucelose/patologia , Brucelose/transmissão , Prevalência , Focas Verdadeiras , Zoonoses/epidemiologia , Zoonoses/microbiologia , Zoonoses/transmissãoRESUMO
A new Brucella species, Brucella microti, has been isolated from wild rodents and found to be pathogenic in mice. The biological relevance of this new mouse pathogen is clear, as it allows us to study Brucella infection in a species-specific model. The course of infection in wild-type (wt) and immunodeficient mice that lack B (Jh), T and B (SCID), or T, B, and NK (SCID.Beige) cells was analyzed over 3 weeks. wt mice completely cleared bacteria from the liver and spleen after that time. However, SCID mice showed a much higher bacterial load in the spleen and liver than wt and Jh mice after 1 week and maintained the same level during the next 2 weeks. All mice tested survived for the 3 weeks. In contrast, the bacterial levels in mice that lacked NK cell activity progressively increased and these mice succumbed to infection after 16 to 18 days. Histopathology analysis of infected mice showed extensive areas of necrotic tissue and thrombosis in liver after 1 week in all infected SCID.Beige mice but were not seen in either SCID or wt animals. These processes were dramatically increased after 21 days, corresponding with the death of SCID.Beige animals. Our results indicate that T and/or B cells are required for the control of infection with the mouse pathogen Brucella microti in liver and spleen but that NK cells are crucial for survival in the absence of B and T cells. In addition, they suggest that controlled granuloma formation is critical to clear this type of infection in wt mice.
Assuntos
Brucella/patogenicidade , Brucelose/imunologia , Hospedeiro Imunocomprometido , Animais , Linfócitos B/imunologia , Carga Bacteriana , Brucella/classificação , Brucella/imunologia , Brucelose/microbiologia , Modelos Animais de Doenças , Feminino , Humanos , Células Matadoras Naturais/imunologia , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Especificidade da Espécie , Baço/imunologia , Baço/microbiologia , Baço/patologia , Linfócitos T/imunologiaRESUMO
AIM: To describe and discuss the merits of various direct and indirect methods applied in vitro (mainly on blood or milk) or in vivo (allergic test) for the diagnosis of brucellosis in animals. METHODS: The recent literature on brucellosis diagnostic tests was reviewed. These diagnostic tests are applied with different goals, such as national screening, confirmatory diagnosis, certification, and international trade. The validation of such diagnostic tests is still an issue, particularly in wildlife. The choice of the testing strategy depends on the prevailing brucellosis epidemiological situation and the goal of testing. RESULTS: Measuring the kinetics of antibody production after Brucella spp. infection is essential for analyzing serological results correctly and may help to predict abortion. Indirect ELISAs help to discriminate 1) between false positive serological reactions and true brucellosis and 2) between vaccination and infection. Biotyping of Brucella spp. provides valuable epidemiological information that allows tracing an infection back to the sources in instances where several biotypes of a given Brucella species are circulating. Polymerase chain reaction and new molecular methods are likely to be used as routine typing and fingerprinting methods in the coming years. CONCLUSION: The diagnosis of brucellosis in livestock and wildlife is complex and serological results need to be carefully analyzed. The B. abortus S19 and B. melitensis Rev. 1 vaccines are the cornerstones of control programs in cattle and small ruminants, respectively. There is no vaccine available for pigs or for wildlife. In the absence of a human brucellosis vaccine, prevention of human brucellosis depends on the control of the disease in animals.
Assuntos
Animais Selvagens , Brucella/classificação , Brucelose/veterinária , Gado , Animais , Técnicas de Tipagem Bacteriana/métodos , Brucella/genética , Brucella/imunologia , Brucella/isolamento & purificação , Brucelose/sangue , Brucelose/diagnóstico , Brucelose/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Programas de Rastreamento , Reação em Cadeia da Polimerase , Saúde Pública , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The recent isolation of Brucella microti from the common vole, the red fox, and the soil raises the possibility of an eventual reemergence of brucellosis in Europe. In this work, the pathogenic potential of this new Brucella species in both in vitro and in vivo models of infection was analyzed. METHODS: The ability of B. microti (as compared to that of the closely related species Brucella suis) to replicate in human macrophages and in human and murine macrophage-like cells was determined. The behavior of B. microti and B. suis was evaluated in vivo in murine models of infection with Balb/c, CD1, and C57BL/6 mice. RESULTS: B. microti showed an enhanced capacity for intramacrophagic replication compared with that of B. suis. Surprisingly, and in contrast to other species of Brucella, 10(5) colony-forming units of B. microti killed 82% of Balb/c mice within 7 days. Infection of spleen and liver with B. microti peaked at day 3, compared with B. suis infection, which peaked at day 7. Sublethal doses of B. microti induced good protection against a subsequent challenge with lethal doses. CONCLUSIONS: In experimental cellular and murine infections, B. microti exhibited a high pathogenic potential, compared with other Brucella species.
Assuntos
Brucella/classificação , Brucella/fisiologia , Brucelose/microbiologia , Macrófagos/microbiologia , Animais , Brucelose/imunologia , Brucelose/mortalidade , Humanos , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Baço/patologiaRESUMO
BACKGROUND: Brucellosis is primarily a zoonotic disease caused by Brucella species. There are currently ten Brucella spp. including the recently identified novel B. inopinata sp. isolated from a wound associated with a breast implant infection. In this study we report on the identification of an unusual Brucella-like strain (BO2) isolated from a lung biopsy in a 52-year-old patient in Australia with a clinical history of chronic destructive pneumonia. RESULTS: Standard biochemical profiles confirmed that the unusual strain was a member of the Brucella genus and the full-length 16S rRNA gene sequence was 100% identical to the recently identified B. inopinata sp. nov. (type strain BO1(T)). Additional sequence analysis of the recA, omp2a and 2b genes; and multiple locus sequence analysis (MLSA) demonstrated that strain BO2 exhibited significant similarity to the B. inopinata sp. compared to any of the other Brucella or Ochrobactrum species. Genotyping based on multiple-locus variable-number tandem repeat analysis (MLVA) established that the BO2 and BO1(T) strains form a distinct phylogenetic cluster separate from the other Brucella spp. CONCLUSION: Based on these molecular and microbiological characterizations, we propose that the BO2 strain is a novel lineage of the newly described B. inopinata species.
Assuntos
Brucella/classificação , Brucella/isolamento & purificação , Brucelose/microbiologia , Pneumonia Bacteriana/microbiologia , Técnicas de Tipagem Bacteriana , Biópsia , Brucella/genética , DNA Bacteriano/genética , Humanos , Pulmão/microbiologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Since 1994, Brucella strains have been isolated from a wide range of marine mammals. They are currently recognized as two new Brucella species, B. pinnipedialis for the pinniped isolates and B. ceti for the cetacean isolates in agreement with host preference and specific phenotypic and molecular markers. In order to investigate the genetic relationships within the marine mammal Brucella isolates and with reference to terrestrial mammal Brucella isolates, we applied in this study the Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) approach. A previously published assay comprising 16 loci (MLVA-16) that has been shown to be highly relevant and efficient for typing and clustering Brucella strains from animal and human origin was used. RESULTS: 294 marine mammal Brucella strains collected in European waters from 173 animals and a human isolate from New Zealand presumably from marine origin were investigated by MLVA-16. Marine mammal Brucella isolates were shown to be different from the recognized terrestrial mammal Brucella species and biovars and corresponded to 3 major related groups, one specific of the B. ceti strains, one of the B. pinnipedialis strains and the last composed of the human isolate. In the B. ceti group, 3 subclusters were identified, distinguishing a cluster of dolphin, minke whale and porpoise isolates and two clusters mostly composed of dolphin isolates. These results were in accordance with published analyses using other phenotypic or molecular approaches, or different panels of VNTR loci. The B. pinnipedialis group could be similarly subdivided in 3 subclusters, one composed exclusively of isolates from hooded seals (Cystophora cristata) and the two others comprising other seal species isolates. CONCLUSION: The clustering analysis of a large collection of marine mammal Brucella isolates from European waters significantly strengthens the current view of the population structure of these two species, and their relative position with respect to the rest of the Brucella genus. MLVA-16 is confirmed as being a rapid, highly discriminatory and reproducible method to classify Brucella strains including the marine mammal isolates. The Brucella2009 MLVA-16 genotyping database available at http://mlva.u-psud.fr/ is providing a detailed coverage of all 9 currently recognized Brucella species.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Brucella/classificação , Caniformia/microbiologia , Cetáceos/microbiologia , Animais , Brucella/genética , Brucella/isolamento & purificação , Brucelose/microbiologia , Geografia , Humanos , Repetições Minissatélites , Nova Zelândia , FilogeniaRESUMO
Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.
Assuntos
Arvicolinae/microbiologia , Brucella/classificação , Brucella/isolamento & purificação , Brucelose/veterinária , Doenças dos Roedores/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Técnicas de Tipagem Bacteriana , Brucella/genética , Brucella/fisiologia , Brucelose/microbiologia , DNA Bacteriano/análise , Genes de RNAr , Genótipo , Repetições Minissatélites/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Recombinases Rec A/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
OBJECTIVES: Because of the suboptimal recovery rate of brucellae from blood, it has been proposed that cultures of bone marrow, liver tissue, and lymph nodes may improve the recovery rate of the organism. Data in support of these recommendations are limited and not clearly convincing, especially that of bone marrow culture. The main purpose of this work was to evaluate the roles of blood, bone marrow, liver, and lymph node cultures in the diagnosis of human brucellosis. METHODS: Blood and bone marrow cultures were evaluated in parallel in 103 cases of human brucellosis using Castaneda's biphasic technique. Simultaneous cultures of blood, bone marrow, liver, and lymph node aspirates were also carried out for 13 of these 103 cases. RESULTS: Blood culture identified 47 (45.6%) cases and bone marrow culture identified 85 (82.5%) cases. Faster recovery of Brucella spp was accomplished with the bone marrow culture (2.8+/-0.7 days, p<0.05). When the results of cultures of blood and bone marrow were compared with each other in the 13 cases, it was found that bone marrow specimens could be sterile (six cases (46%)) when bacteremia was present, but Brucella melitensis was detected in liver aspirate in all these six bacteremic cases. CONCLUSIONS: Our data indicate that it is worthwhile practicing bone marrow culture by conventional biphasic technique for the definitive and rapid diagnosis of brucellosis; this is particularly the case in developing countries where diagnostic facilities by advanced technologies such as automated culture systems with PCR are not available. Bone marrow culturing would be a better gold standard in areas where antibiotic pretreatment is common. Also, adopting the practice of culturing liver/lymph node fluids may enhance bacterial isolation and aid in the establishment of a diagnosis of brucellosis in cases for whom blood and bone marrow cultures are negative.
Assuntos
Bacteriemia/diagnóstico , Brucella/isolamento & purificação , Brucelose/diagnóstico , Brucelose/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/microbiologia , Técnicas Bacteriológicas , Sangue/microbiologia , Medula Óssea/microbiologia , Brucella/classificação , Brucelose/microbiologia , Criança , Pré-Escolar , Meios de Cultura , Feminino , Humanos , Fígado/microbiologia , Linfonodos/microbiologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.
Assuntos
Arvicolinae/microbiologia , Brucella/isolamento & purificação , Brucella/fisiologia , Brucelose/veterinária , Doenças dos Roedores/microbiologia , Animais , Sangue/microbiologia , Brucella/classificação , Brucella/genética , Brucelose/microbiologia , Brucelose/patologia , República Tcheca/epidemiologia , Feminino , Linfonodos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Ochrobactrum/classificação , Ochrobactrum/isolamento & purificação , Doenças dos Roedores/patologiaRESUMO
We reviewed a case of Brucella spondylodiscitis admitted to a referral, university hospital, in Ankara, Turkey. A 75-year-old female was referred to our hospital with low back pain. Previous magnetic resonance imaging yielded cortical destruction of T9-10 and T12-L2 vertebral bodies, focal infectious foci at discs within this range, significant microabscesses at paravertebral areas, which lead to the diagnosis of spondylodiscitis. History of consumption of unpasteurized dairy products led us to first suspect brucellosis yet, the serum agglutination test and blood culture were negative and did mislead us to several other, sometimes invasive, diagnostic tests. The final diagnosis was reached by culturing the specimen obtained through fine-needle aspiration from the paravertebral microabscesses. The exhausting diagnostic journey that started with the suspicion of tuberculosis or malignancy ended with a diagnosis of brucellosis. Brucellosis should be considered in all patients with osteoarthritic complaints in endemic regions, and the "prozone phenomenon" should be kept in mind, before proceeding to high-tech lab tests, imaging, or invasive procedures.
Assuntos
Brucella/patogenicidade , Brucelose/diagnóstico , Discite/microbiologia , Discite/patologia , Reações Falso-Negativas , Idoso , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Brucella/classificação , Brucelose/tratamento farmacológico , Brucelose/microbiologia , Brucelose/patologia , Discite/diagnóstico , Doxiciclina/administração & dosagem , Doxiciclina/uso terapêutico , Quimioterapia Combinada , Feminino , Seguimentos , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Imageamento por Ressonância Magnética , Radiografia , Rifampina/administração & dosagem , Rifampina/uso terapêutico , Testes Sorológicos , Vértebras Torácicas/diagnóstico por imagem , Vértebras Torácicas/patologia , Fatores de Tempo , Resultado do TratamentoRESUMO
By comparing smooth wild-type Brucella spp. to their rough mutants, we show that the LPS O chain restricted the activation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, thus preventing the synthesis of immune mediators that regulate host defense. We conclude that the MAPKs are a target for immune intervention by virulent smooth Brucella.
Assuntos
Brucella/crescimento & desenvolvimento , Brucella/patogenicidade , Ativação Enzimática , Regulação da Expressão Gênica , Macrófagos/microbiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Brucella/classificação , Brucelose/microbiologia , Linhagem Celular , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Virulência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We present the first report of community-acquired human infections with marine mammal-associated Brucella spp. and describe the identification of these strains in two patients with neurobrucellosis and intracerebral granulomas. The identification of these isolates as marine mammal strains was based on omp2a sequence and amplification of the region flanking bp26.
Assuntos
Brucella/isolamento & purificação , Infecções Comunitárias Adquiridas/fisiopatologia , Granuloma/patologia , Adolescente , Adulto , Brucella/classificação , Brucella/patogenicidade , Brucelose/tratamento farmacológico , Brucelose/fisiopatologia , Infecções Comunitárias Adquiridas/tratamento farmacológico , Humanos , Masculino , PeruRESUMO
Human brucellosis is an important animal transmitted disease of man. Although, the cases have been recorded all over the world, the prevalence is higher in developing countries. Lack of sufficient knowledge about the disease among the physicians, its under-diagnosis or misdiagnosis and absence of effective prevention and management strategies are attributed to the widespread of the disease. Increase in the occurrence of animal brucellosis has also resulted indirectly in an increase in the prevalence of human infection. Absence of characteristic clinical symptoms, chronic nature of the infection and difficulty in isolation of the causal agent from the patients make the diagnosis of the disease more difficult. The serological tests employed for diagnosing human brucellosis vary in terms of their sensitivity and specificity. Therefore, a combination of serological tests is desirable. Currently no vaccine is available against human brucellosis, which could check the spread of the disease effectively. It is suggested that clinicians investigate the cases of pyrexia of unknown origin (PUO) for brucellosis. It is desirable that specimens from cases of tuberculosis, typhoid, rheumatoid arthritis, urogenital infections, kala-azar, cirrhosis, bacterial endocarditis, leukemia and filariasis should also be screened for brucellosis in man. The cases of meningitis of unestablished etiology as the cases of human brucellosis are often misdiagnosed as cases of typhoid or tuberculosis.