RESUMEN
Bacteria use the twin arginine translocator (Tat) system to export folded proteins from the cytosol to the bacterial envelope or to the extracellular environment. As with most Gram-negative bacteria, the Tat system of the zoonotic pathogen Brucella spp. is encoded by a three-gene operon, tatABC. Our attempts, using several different strategies, to create a Brucella suis strain 1330 tat mutant were all unsuccessful. This suggested that, for B. suis, Tat is essential, in contrast to a recent report for Brucella melitensis. This was supported by our findings that two molecules that inhibit the Pseudomonas aeruginosa Tat system also inhibit B. suis, B. melitensis, and Brucella abortus growth in vitro. In a bioinformatic screen of the B. suis 1330 proteome, we identified 28 proteins with putative Tat signal sequences. We used a heterologous reporter assay based on export of the Tat-dependent amidase AmiA by using the Tat signal sequences from the Brucella proteins to confirm that 20 of the 28 candidates can engage the Tat pathway.
Asunto(s)
Brucella melitensis , Brucella suis , Sistema de Translocación de Arginina Gemela , Brucella suis/genética , Brucella suis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistema de Translocación de Arginina Gemela/genética , Señales de Clasificación de Proteína , ArgininaRESUMEN
Human brucellosis is a zoonoses caused by bacteria of the genus Brucella. Infection results in subacute or chronic debilitating disease with nonspecific clinical manifestations and is often associated with consuming unpasteurized dairy products. We report 2 cases of brucellosis in male patients who were hospitalized in distinct towns of French Guiana, an overseas territory of France located on the northeastern shore of South America. Both men were citizens of Brazil working as clandestine goldminers in the deep Amazonian rainforest. Characterization of the 2 bacterial isolates revealed that they represent a potential new species of Brucella. Medical practitioners working in contact with wildlife in this region of the world should be aware of the existence of these pathogens and the potential for human infection.
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Brucella , Brucelosis , Animales , Humanos , Masculino , Guyana Francesa/epidemiología , Brucelosis/diagnóstico , Brucelosis/epidemiología , Brucelosis/microbiología , Zoonosis/microbiología , BrasilRESUMEN
We report the first case of brucellosis caused by an isolate whose genome is identical that of a frog isolate from Texas, demonstrating the zoonotic potential of amphibian-type Brucella. Importantly, with such atypical Brucella, correct diagnosis cannot be performed using routine serological tests or identification methods.
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Brucella , Brucelosis , Anfibios , Animales , Brucella/genética , Brucelosis/diagnóstico , Humanos , Pruebas Serológicas , TexasRESUMEN
The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis (CF) patients. Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia. We have recently shown that macrophages are a critical site for replication of B. cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model. Here, we show that ShvR, a LysR-type transcriptional regulator that is important for biofilm formation, rough colony morphotype and inflammation in a rat lung infection model, is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae. ShvR was not essential, however, for bacterial survival and replication in macrophages. Temporal, rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease. ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster, which specifies the synthesis of a lipopeptide with antifungal activity. Mutation of afcE, encoding an acyl-CoA dehydrogenase, has been shown to give similar phenotypes as the shvR mutant. We found that, like shvR, afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish. The closely related B. cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models, including Galleria mellonella and rats. Interestingly, constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc. These data confirm a critical role for afc in acute virulence caused by B. cenocepacia that depends on strain-specific regulatory control by ShvR. We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species, either through pro-inflammatory effects of the lipopeptide AFC, or through AFC-dependent membrane properties.
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Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/patogenicidad , Macrófagos/microbiología , Virulencia/fisiología , Animales , Pez CebraRESUMEN
Brucellosis is a zoonosis caused by bacteria of the Brucella genus. In ruminants, brucellosis causes abortion, followed by chronic infection and secretion of bacteria in milk. In humans, it usually presents as flu-like symptoms, with serious complications if untreated. Epidemiological studies have only recently established that brucellosis can also cause pregnancy complications in women, but the pathogenic mechanisms are unknown. Pioneering studies in ruminants showed that Brucella infect trophoblasts and then colonise the placenta where they grow to high density. A recent study showed that the main zoonotic Brucella species can infect human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT). In this work, we show that Brucella papionis (associated with stillbirth in primates) also infects human trophoblasts. However, it replicates actively in CTB, whereas its replication is very restricted within EVT. We also observed alteration of several trophoblastic functions upon infection by B. papionis or Brucella melitensis (the most prevalent species in human brucellosis). Infection altered the production of hormones, the ability of CTB to form syncytiotrophoblasts, and the invasion capacity of EVT. We also found that infection can spread between different types of trophoblasts. These findings constitute a new step in understanding how Brucella infection causes adverse pregnancy outcomes.
Asunto(s)
Brucella melitensis/patogenicidad , Brucella/patogenicidad , Brucelosis/microbiología , Trofoblastos/microbiología , Brucelosis/patología , Femenino , Humanos , Embarazo , Trofoblastos/patologíaRESUMEN
Despite the benefits, one in three older adults in Ireland has low activity levels. This study examined associations between the local social and built environment and physical activity of older adults to identify age-friendly factors that support physical activity among the aging population. Data were from the population-representative Healthy and Positive Ageing Initiative Age-Friendly City and Counties Survey (N = 10,540). Physical activity was measured using a short-form of the International Physical Activity Questionnaire. Mixed-effects negative binomial regression models were adjusted for known health and sociodemographic correlates of physical activity. Results are reported as unstandardized beta coefficients (ß) with standard errors. Loneliness, community participation, and difficulty in accessing green spaces partially explained the differences in the number of minutes that respondents were physically active. Combined with individual-level behavior change interventions, improvements to the local environment and promoting social connectedness may also be useful in promoting physical activity among older adults.
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Envejecimiento , Ambiente , Ejercicio Físico , Medio Social , Anciano , Ciudades , Humanos , Irlanda , Persona de Mediana EdadRESUMEN
Bacteria of the Burkholderia cepacia complex (Bcc) can cause devastating pulmonary infections in cystic fibrosis (CF) patients, yet the precise mechanisms underlying inflammation, recurrent exacerbations and transition from chronic stages to acute infection and septicemia are not known. Bcc bacteria are generally believed to have a predominant extracellular biofilm life style in infected CF lungs, similar to Pseudomonas aeruginosa, but this has been challenged by clinical observations which show Bcc bacteria predominantly in macrophages. More recently, Bcc bacteria have emerged in nosocomial infections of patients hospitalized for reasons unrelated to CF. Research has abundantly shown that Bcc bacteria can survive and replicate in mammalian cells in vitro, yet the importance of an intracellular life style during infection in humans is unknown. Here we studied the contribution of innate immune cell types to fatal pro-inflammatory infection caused by B. cenocepacia using zebrafish larvae. In strong contrast to the usual protective role for macrophages against microbes, our results show that these phagocytes significantly worsen disease outcome. We provide new insight that macrophages are critical for multiplication of B. cenocepacia in the host and for development of a fatal, pro-inflammatory response that partially depends on Il1-signalling. In contrast, neutrophils did not significantly contribute to disease outcome. In subcutaneous infections that are dominated by neutrophil-driven phagocytosis, the absence of a functional NADPH oxidase complex resulted in a small but measurably higher increase in bacterial growth suggesting the oxidative burst helps limit bacterial multiplication; however, neutrophils were unable to clear the bacteria. We suggest that paradigm-changing approaches are needed for development of novel antimicrobials to efficiently disarm intracellular bacteria of this group of highly persistent, opportunistic pathogens.
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Burkholderia cenocepacia/aislamiento & purificación , Infección Hospitalaria/microbiología , Inflamación/microbiología , Macrófagos/microbiología , Neutrófilos/microbiología , Animales , Infecciones por Burkholderia/inmunología , Complejo Burkholderia cepacia/inmunología , Fibrosis Quística/complicaciones , Humanos , Pulmón/microbiología , Neutrófilos/inmunología , Fagocitosis/inmunología , Pseudomonas aeruginosa/fisiología , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006437.].
RESUMEN
UNLABELLED: Recent breakthroughs in next-generation sequencing technologies have led to the identification of small noncoding RNAs (sRNAs) as a new important class of regulatory molecules. In prokaryotes, sRNAs are often bound to the chaperone protein Hfq, which allows them to interact with their partner mRNA(s). We screened the genome of the zoonotic and human pathogen Brucella suis 1330 for the presence of this class of RNAs. We designed a coimmunoprecipitation strategy that relies on the use of Hfq as a bait to enrich the sample with sRNAs and eventually their target mRNAs. By deep sequencing analysis of the Hfq-bound transcripts, we identified a number of mRNAs and 33 sRNA candidates associated with Hfq. The expression of 10 sRNAs in the early stationary growth phase was experimentally confirmed by Northern blotting and/or reverse transcriptase PCR. IMPORTANCE: Brucella organisms are facultative intracellular pathogens that use stealth strategies to avoid host defenses. Adaptation to the host environment requires tight control of gene expression. Recently, small noncoding RNAs (sRNAs) and the sRNA chaperone Hfq have been shown to play a role in the fine-tuning of gene expression. Here we have used RNA sequencing to identify RNAs associated with the B. suis Hfq protein. We have identified a novel list of 33 sRNAs and 62 Hfq-associated mRNAs for future studies aiming to understand the intracellular lifestyle of this pathogen.
Asunto(s)
Brucella suis/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteína de Factor 1 del Huésped/metabolismo , ARN Bacteriano/metabolismo , Transcriptoma , Animales , Brucella suis/genética , Línea Celular , Regulación Bacteriana de la Expresión Génica/fisiología , Estudio de Asociación del Genoma Completo , Proteína de Factor 1 del Huésped/genética , Ratones , Chaperonas Moleculares , ARN Bacteriano/genética , ARN no TraducidoRESUMEN
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.
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Brucella , Brucelosis/diagnóstico , Brucelosis/microbiología , Viabilidad Microbiana , Manejo de Especímenes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Brucella/efectos de los fármacos , Humanos , Viabilidad Microbiana/efectos de los fármacos , Solventes , Manejo de Especímenes/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Strains of uropathogenic Escherichia coli (UPEC) are the major causative agent of urinary tract infections (UTI), the most common infectious diseases in the world. Their ability to attach and enter into cells in the urinary tract is a limiting step for their pathogenicity. Many studies are thus focussing on these key mechanisms to propose new therapeutic strategies. To facilitate such studies, we developed a fast and high-throughput assay which makes it possible to monitor the interaction of UPEC with cultured human uroepithelial cells. This assay allows measurement of the in vitro association of fluorescently labelled clinical isolates with bladder epithelial cells using flow cytometry in a microplate format. The assay was sensitive enough to detect variations between isolates expressing different adhesins and virulence factors and the inhibitory effect of proanthocyanidins. Thus we have developed a fast and robust assay which allows us to measure variations in the adhesion properties of UPEC to human bladder cells. This novel assay will be valuable for the study of initial steps of pathogenesis in UTI and for the screening or validation of inhibitory molecules.
Asunto(s)
Adhesión Bacteriana/fisiología , Vejiga Urinaria/microbiología , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/fisiología , Adhesión Bacteriana/efectos de los fármacos , Técnicas Bacteriológicas/métodos , Células Cultivadas , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Citometría de Flujo/métodos , Humanos , Fitoterapia , Extractos Vegetales/farmacología , Vejiga Urinaria/citología , Escherichia coli Uropatógena/patogenicidad , Vaccinium macrocarpon , VirulenciaRESUMEN
Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host.
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Brucella/patogenicidad , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Interacciones Huésped-Patógeno/fisiología , Espacio Intracelular/microbiología , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Brucella/metabolismo , Brucelosis/metabolismo , Brucelosis/microbiología , Células Cultivadas , Fibroblastos/química , Fibroblastos/metabolismo , Fibroblastos/microbiología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Técnicas de Inactivación de Genes , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Espacio Intracelular/química , Espacio Intracelular/metabolismo , Ratones , Salmonella , Vacuolas/metabolismo , Vacuolas/microbiología , Factores de Virulencia/metabolismoRESUMEN
OBJECTIVES: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α-converting enzyme baseline and inducible activity profiles. DESIGN: Observational clinical study. SETTING: Mixed surgical/medical teaching hospital ICU. PATIENTS: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α-converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients' monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α-converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α-converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α-converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α-converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α-converting enzyme activity could be induced in healthy volunteer monocytes using an in vitro two-hit inflammation model. Patients with sepsis also displayed reduced shedding of monocyte tumor necrosis factor receptors upon stimulation with lipopolysaccharide. CONCLUSIONS: Monocyte tumor necrosis factor-α-converting enzyme catalytic activity appeared altered by sepsis and may result in reduced shedding of tumor necrosis factor receptors. Changes seemed specific to sepsis and correlated with illness severity. A better understanding of how tumor necrosis factor-α-converting enzyme function is altered during sepsis will enhance our understanding of sepsis pathophysiology, which will help in the assessment of patient inflammatory status and ultimately may provide new strategies to treat sepsis.
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Proteínas ADAM/fisiología , Inflamación/sangre , Monocitos/enzimología , Sepsis/sangre , Proteína ADAM17 , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as "stealth pathogens" that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing "atypical" strains and a highly conserved "classical" core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.
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Evolución Biológica , Brucellaceae/genética , Brucellaceae/patogenicidad , Genoma Bacteriano , Genómica/métodos , Filogenia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , VirulenciaRESUMEN
BACKGROUND: The TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton. RESULTS: Unlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules. CONCLUSIONS: Our results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways.
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Proteínas Bacterianas/metabolismo , Brucella melitensis/metabolismo , Microtúbulos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Factores de Virulencia/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Seudópodos/metabolismo , Factores de Virulencia/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Sepsis, the dysregulated host response to infection causing life-threatening organ dysfunction, is a global health challenge requiring better understanding of pathophysiology and new therapeutic approaches. Here, we applied high-throughput tandem mass spectrometry to delineate the plasma proteome for sepsis and comparator groups (noninfected critical illness, postoperative inflammation, and healthy volunteers) involving 2612 samples (from 1611 patients) and 4553 liquid chromatography-mass spectrometry analyses acquired through a single batch of continuous measurements, with a throughput of 100 samples per day. We show how this scale of data can delineate proteins, pathways, and coexpression modules in sepsis and be integrated with paired leukocyte transcriptomic data (837 samples from n = 649 patients). We mapped the plasma proteomic landscape of the host response in sepsis, including changes over time, and identified features relating to etiology, clinical phenotypes (including organ failures), and severity. This work reveals subphenotypes informative for sepsis response state, disease processes, and outcome; identifies potential biomarkers; and advances opportunities for a precision medicine approach to sepsis.
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Proteoma , Sepsis , Humanos , Sepsis/sangre , Proteoma/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Proteómica/métodos , Masculino , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/análisis , Femenino , Persona de Mediana Edad , Espectrometría de Masas en Tándem/métodosRESUMEN
Brucella abortus is a globally important zoonotic pathogen largely found in cattle hosts and is typically transmitted to humans through contaminated dairy products or contact with diseased animals. Despite the long, shared history of cattle and humans, little is known about how trade in cattle has spread this pathogen throughout the world. Whole genome sequencing provides unparalleled resolution to investigate the global evolutionary history of a bacterium such as B. abortus by providing phylogenetic resolution that has been unobtainable using other methods. We report on large-scale genome sequencing and analysis of B. abortus collected globally from cattle and 16 other hosts from 52 countries. We used single nucleotide polymorphisms (SNPs) to identify genetic variation in 1,074 B. abortus genomes and using maximum parsimony generated a phylogeny that identified four major clades. Two of these clades, clade A (median date 972 CE; 95% HPD, 781-1142 CE) and clade B (median date 150 BCE; 95% HPD, 515 BCE-164 CE), were exceptionally diverse for this species and are exclusively of African origin where provenance is known. The third clade, clade C (median date 949 CE; 95% HPD, 766-1102 CE), had most isolates coming from a broad swath of the Middle East, Europe, and Asia, also had relatively high diversity. Finally, the fourth major clade, clade D (median date 1467 CE; 95% HPD, 1367-1553 CE) comprises the large majority of genomes in a dominant but relatively monomorphic group that predominantly infects cattle in Europe and the Americas. These data are consistent with an African origin for B. abortus and a subsequent spread to the Middle East, Europe, and Asia, probably through the movement of infected cattle. We hypothesize that European arrival to the Americas starting in the 15th century introduced B. abortus from Western Europe through the introduction of a few common cattle breeds infected with strains from clade D. These data provide the foundation of a comprehensive global phylogeny of this important zoonotic pathogen that should be an important resource in human and veterinary epidemiology.
RESUMEN
A 29-year-old Brazilian illegal gold miner developed intermittent fever. Blood cultures were positive for Gram-negative coccobacilli and, after an initial misidentification by an automated identification system, the diagnosis of brucellosis caused by Brucella suis was confirmed. We hypothesize an association with domestic or wild swine exposure. The patient responded well to standard antibiotic therapy of brucellosis. We report the first case of human brucellosis on the Guiana Shield. This report underlines the importance of considering brucellosis in the presence of a fever of unknown origin, even in the Amazonian rainforest area, where several zoonotic diseases might be considered in the differential diagnosis of unexplained fever.
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Brucella suis , Brucelosis , Enfermedades de los Porcinos , Animales , Porcinos , Humanos , Adulto , Brasil , Guyana Francesa , Brucelosis/diagnóstico , Brucelosis/tratamiento farmacológico , Zoonosis/diagnóstico , Sus scrofa , FiebreRESUMEN
Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation.
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Brucella abortus , Brucelosis , Humanos , Brucella abortus/genética , Técnicas de Genotipaje , Genotipo , Tipificación de Secuencias Multilocus/métodos , Brucelosis/epidemiología , Repeticiones de Minisatélite , FilogeniaRESUMEN
The Brucellaceae family comprises microorganisms similar both phenotypically and genotypically, making it difficult to identify the etiological agent of these infections. This study reports the first isolation, identification, and characterization of Pseudochrobactrum saccharolyticum (strain 115) from Latin America. Strain 115 was isolated in 2007 from a bovine in Brazil and was initially classified as Brucella spp. by classical microbiological tests and bcsp31 PCR. The antimicrobial susceptibility of strain 115 was tested against drugs used to treat human brucellosis by minimal inhibitory concentration test. Subsequently, the whole genome of the strain was sequenced, assembled, and characterized. Phylogenetic trees built from 16S rRNA and recA gene sequences enabled the classification of strain 115 as Pseudochrobactrum spp. Phylogenomic analysis using Single Nucleotide Polymorphisms and Average Nucleotide Identity allowed the classification of the strain as P. saccharolyticum. Additionally, a Tetra Correlation Search identified one related genome from the same species, which was compared with strain 115 by analyzing genomic islands. This is the first identification and whole-genome sequence of P. saccharolyticum in Latin America and highlights a challenge in the diagnosis of bovine brucellosis, which could be solved by including the sequencing of 16S rRNA and recA genes in routine diagnostics.