The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound "fixation threads" (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-TrnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (a) aided the genus-wide retention of nodulation, and (b) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

Relationship between clinical-epidemiological parameters and outcomes of patients with COVID-19 admitted to the intensive care unit: a report from a Brazilian hospital.

Background: People in low-income countries, especially those with low socio-economic conditions, are likelier to test positive for SARS-CoV-2. The unequal conditions of public health systems also increase the infection rate and make early identification and treatment of at-risk patients difficult. Here, we aimed to characterize the epidemiological profile of COVID-19 patients in intensive care and identify laboratory and clinical markers associated with death. Materials and methods: We conducted an observational, descriptive, and cross-sectional study in a reference hospital for COVID-19 treatment in the Southern Region of Bahia State, in Brazil, to evaluate the epidemiological, clinical, and laboratory characteristics of COVID-19 patients admitted to the intensive care unit (ICU). Additionally, we used the area under the curve (AUC) to classify survivors and non-survivors and a multivariate logistic regression analysis to assess factors associated with death. Data was collected from the hospital databases between April 2020 and July 2021. Results: The use of bladder catheters (OR 79.30; p < 0.0001) and central venous catheters (OR, 45.12; p < 0.0001) were the main factors associated with death in ICU COVID-19 patients. Additionally, the number of non-survivors increased with age (p < 0.0001) and prolonged ICU stay (p < 0.0001). Besides, SAPS3 presents a higher sensibility (77.9%) and specificity (63.1%) to discriminate between survivors and non-survivor with an AUC of 0.79 (p < 0.0001). Conclusion: We suggest that multi-laboratory parameters can predict patient prognosis and guide healthcare teams toward more assertive clinical management, better resource allocation, and improved survival of COVID-19 patients admitted to the ICU.

Uranium transfer in grasses grown on mining waste and natural soil.

The transfer of radionuclides from soil to the food chain often begins with uptake by plant root system. The roots of most angiosperms showed symbiosis with arbuscular mycorrhizal fungi (AMF) and to understand the transfer process of these toxic elements it is important to consider different physical, chemical and biological factors in soils. In the present study, three grass species (Poaceae), Zea mays, Chrysopogon zizanioides and Aristida setifolia were cultivated with and without organic fertilization in experimental blocks on natural soils, at Fazenda Vargem Formosa (VF) with low uranium (U) contents in the soil, and in the leached ore deposit at the Uranium Concentrate Unit Mine (URA) in Caetité (Uraniferous Province of Lagoa Real - Brazil). In the present study, the biomass production of plants, their rate of root colonization by AMF, the levels of U in soils, roots and leaves, as well as different physico-chemical parameters related to soil fertility were evaluated. The data analysis was performed using Artificial Neural Networks (ANNs), specifically Self-Organizing Maps (SOMs). The levels of available uranium in the soil ranges from 0.33 to 1.11 mg kg-1 in VF and from 177.5 to 475.8 mg kg-1 in URA. The results revealed high percentage of root AMF colonization, even in soils with high U contents. There was an inverse relationship between soil U content and its transfer to the plant organs, with U transfer rates being influenced by plant species and not by soil parameters. C. zizanioides had the lowest transfer factor to the shoot and the highest mass productivity under conditions of high U content in soil. The results indicate that C. zizanioides is an important species for use in the recovery of U mining areas.

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes.

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade.

Ex-vivo mucolytic and anti-inflammatory activity of BromAc in tracheal aspirates from COVID-19.

COVID-19 is a lethal disease caused by the pandemic SARS-CoV-2, which continues to be a public health threat. COVID-19 is principally a respiratory disease and is often associated with sputum retention and cytokine storm, for which there are limited therapeutic options. In this regard, we evaluated the use of BromAc®, a combination of Bromelain and Acetylcysteine (NAC). Both drugs present mucolytic effect and have been studied to treat COVID-19. Therefore, we sought to examine the mucolytic and anti-inflammatory effect of BromAc® in tracheal aspirate samples from critically ill COVID-19 patients requiring mechanical ventilation. METHOD: Tracheal aspirate samples from COVID-19 patients were collected following next of kin consent and mucolysis, rheometry and cytokine analysis using Luminex kit was performed. RESULTS: BromAc® displayed a robust mucolytic effect in a dose dependent manner on COVID-19 sputum ex vivo. BromAc® showed anti-inflammatory activity, reducing the action of cytokine storm, chemokines including MIP-1alpha, CXCL8, MIP-1b, MCP-1 and IP-10, and regulatory cytokines IL-5, IL-10, IL-13 IL-1Ra and total reduction for IL-9 compared to NAC alone and control. BromAc® acted on IL-6, demonstrating a reduction in G-CSF and VEGF-D at concentrations of 125 and 250 µg. CONCLUSION: These results indicate robust mucolytic and anti-inflammatory effect of BromAc® ex vivo in tracheal aspirates from critically ill COVID-19 patients, indicating its potential to be further assessed as pharmacological treatment for COVID-19.

The large mimosoid genus Inga Mill. (tribe Ingeae, Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil.

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.

Burkholderia perseverans sp. nov., a bacterium isolated from the Restinga ecosystem, is a producer of volatile and diffusible compounds that inhibit plant pathogens.

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated CBAS 719 T, CBAS 732 and CBAS 720 were isolated from leaf litter samples, collected in Espírito Santo State, Brazil, in 2008. Sequences of the 16S rRNA, gyrB, lepA and recA genes showed that these strains grouped with Burkholderia plantarii LMG 9035 T, Burkholderia gladioli LMG 2216 T and Burkholderia glumae LMG 2196 T in a clade of phytopathogenic Burkholderia species. Digital DNA-DNA hybridization experiments and ANI analyses demonstrated that strain CBAS 719 T represents a novel species in this lineage that is very closely related with B. plantarii. The genome sequence of the type strain is 7.57 Mbp and its G + C content is 69.01 mol%. The absence of growth on TSA medium supplemented with 3% (w/v) NaCl, citrate assimilation, ß-galactosidase (PNPG) activity, and of lipase C14 activity differentiated strain CBAS 719 T from B. plantarii LMG 9035 T, its nearest phylogenetic neighbor. Its predominant fatty acid components were C16:0, C18:1 ω7c, cyclo-C17:0 and summed feature 3 (C16:1 ω7c and/or C15:0 iso 2-OH). Based on these genotypic and phenotypic characteristics, the strains CBAS 719 T, CBAS 732 and CBAS 720 are classified in a novel Burkholderia species, for which the name Burkholderia perseverans sp. nov. is proposed. The type strain is CBAS 719 T (= LMG 31557 T = INN12T).

Nodulation of the neotropical genus Calliandra by alpha or betaproteobacterial symbionts depends on the biogeographical origins of the host species.

The neotropical genus Calliandra is of great importance to ecology and agroforestry, but little is known about its nodulation or its rhizobia. The nodulation of several species from two restricted diversity centres with native/endemic species (Eastern Brazil and North-Central America) and species widespread in South America, as well as their nodule structure and the molecular characterization of their rhizobial symbionts based on phylogeny of the 16S rRNA, recA and nodC gene, is reported herein. Species representative of different regions were grown in Brazilian soil, their nodulation observed, and their symbionts characterized. Calliandra nodules have anatomy that is typical of mimosoid nodules regardless of the microsymbiont type. The rhizobial symbionts differed according to the geographical origin of the species, i.e. Alphaproteobacteria (Rhizobium) were the exclusive symbionts from North-Central America, Betaproteobacteria (Paraburkholderia) from Eastern Brazil, and a mixture of both nodulated the widespread species. The symbiont preferences of Calliandra species are the result of the host co-evolving with the "local" symbiotic bacteria that thrive in the different edaphoclimatic conditions, e.g. the acidic soils of NE Brazil are rich in acid-tolerant Paraburkholderia, whereas those of North-Central America are typically neutral-alkaline and harbour Rhizobium. It is hypothesized that the flexibility of widespread species in symbiont choice has assisted in their wider dispersal across the neotropics.

Paraburkholderia youngii sp. nov. and 'Paraburkholderia atlantica' - Brazilian and Mexican Mimosa-associated rhizobia that were previously known as Paraburkholderia tuberum sv. mimosae.

Previous studies have recognized South and Central/Latin American mimosoid legumes in the genera Mimosa, Piptadenia and Calliandra as hosts for various nodulating Paraburkholderia species. Several of these species have been validly named in the last two decades, e.g., P. nodosa, P. phymatum, P. diazotrophica, P. piptadeniae, P. ribeironis, P. sabiae and P. mimosarum. There are still, however, a number of diverse Paraburkholderia strains associated with these legumes that have an unclear taxonomic status. In this study, we focus on 30 of these strains which originate from the root nodules of Brazilian and Mexican Mimosa species. They were initially identified as P. tuberum and subsequently placed into a symbiovar (sv. mimosae) based on their host preferences. A polyphasic approach for the delineation of these strains was used, consisting of genealogical concordance analysis (using atpD, gyrB, acnA, pab and 16S rRNA gene sequences), together with comparisons of Average Nucleotide Identity (ANI), DNA G+C content ratios and phenotypic characteristics with those of the type strains of validly named Paraburkholderia species. Accordingly, these 30 strains were delineated into two distinct groups, of which one is conspecific with 'P. atlantica' CNPSo 3155T and the other new to Science. We propose the name Paraburkholderia youngii sp. nov. with type strain JPY169T (= LMG 31411T; SARCC751T) for this novel species.

Cave Drip Water-Related Samples as a Natural Environment for Aromatic Hydrocarbon-Degrading Bacteria.

Restricted contact with the external environment has allowed the development of microbial communities adapted to the oligotrophy of caves. However, nutrients can be transported to caves by drip water and affect the microbial communities inside the cave. To evaluate the influence of aromatic compounds carried by drip water on the microbial community, two limestone caves were selected in Brazil. Drip-water-saturated and unsaturated sediment, and dripping water itself, were collected from each cave and bacterial 16S rDNA amplicon sequencing and denaturing gradient gel electrophoresis (DGGE) of naphthalene dioxygenase (ndo) genes were performed. Energy-dispersive X-ray spectroscopy (EDX) and atomic absorption spectroscopy (AAS) were performed to evaluate inorganic nutrients, and GC was performed to estimate aromatic compounds in the samples. The high frequency of Sphingomonadaceae in drip water samples indicates the presence of aromatic hydrocarbon-degrading bacteria. This finding was consistent with the detection of naphthalene and acenaphthene and the presence of ndo genes in drip-water-related samples. The aromatic compounds, aromatic hydrocarbon-degrading bacteria and 16S rDNA sequencing indicate that aromatic compounds may be one of the sources of energy and carbon to the system and the drip-water-associated bacterial community contains several potentially aromatic hydrocarbon-degrading bacteria. To the best of our knowledge, this is the first work to present compelling evidence for the presence of aromatic hydrocarbon-degrading bacteria in cave drip water.

Purple Sulfur Bacteria Dominate Microbial Community in Brazilian Limestone Cave.

The mineralogical composition of caves makes the environment ideal for inhabitation by microbes. However, the bacterial diversity in the cave ecosystem remains largely unexplored. In this paper, we described the bacterial community in an oxic chamber of the Sopradeira cave, an iron-rich limestone cave, in the semiarid region of Northeast Brazil. The microbial population in the cave samples was studied by 16S rDNA next-generation sequencing. A type of purple sulfur bacteria (PSB), Chromatiales, was found to be the most abundant in the sediment (57%), gravel-like (73%), and rock samples (96%). The predominant PSB detected were Ectothiorhodospiraceae, Chromatiaceae, and Woeseiaceae. We identified the PSB in a permanently aphotic zone, with no sulfur detected by energy-dispersive X-ray (EDX) spectroscopy. The absence of light prompted us to investigate for possible nitrogen fixing (nifH) and ammonia oxidizing (amoA) genes in the microbial samples. The nifH gene was found to be present in higher copy numbers than the bacterial-amoA and archaeal-amoA genes, and archaeal-amoA dominated the ammonia-oxidizing community. Although PSB dominated the bacterial community in the samples and may be related to both nitrogen-fixing and ammonia oxidizing bacteria, nitrogen-fixing associated gene was the most detected in those samples, especially in the rock. The present work demonstrates that this cave is an interesting hotspot for the study of ammonia-oxidizing archaea and aphotic PSB.

Whole Genome Analyses Suggests that Burkholderia sensu lato Contains Two Additional Novel Genera (Mycetohabitans gen. nov., and Trinickia gen. nov.): Implications for the Evolution of Diazotrophy and Nodulation in the Burkholderiaceae.

Burkholderia sensu lato is a large and complex group, containing pathogenic, phytopathogenic, symbiotic and non-symbiotic strains from a very wide range of environmental (soil, water, plants, fungi) and clinical (animal, human) habitats. Its taxonomy has been evaluated several times through the analysis of 16S rRNA sequences, concantenated 4⁻7 housekeeping gene sequences, and lately by genome sequences. Currently, the division of this group into Burkholderia, Caballeronia, Paraburkholderia, and Robbsia is strongly supported by genome analysis. These new genera broadly correspond to the various habitats/lifestyles of Burkholderia s.l., e.g., all the plant beneficial and environmental (PBE) strains are included in Paraburkholderia (which also includes all the N2-fixing legume symbionts) and Caballeronia, while most of the human and animal pathogens are retained in Burkholderia sensu stricto. However, none of these genera can accommodate two important groups of species. One of these includes the closely related Paraburkholderia rhizoxinica and Paraburkholderia endofungorum, which are both symbionts of the fungal phytopathogen Rhizopus microsporus. The second group comprises the Mimosa-nodulating bacterium Paraburkholderia symbiotica, the phytopathogen Paraburkholderia caryophylli, and the soil bacteria Burkholderia dabaoshanensis and Paraburkholderia soli. In order to clarify their positions within Burkholderia sensu lato, a phylogenomic approach based on a maximum likelihood analysis of conserved genes from more than 100 Burkholderia sensu lato species was carried out. Additionally, the average nucleotide identity (ANI) and amino acid identity (AAI) were calculated. The data strongly supported the existence of two distinct and unique clades, which in fact sustain the description of two novel genera Mycetohabitans gen. nov. and Trinickia gen. nov. The newly proposed combinations are Mycetohabitans endofungorum comb. nov., Mycetohabitansrhizoxinica comb. nov., Trinickia caryophylli comb. nov., Trinickiadabaoshanensis comb. nov., Trinickia soli comb. nov., and Trinickiasymbiotica comb. nov. Given that the division between the genera that comprise Burkholderia s.l. in terms of their lifestyles is often complex, differential characteristics of the genomes of these new combinations were investigated. In addition, two important lifestyle-determining traits-diazotrophy and/or symbiotic nodulation, and pathogenesis-were analyzed in depth i.e., the phylogenetic positions of nitrogen fixation and nodulation genes in Trinickia via-à-vis other Burkholderiaceae were determined, and the possibility of pathogenesis in Mycetohabitans and Trinickia was tested by performing infection experiments on plants and the nematode Caenorhabditis elegans. It is concluded that (1) T. symbiotica nif and nod genes fit within the wider Mimosa-nodulating Burkholderiaceae but appear in separate clades and that T. caryophyllinif genes are basal to the free-living Burkholderia s.l. strains, while with regard to pathogenesis (2) none of the Mycetohabitans and Trinickia strains tested are likely to be pathogenic, except for the known phytopathogen T. caryophylli.

Trichoderma asperelloides ethanolic extracts efficiently inhibit Staphylococcus growth and biofilm formation.

Fungi from the widely distributed genus Trichoderma are of great biotechnological interest, being currently used in a vast range of applications. Here, we report that high-molecular weight fraction (HWF) derived from Trichoderma asperelloides ethanolic extract exhibits antibiotic activity against staphylococcal biofilms. The antibacterial and anti-biofilm properties of T. asperelloides extracts were evaluated by well-established assays in Staphylococcus aureus ATCC strains (29213 and 6538) and in one clinical isolate from bovine mastitis. The HWF from T. asperelloides eradicated S. aureus by causing substantial matrix de-structuring and biomass reduction (p < 10-5) at concentrations as low as 2.3 µg mL-1. Additionally, we present ultra-structure analysis by the use of scanning electron microscopy as well as transmission microscopy, which showed that T. asperelloides killed cells through cell wall and membrane disturbance. Remarkably, the HWF from T. asperelloides killed S. aureus and eradicated its biofilms in a greater performance than gentamicin (p < 10-5), a known potent antibiotic against S. aureus. Our results indicate that extract from T. asperelloides may represent a promising candidate for the development of new antibiotics against gram-positive bacteria.

Brazilian species of Calliandra Benth. (tribe Ingeae) are nodulated by diverse strains of Paraburkholderia.

The Chapada Diamantina in NE of Brazil is a biodiversity hotspot and a center of radiation for many Neotropical legume genera, such as Calliandra and Mimosa. The present study aimed to evaluate nodulation in Calliandra species endemic to various environments, and to characterize the diversity of their symbiotic rhizobia using housekeeping (16S rRNA, recA) and plasmid-borne, symbiosis-related (nifH and nodC) genes. The nodulation ability of selected isolates was assessed. All of the 126 bacterial isolates from 18 Calliandra species collected in six different vegetation types were identified as Paraburkholderia according to their housekeeping and symbiosis gene phylogenies. They were grouped in seven clades in relation to the dominant vegetation type in their native environments. The majority, particularly those from highland "campo rupestre" vegetation, were similar to Paraburkholderia nodosa, but had nodC genes identical to the Mimosa symbiont Paraburkholderia tuberum sv. mimosae. The other smaller groups were related to Paraburkholderia diazotrophica and Paraburkholderia sabiae, and some single strains were not close to any known species. The symbionts of Calliandra spp. in NE Brazil are Paraburkholderia strains closely-related to Mimosa symbionts from the same region. NE Brazil is a reservoir of symbiotic Paraburkholderia that have an affinity for genera in the Mimosoid clade.

Cadmium induced changes in Solidago chilensis Meyen (Asteraceae) grown on organically fertilized soil with reference to mycorrhizae, metabolism, anatomy and ultrastructure.

Solidago chilensis Meyen (Asteraceae) is a medicinal important plant with few studies on nutrition and metabolism and none information on cadmium phytotoxicity. The objective of this study was to investigate Cd induced responses on the growth and metabolism in S. chilensis and on arbuscular mycorrhiza (AM). The experiment was carried out in a greenhouse, consisting of a 5 × 4 factorial with five doses of manure (0, 3.5, 7, 14 and 21gdm-3) and four doses of cadmium (0, 25, 50 and 75mgdm-3) applied to a Dystrophic Ultisol. After 250 days of plant cultivation, biomass, nutrient content, photosynthetic rate, guaiacol peroxidase activity, mycorrhizal colonization, glomalin content, anatomical and ultrastucture were evaluated. Plants were significantly affected by interaction of manure and Cd doses with anatomical, ultrastructural, physiological and nutritional modifications. Manure applied into Cd contaminated soil significantly improved mycorrhizal colonization and glomalin production. The highest organic manure dose (21gdm-3) alleviated toxicity symptoms of Cd on S. chilensis.

Diverse genotypes of Bradyrhizobium nodulate herbaceous Chamaecrista (Moench) (Fabaceae, Caesalpinioideae) species in Brazil.

The genus Chamaecrista comprises more than 330 species which are mainly distributed across tropical America, especially in Brazil (256 spp.), the main center of radiation. In this study, nodulation of herbaceous Chamaecrista species that are commonly found growing in different vegetation types in the north eastern Brazilian state of Bahia was assessed together with the diversity of rhizobia isolated from their root nodules. Genetic characterization of the isolates was performed using molecular markers to examine the phylogeny of their "core" (16S rRNA, ITS, recA, glnII, dnaK and gyrB) and symbiosis-related (nifH, nodC) genomes. Nodule morphology, anatomy and ultrastructure were also examined, as was the capacity of the isolates to form nodules on Chamaecrista desvauxii and siratro (Macroptilium atropurpureum). Analysis of 16S rRNA gene sequences demonstrated that the isolates belonged to seven clusters within the genus Bradyrhizobium, and more detailed analyses using sequences of the ITS region and concatenated housekeeping genes grouped the Chamaecrista rhizobia by vegetation type and plant species. These analyses also suggested some potentially novel Bradyrhizobium species, which was corroborated by analyses of their nifH and nodC sequences, as these formed separated branches from all Bradyrhizobium type strains. All the 47 strains tested produced effective nodules on C. desvauxii but none on siratro. Chamaecrista nodules are herein described for the first time in detail: they are indeterminate and structurally similar to others described in the Caesalpinioideae, with infection threads in the invasion and nitrogen fixation zones, and with both infected and uninfected (interstitial) cells in the nitrogen fixation zone.

Burkholderia species are the most common and preferred nodulating symbionts of the Piptadenia group (tribe Mimoseae).

Burkholderia legume symbionts (also called α-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the "Piptadenia group". We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, α-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from ß to α-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species.

Burkholderia diazotrophica sp. nov., isolated from root nodules of Mimosa spp.

Five strains, JPY461(T), JPY359, JPY389, DPU-3 and STM4206 were isolated from nitrogen-fixing nodules on the roots of Mimosa spp. and their taxonomic positions were investigated using a polyphasic approach. All five strains grew at 15-40 °C (optimum, 30-37 °C), at pH 4.0-8.0 (optimum, pH 6.0-7.0) and with 0-1 % (w/v) NaCl [optimum, 0 % (w/v)]. On the basis of 16S rRNA gene sequence analysis, a representative strain (JPY461(T)) showed 97.2 % sequence similarity to the closest related species Burkholderia acidipaludis SA33(T), a similarity of 97.2 % to Burkholderia terrae KMY02(T), 97.1 % to Burkholderia phymatum STM815(T) and 97.1 % to Burkholderia hospita LMG 20598(T). The predominant fatty acids of the five novel strains were summed feature 2 (comprising C(16 : 1) iso I and/or C(14 : 0) 3-OH), summed feature 3 (comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c), C(16 : 0) , C(16 : 0) 3-OH, C(17 : 0) cyclo, C(18 : 1)ω7c and C(19 : 0) cyclo ω8c. The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 63.0-65.0 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and several unidentified phospholipids. The DNA-DNA relatedness of the novel strain with respect to recognized species of the genus Burkholderia was less than 54 %. On the basis of 16S rRNA and recA gene sequence similarities, chemotaxonomic and phenotypic data, the five strains represent a novel species in the genus Burkholderia, for which the name Burkholderia diazotrophica sp. nov. is proposed with the type strain, JPY461(T) ( = LMG 26031(T) = BCRC 80259(T) = KCTC 23308(T)).

Infection cycle of Salmonella enterica serovar Enteritidis in latent carrier mice.

This work reports the distribution of an oral dose of Salmonella enterica serovar Enteritidis (SE) in C57Bl/6-Bcgr mice, to study its pathogenesis in a latent carrier animal. Mice orally inoculated with a high dose of SE developed a latent infection characterized by the absence of clinical symptoms in which the cecum is functioning as a "strategic site" of SE proliferation, releasing bacteria into feces intermittently over the 4-week study. A sequence of disruptions occurred in the small intestine at 1 day postinculation (PI). The microvilli exhibited different degrees of degeneration, which were reversible as the cells became vacuolated. From 2 days PI, SE was detected in the mononuclear phagocytic system, and an exponential growth of the remaining bacteria in tissues was observed until 4 days PI. The production of interferon gamma from 3 days PI is restricting the SE growth, and a plateau phase was observed from 4 to 15 days PI. A recurrence of the bacterial growth in tissue occurred from 15 to 28 days PI, especially in the cecum. Increasing our knowledge about the host-pathogen interaction of adapted pathogens with the ability to develop latency is essential for the development of an efficient strategy for Salmonella control.

Burkholderia symbiotica sp. nov., isolated from root nodules of Mimosa spp. native to north-east Brazil.

Four strains, designated JPY-345(T), JPY-347, JPY-366 and JPY-581, were isolated from nitrogen-fixing nodules on the roots of two species of Mimosa, Mimosa cordistipula and Mimosa misera, that are native to North East Brazil, and their taxonomic positions were investigated by using a polyphasic approach. All four strains grew at 15-43 °C (optimum 35 °C), at pH 4-7 (optimum pH 5) and with 0-2 % (w/v) NaCl (optimum 0 % NaCl). On the basis of 16S rRNA gene sequence analysis, strain JPY-345(T) showed 97.3 % sequence similarity to the closest related species Burkholderia soli GP25-8(T), 97.3 % sequence similarity to Burkholderia caryophylli ATCC25418(T) and 97.1 % sequence similarity to Burkholderia kururiensis KP23(T). The predominant fatty acids of the strains were C(18 : 1)ω7c (36.1 %), C(16 : 0) (19.8 %) and summed feature 3, comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c (11.5 %). The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 64.2-65.7 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized aminophospholipids and phospholipids. DNA-DNA hybridizations between the novel strain and recognized species of the genus Burkholderia yielded relatedness values of <51.8 %. On the basis of 16S rRNA and recA gene sequence similarities and chemotaxonomic and phenotypic data, the four strains represent a novel species in the genus Burkholderia, for which the name Burkholderia symbiotica sp. nov. is proposed. The type strain is JPY-345(T) (= LMG 26032(T) = BCRC 80258(T) = KCTC 23309(T)).