Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation with a short-term transient infection, as well as less severe, but more chronic inflammation and infection persistence. E. coli is a highly diverse organism that has been classified into a number of different pathotypes or pathovars, and mammary pathogenic E. coli (MPEC) has been proposed as a new such pathotype. The purpose of this study was to use genome sequence data derived from both transient and persistent MPEC isolates (two isolates of each phenotype) to construct a genome-based phylogeny that places MPEC in its phylogenetic context with other E. coli pathovars. A subsidiary goal was to conduct comparative genomic analyses of these MPEC isolates with other E. coli pathovars to provide a preliminary perspective on loci that might be correlated with the MPEC phenotype. Both concatenated and consensus tree phylogenies did not support MPEC monophyly or the monophyly of either transient or persistent phenotypes. Three of the MPEC isolates  originated from within the predominately commensal clade of E. coli, referred to as phylogroup A. The fourth MPEC isolate, of the persistent phenotype (ECC-1470), was sister group to an isolate of ETEC, falling within the E. coli B1 clade. This suggests that the MPEC phenotype has arisen on numerous independent occasions and that this has often, although not invariably, occurred from commensal ancestry. Examination of the genes present in the MPEC strains relative to the commensal strains identified a consistent presence of the type VI secretion system (T6SS) in the MPEC strains, with only occasional representation in commensal strains, suggesting that T6SS may be associated with MPEC pathogenesis and/or as an inter-bacterial competitive attribute and therefore could represent a useful target to explore for the development of MPEC specific inhibitors. Escherichia coli [E. coli can cause both severe inflammation with a short-term transient infection, as well as less severe but more chronic inflammation and infection persistence [E. coli is a highly diverse organism that has been classified into a number of different pathotypes or pathovars, including adherent-invasive E. coli (AIEC), avian pathogenic E. coli (APEC), enteroaggregative E. coli (EAEC), enterohemorrageic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), extra-intestinal pathogenic E. coli (ExPEC), and uropathogenic E. coli (UPEC). Mammary pathogenic E. coli (MPEC) has been proposed as a new E. coli pathotype [E. coli have long been acknowledged [E. coli phylogeny that includes MPEC isolates in a comparative dataset that encompasses representative isolates of the various E. coli pathovars. The only published sequence based phylogeny regarding MPEC that we are aware of, uses MLST data and reports a single most parsimonious tree of a set of 165 MP trees and includes only one non-MPEC isolate in the analysis [E. coli phylogroup membership, however, this is not a phylogenetic analysis, and provides no information on relative relationships of the isolates within each phylogroup. Furthermore, this PCR based test has been shown to provide unreliable designations for certain phylogroups [Dairy cattle mastitis, or inflammation of the mammary gland, is the dominant bovine health disorder affecting milk production worldwide, with reported annual losses exceeding two billion dollars in the United States dairy industry –3. A varhia coli , are linsistence , 6. E. cathotype . Distincowledged –10 and sanalysis . There aanalysis  to asseslogroups , and in E. coli isolates that can be divided into two distinct groups: those isolated from intramammary infections (IMI) displaying transient infection characteristics  andexamples ). The puE. coli strains associated with transient IMI, and two strains associated with persistent IMI were evaluated. The strains from transient infections were ECA-727 [E. coli isolates associated with persistent IMI, strain ECC-Z [Two  ECA-727  and ECA- ECA-727 . The strin ECC-Z  and strain ECC-Z , were isin ECC-Z , 6.De novo assembly with Newbler Software) into 1173, 539, 994, and 24 contigs for ECA-0157, ECA-727, ECC-1470, and ECC-Z, respectively. Genome annotation for the four MPEC strains was done by the National Center for Biotechnology Information (NCBI) Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) at http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html. The whole genome shotgun projects for ECA-0157, ECA-727, ECC-1470, and ECC-Z have been deposited at DDBJ/EMBL/GenBank under the accession numbers AHHK00000000, NZ_AHHL00000000, NZ_AHHM00000000, and AHHN00000000, respectively. We compared these four MPEC genome sequences to 56 complete E. coli genome sequences representing a combination of commensal isolates and the following ten pathotypes or pathovars: ABU (Asymptomatic Bacteriuria), AIEC, APEC, EAEC, EHEC, EPEC, ETEC, ExPEC, NMEC , and UPEC. Also included were nine Shigella sp. isolates representing four different species. All of these 65 sequences were obtained from NCBI; their strain names, accession numbers, genome lengths and respective pathotypes are presented in We determined the genome sequences of the four MPEC strains isolated from transient (ECA-O157 and ECA-727) and persistent (ECC-1470 and ECC-Z) bovine mastitis. Roche/454 pyrosequencing, involving single or paired-end reads from the FLX sequencer, was used to determine the sequence of the four MPEC genomes. The genome sequence for ECA-727 did not involve paired end data. The sequences were assembled (http://micans.org/mcl). The pipeline uses Markov clustering (MCL) to assign genes to homologous clusters based on a BLASTp search between all pairs of protein sequences using an E value cut-off of 1e-5. The MCL algorithm was implemented using an inflation parameter of 1.8. Simulations have shown this value to be generally robust to false positives and negatives [2. We excluded any cluster that showed evidence of recombination for any two of the three methods. Using this approach, we excluded 546 clusters (438 remained).Homologous genes among the 69 genomes analyzed were delineated using the MCL algorithm  as impleegatives . Nucleotegatives . For theWe utilized two approaches to phylogenetic reconstruction. The first was a gene tree consensus approach. Here, phylogenetic trees for each MCL gene cluster alignment were obtained using maximum likelihood (PhyML v.3.0), and a consensus of these gene trees obtained using the Triple Construction Method as implemented in the program Triplec . This prFDR = 0.05).Gene Ontology (GO) terms were assigned to all genomes using Blast2GO v.2.5.0 . RelativBasic genome statistics for the four sequenced MPEC strains are summarized in Escherichia coli strains have been classified into an array of different phylogenetic groups, often referred to as phylogroups or phylotypes  [D, E, F) –10, as wD, E, F) . The CleD, E, F)  has beenD, E, F)  and in aD, E, F) . RecentlD, E, F) . Gordon D, E, F)  comparedE. coli strains, and nine strains of Shigella. This involved data for 56 complete genomes of E. coli taken from GenBank representing all the major phylotypes (with the exception of group C for which there were no complete genome sequences at the time this study was conducted) and pathovars, plus draft genome sequences of our four MPEC isolates. The comparative genome sequence data were first screened for putative recombinant loci, which were excluded in the phylogenetic reconstructions and therefore these orthologous loci should represent our best source of data to estimate the true evolutionary history of these set of isolates. We reconstructed the phylogeny using both a concatenated dataset that included all 438 of these non-recombinant orthologs   , and a c62 SNPs) . Both rees (e.g. ), providE. coli virulence genes, and including many more MPEC isolates than are included here, have indicated that the presence and combinations of E. coli virulence loci in MPEC strains varies greatly , 37E. coYersinia pestis is sufficient to render the bacteria completely avirulent [Examination of the genes present in the MPEC strains relative to the commensal strains identified a consistent presence of the type VI secretion system (T6SS) in the MPEC strains, with more occasional representation in commensal strains (36.8% of strains). The type VI secretion system is the most recently described , 39 of tvirulent .E. coli, including for example, strains W and HS included in our analyses [E. coli strains isolated from the ileum of patients with inflammatory bowel disease [E. coli with an adherent-invasive (AIEC) pathotype. PCR based analysis of E. coli associated with mastitis, with similar adherent and invasive activity to AIEC, identified the T6SS hcp gene, a T6SS effector protein, in 88% of transient and 67% of persistent strains [E. coli type six secretion system cluster [E. coli strains from the total of 69 strains included in Figs. E. coli and the MPEC strains. MPEC strain ECA-O157 was excluded because of fragmentation across multiple different contigs. Using this alignment, we performed a ML phylogenetic analysis using PhyML (see E. coli (UMNK88-ETEC). The T6SS sequence similarity is very high between the ETEC and these two MPEC strains . For example, after exclusion of a 1,619bp indel within the IcmF-like protein for strain ECC-Z, identity for the 15 genes used in the phylogeny was 99.9%. This high sequence identity occurred despite the fact these strains are not closely related in the strain phylogeny Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


PhenoMiner, the search engine for these integrated phenotype data, facilitates mining of data sets across studies by searching the database with a combination of terms from four different ontologies/vocabularies . In this study, salt-induced hypertension was used as a model to retrieve blood pressure records of Brown Norway, Fawn-Hooded Hypertensive (FHH) and Dahl salt-sensitive (SS) rat strains. The records from these three strains served as a basis for comparing records from consomic/congenic/mutant offspring derived from them. We examined the cardiovascular and renal phenotypes of consomics derived from FHH and SS, and of SS congenics and mutants. The availability of quantitative records across laboratories in one database, such as these provided by PhenoMiner, can empower researchers to make the best use of publicly available data.Rats have been used extensively as animal models to study physiological and pathological processes involved in human diseases. Numerous rat strains have been selectively bred for certain biological traits related to specific medical interests. Recently, the Rat Genome Database (Database URL:http://rgd.mcw.edu These quantitative phenotype data of wild-type, consomic and mutant rats are available online through PhysGen (http://pga.mcw.edu/). Data in PhysGen are categorized by study projects (Consomic or ENU mutant) with limited searching flexibility. To better facilitate data mining across multiple projects, the Rat Genome Database (RGD) has initiated the PhenoMiner project to integrate quantitative phenotype data from PhysGen projects (http://pga.mcw.edu/) and the National BioResource Project in Japan (NBRP) (http://www.anim.med.kyoto-u.ac.jp/nbr/)  inbred rats were selectively bred from Sprague-Dawley outbred rats for sensitivity to salt-induced hypertension. After developing hypertension, the SS rats progress to proteinuria and kidney pathologies ,2. The Fjp/nbr/) . The higjp/nbr/) . In addijp/nbr/) .The curated quantitative data in PhenoMiner are systematically organized by four ontologies: rat strain ontology (RS) , clinicaUsing salt-induced hypertension as a model, we examined the blood pressure and renal function records in BN, FHH, SS and their derivatives from selective breeding. This article with data retrieval and analysis demonstrates the searching functionality and analytical application of curated data in PhenoMiner.http://www.rgd.mcw.edu/wg/home/rgd_rat_community_videos/introduction-to-the-rgd-phenotypes-and-models-portal) and in a recent publication (http://www.rgd.mcw.edu/phenotypes/) in the Phenotypes & Models Portal at RGD. Data in PhenoMiner are searchable across multiple studies by using the four ontologies: RS, CMO, MMO and XCO (http://www.rgd.mcw.edu/phenotypes/termSelection/selectTerms?ont=RS). The data in http://pga.mcw.edu/). Data in PhysGen are grouped into ‘Consomic’ and ‘ENU Mutant’ projects. The mean arterial blood pressure (MAP) data can be downloaded from ‘Phenotype’ under ‘Consomic’ in the data section. The NBRP phenotype data for all strains are arranged into nine groups to allow users to search strain-associated data by groups. The data used in this article can also be found in the ‘Urine Volume and Urine Electrolyte Values’ group at NBRP Rat.A video tutorial and step-by-step instructions for use of PhenoMiner can be found on the RGD website . These data are now available through PhenoMiner. To retrieve MAP records of these PhysGen data, PhenoMiner searches started with selecting wild-type  strains used in Physgen. The hypertension-resistant BN strain  and three hypertension model strains, FHH , spontaneously hypertensive rat  and SS , were selected to be compared in http://pga.mcw.edu/?module=content&func=DataStatus).Diet and genetic factors play roles in the development of hypertension. The hypertension phenotype of SS rats induced by high salt intake can be modified by diet . The Phy+ level’ and ‘urine volume’ in NBRP rats, we calculated the ‘Na+ excretion (mEq/day)’ for the two strains. The calculated Na+ excretion (mEq/day) for BN/SsNSlc can be referenced as a basal value to compare the Na+ measurements of BN rats under high salt conditions from PhysGen.High salt diets induce both hypertension and renal diseases in SS rats and FHH rats. To look at the effect of salt levels in diet on cardiovascular and renal phenotypes, related data of BN, FHH and SS were retrieved from PhenoMiner and organized into + excretion in 14 consomic strains, 7 from the FHH background and 7 from the SS background, highlighted in publications (http://www.rgd.mcw.edu/phenotypes/).Blood pressure records of consomic rats were searched to find chromosome localizations of genes potentially involved in salt-induced hypertension and renal disease. Complete sets of BN chromosome-substituted consomics only exist among FHH and SS rats in PhenoMiner. In these studies, chromosomes from BN, a hypertension-resistant donor, were transferred to the FHH or SS background and their offspring were phenotyped under high salt diets for SS consomics or ‘high salt 8% + L-NAME’ for FHH consomics. ications ,14. The ications . Among tications . The comA systemic search was made to find congenics and mutants for the seven chromosomes listed in http://www.rgd.mcw.edu/rgdweb/report/reference/main.html? id =2291850 and http://www.rgd.mcw.edu/rgdweb/report/reference/main.html?id=728381). Sequence variations among the sequenced strains in these regions can be analysed using the Variant Visualizer tool (http://www.rgd.mcw.edu/rgdweb/front/select.html) at the RGD website.Transferring chromosome 1 from the BN rat ameliorated salt-induced hypertension in both SS and FHH backgrounds . To narrem2Mcwi-/-Nox4 mutants (RGD: 4139868) resulted in lower MAP and UPE compared with most of the controls and mutants, suggesting its role in salt-induced hypertension and renal damage in SS rats. Nox4 is one of the major producers of reactive oxygen species and has been associated with renal injuries in salt-induced hypertensive rats (em1Mcwi-/-Tcf7l2) (RGD: 5688074) and the MAS1 proto-oncogene (Mas1) mutants (SS-em1Mcwi-/-Mas1) (RGD:5688028) had the highest combined MAP and UPE among mutants, indicating those genes are protective against hypertension and renal injury.The blood pressures records from mutant rats allow us to come down to the gene level in the study of blood pressure regulation. The SS chromosome 1 mutant rat strains were retrieved from PhenoMiner by selecting ‘chromosome 1 mutant’ under the ‘chromosome altered’ folder on the ‘Rat Strains Selection’ page. There were 84 MAP records from SS mutants, including wild-type controls, homozygous mutants and heterozygous mutants in PhenoMiner. ive rats  and in kive rats . Knockinhttp://www.rgd.mcw.edu/wg/physgenknockouts).There are data from SS.LEW chromosome 5 congenic rats curated from two publications ,21 in Phem4Mcwi-/-Nppa) (RGD: 5686725) had the highest MAP, but lowest UPE, and the angiotensin II receptor-associated protein (Agtrap) mutant (SS-em4Mcwi-/Agtrap) (RGD: 5686304) had a medium MAP, but the highest UPE. Detailed analysis of the cardiovascular and renal phenotypes of these mutants and their relationship to human diseases was published by Flister et al. . PhenoMiner data can also be accessed from ontology terms used in data curation. For example, the ‘urine sodium excretion rate’ ontology report page (http://www.rgd.mcw.edu/rgdweb/ontology/annot.html?acc_id=CMO:0000760) lists related phenotype data for all associated RS, CMO, XCO and MMO terms in PhenoMiner.Hypertension is an important health issue in modern society. To study the underlying mechanism of hypertension, rats have been used extensively as a disease model, especially salt-induced hypertension . GeneticPhysGen and NBRP are the two major high-throughput data contributors, and their data are complementary to one another. There are more than 30 000 PhysGen and PhysGen Knockout records imported into PhenoMiner. These are quantitative phenotypes measured from a variety of rat strains, including wild-type parents, consomics, congenics and mutants, with a focus on hypertension and renal diseases. On the other hand, NBRP collects phenotype data under control conditions and as such provides basal values for physiological measurements. The manual annotations in PhenoMiner were curated from more than 290 papers and more than 40% of them report cardiovascular research. RGD intends to expand its literature coverage to capture the complete spectrum of rat physiology research. Future curation targets include development, behaviour, pharmacogenomics and chemical interactions.http://www.rgd.mcw.edu/wg/gerrc), an ongoing community-engaged rat genome-modifying project and data directly submitted from laboratories involved in rat research. In the PhenoMiner user-interface, all records are searchable by controlled vocabularies and can be downloaded for further reference. Using salt-induced hypertension as an example, we demonstrated how the data in Phenominer can be searched and analysed using the functionality available in the tool.The goal of the PhenoMiner project is to provide the research community with organized and retrievable quantitative phenotype data curated from different rat research laboratories. The data currently comprise results from NBRP, the PhysGen projects and the biomedical literature manually curated by RGD. Future content will include expanding manual curation coverage, results from the MCW Gene Editing Rat Resource Centre . Funding for open access charge: The National Heart, Lung and Blood Institute on behalf of the National Institutes of Health (HL64541).This work was supported by the Conflict of interest. None declared.


M. avium-intracellulare complex is apparently common throughout the world, other Non-tuberculous mycobacterial species have been isolated from both immunocompromised and immunocompetent individuals. The increasing number of infections caused by these organisms has made it clinically important to quickly identify mycobacterial species. The diagnosis of a pathogenic versus a non-pathogenic species not only has epidemiological implications but is also relevant to the demands of patient management. Since antibiotic treatment varies according to the species encountered, species identification would reduce the burden of some of these emerging opportunistic pathogens especially in immunocompromised patients and improve their quality of life.The emergence of Acquired Immunodeficiency Syndrome has highlighted the increased incidence and importance of the disease caused by Non-tuberculous Mycobacteria (NTM). While disease due to 16S-23S rRNA intergenic transcribed spacer (ITS) region of Mycobacteria.A total of 91 NTM suspected isolates from four regions of Zambia were included in the study. These isolates were identified using the sequence analysis of the M. intracellulare (27.8%), M. lentiflavum (16.7%), M. avium (14.8%), M. fortuitum (7.4%), M. gordonae (7.4%), M. kumamotonense (3.7%), M. indicus pranii (3.7%), M. peregrinum (3.7%), M. elephantis (1.85%), M. flavescens (1.85%), M. asiaticum (1.85%), M. bouchedurhonense (1.85%), M. chimaera (1.85%), M. europaeum (1.85%), M. neourum (1.85%), M. nonchromogenicum (1.5%).Fifty-four of the 91 (59%) isolates were identified as NTM and these included The study has shown that DNA sequencing of the ITS region may be useful in the preliminary identification of NTM species. All species identified in this study were potentially pathogenic.The online version of this article (doi:10.1186/s12941-014-0059-8) contains supplementary material, which is available to authorized users. Mycobacterium are important causes of respiratory disease, thereby posing an important public health threat to people and animals worldwide. Recently, there has been increased cognisance of a variety of diseases that have been caused by Non-tuberculous Mycobacteria (NTM) [Members of the genus ia (NTM) . The curia (NTM)  and the ia (NTM) . Consistia (NTM) . The numia (NTM)  and curria (NTM) .Although the reservoir of infection in most cases remains unclear, there is a general notion that NTM infections are derived mainly from the environment. NTM are widely distributed in nature and have been isolated from water and soil with water being the major reservoir . There aM. tuberculosis. In addition, treatment and infection control measures vary according to the aetiological species [Non-tuberculous Mycobacteria are often involved in nosocomial outbreaks , althoug species . TherefoM. tuberculosis, NTM are also isolated from these specimens, without any attempt to identify them to species level. Therefore this study was initiated to identify NTM to species level for ease of managing such suspect conditions.Zambia is a high burden country for tuberculosis and patients with chronic pneumonia, lymphadenitis, pyrexia of unknown origin and other chronic infections are evaluated for tuberculosis through microbiological cultures of various clinical specimens. In the process of isolating This was a retrospective study of 91 isolates stored over a period of three and half years from January 2009 to June 2012 from four regions of Zambia . The stored isolates were revived using Lowestein Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT) by standard microbiological procedures . The culMycobacterium tuberculosis complex (17), Rodococcus equi (3), Tsukamurella pulmonis (1), Norcadia carnea (1) and Paenibacillus species (1) as shown in Figure M. intracellulare exhibiting the highest frequency of identity , Southern (3), Western (1) and Eastern (1). M. fortuitum was identified in the Eastern and Lusaka regions, with one and three isolates respectively. All the other 14 species identified were from the region of Lusaka. A map of Zambia showing regions of distribution of various NTM identified in this study is shown in Figure Sequence analysis and identification of the ITS region of the 91 strains showed: NTM species (68), M. intracellulare followed by M. lentiflavum and M. avium. This was in partial agreement with the findings of the study conducted by Buijtels and others [M. fortuitum, was isolated from a clinical case. The other studies conducted in the Western and Northern regions of Zambia [Non-tuberculous Mycobacteria have gained a lot of clinical significance in the last couple of decades in immunocompromised and immunocompetent individuals or patients . Their ud others  in the Ef Zambia  and othef Zambia ,20 were f Zambia .M. intracellulare has been identified as the important species of the Mycobacterium avium complex. It has been identified together with M. avium as a complex because of their close similarities. M. intracellulare has been found to be more pathogenic than M. avium [M. lentiflavum and M. avium have been implicated in clinical disease of immunocompromised as well as immunocompetent individuals [M. lentiflavum has been isolated from various human specimens including pleural effusions, ascites and lung tissue [M. intracellulare, most M. avium species do not multiply in monocytes of healthy individuals [M. fortuitum infrequently cause a variety of diseases including bone and soft tissue infections, lymphadenitis and post-surgical infections and lung disease [M. kumamotonense, M. indicus pranii, M. flavescens, M. bouchedurhonense, M. chimaera, M. europaeum and M. nonchromogenicum were identified and reported for the first time in Zambia. Some of these NTM have been associated with clinical disease [M. indicus pranii is an atypical saprophytic bacterium that has raised a lot of research interest in leprosy immunotherapeutic [M. flavescens has been isolated from the synovial fluid of an AIDS patient [M. bouchedurhonense and M. chimaera have been documented in some respiratory tract infections [M. europaeum was isolated from the sputum samples of an Iranian human immunodeficiency virus-infected patient and a cystic fibrosis patient with chronic pulmonary disease [M. nonchromogenicum has been associated with sarcoidosis [M. avium  and haveM. avium . Other Nividuals ,25. M. lg tissue ,27 and hg tissue . Unlike ividuals . M. fort disease . M. kuma disease ,32 whilerapeutic . M. flav patient , whereasfections . M. euro disease  while M.coidosis .Mycobacterium tuberculosis complex species, Rodococcus equi, Nocardia carnea, Tsukamura pulmonis and Paenibacillus species. Of significance is the identification of Rodococcus equi from a clinical specimen in Zambia. This is the second time Rodococcus equi has been reported in Zambia [Rodococcus equi, Nocardia carnea, Tsukamura pulmonis and Paenibacillus species have been known to cause pulmonary diseases that are similar to tuberculosis [Other organisms which are not NTM that were identified include n Zambia . The orgrculosis -41. ManaThe data supporting the results of this study are included within this article.


Microvirga lotononidis is a recently described species of root-nodule bacteria that is an effective nitrogen- (N2) fixing microsymbiont of the symbiotically specific African legume Listia angolensis (Welw. ex Bak.) B.-E. van Wyk & Boatwr. M. lotononidis possesses several properties that are unusual in root-nodule bacteria, including pigmentation and the ability to grow at temperatures of up to 45°C. Strain WSM3557T is an aerobic, motile, Gram-negative, non-spore-forming rod isolated from a L. angolensis root nodule collected in Chipata, Zambia in 1963. This is the first report of a complete genome sequence for the genus Microvirga. Here we describe the features of Microvirga lotononidis strain WSM3557T, together with genome sequence information and annotation. The 7,082,538 high-quality-draft genome is arranged in 18 scaffolds of 104 contigs, contains 6,956 protein-coding genes and 84 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program. Tradioduction . Howeveroduction , have adLotononis s. l. is grouped within tribe Crotalarieae, has a centre of origin in South Africa and consists of some 150 species, divided into 15 sections [Lotononis s. l. are now recognized at the generic level as Listia, Leobordea and Lotononis s. s. [Listia are of agronomic interest, as they have potential as perennial pasture legumes that are able to reduce groundwater recharge and assist in preventing dry land salinity in southern Australian agricultural systems [Listia spp. produce stoloniferous roots on their lower branches  [Listia species and their associated root-nodule bacteria are highly specific. All studied host species are nodulated by strains of pigmented methylobacteria [Listia angolensis, which is effectively nodulated only by newly described species of Microvirga [Microvirga lotononidis strain WSM3557T is the type strain for this species. Here we present a set of preliminary classification and general features for M. lotononidis strain WSM3557T together with the description of the genome sequence and annotation.is s. s. . Species systems . Listia e found) ,8. The sbacteria ,9,10, excrovirga . MicroviM. lotononidis strain WSM3557T is a motile, Gram-negative, non-spore-forming rod with one to several flagella  [T is able to tolerate a pH range between 6.0 and 9.5 [bacteria . WSM3557 of 41°C . WSM3557 and 9.5 . Carbon  and 9.5 . MinimumM. lotononidis strain WSM3557T nodulates (Nod+) and fixes N2 effectively (Fix+) with Listia angolensis; nodulates and is partially effective on Leobordea platycarpa, Leobordea bolusii and Lotononis crumanina and nodulates but is unable to fix N2  with Leobordea longiflora, Leobordea stipulosa and Lotononis falcata [Phaseolus vulgaris, but is unable to nodulate Crotalaria juncea, Indigofera patens, Lotus corniculatus, Lupinus angustifolius, or Macroptilium atropurpureum [ falcata . It formurpureum .This organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database  and an iMicrovirga lotononidis WSM3557T was grown to mid-logarithmic phase in TY rich medium [h medium  on a gyrh medium .Microvirga lotononidis WSM3557T was generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina [The improved high quality draft genome of Illumina  and 454 Illumina . An IlluIllumina . The iniIllumina , and theIllumina -30 was uIllumina , or sequGenes were identified using Prodigal  as part The genome is 7,082,538 nucleotides with 63.00% GC content  and comp


Citrus species and close relatives by the evaluation of several parameters such as nucleotide substitution (r), nucleotide diversity (π) and the estimated values of transition/transversion bias (R). The observed results indicated the presence of a wide divergence pattern of rDNA in subfamily Aurantioideae. Maximum parsimony (MP) analysis inferred divergence pattern in the Citrus genus. We observed seven strongly supported clades among the subfamily Aurantioideae. We postulate that the present investigation provides a more robust topology of Citrus and its close relatives, which can significantly prove as an additional support to resolve the phylogenetic relationships in Citrus genera. Therefore, sequences of noncoding regions should exhibit more phylogenetically informative sites than the coding regions do, which is in accordance with the present study.The present study aims to analyse phylogenetic relationships, using internal transcribed spacer sequence data of ribosomal DNA (rDNA), across 24  Interestingly, the taxonomy of Citrus is complex and still the precise number of natural species is unclear, mainly because of the sexually compatible relatives.[Citrus viz. Citron (Citrus medica L.), Mandarin (Citrus reticulata Blanco) and Pummelo (Citrus grandis L. Osbeck). The origins of other species are a result of hybridization of these true species. In view of this, taxonomic characterization is critically important for the Citrus genus, which has the widest divergence reported among the fruit species and it is imperative to resolve the phylogeny in order to have a better understanding of the complexity of the genus and to develop resources for the proper sustainable development of this genus.Aurantioideae, a subfamily of family Rutaceae, presents a vast variety of commercially important genera such as elatives. Barrett elatives. performeCitrus. In Citrus, molecular phylogeny at various taxonomic levels has been observed in several earlier studies through application of a wide variety of molecular markers such as SSR,[Several earlier attempts have been made to revisit the intra- and inter-species relationships in Aurantioideae, which hah as SSR, ISSR,[13h as SSR, as well h as SSR,Triticum,[Solanum lycopersicum,[Oryza sativa,[Citrus.[r), nucleotide diversity (π) and the estimated values of transition/transversion bias (R) to provide better and significant understanding of the genetic diversity and phylogenetic relations across 24 studied Citrus species and other species related to the genus. The present investigation provides an additional support for resolving the phylogeny of the complex genus Citrus.Ribosomal DNA (rDNA) as a source of higher genetic variability has been studied extensively for classification and identification at the generic and infra generic levels in plants. ConsequeTriticum, Solanum persicum, Oryza saa sativa, and clos,[Citrus.,17,22 InCitrus and species related to it, which includes the following major groups as listed in Citrus Breeding (NCCB), Huazhong Agricultural University (HZAU), Wuhan, China. Genomic DNA of Citrus cultivars  light. The PCR fragments were excised and purified from the gel, using an E.Z.N.A® Gel Extraction Kit  and were subsequently ligated to a pMD18-T Easy vector as per the manufacturer instructions . The ligation product was transformed into E. coli DH-5α-competent cells, using ampicillin as a selection marker. Three positive colonies from each clone were selected and sequenced by the Uni-Gene Company .In our present research, the entire ITS region (including ITS-1 and ITS-2 of nuclear rDNA and the 5.8S rRNA gene) of rDNA was amplified using the primers ITS1 (5′TCCGTAGGTGAACCTGCGG3′) and (5′TCCTCCGCTTATTGATATGC3′) ITS-4 as previously described. Briefly,http://www.ncbi.nlm.nih.gov/gorf/gorf). The trimmed sequences were further aligned using MUSCLE v3.70+ fix1-2 [R), nucleotide substitutions (r) for each nucleotide pair and cluster analysis among the 24 Citrus genotypes were estimated using MEGA 5.[Citrus genus and its related species. In brief, the MP tree was obtained using the Subtree–Pruning–Regrafting (SPR) algorithm with search level 1 in which the initial trees were obtained with the random addition of sequences (10 replicates). The analysis involved 25 nucleotide sequences corresponding to a total of 943 positions in the final dataset. This approach has been previously followed for resolving the phylogeny of Indian Citrus cultivars.[Atlantia monophylla  as reported in a previous study.[Sequencing chromatograms obtained were analysed and vector sequences were trimmed. In all the sequenced ITS regions, after vector trimming open reading frames (ORFs) were predicted using the National Center for Biotechnology Information (NCBI) Open Reading Frame Finder (+ fix1-2  and the g MEGA 5. We furthg MEGA 5. For the g MEGA 5. The bootultivars. The phylus study.https://www.ncbi.nlm.nih.gov/) database, using BLASTn, which revealed 93%–100% similarity with the previously sequenced ITS regions, providing an evidence for the good trustworthiness of ITS regions sequenced in this study. All the ITS sequences of Citrus and its relative species have been submitted to GenBank databases (www.ncbi.nlm.nih.gov) and can be accessed under accession numbers as referred in Citrus cultivars [In our study, first after subsequent cleaning of the ITS sequence, we performed homology searches against the NCBI GenBank , whereas the lowest one was recorded in C. sinensis cv. Newhall (609 bases). The maximum GC content (64%) and the lowest AT content (35.8%) were observed in the case of C. sinensis cv. Anliu, C. paradisi Macf. cv. Red Marsh grapefruit, Fortunella hindsii Swing. cv. Hongkong Kumquat, C. sinensis cv. Valencia and C. reticulata × C. sinensis (L.) Osbeck cv. Murcott (hybrid). Nevertheless, the lowest GC content (46%) and maximum AT content (54%) were recorded in Poncirus trifoliata (L) Raf. A similar GC content was observed in the sequenced ITS region of Indian cultivars, which supports the observed pattern of GC variation.[Cymbidium species,[Recently, length variation across ITS regions has also been observed in  species. The nuclariation. In view ariation. Sequence species, which suCitrus genus and its closely related species. It is a well-known fact that during DNA sequence evolution, the rate of transitional changes differs quite relatively from the rate of transversional changes, with transitions generally occurring more frequently than transversions. The transition/transversion bias (R) across the combined data was evaluated using Kimura two-parameter analysis with four models  to describe the best substitution pattern (r) for each nucleotide pair was recorded among r (CG ±0.189), revealing high levels of substitutions. However, moderate and lower values of substitution were observed for r  and , respectively.[R = 0.956, which gives a strong support for the dominance of the transitions over transversion in Citrus germplasm. The observed higher transition/transversion (R) rate is in accordance with the recent reports of the observed higher transition/transversion bias (1.158) in the phylogeny in Indian Citrus cultivars recently inferred using ITS.[Citrus germplasm using SSR markers.[We further evaluated the nucleotide diversity value (π), using the Tajima Neutrality test. We obser pattern . The higectively. The tranectively. In our asing ITS. The pres r CG ±0.89, reveaCitrus.[Citrus and its relative species by the MP method, using the SPR algorithm with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates) as implemented in MEGA5.[A. monophylla  as an outgroup species  and Poncirus trifoliata with a bootstrap value of 96.2%. In context, the second clade represents the C. grandis × C. paradisi cv. HB pummelo and C. sinensis × P. trifoliata cv. Citrange. Additionally, C. sinensis cv. Jincheng, C. sinensis cv. Anliu, C. sinensis cv. Valencia and C. reticulata × C. sinensis L. Osbeck cv. Murcott (hybrid) were placed together in the third clade with bootstrap values of 100%. The phylogenetic analysis, as inferred for the ITS sequence data indicated that sweet oranges  showed a close relationship with the hybrid Murcott (C. reticulata × C. sinensis). The observed clades are in strong support with the previously observed clades.[The phylogenetic analysis revealed several well-supported clades with strong bootstrap values. In total, we observed seven strongly supported clades, which were clearly distinguishable among the subfamily d clades.,31,32Fortunella contains the Kumquats. It closely resembles Citrus species, although their morphology is very different. Across the fourth clade, C. paradisi × P. trifoliata cv. Citrumelo, C. aurantium (L.) cv. Daidai, Fortunella hindsii Swing. cv. Hongkong Kumquat and Fortunella crassifolia Swing. cv. Meiwa Kumquat were grouped in a sister clade. Morphologically, Fortunella and Citrus are significantly different from each other. However, there are previous evidences of nested clustering of Fortunella with Citrus species.[Fortunella spp. within Citrus, close to the C. reticulata group, confirming their recent reclassification as C. japonica, using ITS sequence polymorphism. Our ITS rDNA data showed a close evolutionary relationship between Fortunella (Hongkong and Meiwa Kumquat) and sour orange (C. aurantium). Biswas et al. [Fortunella might be less divergent than Citrus at the molecular level than observed in morphology. Moreover, C. medica var sarcodactylis cv. Fingered Citron, C. unshiu Marc. cv. Guoqing No.1, C. jambhiri (L.) Burm. f. cv. Rough lemon and C. limon (L.) Burm. f. cv. Eureka lemon occupies the fifth clade. Several earlier experts hypothesized C. limon to have a complex hybrid origin of Citron and Lime [C. limon was grouped with C. jambhiri and C. medica var sarcodactylis in the MP tree, which supports the close relationship among these species. In a previous study by Jena et al.,[C. jambhiri and C. reticulata and supported the role of C. reticulata as a maternal parent in the hybrid origin of C. jambhiri, which perfectly fits with the phylogenetically observed clade in our analyses.The genus  species.,12 Kyndts et al.  reportedand Lime  or Citroand Lime ,37 or Soand Lime  In our sa et al., they proC. medica. This species did not transmit its cytoplasm to other species but played an important role as a male parent.[C. jambhiri (L.) Burm. f. cv. Rough lemon and C. limon (L.) Burm. f. cv. Eureka Lemon. Furthermore, Citrus reticulata Blanco cv. Ponkan, C. paradisi Macf. cv. Red Marsh grapefruit and C. reticulata × C. paradisi Macf. cv. Nova (hybrid) were grouped jointly as a sixth clade. As it is well known, the origin of grapefruit has been well documented and is considered to have originated most probably from a hybrid between pummelo and sweet orange, perhaps through back introgression to pummelo.[C. paradisi Macf. cv. Red Marsh grapefruit and the C. reticulata × C. paradisi Macf. cv. Nova (hybrid) were grouped together with pummelo, supporting the viewpoint of a backcross with pummelo.[C. grandis, C. grandis L. Osbeck cv. Guan xi Miyon Pummelo, C. grandis L. Osbeck cv. Gao Ban pummelo and C. grandis L. Osbeck cv. Shatian pummelo were grouped with C. reticulata Blanco cv. Bendizao in the last clade. In addition, Shatian pummelo, Gao Ban pummelo and Guan xi Miyon Pummelo (C. grandis) were clustered with C. reticulata (Ponkan mandarin). In contrast, HB pummelo (C. grandis × C. paradisi) is closer to Citrange (C. sinensis × P. trifoliata). It is generally accepted that citrons, mandarin and pummelo are three true species in the genus Citrus.[Citrus; in addition to this, the authors supported that pummelo mitotype was found to be present as the most important cultivated Citrus species. Furthermore, Barkley et al. [The citron mitotype contained only e parent. Indeed,  pummelo.,40 In ou pummelo. For C. gs Citrus.,40 Our ds Citrus. which sus Citrus. stronglyy et al.  reportedCitrus genus and its relatives. The phylogenetic tree of the rDNA supported seven strong clades which were clearly shown among the genus Citrus. Consequently, this study not only corroborated the previous molecular reconstruction of subfamily Aurantioideae, but also strengthened previous claims concerning the evolutionary biology of the genus Citrus.To conclude, the present study presents an effective utilization of rDNA sequence divergence to maximize the possible knowledge of the genetic diversity within the


Enterobacteriaceae (CRE) has increasingly spread worldwide in the past decade. The prevalence and characteristics of CRE in Thailand are unknown. In this study, we conducted a 2-year surveillance of CRE among 12,741 clinical isolates of Enterobacteriaceae at the largest university hospital in Thailand with molecular characterization of beta-lactamase (bla) genes, including carbapenemase genes. The CRE prevalence was 1.4%. blaKPC-13 and blaIMP-14a were the only carbapenemase genes detected among these CRE isolates. blaKPC-13 gene was found in a single isolate of Escherichia coli, Enterobacter cloacae and Citrobacter freundii, and blaIMP-14a was found in four isolates of Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) isolates were resistant to multiple carbapenems at a higher ratio than other CRE species, and thus were further characterized for resistance phenotypes, bla genotypes and molecular epidemiology. Most CRKP isolates harboured multiple bla genes, especially those related to extended-spectrum beta-lactamases. Seven CRKP isolates were resistant to all tested carbapenems, and showed decreased ompK35 and/or ompK36 porin gene expression. Molecular typing of CRKP based on pulsed-field gel electrophoresis (PFGE) demonstrated several unrelated clones. Multilocus sequence typing (MLST) was partially concordant with PFGE results and revealed that ST340, a member of drug-resistant K. pneumoniae clonal complex 258, was the most predominant clone, followed by ST48, ST11 and ST273. The novel ST1645 was identified from this study. ST340 has neither been shown to be predominated among CRKP from other studies, nor been reported in Thailand. Therefore, it emphases a critical concern to monitor and control the spread of CRKP.Carbapenem-resistant  Enterobacteriaceae in many countries, carbapenems have been increasingly used as a common drug of choice . Pr. PrEntereriaceae –9. Resisescribed –13. Othe strains , 15]. H. HEntero strains , 17]..EnterobaK. pneumoniae is known to be the most common species among CRE, hereafter called carbapenem-resistant K. pneumoniae (CRKP). CRKP has been reported in many countries and their molecular epidemiology based on multilocus sequence typing (MLST) demonstrates that various sequence types (STs) are widespread . T. TK. pneountries –24. A reK. pneumoniae during 2001–2010 was upraised from 26% to 39% . Probably related to the surge of ESBL producers, a survey of carbapenem use in Thailand during 2010–2013 showed a significant increase from approximately 2.1 to 3.1 million vials per year. However, very little is known about the molecular epidemiology and genetic characteristics of CRE in Thailand. Here we investigated the prevalence and resistance characteristics of CRE at the largest university hospital in Thailand with an emphasis on the characterization and molecular typing of CRKP.In Thailand, an overall prevalence of ESBL-producing Enterobacteriaceae obtained from both in-patients (68.3%) and out-patients (31.7%) at the Faculty of Medicine Siriraj Hospital, a 2300-bed university hospital in Bangkok (Thailand), over a 2-year period (2009–2011) were investigated in this study including K. pneumoniae (38.7%), Escherichia coli (37.2%), Enterobacter cloacae (6.0%), Proteus mirabilis (4.2%), Salmonella spp. (4.1%) and other species (9.8%). All Enterobacteriaceae isolates were determined for their susceptibility to various antimicrobial agents by standard disk diffusion method using Kirby-Bauer disk  according to clinical breakpoints recommended by the Clinical Laboratory Standards Institute (CLSI) . H. HEnterooduction . Our resenemases , 39]. B. BEnteroEnterobacteriaceae have been reported to spread rapidly in the last decade, the prevalence in Thailand remains very low. This study demonstrated that the rate of blaKPC-carrying isolates was only 0.02% among Enterobacteriaceae and 1.7% among CRE isolates. We also reported previously from this survey that it was the first emergence of blaKPC in Thailand , [, [K. pneon types . ST258 id Russia , but hass source . In addi regions . This mabla genes. Only a few isolates harboured a carbapenemase gene, blaKPC-13 or blaIMP-14a, which were particularly found in Thailand. A decreased porin expression in selected CRKP isolates was not directly concordant with levels of increased carbapenem MICs. Therefore, further investigation is required to better understand the mechanisms that contribute to carbapenem resistance among CRE isolates. The PFGE and MLST studies revealed that multiple clones of CRKP were widespread. CC258, especially ST340, was the most predominant clone. Our findings represent the novel epidemiologic data of CRKP in Thailand and ones should be an alert for continuous surveillance of CRE and CRKP in this region to promptly control these highly resistant bacteria.In conclusion, we report an extensive survey for the emergence of CRE at the largest university hospital in Thailand. The prevalence of CRE was low but should be closely monitored. Most CRE isolates harboured multiple


Nocardia cyriacigeorgica associated with Mycobacterium avium complex (MAC) lung disease caused by M. avium.Pulmonary nocardiosis frequently occurs in immunocompromised hosts and in some immunocompetent hosts with chronic lung disease; however, few reports have described pulmonary nocardiosis with nontuberculous mycobacterial lung infection. Here we report for the first time two cases of pulmonary nocardiosis caused by Nocardia species. He received trimethoprim/sulfamethoxazole, and his symptoms and lung infiltrates improved. Case 2 is that of an immunocompetent 53-year-old Japanese woman with MAC lung disease, who was treated with a combined therapy of clarithromycin, rifampicin, ethambutol, and levofloxacin. MAC sputum culture was negative after 1 year of combined treatment, which was maintained for 2 years. After four treatment-free years, Nocardia species were occasionally isolated from her sputum, although MAC was rarely isolated from sputum cultures over the same period. In both cases, the Nocardia species were identified as the recently defined N. cyriacigeorgica by 16S ribosomal RNA gene sequencing.Case 1 is that of a 72-year-old Japanese man with untreated MAC lung disease, who was diagnosed with rheumatoid arthritis and initiated on methotrexate. After 3 years of methotrexate therapy, the patient remained smear-negative and culture-positive for MAC, but also became smear-positive for N. cyriacigeorgica associated with MAC lung disease caused by M. avium and suggest that N. cyriacigeorgica may be a major infective agent associated with MAC lung disease.We report two cases of pulmonary nocardiosis caused by The online version of this article (doi:10.1186/s12879-014-0684-z) contains supplementary material, which is available to authorized users. Nocardia [Pulmonary nocardiosis is an uncommon pulmonary infection caused by aerobic gram-positive actinomycetes of the genus Nocardia . It occuNocardia -6]. Pul. PulNocaNocardia -4]..NocardiaMycobacterium avium complex (MAC) lung disease is becoming more prevalent . He. HeNocarNocardia species infection: HIV infection , org, orgNocanfection . Preexisnfection . Preexisnfection . It is unfection . Airway Nocardia species isolated from our patients was N. cyriacigeorgica, a recently identified species confirmed by 16S ribosomal RNA gene sequencing. N. cyriacigeorgica was first classified in 2001 by Yassin et al. as a novel species distinguished by molecular and biochemical analysis from previously identified members of the genus Nocardia such as N. asteroides [N. asteroides in fact belonged to N. cyriacigeorgica (27 of 121 cases) [N. cyriacigeorgica is the most frequently isolated strain in Taiwanese patients with pulmonary nocardiosis (10 of 20 cases) [N. cyriacigeorgica infection may be most common in East and Southeast Asia. Larger populations should be studied to improve our understanding of the characteristics of pulmonary N. cyriacigeorgica infection.The 1 cases) . In addi0 cases) . N. cyriNocardia species co-infection may be underestimated in patients with MAC lung disease because Nocardia and MAC infection often develop in immunocompromised hosts and in selected immunocompetent hosts with chronic lung disease. Chest CT findings in MAC lung disease, such as nodular bronchiectatic lesions, often resemble those in Nocardia species infection; thus, clinicians may not suspect the relatively rare Nocardia species co-infection. Nocardia species also grow slowly and positive cultures may take up to 4 weeks to appear; thus, clinicians may not obtain positive culture results for Nocardia species unless they proactively suspect Nocardia infection and incubate the samples for longer than normal.Nocardia isolates in vitro and can be used as alternative antimicrobial agents [N. cyriacigeorgica isolated in Case 2 was resistant to TMP/SMX, according to the CLSI definition of susceptibility. TMP/SMX may be excluded as a candidate drug for treatment of pulmonary nocardiosis in Case 2. However, susceptibility test results do not always correlate with clinical outcomes of treatment for Nocardia [TMP/SMX is widely used in first-line therapy of pulmonary nocardiosis. Amikacin, imipenem, third-generation cephalosporins, minocycline, netilmicin, and amoxicillin-clavulanic acid are also effective against l agents . Howeverl agents . N. cyriNocardia . In factNocardia . TherefoN. cyriacigeorgica in patients with MAC lung disease. Nocardia species co-infection may be underestimated in patients with MAC lung disease and N. cyriacigeorgica may be the main Nocardia species co-incident with MAC lung disease.This is the first report of pulmonary nocardiosis due to Written informed consent was obtained from each patient for publication of this Case report. Copies of the written consents are available for review by the Editor of this journal.


Bacillus amyloliquefaciens (syn. Bacillus velezensis) KCTC 13012, which exhibits a broad spectrum of antagonistic activity against bacteria and fungi and promotes plant growth as well. The genome contains an array of biosynthetic gene clusters for secondary metabolites that are comparable to those in Bacillus amyloliquefaciens subsp. plantarum FZB42T.We report the 4.0-Mb draft genome sequence of  Bacillus and Paenibacillus, are known to encode a variety of biosynthetic gene clusters for secondary metabolites . The average nucleotide identity values with close strains, as calculated by JSpecies (B. amyloliquefaciens subsp. plantarum FZB42T), 98.2% (B. velezensis G341) (B. amyloliquefaciens subsp. amyloliquefaciens DSM 7T), which is in accordance with the recent study showing that the genomes of the B, amyloliquefaciens subsp. plantarum group are highly conserved . Genome sequencing was carried out at the National Instrumentation Center for Environmental Management in Seoul National University  using the Illumina HiSeq 2000 platform. In all, 101-nucleotide (nt) paired-end reads, totaling ~1,000× coverage, were produced from a ca. 470-bp-long genomic library. Quality trimming, filtering by length, and a nrsABCDEF, KCTC 13012 has homologous gene clusters for the biosynthesis of all the other secondary metabolites present in FZB42. Because gene clusters for fengycin and surfactin biosynthesis have been found on several short contigs as fragments, the presence of these two gene clusters was confirmed through mapping sequence reads on the FZB42 reference genome, whereas the complete gene cluster structures for the others were represented on the de novo assemblies. A read-mapping analysis showed that KCTC 13012 lacks a prophage-like region (yotN-yokA) and a segment encompassing nrsF through RBAM_027480, which are present in FZB42. Meanwhile, a prophage-containing contig_8 (53.1 kb) represents a KCTC 13012-specific region. Our results will provide further insight into the genomic basis of the diversity of the B. amyloliquefaciens species and its secondary metabolite biosynthetic potential.With the exception of LHCC00000000. The version described in this paper is version LHCC01000000.The whole-genome shotgun project of KCTC 13012 was deposited at DDBJ/EMBL/GenBank under the accession number


T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients.Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts.T. cruzi/HIV co-infected patients.Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in  T. cruzi/HIV co-infected patients. In this study, Chunap (Chagas urine nanoparticle test) was used for concentration and quantification of T. cruzi antigens in urine of T. cruzi/HIV co-infected patients. Values of more than 105 pg of T. cruzi antigens in urine were observed only in patients with reactivation of Chagas disease. This study shows that antigenuria levels are highly correlated to levels of parasitemia and can be used as a non-invasive technique for monitoring parasitemia levels in T. cruzi/HIV co-infected patients.Reactivation of Chagas disease in people living with HIV is a serious clinical condition that is associated with high mortality. Hence, early diagnosis and treatment can be lifesaving. Although there are not well accepted criteria to identify patients at risk of reactivation, parasitemia levels are usually considered as the best predictor. Microscopy is used in Latin America for detection of parasitemia levels. However, this has low sensitivity, which usually leads to a delay in diagnosis and treatment. Quantitative PCR is used only for research proposes in endemic areas. Antigens in urine (antigenuria) are correlated with parasitemia levels in animal models, as well as in cases of congenital Chagas disease. We believe that antigenuria can also be used for prediction of parasitemia levels in  Trypanosoma cruzi, affects an estimated 7.8 million people in the Americas [T. cruzi/HIV co-infection as a significant public health problem in the Americas.Chagas disease, caused by the protozoan Americas . SimilarAmericas . MassiveT. cruzi infection in the world; with adult seroprevalence figures of up to 30% in urban areas and up to 80–90% in some rural areas [T. cruzi/HIV co-infection in this country.Bolivia has the highest prevalence of al areas . HIV infT. cruzi, immunocompetent patients enter the acute phase, this phase is characterized by high parasitemia, mild and nonspecific febrile illness, and, rarely, life-threatening myocarditis and/or meningoencephalitis [After infection with phalitis . After 2phalitis . Chagas phalitis . HoweverT. cruzi/HIV co-infected individuals develop T. cruzi reactivation. Presentation includes high levels of parasitemia and severe clinical manifestations; usually involving CNS syndromes (50–85%) and/or myocarditis (10–55%) [Toxoplasma gondii reactivation. As such, direct detection of the parasite is needed to confirm the diagnosis. Mortality in patients with meningoencephalitis reaches 80–100%, partly as a consequence of late diagnosis and treatment [An estimated 20% of (10–55%) –12. Altereatment .T. cruzi infection with and without reactivation. Current criteria for reactivation are based on microscopic observation of the parasite in blood, but because of its low sensitivity, this technique detects reactivation when the parasitemia is high [T. cruzi/HIV co-infected patients [Some studies suggest that early diagnosis and treatment with both benznidazole and combination antiretroviral therapy (cART) could be lifesaving in patients with CNS reactivation , 13–14.  is high . By this is high , 16. Fur is high . Quantitpatients .T. cruzi/HIV co-infected patients from Brazil demonstrated that the majority of T. cruzi/HIV co-infected patients have substantially higher parasitemia levels when compared to immunocompetent T. cruzi-infected individuals. Some of these patients had detectable parasitemia by microscopy even in the absence of symptoms [The qPCR is used to monitor levels of parasitemia as well as risk of reactivation in immunocompromised individuals after organ transplantation in the USA ,18. Howesymptoms . These asymptoms .T. cruzi antigens has been shown to correlate with parasitemia levels in animals [High levels of parasitemia may represent an intermediate phase that precedes clinical reactivation. In such circumstances, preemptive treatment may be justified , 15. How animals  and coul animals –25.D < 10−12 M) within minutes [A novel nanotechnology based on the use of nano-porous particles that contain high affinity chemical baits (trypan blue) in the inner core is proposed for concentration and preservation of antigens in urine –25. This minutes –26.T. cruzi infection in HIV co-infected is needed. This tool could lead to early treatment and may be lifesaving. In this study, we demonstrate that levels of T. cruzi antigens determined by Chunap are associated with levels of parasitemia in T. cruzi/HIV co-infected patients and could be a valuable non-invasive tool for monitoring Chagas disease reactivation in HIV co-infected patients.A sensitive but relatively simple noninvasive test for monitoring The protocols were approved by the institutional review boards of the study hospitals , Asociacion Benefica Prisma  and the Johns Hopkins University .T. cruzi-infected and 24 T. cruzi uninfected) from Cochabamba and Santa Cruz, Bolivia. A written informed consent was obtained from all participants. The diagnosis of HIV infection was performed according to the Bolivian National Control Program of HIV/AIDS, and was based on detection of specific antibodies by an ELISA test  and Western blot . Blood and urine samples were obtained early on in hospitalization and none had received T. cruzi treatment. Confirmation of T. cruzi infection was based on positive results by 2 or more of the following commercial tests: Chagatest ELISA , Chagatest ELISA recombinant v 3.0 , and the indirect hemagglutination test (IHA) .We evaluated 55 samples of HIV patients . We therefore used a classification of high HIV load as follows: 1) <5000 copies/ml, 2) ≥5000–30 000 copies/ml, and 3) >30 000 copies/ml , 28. TheT. cruzi infection was determined by detection of circulating parasites by micromethod. In this technique, blood samples are collected in 4–6 heparinized microhematocrit tubes, centrifuged and the buffy coat layer is examined microscopically for parasites [Reactivation of arasites . Quantifarasites –34. PatiPatients were asked to provide the first urine of the day before ingestion of liquids, where midstream specimens were collected. Urinalysis was done using urine test strips . Mean values of urine specific gravity was within normal levels . Urine samples (10 mL) were immediately centrifuged after collection at 3000 rcf for 10 min and the supernatant was stored in liquid nitrogen or -80°C until use. For antigen detection the supernatant was adjusted to pH 5–6 with 1M HCl.Poly N-isopoprylacrylamide (NIPAm) particles coupled with trypan blue dye (Poly (NIPAm/TB)) were synthesized as previously described –26. UrinT. cruzi lipophosphoglycan (LPG) mouse monoclonal antibody , diluted 1:250 in PBS with 0.2% I-Block and 0.1% Tween 20. After six washing steps with PBS supplemented with 0.1% Tween 20, antigens were incubated with peroxidase conjugated goat anti-mouse IgM  diluted 1:5000 in PBS supplemented with 0.2% I-Block and 0.1% Tween 20, for 60 minutes at room temperature. The molecular weight was determined using MagicMark XP Western Protein Standard . Each sample was run twice. Visualization of antigenic bands was done using an enhanced chemiluminescence system . The trypomastigote excretory-secretory antigen (TESA) was used to develop a calibration curve. This antigen was harvested from cell cultures of T. cruzi Y strain in LLC-MK2 cells, as previously described [2 = 0.95)  was considered as a positive result. In each experiment we included a negative control  and a positive control . The Chunap was carried out by a laboratory biologist who was blinded to the Chagas status of the patient.Aliquots of 20 μl of re-suspended antigens were heated to 100°C for 7 min. Electrophoresis and Western Blot analysis of the antigens were performed as previously described . Brieflyescribed . The cal = 0.95) . AntigenqPCR was performed to evaluate levels of parasitemia. DNA was purified from 500 μl of blood clot samples as previously described , 33. TheT. cruzi antigen concentration and parasitemia levels was evaluated using a linear regression model unadjusted and adjusted by sex, age (years), antiretroviral treatment (yes versus no), HIV load and immune status (CD4+ and CD8+ T cell counts). The use of a sample size of 31 T. cruzi/HIV co-infected patients with a significance level of 0.05 gives a statistical power of 72% to determine associations between levels of antigenuria and parasitemia. This sample size gives a power below 70% to determine other statistical associations.STATA 13 software was used for all statistical analysis. Parasitemia levels determined by qPCR were evaluated in a logarithmic scale. Differences in mean levels of parasitemia, CD4+ and CD8+ T cell counts, and HIV load between patients with and without reactivation were evaluated by Student’s t-test with equal and unequal variances. Receiver Operating Characteristic (ROC) analysis was used to determine the sensitivity and specificity of different cut-offs of antigenuria and parasitemia levels for the diagnosis of reactivation of Chagas disease using microscopy as gold standard. The association between urine T. cruzi infected and 24 T. cruzi non-infected). Mean age was 36.8 years (SD: 15.1 years). Mean levels of HIV loads were considerably higher  [The nested-case control study consisted of 55 HIV patients (31 pies/ml) , 28, whipies/ml) . TubercuT. cruzi antigens by 100 fold as previously described [T. cruzi/HIV co-infected patients  among the cases with reactivation or high parasitemia, 91.7% (11/12) among cases with moderate parasitemia, and 41.7% (5/12) among patients with negative parasitemia , 95% (18/19) and 74% (23/31), respectively. See In this study poly (NIPAm)/TB nanoparticles were used to increase the effective sensitivity of western blot analysis in the detection of escribed . Bands opatients . Bands oasitemia . The perMean levels of parasitemia were significantly different between patients with reactivation of Chagas disease  and patients without reactivation but with positive qPCR  (p = 0.003)  Fig 2A)Fig 2A). 2 = 0.84) (2 = 0.84) (A linear relationship was observed between antigenuria and parasitemia levels in both the unadjusted and adjusted regression model . Interes = 0.84) . Similar = 0.84) .Among patients with Chagas disease, mean levels of CD4+ T-cell counts were significantly lower in patients with reactivation  compared to patients without reactivation   . ClinicaT. cruzi infection in urine of HIV patients.Reactivation of Chagas disease in HIV patients is a serious medical condition, which is often life-threatening. Early diagnosis of Chagas reactivation and prompt treatment can be life-saving. However, current methods are based on microscopy that have sub-optimal sensitivity that do not detect reactivation until parasite loads are very high. A sensitive, non-invasive test is needed to monitor increase in parasitemia levels and predict risk of reactivation. In this study we introduce the potential use of a nanoparticle-based tool to monitor No differences were observed in HIV loads, CD4+ cell and CD8+ cell count, and presence of other co-infections between HIV patients with and without Chagas disease. Among patients with positive serology for Chagas disease, patients with reactivation of Chagas disease had low levels of CD4+ T-cells counts compared to patients without reactivation, as previously observed . The perClinical manifestations related to Chagas disease were observed in the nervous system , 10. In T. cruzi lipophosphoglycan (LPG) was used in this study for antigen detection. The T. cruzi LPG is a surface glycoconjugate which is composed mainly of glucosamine, sialic acid and galactosamine in the carbohydrate portion, and of alkylacylphosphatidylinositol in the lipid portion [An IgM monoclonal antibody against  portion . Althoug portion . This an portion .M. tuberculosis. We hypothesize that the presence of false positives could be explained by the existence of other co-infections. In the case of tuberculosis, the mycobacterial lipoarabinomannan has been detected in urine samples of HIV co-infected patients [Leishmania, Trichmonas, and Pneumocystis, but the anti-IgM LPG antibody used in this study does not show cross-reaction with the LPG from Leishmania and Trichomonas species [Toxoplasma gondii, and bacteria and fungi such as M. tuberculosis and Cryptococcus neoformans.The percentage of antigenuria positives detected by Chunap (74%) among those with positive serology is similar to that reported before in chronic infected patients (60%-84%) –40. Two patients . The lip species . HoweverDefinition of a parasite load threshold for prediction of reactivation could be used as a guide in the use of anti-trypanosomal therapy. The early increase in parasitemia may not be symptomatic as previously described in one prospective study , so moniT. cruzi, and by the inability to distinguish DNA from living and dying parasites [Levels of parasitemia, determined by qPCR, were higher in patients that had reactivated Chagas disease than in those without reactivation. However, there was a high variability of parasitemia levels between individuals. This observation was also reported by a previous study  . The higarasites . Althougarasites .In this study, reactivation was defined as the detection of circulating parasites in blood by microscopy with or without the presence of clinical manifestations . This deIn the guinea pig model of Chagas disease and congenital Chagas disease, the presence of antigens in urine is correlated with high levels of parasitemia , 26. In Antigenuria levels were significantly higher in patients with < 200 CD4+ T-cells, but only in the adjusted model, suggesting possible confounding effects of HIV loads, treatment, age, and sex. In contrast to a previous study, we could not find statistically significant differences between levels of parasitemia or antigenuria, and HIV load and CD8+ T-cells levels . LimitatAmong patients with Chagas disease, young age was found to be a risk factor for high antigenuria levels in the adjusted model. However, this association could be influenced by the time of diagnosis of HIV infection and ART.T. cruzi antigens from degradation [The Chunap reached sensitivity levels comparable to the more expensive and time consuming standard of care micromethod and qPCR. The feasibility data presented herein provide evidence that Chunap is a promising tool to improve current Chagas diagnostic algorithm in clinical settings. Furthermore, in a previous study we demonstrated that Chunap protect radation .In future studies, poly(NIPAm/TB) particles will be magnetized in order to simplify the clinical diagnostic process and extend accessibility. Sensitivity can be further improved by increasing the volume of urine analyzed. In conclusion, an antigen urine test for monitoring Chagas reactivation in HIV patients would be highly desirable for these reasons: a) levels of antigens in urine are related to levels of parasitemia b) antigenuria levels are less variable compared to levels of parasitemia c) urine is a preferred, less-infectious, non-invasively collected biological fluid that is more acceptable by patients , and d) antigen testing can be scaled to a rapid, point of care test that can be performed in low-equipped laboratories.Jean Cabeza, Roni Colanzi, Daniel Lozano, Gonzalo Borda, Gerson Galdos, Lisbeth Ferrufino, Louisa Messenger, Rosmery Gross, Leny Sanchez, Omar Gandarilla, Maurus Dorn, and Helena Jahuira.S1 Fig(TIF)Click here for additional data file.S1 ChecklistT.cruzi/HIV co-infected patients.Use of Chunap for detection of Chagas disease in (PDF)Click here for additional data file.S1 DiagramT.cruzi/HIV co-infected patients. * Confirmation of T. cruzi infection was based on positive results by 2 or more serological tests for detection of anti- T. cruzi IgG antibodies. ** Reactivation of Chagas disease was based on the detection of circulating parasites by microscopy.Use of Chunap for detection of Chagas disease in (TIF)Click here for additional data file.


Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N6-methyladenine (m6A) methylated motifs, one N4-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication.  MethyMethylation in eukaryotes has been well studied and is known to mediate diverse processes including growth, development, and disease . In prokDespite the important implications of bacterial methylation, its distribution, diversity, and functional consequences have not been extensively investigated. This paucity of data can, in part, be attributed to technological limitations. Methylation studies in eukaryotes have been facilitated by the development of detection methods for m5C, including bisulfite conversion, which allows for genome-wide modification analyses. Comparable methods have not been available for the detection of m6A and m4C until recent advances in sequencing technology. SMRT sequencing couples whole-genome sequencing with the simultaneous detection of base modifications using kinetic signals during DNA polymerization . This neSalmonella enterica is the leading cause of death and hospitalizations due to foodborne pathogens each year [m6ATC motif by the MTase Dam is an essential factor in the virulence of Salmonella, and that a lack of methylation leads to attenuation in animal models [Salmonella also may have implications for virulence, pathogenicity, and other functions. Here, we sequenced and closed the genomes of six Salmonella enterica isolates from five serovars. We then analysed their methylomes, along with the methylomes of four additional serovars that we sequenced previously [Salmonella serovars.ach year . Previoul models . Subsequl models  and the l models . In addil models , and thel models . Thus, ieviously , 20–22, Salmonella enterica subs enterica from our in-house strain collection at the FDA-CFSAN. These included Salmonella enterica subs enterica serovar (S. Bareilly), S. Abaetetuba, S. Abony, S. Anatum, S. Bredeney, S. Montevideo, and two isolates of S. Enteritidis. We also included data from four serovars we sequenced previously, S. Javiana, S. Typhimurium, S. Heidelberg, and S. Cubana [We selected five serovars of . Cubana , 20–22 . All samples were analyzed at the exponential stage of growth.DNA was sheared to approximately 10 kb using a Covaris g-TUBE . SMRTbell 10 kb template libraries were prepared using DNA Template Prep Kit 2.0 and the Low-Input 10 kb Library Protocol . In brief, DNA was concentrated, repaired, ligated to hairpin adapters, and purified. Incompletely formed SMRTbell templates were digested with a combination of Exonucleases III and VII. Adapters were annealed, and SMRT sequencing was carried out on the PacBioRS II  using standard protocols.De novo assembly was performed using the Hierarchical Genome Assembly Process (HGAP) with default parameters [de novo assembly and final consensus and accuracy scores are determined using the Quiver consensus algorithm. HGAP outputs assemblies with overlapping regions at the ends. Coordinates of this region were identified using dot plots in Gepard [http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). Prophages were detected using PHAST [Analysis of sequence reads was implemented using SMRT Analysis 1.10 and the SMRT Portal 2.0 platform (Pacific Biosciences). rameters . HGAP con Gepard , and trin Gepard  (http://ng PHAST . Only prhttp://www.pacb.com/pdf/TN_Detecting_DNA_Base_Modifications.pdf. Interpulse durations (IPDs) were measured based on the kinetic signals [https://github.com/PacificBiosciences/motif-finding. The observed bimodal distribution of kinetic data, resulting from modified and unmodified positions, was then used to determine a more stringent QV threshold (http://www.ncbi.nlm.nih.gov/sra) (http://rebase.neb.com/rebase/rebase.html).Motif Detection and Analysis was also carried out using SMRT Analysis 1.1 and the RS_Modification_and_Motif_Analysis.1 protocol as described at  signals  and proc signals . At eachhreshold . Only sigov/sra)  and MTasSalmonella chromosomes ranged from 4,547,600 – 4,977,480 bp, plasmid sizes ranged from 3,609–221,009 bp . This MTase is usually active in Salmonella, although rarely active in E. coli, and is not thought to be an essential gene [m6ATC motifs could only be performed in eight of the eleven isolates: two were orphan MTases, and the remaining were common Type II enzymes. In multiple serovars, we identified candidate enzymes that have the potential to methylate this motif . Furthermore, as more genome sequence data and PacBio methylation data appear, our ability to predict recognition sequences from sequence data alone is growing. Already, rules are becoming apparent for predicting the specificity of Type IIG enzymes [We cannot completely rule out the possibility that DNA MTase genes exist that show no similarity to characterized MTase genes. However, with methylation data from more than 700 genomes available and almost 2,500 characterized and 50,000 putative MTase genes identified in REBASE, the chances of finding a completely new way of methylating DNA are getting increasingly smaller. In particular, we rarely come across a case where we can be certain that there are insufficient MTases to account for the observed patterns of methylation. However, in Salmonella enterica subsp. enterica serovar Heidelberg CFSAN002064, the methylated motif ACC enzymes .Lactococcus [m6ATGN5G4mGC, is exhibited by the specificity subunit of the SenAboIII system. This example of cooperation between an m6A methylase and an m4C methylase is quite rare and has only been infrequently observed previously .Most of the novel motifs observed in each serovar were modified by Type I RM systems . Type I tococcus . One intSalmonella, suggesting that these systems may have been acquired via HGT. For example, M.SbaUII from S. Bareilly, which methylates the motif Cm6AGCTG, is most similar to an MTase found in Pectobacterium. Currently, we are building a robust Salmonella phylogeny, including representatives of other Enterobacteriaceae genera, to test these and other evolutionary hypotheses.Unique motifs found among closely related taxa may be the result of horizontal gene transfer (HGT). Studies have demonstrated that HGT accounts for the movements of RM systems based on evidence of codon usage bias  and diffTGCm6AT sites across the genome were methylated, and 89.3–100% of CAGm6AG sites were methylated Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Glenrosa J. Watson et H.L. Fisher emend. V. Srinivasan inside silica-rich nodules from the Cenomanian of the Font-de-Benon quarry, Charente-Maritime, western France. Remains are preserved in three dimensions and mainly consist of fragmented leafy axes. Pollen cones of this conifer are for the first time reported and in some cases remain connected to leafy stems. Histology of Glenrosa has not previously been observed; here, most of internal tissues and cells are well-preserved and allow us to describe a new species, Glenrosa carentonensis sp. nov., using propagation phase-contrast X-ray synchrotron microtomography, a non-destructive technique. Leafy axes consist of characteristic helically arranged leaves bearing stomatal crypts. Glenrosa carentonensis sp. nov. differs from the other described species in developing a phyllotaxy 8/21, claw-shaped leaves, a thicker cuticle, a higher number of papillae and stomata per crypt. Pollen cones consist of peltate, helically arranged microsporophylls, each of them bearing 6–7 pollen sacs. The new high resolution tomographic approach tested here allows virtual palaeohistology on plants included inside a dense rock to be made. Most tissues of Glenrosa carentonensis sp. nov. are described. Lithological and palaeontological data combined with xerophytic features of Glenrosa carentonensis sp. nov. suggest that this conifer has been adapted to survive in harsh and instable environments such as coastal area exposed to hot, dry conditions.We report exceptionally well-preserved plant remains ascribed to the extinct conifer  Glenrosa J. Watson et H.L. Fisher emend. V. Srinivasan [Glenrosa is characterized by an unusual stomatal arrangement inside crypts. Stomatal crypts consist of ampulla-shaped pits that are sunken in the mesophyll and contain stomatal apparatuses. The genus was first erected to include two species from the lower Cretaceous of Texas, Glenrosa texensis and Glenrosa pagiophylloides [Brachyphyllum texense and Sequoia pagiophylloides by Fontaine [Glenrosa texensis was described from the Glen Rose Formation and the Trent’s Reach locality, which are late Aptian–earliest Albian and Barremian–earliest Aptian in age respectively. Glenrosa pagiophylloides was only reported from the first [Glenrosa hopewellensis and Glenrosa virginiensis from the middle Albian Patapsco Formation of Virginia [Glenrosa nanjingensis from the late Early Cretaceous Gecun Formation of China [Glenrosa falcata from the upper Barremian of La Huérguina Formation in Spain [Glenrosa species were based only on highly compacted and isolated cuticle remains, and internal histology of leaf tissues has not been described. Likewise, attached reproductive structures have not been found, although isolated cone scales and microsporophylls without in situ pollen sacs have tentatively been assigned to Glenrosa [Glenrosa was tentatively assigned to the Cheirolepidiaceae [The Cretaceous conifer inivasan –2 shows inivasan –8. Glenrylloides , that weFontaine . Glenroshe first . Four otof China , and Glein Spain . All preidiaceae . Howeveridiaceae  noted thGlenrosa sp. have been previously reported from upper Albian and Cenomanian deposits of many localities of Charente-Maritime and Charente . The plant-bearing nodules being large and dense, this work required to test new tomographic protocols combining high energy and multiscale approaches. We discuss the interest and the limits of the synchrotron microtomography, and compare G. carentonensis sp. nov. with other Glenrosa species. The only known other Cretaceous conifer bearing stomatal crypts is Sedites rabenhorstii (Geinitz) Kunzmann from the upper Turonian of the Bohemian Cretaceous Basin [Glenrosa.In western France, fragmented cuticles of leafy axes ascribed to us Basin . Given tus Basin , we alsoOrbitolina) and vertebrates [Catopygus carinatus Goldfuss, Mecaster sp. Pomel), foraminifers (c. Orbitolina conica d’Archiac), and unidentified sponge spicules [Brachyphyllum Brongn. emend. Harris, Frenelopsis (Schenk) emend. J. Watson, Geinitzia Endl., and Glenrosa J. Watson et H.L. Fisher emend. V. Srinivasan [Orbitolina conica and Catopygus carinatus, a Cenomanian age was assigned to the flints.In Charente and Charente-Maritime, the upper Albian–Cenomanian series are divided into seven units, A to G  of Cadeuil [Albian–Cenomanian deposits yielding plant fossils have been reported from several localities of western France such as Charente-Maritime , Dordogn Cadeuil , Font-de Cadeuil ) and Les Cadeuil ); the lo Cadeuil ) and La  Cadeuil ), Bois-V Cadeuil ); and thGlenrosa, as determined by diagnostic helical phyllotaxy and the presence of stomatal crypts, are the main component of the flora preserved in the flint nodules. It represents the first example of silicified leafy stems and male cones of Glenrosa discovered to date. Three pollen cones have been discovered, two of them remain connected with leafy axes. Plants remains are preserved as silica permineralizations, with three dimensions of external and internal microstructures. The flint nodules are housed in the collection of Géosciences Rennes, CNRS UMR 6118, Université Rennes 1 . Seven of them have been investigated (SIL_ARC_1 to SIL_ARC_7). Fourteen leafy stems and the three pollen cones have been investigated.The plant-bearing flints were collected from the Cenomanian of the Font-de-Benon quarry between the villages of Archingeay and Les Nouillers, Charente-Maritime, western France . HoweverThe specimens were scanned at beamlines BM05 and ID19, at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The dense flint nodules were examined using propagation phase-contrast X-ray synchrotron microtomography (PPC-SRμCT). For studying arthropod inclusions in low density and absorption opaque amber, Lak et al.  developeThe distance between the sample stage and the source is 55 m and 145 m, at BM05 and ID19, respectively. ID19 is characterized by a higher coherence of the beam than BM05 and by more flux at higher energy, allowing to work on larger specimens at higher resolution than BM05. In total, seven different setups have been used . We usedThe three dimensional reconstructions of specimens were achieved with the software VG Studio Max 2.2 . The virtual dissection and segmentation were made using 3D region growing and manual selections.Order—ConiferalesIncertae sedisFamily—Glenrosa Watson et Fisher, 1984 emend. Srinivasan, 1992Genus—Glenrosa carentonensis sp. nov. Moreau, Néraudeau, Tafforeau et DépréSpecies—Etymology. Derived from the latin name (Carentonia) of the river Charente.Holotype. SIL_ARC_2_1. Here designated. Figs Paratypes. SIL_ARC_2_2, SIL_ARC_2_3, SIL_ARC_3_1, and SIL_ARC_7_1. Here designated. Figs Type locality. Font-de-Benon quarry, Archingeay-Les Nouillers, Charente-Maritime, western France.Type horizon. Cenomanian flints exposed at the top of the stratigraphic section in the Font-de-Benon quarry.Shoots bearing helically arranged leaves. Leaves, claw-shaped, and thick. Leaf apices obtuse to sub-acute. Leaves keeled with a thick cuticle. Leaves amphistomatic with abundant stomatal crypts. Stomatal crypts, 8–18 per leaf, and randomly distributed. Crypts as wide as deep, with circular to oval apertures, and partially occluded by interdigitate, elongated papillae. Papillae up to 48 per crypt. Stomata, 7–12 per crypt, and basally packed. Epidermis thinner on adaxial surface, and with polygonal ordinary cells. Hypodermis thin, and mainly developed on the adaxial surface. Palisade parenchyma with one (rarely two) layer(s) of elongated cells. Spongy parenchyma well-developed on abaxial and adaxial sides with lacunae and large, rounded cells. Transfusion tracheids, abundantly pitted, well-developed and limited to the abaxial side of leaves. Single vascular bundle per leaf. Continuous vascular cylinder in the stem. Tracheids elongated, narrow and bearing regularly arranged pits. Rays abundant, mainly uniseriate, 3–20 cells high. Pith with large parenchyma cells. Pollen cones spherical to ovoid, slightly longer than wide. Microsporophylls imbricate, helically arranged, peltate, and up to 40 in number per cone. Microsporophyll stalks elongated, exmedially attached to the head and triangular in transversal plane. Microsporophyll heads flat to convex, and rhomboidal. Pollen sacs 6–7 per microsporophyll.Shoots are up to twice branched. Branches arise at 30–70°. The longest specimen is 50 mm long. Shoots are up to 7.0 mm in diameter. Leaves are helically arranged Figs ; 5A–5C. 2). Abaxial surface shows a higher number of stomatal crypts than adaxial surface. Crypts are generally as wide as deep, or slightly deeper than wide. Stomatal crypts are 80–200 μm in diameter, and 80–220 μm deep  has proved to be an efficient, non-destructive technique for the study of inner structures of fossil specimens (e.g. ). In the and SEM –7, 12. HSize of samples being a factor limiting the resolution of the tomographic data, more information would be obtained with invasive approaches such as micro-sampling of leafy stems and cones. The large size of the flint nodules precluded the use of nanotomography as previously described by Moreau et al. . HoweverGlenrosa species show helical leaf arrangement, the phyllotaxy varied greatly and only G. carentonensis sp. nov. shows an 8/21 phyllotaxy  and G. er ones . AltSedites rabenhorstii (Geinitz) Kunzmann from the upper Turonian of the Bohemian Cretaceous Basin [Banksia, Blossfeldia, and Nerium produce stomatal crypts and inhabit a range of environments. Only very pronounced or deep encryptions are associated with significant drought [Glenrosa may have similarly functioned as an adaptation to dry climate conditions.Among conifers, the only other taxon with stomatal crypts is us Basin . Among eus Basin –45. For  drought , which s2 diffusion [G. carentonensis sp. nov, may suggest that this conifer was probably not only adapted to withstand desiccant conditions but also adapted to tolerate open and weakly protected habits that should be occasionally instable or disturbed.The role of papillae in these crypts has also been debated with respect to environment. They have long been interpreted as reducing water-loss , althougiffusion , and dusG. carentonensis sp. nov., probably contributed to offset the water-loss, particularly in dynamic conditions such as fluctuating insolation.Studies in living angiosperms showed that the location of stomata within the stomatal crypts influences transpiration . Roth-NeG. carentonensis sp. nov. may be a benefit to tolerate habitat with extreme sunlight exposition and hot temperatures.The role of hypodermis can be to decrease the ultraviolet penetration in the leaves, allowing the thermal load to be reduced , 50. TheGlenrosa-bearing beds has been deposited in coastal environment highly influenced by marine inputs and partially or occasionally open to the sea  the role of crypts can be also to facilitate the diffusion of CO2 to the sites of assimilation. They explained that this is particularly specified for thick leaves. We cannot exclude that stomatal crypts of G. carentonensis sp. nov. had a multi-function combining the protection of the stomata, the control of the transpiration and the regulation processes of the CO2.In many localities of Charente-Maritime, lithological and palaeontological data support that the Cenomanian u et al.  demonstrG. carentonensis sp. nov. inside the flints of Charente-Maritime suggests that this species was one of the main component of coastal conifer-dominated forests, being so well-adapted to survive in this harsh and instable environments. The other conifers that co-occurred with G. carentonensis sp. nov. inside the flints also support this hypothesis. Brachyphyllum Brongn emend. Harris and Frenelopsis (Schenk) emend. J. Watson also show xeromorphic features generally present among plants inhabiting terrestrial environments highly influenced by marine or haline inputs [The abundance of e inputs , 12, 51.