Cleisostoma yersinii J. Ponert & Vuong, is described and illustrated based on the material collected in the Hon Ba Nature Reserve in southern Vietnam. In addition to conventional (macro)morphological examination we comparatively investigated root and leaf anatomy (using light and fluorescent microscopy), assessed nectar characteristics , determined nuclear genome size (using DNA flow cytometry) and reconstructed phylogenetic relationships (using nrITS sequences). Cleisostoma yersinii differs from its putative closest relative C. birmanicum in wider and shorter leaves, larger flowers, distinct lip with S-shaped tip of the mid-lobe, and a shallow spur with two large nectar sacks separated by prominent calli and septum. Nectar is sucrose-dominant and very rich in sugars. Stomata are developed on both sides of the leaf and have prominent hyperstomatal chambers and substomatal cavities. Roots with well-developed exodermis and tracheoidal idioblasts are covered by a two-layer Vanda-type velamen. Chloroplasts occur not only in the cortex but are also abundant in the stele. Mean 1C-value was estimated to 2.57 pg DNA. An updated identification key is provided for SE Asian sections and all Vietnamese species of Cleisostoma.A new species,  Cleisostoma Blume is a taxonomically challenging group of orchids native to tropical and subtropical regions of the Indian Subcontinent, SE Asia, China and some of the Western Pacific islands )eridinae . PossiblVanda type”  . Originally collected in Vietnam, Khánh Hòa province, Cam Lâm District, Hòn Bà Nature Reserve, roadside in submontane cloud forest, alt. 1380 m, 7. 3. 2012, leg. J. Ponert, T. Q. Tam, T. B. Vuong, R. Rybková, P. Vacík & K. Petrželka.Syntype:—A cultivated specimen from the same collection, pressed on 24 September 2014, Jan Ponert 717 (PRC!: 455378)Perennial with monopodial growth. Stem 0.7–0.8 cm in diameter, up to 30 cm in studied specimens but most likely longer in mature plants in the field, with 1–2 cm long internodes, entirely clothed by tubular leaf sheaths. Roots adventitious, aerial, glabrous, 4.0–5.0 mm in diameter, covered by two-layered velamen. Velamen white in dry state but translucent when wet, revealing the green cortex; pneumatodes linear-elliptic, white . GrowingThe species is named after Alexandre Emile Jean Yersin (1863–1943), a Swiss-French physician and bacteriologist who significantly contributed to the exploration of the Hon Ba mountain area.Cleisostoma roots spread along the ground surface, occasionally reaching more than 1 m. Surface of the soil was largely bare, with only a few lichens and mosses.Plants were found growing on a roadside at elevation about 1380 m a.s.l. The surrounding vegetation was formed by a primary submontane evergreen forest, with many epiphytes. During our field work (February–March) the site was humid with high soil and air moisture, and frequent clouds. Plants were growing directly on bare mineral soil (clayey-sandy weathered residue of granite) exposed during the building of the road in 2003. Ophioglossum reticulatum L. and Psilotum nudum (L.) P.Beauv. were the most abundant species recorded on the site. Other orchids growing in sympatry were Pholidota leveilleana Schltr. , Cleisostoma birmanicum , Anoectochilus annamensis Aver., Cymbidium erythrostylum Rolfe, Dendrobium uniflorum Griff., Thrixspermum annamense (Guillaumin) Garay and Thrixspermum sp. .Cleisostoma yersinii in full flower was found by TBV at elevation about 1500 m a.s.l. in September 2014  of saccharides per mg of nectar, which corresponds to 74.51 ± 11.48% (w/v). Three different saccharides were detected: sucrose, glucose and fructose . SucroseVanda type  = 370.98, p < 0.001, η2 = 0.97). The highest density on the abaxial side was observed in C. paniculatum while C. racemiferum and C. yersinii had the lowest density  S. subulatum Blume, and 3) the remaining five species.Flow-cytometric analyses resulted in high-resolution fluorescence histograms with distinct peaks of both the studied sample and internal reference standard (coefficients of variation of G0/G1 fluorescence peaks below 4%) and little background. 1C-values of seven analyzed species ranged from 1.80 pg to 2.67 pg, spanning nearly 1.5-fold range . There wCleisostoma yersinii clustered clearly together with C. birmanicum, with posterior probability (PP) 1.00 and bootstrap support 100% in maximum parsimony (MP) and maximum likelihood (ML) trees. Cleisostoma striatum (Rchb.f.) N.E.Br., another representative of the sect. Echioglossum (Bl.) Seidenf., was placed together with C. paniculatum (Ker Gawl.) Garay  as a sister group to the C. birmanicum/yersinii clade . Evolutionary relationships of this group to other members of the genus are rather unclear. In general, two separate clades were resolved . The first clade included, in addition to the species of the section Echioglossum and several other Cleisostoma species, also members of related genera Pelatantheria and Schoenorchis while the second clade included, in addition to Cleisostoma, also species of genera Cleisocentron, Diploprora, Malleola, Omoea, Robiquetia, Sarcoglyphis, Smitinandia, Stereochilus and Uncifera.The parameters for sequence alignments are given in C. yersinii and a few habitually similar sterile plants growing on a roadside together with the type specimen were observed by us. Nevertheless, orchids are known to occur in small and often very sparse populations frequently [Thuniopsis L.Li, D.P.Ye & Shi J.Li which was described in 2015 based on the single herbarium specimen prepared from cultivated plant and only one locality is known in nature to the date [Kalimantanorchis Tsukaya was described in 2011 based on the single discovered plant [Bulbophyllum nocturnum J.J.Verm., de Vogel, Schuit. & A.Vogel was described based on the three herbarium specimens, nevertheless these were collected from the single cultivated plant collected a few years ago in the wild [Dracula deniseana Luer, Dracula saulii Luer & Sijm, Dracula vinacea Luer & R. Escobar, Lepanthes cordeliae Luer, Lepanthes persimilis Luer & Sijm, Masdevallia princeps Luer, Porroglossum tripollex Luer and many others).Only two flowering individuals of equently –46. Thisthe date . The gened plant . Bulbophthe wild  and manythe wild . AnotherCleisostoma yersinii is most closely related to C. birmanicum, which occurs from Myanmar through Thailand and Vietnam to Chinese Hainan [C. yersinii is clearly derived from that of C. birmanicum. Taxonomically-informative characters of both species are summarized in e Hainan ,50. BothC. yersinii is unique among all members of its genus. The function of a spur in directing pollinators is substituted by narrow openings of two nectar sacks that are formed by large lip calli and a septum, and located at the base of a shallow spur. In addition, there is only a single short tail (arista) at the tip of the flower lip of C. yersinii while two distinctly longer tails are present in C. birmanicum. The newly described orchid shares with C. birmanicum the three-lobed lip, but the lip of C. yersinii is much more complex and bears prominent calli that allow a floral spur to be reduced.Shape of the lip Figs  and leafC. birmanicum, we classify C. yersinii in sect. Echioglossum. Other species belonging to this section are C. javanicum (Blume) Garay, C. minax (Rchb.f.) Seidenf. and C. striatum. The placement of C. yersinii to sect. Echioglossum was clearly supported also by nuclear and plastid sequence data: the newly described species formed a well-supported clade with C. birmanicum  SE Asian sections (building on ) and  Benth. ex Rolfe [Microcoelia macrantha (H.Perrier) Summerh. [Dendrophylax and Microcoelia belong to the subtribe Angraecinae [C. yersinii belongs to the subtribe Aeridinae, indicating that ∩-thickened cell walls in roots may be more widely distributed in the tribe.The majority of investigated anatomical characters of ex Rolfe  and MicrSummerh. . Both Deraecinae  while C.Echioglossum had stomata on both sides of leaves, while in C. paniculatum (sect. Paniculatum) and C. racemiferum (sect. Cleisostoma) stomata were present on abaxial side only. Hypostomatic species clearly prevail over the amphistomatic ones in the Aeridinae [Echioglossum.Another interesting observation concerns the stomatal density of leaves. The two examined species of sect. eridinae ,56. Howeeridinae . More spC. yersinii were found growing as terrestrial on bare mineral soil exposed by road building  grew there in sympatry. Primary habitats of the new orchid species are most likely trunks of trees; a single epiphytic plant was recorded close to locus classicus in 2014.Several plants of ing Figs  and 2. Hs cortex . A well-C. yersinii as “Data Deficient” according to the IUCN Red List categories [The species is known only from the type locality and a single plant observed two years later close to the original site. This could indicate its rarity although new localities will likely be found in the little explored and hardly accessible Hon Ba mountains. At this stage of investigation, we regard tegories .C. yersinii is a narrow endemic of the Hon Ba Nature Reserve. In such a case, the species would qualify as “Threatened” because Hon Ba forests are under high pressure through illegal logging.The Hon Ba Mts. is isolated from most other mountain ranges by quite extensive lowlands. The only mountains connected by more or less continuous mountain ridge are the Da Lat and the Hon Giao areas, both of which have been studied relatively intensively in the past, with no records of the newly described species. This may indicate that The lack of fruits from flowers not subjected to hand-pollination under the greenhouse conditions suggests allogamy. This assumption is supported by relatively large and colorful flowers with complicated morphology that would unlikely evolved without interactions with pollinators .C. yersinii, is common in Orchidaceae, although raffinose and some other minority sugars have also been found in other orchid species [C. yersinii in concentrations under our detection limit.Sugar-rich nectar with dominant sucrose and lower amounts of fructose and glucose, as detected in  species . HoweverC. yersinii most likely originated by spontaneous or plant-induced hydrolysis of sucrose, as previously reported in other plants [C. yersinii that have relatively wide, suborbicular nectar sacs.Nectar composition is known to be affected by pollinator feeding ,61, eithr plants . In somer plants . It is, C. yersinii. Butterflies are also unlikely to pollinate the flowers because of a massive and wide viscidium. The most likely pollinators of the newly recognized species seem to be bees. This group of insects prefers nectar with dominant sucrose whereas flies and several Hymenoptera prefer hexoses-dominant nectar [see Apis and Bombus) are known to favor more concentrated nectar [C. yersinii. Field observations are, however, necessary to conclusively determine the identity of pollinators.As indicated above, variation in nectar sugar composition may be very high under natural conditions ,68–70, od nectar , providiS1 FigScale bars 1 mm. A specimen cultivated in the Prague Botanical Garden collected as holotype. Photo J. Ponert.(TIF)Click here for additional data file.S2 FigPrimary submontane evergreen forest at elevation about 1500 m a.s.l. Photo T.B.Wuong.(TIF)Click here for additional data file.S3 FigThe first dominant peak corresponds to sucrose while the second and the third minority peaks correspond to glucose and fructose, respectively.(TIF)Click here for additional data file.S1 TableCleistostoma species.Accession numbers in the living collection of the Prague Botanical Garden and original localities are provided for the newly sequenced (DOC)Click here for additional data file.


Mycobacterium abscessus subsp. massiliense, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from Polybia dimorpha Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different M. abscessus subsp. massiliense strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with M. abscessus subsp. massiliense induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the M. abscessus subsp. massiliense cell wall and reduce 40–90% of the bacterial load both in vitro and in vivo. The presented results encourage further studies on the use of Polydim-I as one of the components for M. abscessus subsp. massiliense treatment. Mycobacterium abscessus subsp. massiliense is a rapid growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases and is associated with soft tissue infections that are caused by contaminated hospital equipment and solutions following small invasive procedures or intramuscular injections olydim-I . De novoThus, Polydim-I was synthesized at Aminotech Development and Technology LTDA. The purity (> 99%) and the correct sequence of the peptide were verified and confirmed through mass spectrometry upon receipt using the same parameters that were used for the natural compound.M. abscessus subsp. massiliense from an epidemic outbreak and two reference strains resulting in a MIC of 60.8 μg/mL, while treatment at a concentration of 15.2 μg/mL inhibited 55 to 68% of M. abscessus subsp. massiliense strains growth (data not shown). To determine whether the mycobactericidal effect of Polydim-I could influence the shape of the cell envelope, scanning electron microscopy (SEM) of M. abscessus subsp. massiliense GO 06 exposed to the peptide was performed. As shown in M. abscessus subsp. massiliense to the antibiotic CLR, which prevents bacterial growth through the inhibition of protein synthesis, did not result in visible morphological changes  and treated with 7.6 μg/mL of Polydim-I. All tested strains behaved similarly, and as shown in M. abscessus subsp. massiliense isolate GO 06 shown in To further investigate whether a combination of mycobacteria infection and peptide treatment could affect the membrane integrity of peritoneal macrophages, these cells were infected with six different As reasoned above, Polydim-I activity might be associated with the disruption of cell membrane integrity. Nonetheless, to be a potential candidate as an antimicrobial agent, this compound must show selective toxicity (12). Consequently, we analyzed whether Polydim-I interferes with eukaryotic cell membranes using J774 macrophage cell line treated with 7.6, 15.2, 60.8, and 121.6 μg/mL of Polydim-I for 24 hours using LDH release assay. Polydim-I started to present cytotoxicity on J774 cells at concentrations above 121.6 μg/mL (10%) . HoweverM. abscessus subsp. massiliense strain GO 06 (106 CFU) provided an adequate model of infection with the bacterial load maintained until 30 days post infection [in vivo, IFN-γKO mice, that have increased susceptibility to mycobacterial infection, were infected with M. abscessus subsp. massiliense. Eighteen days post infection, these animals were treated for 8 days with CLR (200 mg/kg/mLW) or one of three different doses of Polydim-I  for 8 days. M. abscessus subsp. massiliense and the different treatment schemes. As shown, Polydim-I treatment at 2 mg/kg/mLW showed significant reduction of the bacterial load in all analyzed organs. The peptide treatment at this concentration reduced the bacillary load of the susceptible animals by up to 90% in the lungs and spleen . The in vivo selective action presented by Polydim-I could be due to the presence of two arginine residues present in its N-terminus. These results are promising because for other wasp AMPs, particularly mastoparans such as MP-AF, MP-A, MP-B, MP-D, MP-M, and MP-V, high cell toxicity through hemolysis was observed [Macrophages are the main targets of mycobacteria and are morphologically and functionally well characterized; hence, these cells were used in cytotoxicity assays (J774 macrophages) and to evaluate ic cells , but theic cells , and thiic cells . In addiic cells , 35 formic cells . In contobserved ; howeverM. abscessus subsp. massiliensi, previously known as M. massiliense [Treatment of infected macrophages with 7.6 μg/mL of Polydim-I decreased approximately 50% of the bacterial load. Although few studies have used infected macrophages to test new drugs against mycobacteria , no sciesiliense . We hyposiliense  in ordersiliense . TherefoM. abscessus subsp. massiliense, we used the mouse model of Lenaerts and colleagues [M. tuberculosis. IFN-γKO mice are highly susceptible to mycobacteria infections [M. abscessus subsp. massiliense [M. abscessus subsp. massiliense using 2 mg/kg/mLW of Polydim-I substantially reduced the bacterial load in the lungs, spleen and livers  and oral macrolides (clarithromycin and azithromycin). Consequently, the ATS/IDSA (American Thoracic Society/Infectious Disease Society of America) recommends that at least two parental drugs and one oral drug should be used to treat infections to avoid drug resistance development [M. abscessus subsp. massiliense infections because of the lower risk of resistance development and the lower cost of production.elopment . These delopment , and theelopment . Novel din vivo. Additionally, peptides could combine to host proteins and generate specific humoral immune responses that could impair repetitive use of the peptide [Some important aspects about the use of AMPs in general should be pointed out as for example they could be degraded faster than other anti-microbial agents due to the action of tissue peptidases/proteinases  peptide –49.M. abscessus subsp. massiliense cell wall and reduced the bacterial load both in vitro and in vivo. These results encourage further studies on the use of Polydim-I and its derivative peptides as a component in the M. abscessus subsp. massiliense infection treatment formulation.In conclusion, this study was the first to demonstrate the therapeutic potential of a peptide derived from wasp venom to treat mycobacteria infections. Polydim-I acts on the S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 FigMycobacterium abscessus subsp. massiliense isolate GO 06 (MOI 10:1); and (D) macrophages infected with M. abscessus subsp. massiliense isolate GO 06 and treated with Polydim-I peptide.Macrophage morphology after Polydim-I treatment and infection. Instant-Prov staining of: (A) Untreated macrophages; (B) Polydim-I peptide-treated macrophages; (C) macrophages infected with (TIF)Click here for additional data file.S6 FigBALB/c mice were treated with 2 mg/kg/mLW (C and D) or 20 mg/kg/mLW (E and F) and 48 hours later the livers were processed and stained with HE. As controls, mice were treated with PBS (A and B). The results are presented at 40 x and 100 x magnification.(TIF)Click here for additional data file.


Brassica rapa and B. oleracea, characterization of mTEs and building a curated database are prerequisite to extending their utilization for genomics and applied fields in Brassica crops.MITE, TRIM and SINEs are miniature form transposable elements (mTEs) that are ubiquitous and dispersed throughout entire plant genomes. Tens of thousands of members cause insertion polymorphism at both the inter- and intra- species level. Therefore, mTEs are valuable targets and resources for development of markers that can be utilized for breeding, genetic diversity and genome evolution studies. Taking advantage of the completely sequenced genomes of Brassica species. At present, BrassicaTED has datasets for 41 mTE families, including 5894 and 6026 members from 20 MITE families, 1393 and 1639 members from 5 TRIM families, 1270 and 2364 members from 16 SINE families in B. rapa and B. oleracea, respectively. BrassicaTED offers different sections to browse structural and positional characteristics for every mTE family. In addition, we have added data on 289 MITE insertion polymorphisms from a survey of seven Brassica relatives. Genes with internal mTE insertions are shown with detailed gene annotation and microarray-based comparative gene expression data in comparison with their paralogs in the triplicated B. rapa genome. This database also includes a novel tool, K BLAST (Karyotype BLAST), for clear visualization of the locations for each member in the B. rapa and B. oleracea pseudo-genome sequences.We have developed BrassicaTED as a unique web portal containing detailed characterization information for mTEs of B. rapa and B. oleracea. The database will promote the development of desirable mTE-based markers, which can be utilized for genomics and breeding in Brassica species. BrassicaTED will be a valuable repository for scientists and breeders, promoting efficient research on Brassica species. BrassicaTED can be accessed at http://im-crop.snu.ac.kr/BrassicaTED/index.php.BrassicaTED is a newly developed database of information regarding the characteristics and potential utility of mTEs including MITE, TRIM and SINEs in  B. rapa genome are occupied by transposons[trans-activation by long and complete counterpart autonomous TEs[Transposable elements (TEs) are ubiquitous and compose a large fraction of most of eukaryotic genomes. For example, more than 80% of the maize genome and 40% nsposons. TEs cannsposons-5. Althonsposons. Miniatumous TEs,7-9. Themous TEs familiesBrassica genome[Brassica[TRIMs are derivatives of retrotransposons from which the coding domain for transposition has been lost. TRIMs have terminal direct repeats (TDRs) (100–250 bp) flanked by 5-bp target site duplications (TSDs) and contain an internal sequence that begins with the signature region of the primer binding site (PBS) of tRNA-methionine and ends with a polypurine tract (PPT) motif,11,12. Sa genome,11,13. TShort interspersed elements (SINEs) are non-autonomous, non-LTR retrotransposons that are transcribed by RNA polymerase III and widely distributed in eukaryotic organisms,14. SINEStowaway superfamily members recognize 2-bp (TA) nucleotides and duplicate these at their terminal region via insertion. Similarly, Tourist superfamily members produce unique TAA duplications, hAT superfamily members produce 5-, 6-, or 8-bp TSD, Mutator superfamily members produce 9- or 10-bp TSD, and En/Spm superfamily members produce 3-bp non-unique TSD[MITEs are small, non-autonomous class II DNA transposons that are AT-rich and have DNA structures characteristic of TEs, like terminal inverted repeats (TIRs) and TSD of variable lengths. Based on their TSD, most MITEs have been classified into several superfamilies. ique TSD. Availabique TSD.mTEs are stably maintained and highly dispersed throughout the genome, often in association with genic regions, where they can alter gene structure and function,23. InseBrassica (family Brassicaceae) is composed of more than 3700 species including important vegetable and oil-seed crops such as kimchi-cabbage, cabbage, and oilseed rape [B. rapa was the first Brassica vegetable to be sequenced and annotated, revealing recent genome triplication at 13–17 MYA, after divergence from Arabidopsis thaliana 20 MYA[A. thaliana genome is occupied by TEs[B. rapa and B. oleracea genome is derived from TEs[Brassica genome[B. rapa were identified using in silico MITE identification tools[Brassica species have not been developed or included in any databases. Furthermore, there is no resource with information regarding the distribution of TRIM, SINE and MITE families in various genomic regions for the Brassica species. Therefore, we aimed to establish a unique web portal containing detailed characterization of TEs in Brassica species. As a starting point, we have investigated the MITE, TRIM and SINE families of B. rapa and B. oleracea at the whole-genome scale and developed a database, BrassicaTED (Brassica Transposable Elements Database), for effective utilization of MITE, TRIM and SINEs in the Brassica genome.The genus . napus). B. rapad by TEs,37, arouon tools. HoweverB. rapa and B. oleracea genomes. This database has three-tier architecture, namely a client tier, a middle tier and a database tier  and gene annotation information (version 1.2) were obtained from the publically available database[Brassica[B. rapa pseudo-chromosome sequences. The physical position of each MITE on the B. rapa pseudo-chromosome was described using a custom Perl script. A total of 5894 MITEs (Br-members) were recently identified in the B. rapa genome[B. rapa genome.database. We inclaffolds 23 Mb and [Brassica,43-45. CB. oleracea pseudo-chromosome sequences (version 1.0)[B. oleracea genome. Similar to B. rapa, a total of 6026 members (Bo-Members) were annotated based on physical position and gene annotation information for B. oleracea[B. oleracea[The recent availability of 385 Mb of ion 1.0),47 allow oleracea. This wo oleracea.B. rapa BAC end sequences and 434 Mb of B. oleracea shotgun sequences,[B. rapa and B. oleracea[B. rapa and B. oleracea. In total, 1393 and 1639 copies from the five TRIM families were identified from B. rapa and B. oleracea whole-genome pseudo-chromosome sequences, respectively  and 656 (40%) members reside in or within 2 kb of a gene in the B. rapa and B. oleracea genome, respectively. This information will be highly valuable for marker development and characterization of target regions that have TRIM element insertion.In a previous study, we characterized four TRIM families  and their members in 96 Mb of quences,. Now, we oleracea. TRIM elB. rapa and B. oleracea genomes[B. rapa and B. oleracea, respectively, were present in close association with genic regions (with < 2 kb of a gene) , salt (250 mM NaCl), drought (air-dry) and ABA (100 μM)[BrassicaTED includes information regarding paralogous genes in database. Single,(100 μM).This section includes tools for basic bioinformatics analysis to visualize or map the query sequence onto the chromosome region and perform similarity searches. A sequence extractor tool is also provided to extract target sequences for further analysis, such as for MIP primer development or related gene structure identification Figure .B. rapa and B. oleracea. We have also included A. thaliana genome sequence for use in K BLAST.We developed a new tool, which we call K BLAST (Karyotype BLAST), to map the unique query sequence (s) onto homologous chromosomal regions and visualize the resulting distribution pattern. Users can visualize the locations of many different query sequences on each chromosome in different colors using K BLAST. It is a straightforward approach to inspect the distribution or dispersion of any DNA sequences (e.g. genes or repeats) on the chromosomes of B. rapa, B. oleracea, and A. thaliana.The tool panel offers the standard NCBI BLAST program for sequence similarity searches against the genome sequences. Users can match nucleotide or protein sequence (s) against database sequences such as whole-genome sequences of B. rapa and B. oleracea. This tool also provides an optional input box to add flanking region length for input start and end positions.We incorporated a sequence extractor tool using Python script. This can be utilized by entering single or multiple start and end positions in column tab-delimited format in the input query box for specific regions on the selected chromosome of Brassica genome. We will keep adding newly identified mTE elements to our database.BrassicaTED provides three sections  with user-friendly navigation. First, there are three parts of the Browse section: one each for MITEs, TRIMs and SINEs. Users can choose any MITE family in the list box and click ‘submit’ to enter the area pertaining to that family  are valuable targets for genomics and phylogenetic studies. Comprehensive analysis and tools are desirable for effective utilization of mTEs for molecular studies, and were lacking. We developed BrassicaTED web as an interface for extensive characterization of MITEs, TRIM and SINEs in the Brassica genomes contain triplicated homologous counterparts relative to the genome of A. thaliana[. Recent investigation of BraSto-2 suggested that MITEs may have important roles in triplicated Brassica genome evolution[ thaliana,50. Recevolution. Brassicvolution,30,44. mBrassica genome and >50-85% of them are related to genic regions. The accumulated IP marker information from 289 MITE targets  [B. rapa and A. thaliana. Though recently reported database for plant MITEs, P-MITE, has data for MITE families from 41 plant species, it does not provide specific information regarding MITE insertion positions in the B. rapa and B. oleracea genomes. By contrast, BrassicaTED is built to establish an interactive web platform for all types of mTEs and includes in-depth analysis of 41 mTE families in B. rapa and B. oleracea, as well as in A. thaliana. In addition, BrassicaTED provides tools to extract gene sequences and compare the expression of paralogous genes using the 24 k microarray datasets.In sum, we have characterized members of 41 mTE families using the whole-genome pseudo-chromosome sequences of ome data. SimilarBolbase) was desiBrassica species, especially from B. rapa and B. oleracea. This will be an important repository to promote the utilization of mTEs and the elucidation of the effects of mTEs on genome evolution in Brassica species. Unlike other publically available Brassica databases, BrassicaTED has a unique user-friendly visualization tool (K-BLAST) and a microarray expression data comparison tool for B. rapa. BrassicaTED will be a valuable storehouse for scientists and breeders who work on Brassica species and will be continuously updated as more data is uncovered.BrassicaTED is a new database exclusively made with mTEs of http://im-crop.snu.ac.kr/BrassicaTED/index.php. The database is optimized for Internet Explorer, Mozilla Firefox, Google Chrome and Safari.Datasets in BrassicaTED are freely accessible for research purposes for non-profit and academic organizations at The authors declare that they have no competing interests.PS and TJY developed the methodology and conducted the study. JM, PS, SCL and BSC participated in the database and bioinformatics tool development. JM, PS, NS, SL and TJY drafted the manuscript, which was revised by all authors. All authors read and approved the final manuscript.


Professional mononuclear phagocytes such as polymorphonuclear neutrophils (PMN), monocytes, and macrophages are considered as the first line of defence against invasive pathogens. The formation of extracellular traps (ETs) by activated mononuclear phagocytes is meanwhile well accepted as an effector mechanism of the early host innate immune response acting against microbial infections. Recent investigations showed evidence that ETosis is a widely spread effector mechanism in vertebrates and invertebrates being utilized to entrap and kill bacteria, fungi, viruses, and protozoan parasites. ETs are released in response to intact protozoan parasites or to parasite-specific antigens in a controlled cell death process. Released ETs consist of nuclear DNA as backbone adorned with histones, antimicrobial peptides, and phagocyte-specific granular enzymes thereby producing a sticky extracellular matrix capable of entrapping and killing pathogens. This review summarizes recent data on protozoa-induced ETosis. Special attention will be given to molecular mechanisms of protozoa-induced ETosis and on its consequences for the parasites successful reproduction and life cycle accomplishment. Professional mononuclear phagocytes, such as polymorphonuclear neutrophils (PMN), monocytes, and macrophages, are considered as the first line of defence of the early host innate immune response , 2. Theiα, IL-1β, CC, and CXC chemokines  . Us. Us Eimeructures . Meanwhi Figures  24, 27], 27 Eime species , 24. Giv species , 109, itay occur , 23, 24.icropyle , 24. Theructures . So far, Eimeria-induced NETosis is not entirely understood, so far. Enzyme activity measurements and inhibition studies revealed a key role of NOX, NE, and MPO in Eimeria-triggered NET formation   Besnoiti B. besnoiti tachyzoites mainly proliferate within endothelial cells during the acute phase, these parasitic stages are released via cell lysis in close proximity to endothelium and are exposed to blood contents, such as leukocytes. Several reports have shown that NETs themselves interact with endothelium and may cause endothelial damage or dysfunction [ B. besnoiti tachyzoites, and NETs are quite likely. Accordingly, Maksimov et al. [Given thatfunction –131. Sinfunction , 132, inv et al.  recently Neospora caninum have shown that bovine PMN exposed to viable tachyzoites also result in strong NETosis .Recent NET-related investigations on the closely related cyst-forming apicomplexan protozoa NETosis . With reCryptosporidium parvum is an euryxenous apicomplexan parasite with worldwide distribution and high zoonotic potential, mainly affecting young children, immunocompromised humans, and neonatal livestock [ in vivo [ C. parvum stages [ in vivo infection. In vitro infection experiments additionally showed the negative impact of NETs on host cell invasion since infection rates were significantly reduced when using PMN-preexposed C. parvum stages [ Eimeria-induced NETosis, inhibition experiments revealed C. parvum-triggered NET formation as dependent on intracellular Ca2+ release and ERK 1/2 and p38 MAPK-mediated signalling pathways [ C. parvum sporozoite-exposed bovine PMN showed increased gene transcription of proinflammatory molecules, some of which were recently shown as potent NET inducers  [ivestock . Typicalivestock , 134. Afivestock . Recent  in vivo , 137. Mum stages . Parasitm stages . Given tm stages , 139, thm stages . The facm stages . The colm stages . In agrepathways . Interesd TNF-α) , 141 and Leishmania spp. represent a major health problem and according to the WHO [ Leishmania species [ Leishmania species. Guimarães-Costa et al. [ Leishmania amazonensis, L. major, and L. chagasi were capable of triggering NET formation. Additionally, Leishmania-triggered NETosis seems not entirely stage-specific, since both promastigotes  and amastigotes  promoted NET formation in vitro and in vivo [ Leishmania-mediated NETosis. Thus, Leishmania-derived lipophosphoglycans (LPG) were suggested as the main trigger of NET release since these molecules also induced NETs in a purified form. The former authors showed that NETs possessed detrimental effects on parasites as NET-entrapped L. amazonensis promastigotes exhibited decreased viability [ L. amazonensis, L. major, L. braziliensis, and L. mexicana.Infections with the WHO  10% of t the WHO , 143. Le species –144. Par species –149 and a et al.  showed f in vivo , 150–152 in vivo  providediability . Authorsiability . The leiiability  demonstr L. donovani, Gabriel et al. [ Leishmania-lipophosphoglycan- (LPG-) dependent NET induction reported by Guimarães-Costa et al. [ L. donovani. When using genetically modified L. donovani promastigotes Gabriel et al. [ L. donovani maintained its viability in the presence of NETs, whilst mutant parasites lacking LPG were efficiently killed by these extracellular structures [In case ofl et al.  reporteda et al.  was not l et al.  observedructures . Leishmania parasites trigger not only the classical ROS-dependent NETosis as previously demonstrated but also a ROS-independent form, named as early/rapid vital NETosis [ Leishmania-triggered NETosis, in which NET formation takes place after 5–15 min of activation without affecting PMN viability [A more recent study revealed that NETosis . During iability , 68, the Leishmania spp. seem capable of evading NET killing by firstly blocking the oxidative burst activity of PMN or even by resisting microbicidal activity of NETs [ L. infantum promastigotes express the enzyme 3′-nucleotidase/nuclease which was previously described to be involved in parasite nutrition and infection and was proven to be part of the ability of promastigotes to escape NET-mediated killing. A recent investigation has shown that a salivary component of the sand fly insect that transmits leishmaniasis may also play a role in the survival of Leishmania in the definitive hosts, by modulating their innate immune system. A molecule named Lundep from the salivary gland of Lutzomyia longipalpis was recently described as an endonuclease with NET-destroying properties in humans [ L. major survival rates [ L. major infection.Regarding NET-related evasion strategies of Trypanosomatidae parasites, of NETs , 150. Mo of NETs  showed tn humans . In the al rates . Furtheral rates  measuredal rates . Lundep al rates . In conc Trypanosoma cruzi. Approximately eight million people are affected by this tropical disease in the Americas and an average of 12,000 deaths per year is known to occur due to American trypanosomiasis [ in vitro that T. cruzi is able to trigger NETs in a dose-, time-, and ROS-dependent manner. In agreement with reports on Eimeria spp. and B. besnoiti [ T. gondii and Leishmania spp. [ T. cruzi stages was not affected by NETs, but NETosis significantly impaired the parasite host cell infectivity. In fact, NETs components as NE may affect T. cruzi infectivity, since this enzyme appears to be involved in increased trypanocidal activity and in the reduction of trypomastigote release by prestimulated infected macrophages [ T. cruzi-triggered NETosis is a TLR2- and TLR4-dependent process. Moreover, the study showed that not only viable T. cruzi trypomastigote forms but also soluble antigens and killed T. cruzi parasites induced NET release in human PMN. In vivo murine studies indicated the relevance of NETosis for the outcome of trypanosomiasis since significantly decreased parasites numbers were found in the blood system of those animals which had previously been infected with NETs-pretreated parasites [American trypanosomiasis or Chagas disease is caused by the protozoan parasitesomiasis . It is wsomiasis , 155. Resomiasis  demonstrbesnoiti , 24, 59 nia spp. , 51, therophages , 156. Adarasites . in vivo data have now proven the existence and importance of this early host innate effector mechanism. However, there is still a total lack of information on parasite-derived ligands triggering ETosis. Taking into account that in most cases ET formation is considered as a species- and stage-independent process, rather ubiquitary occurring molecules may represent parasite-derived target molecules of ETs. Moreover, recent data revealed that other leukocytes such as monocytes, macrophages, basophils, mast cells, and eosinophils also perform ETosis upon pathogen encounter. However, respective data on parasite-induced mechanism are scarce. Furthermore, ET-related research mainly focused on the leukocytes aptitude to impact the parasites life cycle, but not on the propensity of parasitic stages to develop counter mechanisms for ETs avoidance. While a bunch of data is available on bacterial nucleases or other counter mechanisms, parasites have been neglected on this topic. Taken together, we call for more parasite-related studies in the exciting field of ETosis.During the last years a vast amount of data on protozoan-mediated ETosis was published strengthening the role of this effector mechanism in the defence of parasitic infections. Several


Vulpes vulpes) is a widespread species, harbouring many pathogens relevant for humans and pets. Indeed, Anaplasma spp., Ehrlichia canis and Rickettsia spp. among the bacteria and Hepatozoon canis as well as Babesia sp. among the parasites have been the focus of several studies.The red fox (Babesia microti-like pathogens were found in 50%, while H. canis was detected in 58.3% of the samples. The spleen was more useful for detection of H. canis, whereas B. microti-like parasites were more frequently found in the blood. Bacteria could not be confirmed in any of the cases to demonstrate the occurrence of such tick-borne pathogens using PCR and sequencing on blood and spleen samples.In a cohort of 36 foxes shot on one day in the north-eastern part of Austria, B. microti-like and H. canis parasites raised many questions, because these infections have never been found autochthonously in dogs. Furthermore in the case of H. canis the main vector tick, Rhipicephalus sanguineus, is absent in the sampling area, leaving space for further hypotheses for transmission such as vertical transmission, transmission via ingestion of prey animals or other vector ticks. Further studies are needed to evaluate the risks for pets in this area. PCRs delivered differing results with the different tissues, suggesting the use of both spleen and blood to obtain an integral result.The occurrence of  Vulpes vulpes) are among the most widely distributed mammals in the world and are invading many urban areas due to a good adaptation to human environments, and to rabies vaccination [Anaplasma phagocytophilum [Hepatozoon canis [Babesia sp. [Ehrlichia canis [Rickettsia spp. [Red foxes , Ehrlichia canis, Hepatozoon canis and Rickettsia sp. were used (Table http://genedoc.software.informer.com/2.7/) and blasted on GenBank® (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Foxes shot on 18 January 2014 in the district of Gänserndorf (in the northeast of Lower Austria) were further processed on the same day. From the 36 foxes, 35 spleen samples and 17 blood samples were obtained. Extraction of DNA from blood and tissue was performed as previously described [Fox were shot during routine hunting events under the restrictions of the game laws of the province of Lower Austria.B. microti-like pathogen-positive foxes, 21 foxes harbouring H. canis and four foxes with double infections (Table Babesia sp. nested) in blood and spleen detected 13 (76.5% of the blood samples) and 11 (31.4% of the spleens) B. microti-like pathogens, respectively. Sequences of these pathogens showed 98–100% similarity to B. sp. “Spanish dog” (e.g. GenBank® accession no. AF188001.1 or EU583387.1). Using the Hepatozoon-specific primers, 21 foxes tested positive for H. canis. The investigation of the spleen samples identified 18 positive results (51.4%), whereas in the blood samples only six positive results (35.3%) were found. Seven more PCR products, positive on the gel, provided no conclusive sequence data, and therefore were noted as false positives. All conclusive sequences delivered 99–100% similarity to H. canis found in GenBank® .The investigation of the blood and spleen samples identified 18 Anaplasma sp., E. canis or Rickettsia spp. be detected.In none of the blood or spleen samples could Babesia microti-like parasites [Foxes are known to be major reservoirs for arasites . The higarasites , Portugaarasites  and Spaiarasites .H. canis foxes in Austria are in concordance with four positive foxes out of nine found in Slovakia [H. canis is not found endemically in dogs in these areas, nor is R. sanguineus known to occur autochthonously [H. canis has already been found in dogs in areas lacking the main vector tick in Germany [The 58.3% positive Slovakia , 45.2% iSlovakia , 16 out Slovakia  or 8% inSlovakia . To datehonously ,19,21,23 Germany ,20 and H Germany ,22.Babesia microti-like pathogens and H. canis could be found in a relatively small set of fox samples originating from north-eastern Austria. Especially, the occurrence of H. canis in considerable numbers in this population so far north raises many questions such as the potential impact on domestic animals, reservoirs and infection pathways. Moreover, the main vector tick, Rhipicephalus sanguineus, is absent in the sampling area. Therefore other transmission pathways such as vertical transmission, transmission via ingestion of preyed animals or other vector ticks need to be evaluated.Foxes represent a good reservoir for several zoonotic and pet-relevant diseases. In terms of blood parasites this seems more the rule than the exception. Human- and pet-relevant agents such as B. microti-like as well as H. canis pathogens have to be recognized as an unnoticed threat in northern areas. The use of piroplasm PCRs could help to identify both B. microti-like and H. canis pathogens prior to screening, followed by PCRs with species-specific primers.Thus foxes have to be considered during treatment strategies and


Mesorhizobium ciceri bv. biserrulae strain WSM1271T was isolated from root nodules of the pasture legume Biserrula pelecinus growing in the Mediterranean basin. Previous studies have shown this aerobic, motile, Gram negative, non-spore-forming rod preferably nodulates B. pelecinus – a legume with many beneficial agronomic attributes for sustainable agriculture in Australia. We describe the genome of Mesorhizobium ciceri bv. biserrulae strain WSM1271T consisting of a 6,264,489 bp chromosome and a 425,539 bp plasmid that together encode 6,470 protein-coding genes and 61 RNA-only encoding genes. The productivity of sustainable agriculture around the world is heavily dependent on the provision of bioavailable nitrogen (N) . The demIn contrast, the demand for N by leguminous plants can be sustainably met through the biological process of N fixation that occurs following the successful formation of an effective symbiosis. This symbiotic nitrogen fixation (SNF) process can account for approximately 70% of the bioavailable nitrogen supplied to legumes .Biserrula pelecinus L., which is an annual herbaceous legume native to the Mediterranean basin that was introduced into Australian soil in 1994 [Mesorhizobium ciceri bv. biserrulae type strain WSM1271T (= LMG23838 = HAMBI2942) [B. pelecinus L [in situ evolution of a diverse range of competitive strains capable of nodulating B. pelecinus L. compromised optimal N2-fixation with this host. This rapid emergence of less effective strains threatens the establishment of this legume species in the Australian agricultural setting. The sub-optimal strains appear to have evolved from indigenous mesorhizobia that acquired the island of genes associated with symbiosis from the original inoculant, WSM1271T, following a horizontal gene transfer event [One legume that has many beneficial agronomic attributes is  in 1994 . The ben in 1994 ,3. This MBI2942) . Australer event .M. ciceri bv. biserrulae strain WSM1271T are presented together with the description of the complete genome sequence and its annotation.In this report, a summary classification and a set of general features for M. ciceri strain WSM1271T is a motile, Gram-negative, non-spore-forming rod  broth [ming rod  in the oM. ciceri bv. biserrulae strain WSM1271T in a 16S rRNA sequence based tree. This strain clustered in a tight group, which included M. australicum, M. ciceri, M. loti and M. shangrilense and had >99% sequence identity with all four type strains. Our polyphasic taxonomic study indicates that WSM1271T is a new biovar of nodulating bacteria [The organism tolerates a pH range between 5.5 and 9.0. Carbon source utilization and fatty acid profiles have been described before . MinimumM. ciceri bv. biserrulae strain WSM1271T has an extremely narrow legume host range for symbiosis only forming highly effective nitrogen-fixing root nodules on Biserrula pelecinus.L. This strain also nodulates the closely related species Astragalus membranaceus but does not nodulate 21 other legume species nodulated by Mesorhizobium spp [B. pelecinus L. growing in undisturbed landscapes in the Mediterranean basin, and is an important example of a highly specific legume host-root nodule bacteria relationship in an annual herbaceous legume used as a forage species in agriculture.bium spp . The higThe Joint Genome Institute (JGI) operated by US Department of Energy (DOE) sequenced, finished and annotated WSM1271 as part of the Community Sequencing Program (CSP). The genome project is deposited in the Genomes OnLine Database . The finM. ciceri bv. biserrulae strain WSM1271T was grown to mid logarithmic phase in TY rich medium [h medium  on a gyrh medium .M. ciceri bv. biserrulae WSM1271T using a combination of Illumina [The Joint Genome Institute (JGI) generated the draft genome of Illumina  and 454 Illumina . The seqIllumina . The iniIllumina , and theIllumina  was usedIllumina , or sequGenes were identified using Prodigal  as part The genome is 6,690,028 bp long with a 62.56% GC content  and comp


The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem.To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization. As the cost of sequencing has dropped, the number of sequencing projects available in the GOLD database  has incrSingle-molecule sequencing is a challenging problem that has not, until recently, resulted in a commercial product. Released in 2011, the PacBio RS is the only long-read, single-molecule sequencer currently available. In contrast to competing nanopore approaches -11, the Escherichia coli K12 MG1655. This genome can only be assembled into a single contig when the read length exceeds the size of the longest repeat in the genome, a multi-copy rDNA operon. The rDNA operon, sized around 5 to 7 kbp, is the largest repeat class in most bacteria and archaea  is available. Reference-based validation was performed using the GAGE scripts and metrics [E. coli O157:H7 the reference E. coli O157:H7 Sakai [GenBank:NC_002127], [GenBank:NC_002128], [GenBank:NC_002695] was used. For F. tularensis, the reference F. tularensis subsp. Holarctica OSU18 [GenBank:NC_008369] was used. For S. enterica Newport, the reference S. enterica Newport SL254 [GenBank:NC_011079], [GenBank:NC_011080], [GenBank:NC_009140] was used. For S. enterica Newport, Illumina reads and contigs corresponding to two short plasmids (<10 kbp) and the phiX control were removed prior to validation. Reference-free validation was performed with FRCbam, B. trehalosi [PRJNA157929], M. haemolytica [PRJNA212438], S. enterica [PRJNA51643], E. coli O157:H7 [PRJNA63279], and F. tularensis [PRJNA212941]. The Illumina MiSeq E. coli K12 sequencing data are available from the Illumina Scientific Data Website [The sequencing data and assemblies for novel strains generated for this study have been deposited in NCBI under the following accessions:  Website .bp: base pair; CCS: Circular consensus sequencing; CLR: Continuous long read; indel: Insertion/deletion; PBcR: PacBio corrected read; QV: 'Phred’ style consensus quality value; SNP: Single-nucleotide polymorphism.The authors declared that they have no competing interests.SK and AMP conceived the project. SK implemented the algorithms and ran experiments. SK and AMP wrote the manuscript and generated figures. GPH ran assembly experiments and validated sequencing. DR performed manual validation of assemblies. DH, JB, DSM, NHB, and TS isolated strains and provided sequencing. All authors read and approved the final manuscript.Repeat Stats. Listing of repeat count and maximum repeat size for all complete genomes.Click here for fileReport.log.krona. An interactive Krona [ve Krona  chart deClick here for fileValidationResults. Reference-free assembly validation. The columns are as described in Table Click here for file


These bacteria may cause paucisymptomatic bacteremia and endocarditis in humans. Bartonella species are known to cause afebrile bacteremia in humans and other mammals, including B. quintana, the agent of trench fever, and B. henselae, the agent of cat scratch disease. Reports have indicated that animal-associated Bartonella species may cause paucisymptomatic bacteremia and endocarditis in humans. We identified potentially zoonotic strains from 6 Bartonella species in samples from patients who had chronic, subjective symptoms and who reported tick bites. Three strains were B. henselae and 3 were from other animal-associated Bartonella spp. . Genomic analysis of the isolated strains revealed differences from previously sequenced Bartonella strains. Our investigation identifed 3 novel Bartonella spp. strains with human pathogenic potential and showed that Bartonella spp. may be the cause of undifferentiated chronic illness in humans who have been bitten by ticks.Certain  Bartonella spp. cause varied and multifaceted human diseases, including cat scratch disease (B. henselae), Carrion’s disease (B. bacilliformis), trench fever (B. quintana), endocarditis (B. quintana and B. henselae) (B. quintana and B. henselae), and hepatic peliosis (B. henselae). Bartonella spp. can also cause prolonged intra-erythrocytic bacteremia in both humans and animals .In this context, we screened for the presence of During January–June 2013, we conducted a study of a cohort of 66 French patients who had consulted their doctors for chronic symptoms appearing after a tick bite. The entire study protocol was approved by the ethics committee of the Institut Federatif de Recherche 48 under reference >3 months before the study. All patients associated symptom onset with tick bites that occurred during 2008–2012 . At sympWe collected blood samples from each patient in EDTA-containing sample tubes. For a control population, we used anticoagulated blood samples from 70 anonymous healthy blood donors from Paris (France). All samples (control and patients) were tested simultaneously. Bartonella spp., 100 μL of blood samples from patients or healthy donors were directly plated onto sheep blood agar plates and incubated at 35°C in a humidified atmosphere with 5% CO2 for 45 days. The plates were assessed daily from days 7–45 before the culture was deemed negative   default parameters.We extracted genomic DNA from each isolated strain by using the EZ1 automated extraction system , following the manufacturer’s recommendations. Bacterial genomic DNA was sequenced by using the Nextera XT DNA sample prep kit  and a 2×250 paired-end protocol with the MiSeq pyrosequencer (Illumina), according to the manufacturer’s instructions. We aligned each genome by using Mira version 3.2 software in the mapping mode (Bartonella strains by using GGDC software (Single-nucleotide polymorphisms (SNPs) among genomes were identified by using SNIT software , we had access to 2 blood samples that were taken at a 1-month interval. B. henselae was grown from the 2 samples, with similar bacteremia , suggesting chronic bacteremia.Bacteremia in the Bartonella  were classified within the B. henselae species on the basis of both their phylogenetic position and gltA and rpoB sequence similarities  DNA in 96 cases, including 2 co-infected with B. henselae (B. vinsonii berkhoffii (an agent of canine bacteremia and endocarditis) DNA in 24 cases (B. henselae and 2 cases of B. melophagi (http://www.mediterranee-infection.com/article.php?laref=14&titre=collection-de-souches) and are available upon request under references B546, B547, B548, B549, B550, and B551 for isolates MVT01, MVT02, MVT03, MVT04, MVT05, and MVT07, respectively.This work is similar to that of E.B. Breitschwerdt et al. . B. schoenbuchensis is normally found in deer, elk, and cattle  is particularly arduous and may require 6 weeks of doxycycline treatment together with 3 weeks of gentamicin, as these are the only antimicrobial drugs known to be effective in eradication of Bartonella (Bartonella spp. can also cause endocarditis, including B. quintana and B. henselae; therefore, reports of rare cases of endocarditis attributed to zoonotic Bartonella such as B. kohlerae, B. alsatica, Candidatus B. mayotimonensis, B. vinsonii, or B. elizabethae may actually be the final manifestation of asymptomatic bacteremia, similar to that reported by our infected patients (The significance of these Bartonella other than B. quintana in the blood of patients with poorly qualified syndromes. These results indicate that zoonotic Bartonella spp. infection may cause undifferentiated chronic illness in humans.In summary, our major finding is the isolation of zoonotic


Mesorhizobium strain MAFF303099 and M. huakuii. To define underlying causes for the rather striking difference in host specificity between M. huakuii strain 7653R and MAFF303099, several probable determinants also require comparison at the genomic level. An improved understanding of mobile genetic elements that can be integrated into the main chromosomes of Mesorhizobium to form genomic islands would enrich our knowledge of how genome dynamics may contribute to Mesorhizobium evolution in general.Evidence based on genomic sequences is urgently needed to confirm the phylogenetic relationship between Mesorhizobium genomes. Genomes of 7653R and MAFF303099 were found to share a large set of orthologs and, most importantly, a conserved chromosomal backbone and even larger perfectly conserved synteny blocks. We also identified candidate molecular differences responsible for the different host specificities of these two strains. Finally, we reconstructed an ancestral Mesorhizobium genomic island that has evolved into diverse forms in different Mesorhizobium species.In this study, we sequenced the complete genome of 7653R and compared it with five other M. huakuii. Differences in nodulation factors and secretion systems T3SS, T4SS, and T6SS may be responsible for the unique host specificities of 7653R and MAFF303099 strains. The plasmids of 7653R may have arisen by excision of the original genomic island from the 7653R chromosome.Our ortholog and synteny analyses firmly establish MAFF303099 as a strain of The online version of this article (doi: 10.1186/1471-2164-15-440) contains supplementary material, which is available to authorized users. Mesorhizobium, whose growth rate is intermediate between that of genera Rhizobium and Bradyrhizobium, is one of the largest genera; it presently comprises 24 species found primarily in Asia, Europe, the Mediterranean region, and Africa [Mesorhizobium huakuii and M. loti were two of the first species identified in the genus. The first known strain of M. huakuii was isolated from a winter-growing green manure crop, Astragalus sinicus, in Hubei, China in the 1940s by Huakui Chen [Rhizobium huakuii by Wenxin Chen [Rhizobium huakuii was later classified into Mesorhizobium gen. nov. and consequently renamed M. huakuii[M. huakuii is a narrow-host-range rhizobium: it only induces indeterminate-type nitrogen-fixing nodules on the roots of A. sinicus, an economically important forage and green manure crop grown throughout eastern Asia in winter. The M. huakuii strain 7653R has been studied extensively and has been applied in sustainable agriculture for many years [Rhizobia are nitrogen-fixing soil bacteria constituting around 100 known species classified into 13 genera . Mesorhixin Chen . Rhizobi. huakuii. M. huakMesorhizobium strain was M. huakuii bv. loti MAFF303099, initially considered a strain of M. loti[glnA, glnII, and recA) have suggested instead a closer phylogenetic relationship with strains of a different species, M. huakuii, prompting the hypothesis that MAFF303099 is a strain of M. huakuii[M. loti strain R7A by the JGI GEBA project and various research findings related to R7A, such as genomic island mobility [M. huakuii.The first completely sequenced f M. loti. Compara. huakuii. Whole-gmobility , the Nifmobility , and themobility , 15, havM. huakuii, they display drastically different host preferences. Strain 7653R forms specific symbiosis with A. sinicus, whereas MAFF303099 forms determinant-type globular nodules and performs nitrogen fixing on several host plants of Lotus, including L. japonicus and L. corniculatus[Although MAFF303099 and 7653R may both be strains of the same species—niculatus. Our thiM. loti R7A have their nodulation and nitrogen-fixation genes concentrated in a long DNA region called a symbiosis island on their main chromosomes [M. ciceri bv. biserrulae WSM1271 [M. australicum WSM2073 [M. opportunistum WSM2075 [Nodulation and nitrogen-fixation genes show remarkably different genomic locations in different genomes. While MAFF303099 and omosomes , the coromosomes . Interes WSM1271 , M. aust WSM2073 , 20 and  WSM2075 , 21, shoM. huakuii 7653R genome consisted of three circular replicons of 6,364,365 bp (chromosome), 193,835 bp (plasmid pMhu7653Ra), and 323,475 bp (plasmid pMhu7653Rb)  are summarized in Table Our 6,881,675-bp assembly of the We predicted 7,205 protein-coding genes in the 7653R genome, a number essentially identical despite the different genome sizes to the number predicted in MAFF303099 7,281 genes) . 7653R w genes . M. huakuii on the basis of comparative analysis of a few conserved genes in MAFF303099 and M. huakuii strains [MAFF303099 has been hypothesized to be a strain of  strains . The avaMesorhizobium genomes . Of these groups, 3,991 (54%) were found to be conserved across all five genomes, with each group represented by at least one gene in each of the five genomes. We termed this subset of orthologous groups the core genome of Mesorhizobium. An additional 805 (11% of orthologous groups) were observed to be present in four of the five genomes  using a few complementary approaches. For convenience of comparison, we considered dnaA to be the start position of the main chromosome and repABC to be the start position of plasmids in all five genomes. Mauve alignment [Mesorhizobium strains  is needed for the biosynthesis and transport of NFs that induce nodule organogenesis. A total of 21 nodulation genes (2 nol genes and 19 nod genes) were identified in the 7653R chromosome and plasmids, while 33 nodulation genes were located in MAFF303099  were identified in a 140-kb genomic region of the pMhu7653Rb plasmid of 7653R  and 12 nif genes  . Specifically, the 10 35 genes . In sharnod gene nodH, required for NF synthesis in 7653R [nod genes in MAFF303099  had a substantially higher percentage identity (PID) with its ortholog in R7A than with its ortholog in 7653R . Taken together, 7653R and MAFF303099 have very different numbers of nodulation genes. Indeed, different nodulation genes are required for NF synthesis in these two strains. Of the 21 nod genes identified in 7653R, 12  are possibly key elements involved in the synthesis of the core NFs of 7653R [M. loti strain R7A, nod genes organized in seven transcriptional units—noeKJ, nodZnoeLnolK, nodS, nodACIJnolO, nodB, nolL, and nodM—are needed for NF synthesis [Second, the numbers of nodulation genes putatively participating in NF synthesis were found to be different between these two strains. The in 7653R , had no of 7653R  , EPSs , exopolysaccharides (EPSs), lipopolysaccharides (LPSs), and capsular polysaccharides (KPSs or K-antigens), are necessary for establishing successful symbiosis with their hosts to form effective root nodules . ComparaProteins secreted by some rhizobial strains play an important role in infection of leguminous plant roots and establishment of a mutually beneficial symbiosis. Different types and numbers of protein secretion systems are present in almost all rhizobial strains. By means of similarity searches using protein secretion genes identified in other Gram-negative bacteria as queries, we identified 101 genes related to secretory processes in the 7653R genome. These genes and proteins are involved in 12 putative protein secretion systems: a general export pathway, four separate type-I systems, a twin-arginine translocase secretion system, one functional type-III system (T3SS), two type-IV systems (T4SSs), one type-V autotransporter, and two putative type-VI secretion systems (T6SSs) , only 7653R has symbiotic plasmids. In contrast, all other strains either have no plasmids, or their plasmids do not contain genes involved in symbiosis. Thus, while the nodulation and nitrogen-fixation genes are localized on the plasmids as a symbiosis island in 7653R, they are localized on the main chromosomes of the other four strains. Global genome alignment between 7653R and the other genomes revealed that the symbiosis islands are positioned in a synteny gap region that corresponds to the genome-specific region in MAFF303099 and the gap in 7653R  of all transposase genes identified in the entire contigs of R7A are found in the symbiosis island of contig 3. In contrast, the GEIs of the other three s by HGT . Our cluRhizobiales[Bradyrhizobium and Mesorhizobium strains with a few or no relatively small plasmids are typically cited as examples, although the sole evidence for this viewpoint is the presence of ITR plasmid gene clusters and other plasmid genes on the chromosomes of these species. Aside from plasmid genes shown on chromosomes, further evidence based on genomic structure, nucleotide composition, and transposase genes was used in this study to infer a possible evolutionary pathway explaining GEI formation  composed of clusters of tra and trb genes  seems to be necessary for integrase-dependent excision and horizontal transfer of GEIs [tra and trb genes scattered on them. The existence of the same set of tra and trb genes with high similarities in strains MAFF303099, WSM1271, WSM2075, WSM2073, and R7A [Mesorhizobium strains contained a functional conjugation/type-IVA secretion system. Plasmid pMhu7653Ra of 7653R, however, has only a few tra genes and no trb gene. Integrated mobile elements should theoretically be inactivated or lose genes related to plasmid mobilization or transfer, such as tra and trb. It is difficult to judge whether the IVA systems are inactive or if some of the key tra-trb genes have already been deleted from the GEIs of MAFF303099, WSM1271, WSM2075, and WSM2073. To determine what happened to the tra-trb genes on the GEI of 7653R chromosome before excision, further bioinformatics analysis and experimental evidence are needed.In  of GEIs . A similM. huakuaii 7653R genome. We used this genome sequence to examine the phylogenetic position of MAFF303099, a strain whose phylogenetic position has been debated. These two strains share a large set of orthologs and, most importantly, a conserved chromosomal backbone and even larger perfectly conserved synteny blocks. Our ortholog and synteny analyses have firmly placed MAFF303099 as a strain of M. huakuii, as is 7653R.Whole-genome sequencing has proven valuable and critical for refining the phylogenetic positions of a series of rhizobial strains . In thisM. huakuii, they exhibit important differences in symbiotic phenotypes and thus belong to different symbiosis variants  [M. huakuii strains 7653R and MAFF303099 that were focused on the number and arrangement of genes responsible for synthesizing NFs and secretion systems T3SS, T4SS, and T6SS. In conjunction with NFs, these secretion systems may contribute to the establishment of differential host specificity.Although 7653R and MAFF303099 are both strains of biovars) . This plbiovars) –51. FurtMesorhizobium chromosome, with the GEIs of the genus showing different degrees of variability after divergence from the mesorhizobial ancestor. A similar phenomenon has been observed in Bradyrhizobium japonicum strains. Various lines of evidence support past horizontal insertion of GEIs into the ancestral genome of B. japonicum USDA110, and comparative genomic hybridization profiles show that GEIs may be highly dynamic entities in B. japonicum genomes [Bradyrhizobium and Mesorhizobium species have very large chromosomes with few plasmids [Mesorhizobium species has enabled analysis of the global changes between them after the acquisition and integration of the ancestral ITR plasmid. An improved understanding of these variations should improve our understanding of how genome dynamics can contribute to bacterial evolution in general.Our results strongly suggest a common site-specific GEI localization mechanism in the ancestral  genomes . The abiplasmids . The recMesorhizobium genomes. Additionally, homologs of nodulation and nitrogen-fixation genes on the other four GEIs are found on the two plasmids of 7653R. Moreover, it has been reported that GEIs can excise themselves spontaneously from the chromosome and form plasmids with the acquisition of functions for autonomous replication  or can be transferred to other suitable recipients [7653R plasmids possess the same characteristics as the GEIs of the other four Mesorhizobium huakuii 7653R was cultured for 3 days at 28°C in trypticase-yeast extract medium. Cells of 7653R were harvested by centrifugation, with total DNA prepared using a Genomic DNA Mini Preparation kit.de novo sequencing of the 7653R genome, a combined strategy comprising Solexa sequencing on an Illumina GAIIx platform was carried out by BGI . As a result, 367 contigs were generated with a 29-fold median coverage depth.For de novo prediction of genes, rRNA genes, and tRNAs. Clusters of Orthologous Groups (COG) annotation was performed using RPS-BLAST against the CDD database [E-value < 1 × 10−5, identity > 30%, coverage > 70%, and bit score > 60) was used for KEGG [Sequence assembly was performed using SOAPdenovo , with PCdatabase , and Gendatabase . A bidirfor KEGG  annotatiM. huakuii bv. loti, NC_002678, NC_002679, and NC_002682; M. ciceri, NC_014923 and NC_014918; M. opportunistum, NC_015675; M. australicum, NC_019973). The sequences were organized according to their chromosomal origins of replication for intuitive comparison. Sequences of three contigs from R7A were obtained from the JGI Genome Portal (Project ID: 404030). Genome sequence alignments were created using MUMmer, ACT, and Mauve software.The complete nucleotide sequences of strains MAFF303099, WSM1271, WSM2075, and WSM2073 were obtained from GenBank (accession numbers: Mesorhizobium genomes. Quartets of orthologous proteins (quartops) in all pairwise genome comparisons were considered to constitute the ‘core’ genome. Proteins with no homologs in the other four Mesorhizobium genomes were defined as differential genes.The OrthoMCL  approachM. huakuii 7653R have been submitted to GenBank under the following assigned accession numbers: Mesorhizobium CP006581; Mesorhizobium_1 CP006582; Mesorhizobium_2 CP006583.Complete genome sequences of Table S1 The numbers and types of tRNAs in five mesorhizobial genome. Table S4 Similarities (%) for nitrogen fixation genes of 7653R, R7A, WSM1271, and WSM2075 in comparison with those of MAFF303099. Table S5 Similarities (%) of EPS biosynthesis genes of 7653R, WSM1271, WSM2075, and WSM2073 in comparison with those of MAFF303099. Table S6 Similarities (%) of LPS biosynthesis genes of 7653R in comparison with those of MAFF303099, WSM1271, WSM2075, and WSM2073. (PDF 187 KB)Additional file 1: Tables S1, S4, S5, and S6: Mesorhizobium huakuii 7653R used for hierarchical clustering analysis. (XLSX 148 KB)Additional file 2: Table S2: A list of 210 conserved genes in P-values for t-test of significance. (XLSX 9 KB)Additional file 3: Table S3: Figure S1 Nodulation genes participating in NF synthesis. nodE and/or nodF from the 7653R chromosome, pMhu7653Rb, or both participate in synthesis of NFs. Figure S2 Lipopolysaccharide biosynthesis pathway in Mesorhizobium huakuii 7653R. Biosynthesis substrates and products and key enzymes of each step are indicated. Figure S3 ACT visualization of 7653R, R7A, and MAFF303099 chromosomes and plasmids. Genomic alignment of strains 7653R, R7A, and MAFF303099 was performed using ACT [Figure S4 Genomic islands (GEIs) predicted for the four Mesorhizobium strains by IslandViewer. GEIs are shown for MAFF303099 (A); WSM1271 (B); WSM2073 (C); and WSM2075 (D). Genomes in EMBL or GENBANK format are used. The green ellipse indicates the position of the GEI, which is the same as the symbiosis island on each chromosome. Figure S5 Comparison of codon usage among genomic islands (GEIs) in Mesorhizobium. Codon usage patterns were compared between GEIs and the remaining chromosomes. Lysine codon usages are not included because of the huge variability. (PDF 1 MB)Additional file 4: Figures S1 to S5: sing ACT . Red conMesorhizobium huakuii 7653R and MAFF303099. (XLSX 138 KB)Additional file 5: Table S7: Proteins related to the type-III secretion system in


Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM) in China as a result of rapid radiation. In order to overcome the difficulty of morphological classification of this genus, we surveyed three plastid regions  and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula species across 18 sections, to assess their discriminatory power as barcodes. We found that ITS alone or combined with plastid regions showed the best discrimination across different infrageneric ranks and at species level. We suggest rbcL + matK + ITS as the first choice at present to barcode Primula plants. Although the present barcoding combination performed poorly in many closely related species of Primula, it still provided many new insights into current Primula taxonomy, such as the underlying presence of cryptic species, and several potential improper taxonomic treatments. DNA barcoding is one useful technique in the integrative taxonomy of the genus Primula, but it still requires further efforts to improve its effectiveness in some taxonomically challenging groups.The genus  There is a critical need for rigorously delineated species for many theoretical studies and practical applications . HoweverPrimula L. is an extraordinarily species-rich group within the east Himalaya-Hengduan Mountains (HHM) in China. The genus consists of about 500 species with over 300 of these found in China, and most of them (approximately 200 species) are restricted to populations in Southwest China, and are mainly confined to the HHM [Primula species and/or lineage diversity in China occurred no more than 10 Mya [Pedicularis, Rhododendron, Gentiana and Saussurea), Primula is a taxonomically challenging group because: 1) many key diagnostic features are tiny and empirical, and cannot be determined correctly by non-specialists, these features include the shape of calyx and bracts [Primula section Minutissimae) are too small in size to separate; and 3) frequent hybridization or introgression can confuse the Primula species boundaries. Primula species, even distantly related ones, can be hybridized readily in greenhouse conditions [Primula species in the HHM and adjacent area have been described a number of times in recent years [Primula is still underestimated. Although monographs describing Primula do exist [Primula species and discovering cryptic species or lineages in the genus is urgently required. Despite the promise of DNA barcoding, only a few studies have used it in plant groups that have a high diversity in the HHM or in neighboring regions [ the HHM . The HHM the HHM . The excn 10 Mya , and mayd bracts ; 2) manynditions  and in tnditions –14. In ant years –20. Thisdo exist ,11,21,22 regions –27.Primula provides a good opportunity to answer these questions. In the current study, we sampled 66 species representing 18 sections of Primula in China; these contained many closely related groups. The discriminatory ability of three common plastid barcoding candidates  and nuclear regions (ITS and ITS2) were evaluated.Although the limited ability of DNA barcoding to discriminate species in large genera is well known, the following questions are still unclear: 1) to what extent could DNA barcoding discriminate infrageneric levels  within large genera? 2) Could DNA barcodes discriminate between certain closely related species pairs? 3) In rapidly evolved genera, could DNA barcoding reveal cryptic species? As a typical rapidly evolved plant taxon in the HHM, the genus All samples employed in this study are not endangered nor protected in the sampled area, and none of the sampled locations are privately owned or protected by any law. No specific permits were required for the described field studies.Primula species from 18 of the 24 sections of the genus in China recognized by Hu [Omphalogramma delavayi Franch. as an outgroup [P. secundiflora Franch. and P. Poissonii Franch., across their ranges to allow for their intraspecific variability.During this study we examined a total of 227 accessions of 66 ed by Hu . We usedoutgroup ,29. In ooutgroup , more inProliferae was exhaustively sampled in this study. There are approximately 23 species in this section [P. burmanica Balf. f. et Ward, in the section was recently discovered on the south side of Ailao Mountain in Simao (Szemao) region, China . We collected 84 accessions representing all species of the section in China except P. stenodonta Balf. f. ex W. W. Smith et Fletcher. In addition, we selected several of the most closely related species groups in the genus, such as P. chungensis Balf. f. et Ward vs. P. cockburinana Hemsl., P. ovalifolia Franch. vs. P. tardiflora C. M. Hu, P. prattii Hemsl. vs. P. pulchella Franch., P. fasciculata Balf. f. et Ward vs. P. munroi ssp. yagongensis (Petitm) W. W. Smith et Forr., and the P. poissonii complex. Collection details, voucher numbers, taxonomy, and GenBank accession numbers are listed in To test the effectiveness of DNA barcoding in more closely related groups, section  section . In Chin section ,8,21, anrbcL and matK), one intergenic plastid spacer (trnH-psbA), the nuclear ribosomal internal transcribed spacer  and the internal transcribed spacer2 (ITS2), were evaluated in this study. RbcL was amplified using the primer combination (rbcLa_f and 724R) as suggested by Fay et al. [matK was achieved using the primer pair 3F-KIM and XF  protocol of Doyle & Doyle . Five cay et al.  and Kresy et al. , respect and XF . For the pair-wise genetic distance (PWG-distance) method, the genetic pairwise distance was determined by MEGA6 using the Kimura two-parameter distance model (K2P) with pairwise deletion of missing sites [Sequences for each marker were aligned with Muscle 3.8  and thenng sites . Three png sites ,42, we png sites ,43.To test whether accurate species assignments can be made among the samples using a single marker or combinations of markers, we used another two distance-based methods the ‘best match’ (BM) and ‘best close match’ (BCM) using the TaxonDNA/Species Identifier 1.7.7-dev3 program . BM assiFor the tree-building method, we calculated the proportion of monophyletic groups using a Neighbor-Joining (NJ) tree. The test was performed using PAUP* v4b10 with the K2P model . If all rbcL, matK, trnH-psbA) were successfully amplified across all individuals, but amplification of ITS failed in two Primula species (P. virginis Lévl. and P. duclouxii Petitm.) and one accession of P. gemmifera Batal.  in this study (trnH-psbA). The proportion of variable sites were the lowest for rbcL and highest for ITS2. RbcL exhibited the lowest intra-specific and/or inter-specific divergence as well, whilst trnH-psbA showed the highest intra-specific divergence (0.87%), followed by ITS2 (0.80%). However, the greatest interspecific distance was found in ITS2 (12.73%), followed by trnH-psbA (11.69%). The box-and-whisker plots . In contrast, rbcL provided the lowest discrimination rate (24.24%). Of all the combinations tested, the proportion of the barcoding gap of the core barcode combination (rbcL + matK) was the lowest (42.42%)  followed by rbcL + matK + ITS and rbcL + trnH-psbA + ITS (65.63%). TrnH-psbA and ITS2 individually and/or combined with other plastid markers did not perform well enough to discriminate Primula species in this study (rbcL + matK + trnH-psbA and rbcL + matK + ITS2 could identify 36 Primula species (56.65%), while rbcL + matK + ITS performed better and identified 65.63% of the Primula species.The local barcoding gap, with an interspecific distance larger than the intraspecific distance for a species, directly reveals the species discrimination ability of barcodes. The proportion of the local barcoding gap varied between the regions tested Figs . ITS sho(42.42%) , while ris study . For exarbcL + matK correctly identified 72.24% of specimens, which was only slightly better than rbcL + trnH-psbA (71.36%). For three-locus combinations, matK + trnH-psbA + ITS, rbcL + trnH-psbA + ITS, and rbcL + matK + ITS provided similar discrimination rates , followed by rbcL + matK + trnH-psbA (78.41%). In addition, combinations with ITS2 always produced a lower identification rate compared to combinations with ITS , while rbcL + matK + trnH-psbA + ITS was the best one with a 64.06% discrimination rate, followed by rbcL + matK + ITS and matK + trnH-psbA + ITS (60.94%)  .rbcL and trnH-psbA each only identified four sections  (rbcL + matK) only successfully identified five sections , followed by the combinations rbcL + matK + trnH-psbA + ITS2, rbcL + matK + trnH-psbA + ITS and rbcL + matK + trnH-psbA, which all identified the same eight same sections as ITS. In contrast, rbcL + matK + ITS was the best combination, and was able to discriminate nine sections (including section Proliferae)  according to the revised classification of Primula [Auriculastrum correctly.When we considered the previously recognized infrageneric taxa , rbcL asections . The disiolares) . Among tliferae) . Our sam Primula , neverthProliferae is an example containing closely related taxa that is suitable for testing the discriminatory performance of DNA barcoding. Using the tree-building method, the core barcode (rbcL + matK) could only correctly identify P. smithiana Craib with a relatively high bootstrap value (i.e. over 70%), whereas ITS alone could distinguish five species  in this section . Although the three groups could be easily distinguished by rbcL + matK + ITS, the species within each group were difficult to discriminate using the current barcodes singly and/or in combination. For example, the P. poissonii complex is resolved as monophyletic with high support by rbcL + matK + ITS, however, only two narrowly distributed species  could be readily distinguished . However, we found that a failure often occurred in the most closely related species groups ; these included P. moupingensis Franch., P. bella Franch., P. fasciculata, P. malvacea Franch. and P. yunnanensis Franch.  to 523 bp (P. polynuera Franch.), so there were a large number of gaps in the alignment matrix. Based on tree-building analysis, trnH-psbA identified 42.19% of species; this was the best among the plastid regions but lower than the nuclear markers (ITS)  in  species . The corf 37.88% . One of rs (ITS) . The comsolution , which dPanassia [Ficus [Lysimachia [Sisyrinchium [rbcL + matK + ITS and matK + trnH-psbA + ITS all distinguished 60.94% of monophyletic species, which was the best discrimination performance (Appendix S2). Therefore, as suggested by Yan et al. [rbcL + matK + ITS should be the first choice to barcode Primula plants. Compared with primer problems associated with ITS, ITS2 has conserved regions for designing universal primers, and can be readily amplified in various groups [The strong identification ability of ITS has been verified based on a comprehensive study , even inPanassia , Ficus [a [Ficus , Lysimacsimachia , and Sisrinchium . In thisn et al. , rbcL + s groups . HoweverPrimula species concentrated in the HHM region in China [Primula has always been divided into subgroups, usually with the rank of section [Primula were adopted by Hu [DNA barcoding should be able to help identify some groups within large genera, thus reducing the time required for morphological studies to produce definitive species lists. Although it is well known that DNA barcoding has difficulties in resolving closely related species, it is not clear whether such barcoding could identify samples correctly to section level within large genera. There are more than 200 in China . Primula section ,50. In a section . Twenty-ed by Hu .Auganthus, section Souliei, and section Soldanelloides) together with the monotypic section Pycnoloba were each represented by one species in the current study. Considering the fact that the phylogeny of many sections and their close relatives, such as section Soldanelloides, Minutissimae, and Souliei, still lack detailed studies [Primula. In addition to barcoding discriminatory ability, the infragenetric classification system will also influence the results. A reliable and well-recognized infrageneric rank in a large genus is a prerequisite for applying DNA barcoding. Therefore, a number of new sectional delimitations will be necessary in the genus Primula [In this study, DNA barcoding performed well for distinguishing sections, and could resolve nine of the current 18 sections . However studies ,29, the  Primula ,28.Primula section Proliferae is a well-delimited and natural group characterized by numerous whorls of flowers resembling candelabra [P. miyabeana (endemic to Taiwan), they are narrowly distributed in southwest China [P. poissonii complex, which consists of P. anisodora, P. wilsonii Dunn and P. poissonii, and the P. beesiana group with P. beesiana Forr., P. bulleyana Forr., P. burmanica and P. pulverulenta Duthie. This complex section provides a good example to test the discriminatory ability of candidate barcodes in closely related species, especially those formed through rapid evolutionary radiation.The ndelabra . It is mndelabra . In Chinst China . This seProliferae (discrimination rate of 52.63%), in the current study it confirmed the monophyly of section Proliferae (tree-building method)  , and divbarcodes , but onl species ,11,21,51trnH-psbA, ITS) it is almost impossible to separate very closely related species formed through rapid radiation. Therefore, species-specific barcodes need to be developed for difficult taxa [waxy and leafy) [It is well known that using the universal DNA barcode   or even Traditional taxonomy mainly depends on morphological diagnosis, and it should be corroborated by other sources of data, such as geographical, ecological, reproductive and DNA sequence information . HoweverP. tardiflora, P. prattii, and P. cockburiana each experienced peripheral isolated speciation from their widely distributed relatives (putative parents)  [By surveying the non-monophyletic taxa at species level and examining genetic distance , Table 2ngensis) . The barngensis) . It is angensis) . In the ngensis) ,21, but P. bella examined in this study is an excellent example of over-lumping in traditional taxonomy, as the species appeared polyphyletic and exhibited unexpectedly large intraspecific divergence (P. bella [P. bella GXJ096 (voucher: Hao & Yan 956) should be raised to species status (P. cyclostegia Hand.-Mazz.) on the basis of its genetic profile, although additional work is essential to validate this as a robust species. A similar situation is also probably the case for P. denticulata Smith.Imperfect taxonomy in several plant and animal taxa has been detected by DNA barcoding   , Fig 4, Primula have been reported recently [Primula species. In the current study, underlying hybridization might occur in P. anisodora and its most close relative P. poissonii. They were found in the same populations, and a putative hybrid (P. poissonii Y640) was also discovered . Another plastid marker, trnH-psbA, varied in size and exhibited lower discrimination compared to ITS, suggesting that it is not a suitable barcode for studies of Primula. In contrast, ITS showed the best discriminatory ability of all the single markers tested, discriminating 65.63% and 60.94% of species (according to the PWG-distance method and tree-building method) when combined with rbcL + matK, which performed best among all three-locus combinations. We propose that rbcL + matK + ITS should be treated as the first local barcode in the genus Primula at present, although its discrimination rates with respect to infrageneric rank and separating closely related Primula species are limited.Primula taxonomy, such as detecting potential cryptic species, and revealing several probably improper taxonomic treatments. Obviously, it is difficult to resolve all closely related groups based on the current limited and relatively conserved molecular markers, especially in taxa such as Primula, which have experienced recent rapid radiation. Other more rapidly evolved molecular markers should be incorporated into future DNA barcoding projects, for example low or single copy nuclear genes, nuclear SNPs, nuclear SSRs [Primula section Proliferae using RAD , and exted DNA, . OverallS1 FigAsterisks along branches indicate monophyletic species with bootstrap values above 70%. Accessions are suffixed by sample ID. Monophyletic sections are highlighted with grey shading.(PDF)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Intensive care unit (ICU)-acquired infections are a challenging health problem worldwide, especially when caused by multidrug-resistant (MDR) pathogens. In ICUs, inanimate surfaces and equipment  may be contaminated by bacteria, including MDR isolates. Cross-transmission of microorganisms from inanimate surfaces may have a significant role for ICU-acquired colonization and infections. Contamination may result from healthcare workers’ hands or by direct patient shedding of bacteria which are able to survive up to several months on dry surfaces. A higher environmental contamination has been reported around infected patients than around patients who are only colonized and, in this last group, a correlation has been observed between frequency of environmental contamination and culture-positive body sites. Healthcare workers not only contaminate their hands after direct patient contact but also after touching inanimate surfaces and equipment in the patient zone (the patient and his/her immediate surroundings). Inadequate hand hygiene before and after entering a patient zone may result in cross-transmission of pathogens and patient colonization or infection. A number of equipment items and commonly used objects in ICU carry bacteria which, in most cases, show the same antibiotic susceptibility profiles of those isolated from patients. The aim of this review is to provide an updated evidence about contamination of inanimate surfaces and equipment in ICU in light of the concept of patient zone and the possible implications for bacterial pathogen cross-transmission to critically ill patients. Intensive care unit (ICU)-acquired infections are a major cause of morbidity and mortality worldwide . Infectipatient zone and healthcare area have been proposed as a user-centered, geographically related model designed to improve hand hygiene compliance by healthcare personnel during their daily workflow [Staphylococcus aureus (MRSA) and vancomicin-resistant enterococci (VRE) from prior room occupants. Patients were screened on admission and weekly for MRSA and VRE carriage. Patients occupying rooms of carriers showed a significantly higher risk of acquisition of MRSA  and VRE . This increased risk was still observed after correction for other variables  [Pseudonas aeruginosa  and the second most important independent risk factor for Acinetobacter baumannii acquisition , after mechanical ventilation [A growing body of evidence supports the contribution of inanimate surface and equipment contamination for transmission of pathogens to ICU patients. Healthcare workers’ hands are the major vector of cross-transmission of pathogens, with an estimated 20 to 40 % of nosocomial infections arising from cross-infections via healthcare personnel hands , 23. Bacworkflow . The patworkflow . Healthcworkflow , 26, 28.workflow . In a raworkflow . Notablyworkflow . Figure workflow , 27. In workflow , 31. Morworkflow , MDR isoworkflow . These dworkflow . Howeverworkflow , 32. Morworkflow , 34. Bacworkflow . The molworkflow . Notablytilation . Biofilmtilation . It may tilation , 28.Fig.ICU patients are exposed daily to a number of monitoring devices and support equipment. Invasive devices are defined as those interrupting skin and mucosal integrity or being in direct contact with patient’s blood and mucosa . The association between invasive devices and nosocomial infections has been clearly established  . A numbeP. aeruginosa was the most commonly identified Gram-negative species [Manually cleaned, reusable electrocardiography (ECG) lead wires are widely used in ICU. They are placed on direct contact with intact skin, but they may take close proximity with wounds, intravenous lines, surgical dressings, and injured areas. Contamination of ECG lead wires has been reported during an outbreak of VRE in a burn unit , but it  species . Use of  species .S. aureus was the leading Gram-positive pathogenic species including two MRSA isolates. Acinetobacter spp. were the leading Gram-negative pathogenic bacteria, including one isolate of A. baumannii resistant to all tested antimicrobials except colistin. Participants were asked to clean stethoscopes according to their preferred method, with alcohol swabs resulting in the leading adopted method. After cleaning, 2 % of diaphragms and 7 % of earpieces were still contaminated. When anonimously answered, compliance with cleaning procedures of stethoscopes was higher among nurses (91 % of those interviewed cleaned them after every use) compared with doctors (only 30 % of those interviewed cleaned them after every use) [Whittington et al.  investigery use) .S. aureus and P. aeruginosa were specifically searched. The highest contamination rate for S. aureus was observed on Y-pieces (86.7 %) followed by handrails (64.7 %). The highest contamination by P. aeruginosa was reported for water trap surfaces, with no positive cultures for mechanical ventilator surfaces. Contamination rate increased over time, with the highest contamination rate observed after 8 h from the initial surface disinfection. Notably, P. aeruginosa contamination electively involved the breathing circuit and persisted, especially on water trap surfaces, following 75 % alcohol treatment. Contact with ground surface by water traps may explain this observation [Sui et al.  investigervation .A. baumannii, K. pneumoniae, P. aeruginosa, Stenotrophomonas maltophilia) were detected in 39 % of cultures, and a VRE isolate was cultured on one occasion (3 %). Notably, when a resistant Gram-negative species was cultured from the radiograph equipment, the same species was almost always isolated in surveillance or clinical cultures of at least one ICU patient. During the intervention period, promotion of infection control measures resulted in a significant reduction of radiograph equipment contamination, with a decrease of both Gram-positive- and Gram-negative-resistant strains. Radiograph equipment may represent a reservoir for bacteria, including MDR species, in ICU. Equipment and technicians may cross different patient zones during a day, with significant contribution to patients cross-transmission of pathogens when inadequate hygiene measures are undertaken before entering a patient zone. Educational interventions may increase awareness of this potential risk, and radiograph technicians should be involved in infection control programs.Levin et al.  investigS. aureus [P. aeruginosa [Corynebacterium spp. [Acinetobacter spp. [Bacillus spp. [K. pneumoniae originating from contaminated ultrasound-coupling gel [P. aeruginosa caused by contaminated transesophageal echocardiography equipment [The use of point-of-care ultrasound (US) has greatly increased as part of diagnosis and management of critically ill patients in both the ICU and emergency department. Moreover, several sterile invasive maneuvers are now performed under US guide , posing issues about decontamination and covering of the equipment. All the elements of the ultrasound machine may be contaminated by microorganisms, including probes, keyboards, cords, control settings, gel, and gel bottles , 47, 48.. aureus  . Different studies investigated the contamination of outer surfaces of medical charts in ICU, with an observed contamination rate as high as 80–90 % , 59, 60.patients . NotablyS. aureus. Non-fermenting species were the leading Gram-negative bacteria [A. baumannii has been recovered from mobile phones and corresponding hands. One clone was responsible for patient colonization [Mobile phones are the most commonly used non-medical portable electronic devices in ICU. They are not only used for communication but also for web consultation and use of applications for patient care . Unlike fixed phones, of which contamination was also reported , mobile bacteria . In a stnization . Hand conization .As evidence of the role of environmental contamination for cross-transmission increases, the need for objective monitoring of the cleaning process has emerged, especially in ICU. Objective assessment provides immediate feedback and opportunities to improve hygiene procedures and educational intervention for cleaning staff and healthcare workers. In 2010, the Centers for Disease Control and Prevention (CDC) developed a tool kit providing guidance for development of a program to improve environmental hygiene . Five obInanimate surfaces and equipment in ICU are heavily contaminated by bacteria, including MDR species. Bacterial contamination may contribute to ICU-acquired colonization or infection, but further studies are needed to evaluate this correlation. Clinicians and researchers should be aware of the risk of cross-transmission of pathogens from inanimate surfaces in order to adopt appropriate infection control measures.