Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens  with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes.The fungal genus S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE’s) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style.The genomes of Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.Comparative genomic data suggest a unique ecological shift in the The online version of this article (doi:10.1186/1471-2164-15-943) contains supplementary material, which is available to authorized users. Sporothrix includes about 60 species found on all inhabited continents mainly occurring as environmental saprobes, living in association with plants or decaying matter. One lineage within the genus is composed of at least four pathogenic species associated with human and animal sporotrichosis: Sporothrix schenckii sensu stricto, S. brasiliensis, S. globosa, and S. luriei[S. schenckii are globally endemic [Sporothrix schenckii, detailed studies demonstrated that most outbreak isolates were actually S. brasiliensis[The fungal genus . luriei–6. Subcu endemic , 2. Addi endemic –10. DuriSporothricosis is classically associated with rural activities such as agriculture, floriculture or hunting, but more recently felines have emerged as source of human infection. The most common clinical form is a chronic subcutaneous/lymphocutaneous disease acquired after inoculation of fungal material into the skin. Extracutaneous and disseminated forms secondary to cutaneous infection have been described in patients who are immunocompromised as a result of AIDS, chronic alcoholism and diabetes . Rarely,Sporothrix exhibit a thermo-dimorphic phenotype: in its saprophytic stage or in in vitro culture at 25°C the fungus grows with its filamentous form characterized by hyaline, septate hyphae with sympodial conidiogenous cells that produce two types of spores: hyaline conidia that form clusters and brown, thick-walled spores that are distributed perpendicularly alongside the hyphae. During the parasitic stage, the fungus is found as cigar-shaped yeast cells that can also be obtained in vitro by switching the temperature to 37°C [Blastomyces dermatitidis, B. gilchristii, Histoplasma capsulatum, Paracoccidioides brasiliensis, P. lutzii, Coccidioides immitis and C. posadasi. However, all of these other dimorphic fungi are members of the order Onygenales, phylogenetically distant from Sporothrix in the Ophiostomatales [drk1) regulates the transition of mycelium to yeast and consequently the maintenance of virulence in B. dermatitidis and H. capsulatum[S. schenckii, shows 65% of identity with its ortholog in B.dermatitidis and seems to be highly expressed during the yeast stage [The pathogens of the genus  to 37°C . This di to 37°C , 14 and omatales . The lonpsulatum. This gest stage .Sporothrix remains scant. The cell surface of pathogenic fungi plays a key role in the host-fungus interplay, mediating various processes associated with pathogenesis. The fungal cell wall is mainly composed of glycoconjugates: structural polysaccharides such as chitin and β-glucans, and cell wall glycoproteins [S. schenckii cell wall and their relevance for the host-fungus interaction and stimulation of the host immune system was reinforced by recent studies [S. schenckii conidia and yeast cells being produced in vitro or in vivo during infection [Besides dimorphism and thermo-tolerance, current knowledge about virulence factors of proteins . Few pro studies , 20. Hownfection . Melaninnfection , 23.Sporothrix seem to behave in the host remarkably different from Ophiostoma species, suggesting a fundamental habitat shift from a plant- to a mammal-associated life style [S. schenckii and their relatives [Sporothrix species, which are phenotypically similar to human/animal pathogenic Onygenales but philogenetically closely related to plant-associated Sordariomycetes. To address these questions, we performed a comparative genomic analysis of the pathogens with 14 other fungi, either dimorphic pathogens or plant-associated Sordariomycetes. Of these, we chose the closest relative to the Sporothrix lineage, Grosmannia clavigera, for more detailed comparison. G. clavigera is a tree-pathogenic and insect-associated fungus from a related genus from the Ophiostomatales order [Dendroctonus ponderosae, which affects commercial conifer forests, parks, protected areas and urban forests across North America [Sporothrix lineage has led to expansion of some protein domains and lack of genes associated with plant biomass decay when compared to other Sordariomycetes, which can be interpreted as an adaptation from plant to an animal associated life style.Members of the pathogenic lineage in fe style . Remarkaelatives , 25. Thees order . It is a America . These gS. schenckii and S. brasiliensis genomes were each pyrosequenced to ~20x coverage. The S. schenckii genome (strain 1099–18) yielded 16 scaffolds with N50 of 4.3 Mb, containing 237 contigs comprising a total size of 32.4 Mb. The S. brasiliensis genome (strain 5110) yielded 13 scaffolds with N50 of 3.8 Mb, containing 601 contigs, had a total genome size of 33.2 Mb, and shared similar genomic characteristics with G. clavigera[n were found at 5’ or 3’ terminal ends of 5 out of 13 scaffolds in the S. schenckii and 7 out of 13 scaffolds in the S. brasiliensis genome. Terminal repeats were found in both ends of 1 and 3 scaffolds of S. schenckii and S. brasiliensis respectively, revealing the presence of complete linear chromosomes. Pair-wise genome alignments showed that both Sporothrix species are highly syntenic sharing 97.5% average sequence identity  [Sporothrix genomes than in those of G. clavigera. The tRNA contents revealed a great discrepancy among the analyzed fungi; G. clavigera harbors at least 2-fold more tRNAs than Sporothrix genomes . A total of 4,788 genes were found in all three genomes and 2,001 were found to be Sporothrix-restricted genes, indicating a high content of specific genes in the Sporothrix lineage. A total of 1,549 and 508 genes were considered orphan sequences in S. schenckii and S. brasiliensis, respectively   and 50–5 (53.4%) . We deteS. schenckii and S. brasiliensis in a monophyletic clade closest to G. clavigera being apart from other Sordariomycetes  and KMU2052 (AB568600) (data not shown). The mitochondrial genome assembly of S. brasiliensis strain 5110 spans 36 Kb but covers only 71-75% of the three S. schenckii mentioned genomes before. Despite the high similarity between the two analyzed mitochondrial genomes (99% of identity), S. brasiliensis harbors parasitic group-I intron encoding homing endonucleases (HE’s) which is responsible for the higher mitochondrial genome size in this species. Those elements were detected in the cytochrome C oxidase 1, ATP synthase subunit 6 and between NADH dehydrogenase subunits 2 and 3 ORF’s  shared 75%, 84% and 78% of identity to Madurella mycetomatis (YP_006576197), Fusarium graminearum (YP_001249331) and F. solani (YP_005088115), respectively. The S. brasiliensis mitochondrial GIY-YIG HE (SPBR09426 and SPBR09429) is highly conserved among other Sordarimycetes, sharing 72% and 77% of identity to Podospora anserina (NP_074919) and Ceratocystis cacofunesta (YP_007507073), respectively . C. cacofunesta contains 37 intronic ORFs, thus being responsible for one of the largest mitochondrial genomes among Sordariomycetes [S. cereviseae, P. anserina, Neurospora crassa, Ophiostoma and Aspergillus[The mitochondrial genome assembly of ergillus–36. AccoS. schenckii and S. brasiliensis genomes, respectively  [Trichoderma and 0.1% of Fusarium graminearum genomes assemblies [Transposable elements (TEs) comprise 0.34% and 0.62% of the lmost 6% –40. Diffii (16%) . Althougsemblies , 42.TablSporothrix genome, all classes of TEs have been detected with large variation in number and diversity between S. schenckii and S. brasiliensis. Two major types of retrotransposons, LINEs and LTRs, were found in S. schenckii and S. brasiliensis, but no SINE elements were found  as a replacement strategy. Additionally, we didn’t detect any GH72 genes in Sporothrix genomes although those genes were found in all remaining analyzed fungi  binding characteristics. According to PFAM and CAZy counts, a marked expansion of the LysM domain PF01476 (CAZy CBM family 50) was detected in the Sporothrix chitin exposed upon cell damage, and in this way it may protect itself against recognition of this polymer by keratinocytes. LysM effector (Ecp6) from Cladosporium fulvum was characterized as a virulence factor of this phytopathogenic fungus on tomato plants. Carbohydrate binding assays have shown that Ecp6 specifically binds to chitin, the major constituent of the fungal cell wall, acting as a PAMP upon recognition by the plant during fungal invasion. The presence of chitin-binding effector Ecp6 in the apoplast masks the perception of chitin by plant receptors preventing the activation of defense responses [Chitin is a linear polymer of β--linked GlcNAc, and is one of the major components of fungal cell wall. The absence of chitin in mammalian cells makes this polymer a potential targets for the innate immune system . Chitin esponses . In addiesponses . DermatoC. neoformans[Sporothrix lineage when compared to other Ascomycetes , enoylreductase (IPR020843) family , an evidence of detoxification via mycotoxin pump [Sporothrix lineage  methyl-transferase [P. brasiliensis and H. capsulatum, for which the tBLASTn tool yielded hits with high homology. Since the number of degenerate TEs and overall evidence of transposon activity in Sporothrix spp. genomes is low, it would be interesting to identify which pathway is responsible for TE suppression in this genus instead of RIP. We also note the absence of a mus-18 homologue for the alternative, UV damage-related nucleotide excision repair pathway [Neurospora crassa.The basic mechanisms of DNA repair in nsferase . This sunsferase  pointed  pathway , found iH. capsulatum[C. albicans and S. cerevisiae[Sporothrix. Given that Ryp1 and the hybrid histidine-kinase Drk1 are two determinants of mold-to-yeast transition in dimorphic fungi, and that Sporothrix also has homologues for the latter [Among transcription factors, the recent discovery of the Ryp1 protein in psulatum is of inrevisiae are all e latter , it seemN– and/or O- linked to the protein core [S. schenckii and S. brasiliensis.The cell wall of fungi is a dynamic organelle, which is constantly adjusted depending on environmental insults. Fungal cell wall components are considered relevant for virulence, have antigenic properties and participate in the modulation of the host immune response by being recognized by innate immunity receptors . Currentein core . DespiteSporothrix demonstrated the presence of a main antigen on both species, known as Gp70, a secreted antigen that is also present on the cell surface acting as an adhesin [S. schenckii and S. brasiliensis were investigated with different predictors to ascertain the presence of these cell wall components.Previous studies searching for adhesins and antigens in cell extracts of  adhesin –77. The in silico comparative analysis was performed in order to determine the putative adhesins and/or cell surface proteins bearing a GPI-anchor. According to ProFASTA and FungalRV, S. schenckii harbors 68 and 61 surface proteins, respectively, that have adhesin properties (n = 129). Of these, 12 were found by both predictors , respectively, 11 of which were predicted by both algorithms. For this species, a total of 106 unique predicted proteins , could be ascribed to each of the seven chitin synthase classes known (I to VII)  was originally obtained from the Mycology Section, Department of Dermatology, Columbia University, New York, isolated from a patient manifesting subcutaneous sporotrichosis, and has been widely used in experiments of cell wall composition and virulence studies in mice models [Sporothrix brasiliensis strain 5110 (ATCC MYA-4823) was isolated from a feline skin lesion in the epidemic area of sporotrichosis in Rio de Janeiro, Brazil, presenting high virulence in mouse model [e models , 106. Spse model . MyceliaSporothrix schenckii and S. brasiliensis genomes were sequenced using next generation 454 pyrosequencing (Roche). Shotgun and paired-end 3 kb inserts libraries were constructed and sequenced in the 454 GS FLX platform according to Roche’s protocols at the Computational Genomics Unity of the National Laboratory for Scientific Computing . Genomic assemblies were carried out using Newbler and Celera Assembler. Sequence gap filling and the removal of contigs corresponding to rDNA genes were manually done, decreasing the numbers of scaffolds and contigs. The assembled scaffolds generated by the two species were aligned and oriented using MAUVE [http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=lalign).ng MAUVE . SimilarG. clavigera strain kw1407/UAMH 11150 [G. clavigera proteome were aligned to the S. brasiliensis and S. schenckii assembled genomes using Exonerate  with the model protein2genome. Gene predictions (SNAP and AUGUSTUS) and protein (EXONERATE) alignments were used as input in order to identify consensus gene structures using EVidenceModeler [S. brasiliensis and S. schenckii. Alignments were made by Blastp and the lowest e-value was used to consider homologous sequences. Next, loci identified in S. schenckii and S. brasiliensis genomes were blasted against the genome libraries of 14 selected fungi  to infer the putative orthologues. Gene products were categorized according to biological process, cellular component and molecular function using GeneOntology (GO) using Blast2GO. Secreted proteins were identified using SignalP3.0  using hidden Markov model.Gene predictions were performed using three different approaches: SNAP , AUGUSTUMH 11150  as refereModeler  and valihttp://www.girinst.org/repbase/) [http://transposonpsi.sourceforge.net/); c) reverse position-specific BLAST algorithm (RPSBLAST) against Conserved Domain Database (CDD) version March 2013 [Sporothrix genome. These subsets were built from NCBI Non Redundant (NR) database version March 2011 using particular description terms related to transposable elements including . In addition, the Tandem repeat finder (TRF) algorithm was used for finding tandem repeats [The prediction of mobile genetic entities was performed by similarity searches using the following approaches and databases: a) Nucleotide Blast against Repbase version 17.10 (epbase/) , Dfam daepbase/)  and Gypsepbase/) ; b) PSI-rch 2013 ; and d)  repeats . Transpo repeats . All res repeats  was usedhttp://www.cazy.org; [Whole genome gene families were identified using InterproScan combined with Pfam domain assignments. Annotation of carbohydrate-active enzymes was performed in a two-step procedure where the translated protein sequences were compared to the full length sequences derived from the Carbohydrate-Active enZymes (CAZy) database  using Bazy.org; . The queazy.org;  using hiSporothrix lineage were calculated based on hypergeometric comparisons (P < 0.05) and the reported p-values are adjusted for multiple testing using q-value [Sporothrix and other compared fungi were individually analyzed. Protein sequences of LysM, PKS enoyl reductase, Ras, Rab and Rho small gtpases we individually aligned using ClustalW [Gene families were determined using OrthoMCL approach comparing with other 13 fungi  were performed using two different datasets: first we performed the comparisons between ng MAFFT  and retrng MAFFT  in orderng MAFFT  and the ng MAFFT . Divergeng MAFFT  program ng MAFFT  considerng MAFFT .S. schenckii and S. brasiliensis were performed for prediction of GPI-anchor secretion signal and transmembrane domain identification. The SignalP 4.0 server  was applied for prediction of the presence and location of signal peptide cleavage sites in amino acid sequences, using the method of Input sequences, which do not include TM regions. Then, the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) was used for prediction of transmembrane helices in proteins, and finally the Big-PI fungal predictor [Two programs were used for the prediction of cell wall proteins/adhesins: ProFASTA  and Fungredictor  was usedSaccharomyces cerevisiae. All of these sequences were blasted against the S. schenckii and S. brasiliensis databases in order to correctly identify homologous protein sequences. To narrow down the list, we focused the analysis on 17 genes that are necessary for autophagosome biogenesis in yeast [S. cerevisiae[S. schenckii and S. brasiliensis databases.The initial tool used for this annotation was the KEGG automatic classification, which adequately identified genes involved in peroxisome biogenesis. However, the automatic annotation algorithms only picked up a few genes involved in autophagosome biogenesis and endocytosis, so different approaches were necessary. For the genes involved in autophagy, we started by collecting on the SGD all protein sequences annotated as involved in autophagy and autophagosome biogenesis in in yeast  plus thorevisiae. All proThe data sets supporting the results of this article are included within the article and its additional files.Core genes for general and secondary metabolism.(DOCX 56 KB)Additional file 1:Table S1. Information about the 25 genomes retrieved for phylogenomic inference. Table S2. Pfam domain expansions/contractions of Sporothrix schenckii and S. brasiliensis. Table S3. SMART domain expansions/contractions of Sporothrix schenckii and S. brasiliensis. Table S4. INTERPRO domain expansions/contractions of Sporothrix schenckii and S. brasiliensis. S5. Comparative genomic analysis of carbohydrate active enzymes (CAZy) of Sporothrix schenckii and S. brasiliensis. Table S6. Putative adhesins of S. schenckii. Table S7. Putative adhesins of S. brasiliensis. Table S8. Cell wall and adhesin-related genes characterized in Sporothrix schenckii and S. brasiliensis. Table S9. Glucan synthase and glucanase genes identified in Sporothrix schenckii and S. brasiliensis. Table S10. Chitin synthase and chitinase genes identified in Sporothrix schenckii and S. brasiliensis. Table S11. Genes involved in N-linked glycosylation in Aspergillus nidulans and Neurospora crassa, and their putative orthologs in Sporothrix schenckii and S. brasiliensis. Table S12. Enzymes activities related with protein glycosylation in Aspergillus nidulans and Neurospora crassa, and their putative orthologs in Sporothrix schenckii and S. brasiliensis. Table S13. Genes involved in O-linked glycosylation in Aspergillus nidulans and Neurospora crassa, and their putative orthologs in Sporothrix schenckiia and S. brasiliensis. Table S14. Genes involved in GPI-anchor elaboration in Aspergillus nidulans and Neurospora crassa, and their putative orthologs in Sporothrix schenckii and S. brasiliensis. Table S15. Melanin biosynthesis pathway and putative orthologs in Sporothrix schenckii and S. brasiliensis. Table S16. Comparative analysis of core genes related to amino acids, Secondary, Energy, Cofactor and Vitamin metabolisms of Sporothrix schenckii and S. brasiliensis. Table S17. Genomic identification and classification of phospholipases A, C, and D enzyme families in Sporothrix schenckii and S. brasiliensis genomes. Table S18. Homologs of vitamin and cofactor genes presented analyzed by FUNGIpath in both Sporothrix species. Table S19. Orthologous genes related to catabolism and transport in Sporothrix schenckii and S. brasiliensis.(XLS 742 KB)Additional file 2:Sporothrix. The LysM domains are displayed along the taxa (red bars), chitin binding module type 1 (CB1) (blue bars) plus catalytic sites identified (glycoside hydrolase - GH or Pectin Lyase – PL). Gene paralogous duplications are highlighted by red boxes. (PDF 220 KB)Additional file 3: Figure S2: Phylogenetic distribution of LysM domains containing proteins in Sporothrix lineage. Clades harboring Sporothrix and dimorphic fungi are highlighted in red. (PDF 3 MB)Additional file 4: Figure S3: Unrooted maximum likelihood tree of Ras Small GTPase proteins (IPR020849) family shows high diversification in the Sporothrix lineage. Clades harboring Sporothrix and dimorphic fungi are highlighted in red. (PDF 2 MB)Additional file 5: Figure S4: Unrooted maximum likelihood tree of Rho Small GTPase proteins (IPR003578) family shows high diversification in the Sporothrix duplications (blue boxes). (PDF 34 KB)Additional file 6: Figure S1: Gene tree and species tree reconciliation of LysM domain-containing genes showing specific Sporothrix lineage. Clades harboring Sporothrix and dimorphic fungi are highlighted in red. (PDF 3 MB)Additional file 7: Figure S5: Unrooted maximum likelihood tree of Rab Small GTPase proteins (IPR003579) family shows high diversification in the Sporothrix (blue boxes). (PDF 118 KB)Additional file 8: Figure S6: Gene tree and species tree reconciliation of small GTPase Ras gene family showing independent gene losses in other species and the increased copy number in Sporothrix (blue boxes). (PDF 129 KB)Additional file 9: Figure S7: Gene tree and species tree reconciliation of small GTPase Rho gene family showing independent gene losses in other species and the increased copy number in Sporothrix (blue boxes). (PDF 204 KB)Additional file 10: Figure S8: Gene tree and species tree reconciliation of small GTPase Rab gene family showing independent gene losses in other species and the increased copy number in S. schenckii (n = 118) and (B) S. brasiliensis (n = 106). The relative percentage of putative adhesins and/or GPI- anchored proteins, predicted by either ProFASTA or Fungal RV, is shown as well as the proteins in common by both predictors. (PDF 75 KB)Additional file 11: Figure S9: Chart pies showing the efficiency of the algorithms used to predict the putative adhesins and/or cell wall GPI-anchored proteins of (A) Sporothrix spp. chitin synthases. The Mega 4 software package was employed, using ClustalW for sequence alignment. Construction of the phylogenetic tree was done by the neighbor-joining method using 1000 replications. The seven chitin synthases identified for both, Sporothrix brasiliensis and S. schenckii, cluster within the seven chitin synthase classes (I to VII) previously reported [Additional file 12: Figure S10: Phylogenetic tree of relatedness of reported . GenBankS. schenckii and S. brasiliensis based on melanin biosynthetic pathways described in other pathogenic fungi. The putative enzymes identified in the genomes of S. schenckii and S. brasiliensis are indicated in circles. Locus tags, Gene products, numbers of exons, size of transcripts, estimated protein sizes and current annotations are listed. (PNG 412 KB)Additional file 13: Figure S11: Melanin biosynthesis pathways for DHN-Melanin, DOPA-melanin and pyomelanin proposed for


Drosophila melanogaster. And whether Dyl involved in insect post-embryonic development and its signal transduction are poorly understood. Here, phylogenetic analysis revealed that dyl displayed one-to-one orthologous relationship among insects. In Tribolium castaneum, dyl is abundantly expressed at the late embryonic stage. Tissue-specific expression analysis at the late adult stage illustrated high expression of dyl in the fat body and ovary. Knockdown of dyl resulted in the defects in larval epidermal pigmentation and completely blocked the transitions from larval to pupal and pupal to adult stages of T. castaneum. We further discovered that dyl RNAi phenotypes were phenocopied by blimp-1 or shavenbaby (svb) silencing, and dyl was positively regulated by blimp-1 through svb in T. castaneum. These results suggest that Dyl functions downstream of Blimp-1 through Svb for larval epidermal pigmentation and metamorphosis. Moreover, ftz-f1 was down-regulated after RNA interference (RNAi) suppressing any of those three genes, indicating that Ftz-f1 works downstream of Dyl to mediate the effects of Blimp-1, Svb and Dyl on metamorphosis in T. castaneum. This study provides valuable insights into functions and signaling pathway of insect Dyl.Dusky-like (Dyl) is associated with the morphogenesis of embryonic denticle, adult sensory bristle and wing hair in  Drosophila melanogasterD. melanogasterDusky (Dy), Dusky-like (Dyl) and Miniature were identified as the transmembrane proteins containing a zona pellucid (ZP) domain in in situ hybridization and immunostaining showed that dy and miniature are expressed in pupal wings by 28 hours after puparium formation (APF), but the expression of dyl could not be detected in pupal wings at the same stageD. melanogaster, either of which mutation reduced the size of the whole wings by decreasing the size of individual wing epidermal celldyl mutant embryos were characterized by very small, unhooked denticles, often with a split extremelydyl in adults resulted in stub bristles with pigmentation abnormalitiesdyl mutation caused split, thinned, multiple and often very short hairs and a unique planar cell polarity phenotype of adult wing in D. melanogasterD. melanogaster, which provides limited information about dyl functions in the insect. On the other hand, a confused problem has emerged among the current studies that dy is evolutionarily more close to dyl than miniature, whereas dy shows similar expressions and functions with miniature. Thus, investigation of the functions of Dyl in the insect will help to clarify the functional relationship among Dyl, Dy and Miniature.RNA dyl was abolished in shavenbaby (svb) mutant embryos of D. melanogasterSvb is a key selector gene that integrates Wingless and DER pathways to control epidermis differentiationminiature, singed and forked in embryonic denticles of D. melanogastersvb and dyl in insects is unclear up to now. Moreover, additional upstream genes and downstream genes of Dyl should be identified in the insect.At the transcriptional level, the epidermal expression of dyl but also blimp-1 and ftz-f1 transcripts were down-regulated after RNA interference (RNAi) suppressing the G protein-coupled receptor gene, methuselah-like1 (Tcmthl1)  of Tribolium castaneumdyl, blimp-1 and ftz-f1. In D. melanogaster, blimp-1 RNAi caused lethality at the pupal stages in most of the observed animalsftz-f1 from 6 hftz-f1 by 10 h APFD. melanogasterLeptinotarsa decemlineata (Say)dyl might be associated with blimp-1 and ftz-f1 and involved in the metamorphosis of insect.Recently, we discovered that not only T. castaneum by RNAi in this study. Furthermore, we explored how dyl relates to Blimp-1, Svb and Ftz-f1. These Results indicate that Dyl, Svb and Blimp-1 are essential for larval epidermal pigmentation and metamorphosis in T. castaneum.To address these issues, we investigated the function of Dyl in Drosophila dyl as a reference, 20 homolog genes were obtained from the genome database of D. melanogaster, T. castaneum, Anopheles gambiae, Bombyx mori, Apis mellifera, Acyrthosiphon pisum and Pediculus humanus humanus. Phylogenetic analysis revealed that dyl has the orthologue in each insect and shows one-to-one orthologous relationship among insects. In addition, its two paralogues, dy and miniature, also have this orthologous relationship in insects, respectively. And dyl is evolutionarily close to dy but far away from miniature  analysis. In T. castaneum, dyl reached peak expression in the late embryonic stage, while its pupal and adult expression levels were very low , dsRNAs of dyl were injected into larvae and pupae of T. castaneum. The qRT-PCR analysis indicated that the mRNA levels of dyl were significantly reduced after dsRNA injection  was down-regulated by nearly 89% compared with IB after dyl knockdown  was silenced at larval and pupal stages in T. castaneum, respectively. Knock-down of svb resulted in arrested development at the larval stage of T. castaneum. These larvae have abnormally pigmented epidermis, could not molt into pupae, and then died at larval stage. Pupal RNAi of svb also led to the failure to initiate the eclosion of T. castaneum , singed (Accession number: TC006673) and forked (Accession number: TC005627) were declined by svb RNAi  into larvae or pupae depleted about 90% mRNA of itself in T. castaneum. Reduced blimp-1 levels caused developmental arrest at the larval stage of T. castaneum. The treated larvae with abnormally pigmented epidermis could not molt into pupae. Injection of blimp-1 dsRNAs into pupae led to the failure to start the eclosion ftz-f1 resulted in arrested development at embryonic, larval and pupal stages in D. melanogasterftz-f1 by RNAi also caused arrested development at larval stageftz-f1 is essential for metamorphosis. Therefore, the effects of Dyl, Svb and Blimp-1 on metamorphosis were modulated by Ftz-f1 in T. castaneum. It is concluded that Dyl functions downstream of Blimp-1 through Svb but upstream of Ftz-f1 for metamorphosis , total RNA was isolated from the eggs, larvae, pupae and adults of T. castaneum. And 1 μg of total RNA was converted to cDNA by Moloney Murine Leukemia Virus reverse transcriptase (TaKaRa) and an Oligo(dT)18 primer (TaKaRa).Using RNAisoD. melanogaster dy, dyl and miniature (http://flybase.org/), we searched the genome databases of these insects, including T. castaneum (http://beetlebase.org/), A. gambiae (https://www.vectorbase.org/index.php), B. mori (http://silkworm.genomics.org.cn/), A. mellifera (http://hymenopteragenome.org/beebase/), A. pisum (http://www.aphidbase.com/aphidbase/), P. h. humanus (https://www.vectorbase.org/index.php) and National Center for Biotechnology Information . These sequences were aligned with Clustal Omega . Then the Neighbour-joining tree was reconstructed by MEGA 5 using the bootstrap method with 1000 replications To check the gene expression profiles, qRT-PCR was performed with FastStart Universal SYBR Green Master (ROX) (Roche). The data are expressed here as the relative mRNA levels normalized to a control gene, vermillion (ver) dsRNA or an equal volume of buffer only (IB). Knock-down levels of the target genes were examined by qRT-PCR at the fifth day after injection. These experiments were performed for three biological replications.RNAi was performed as previously9p < 0.05, and “**” indicates p < 0.001.The mean values of the RNAi-treated insects versus the mean values of the control insects were compared using the one-way ANOVA program of SPSS version 13.0. All the data are presented as the mean ± standard error. “*” indicates How to cite this article: Li, C. et al. Dusky-like is required for epidermal pigmentation and metamorphosis in Tribolium castaneum. Sci. Rep. 6, 20102; doi: 10.1038/srep20102 (2016).


Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis are widely used as probiotics in the food industry. Although numerous studies have revealed the properties and functionality of these strains, it is uncertain whether these characteristics are species common or strain specific. To address this issue, we performed a comparative genomic analysis of 49 strains belonging to these three bifidobacterial species to describe their genetic diversity and to evaluate species-level differences. There were 166 common clusters between strains of B. breve and B. longum, whereas there were nine common clusters between strains of B. animalis and B. longum and four common clusters between strains of B. animalis and B. breve. Further analysis focused on carbohydrate metabolism revealed the existence of certain strain-dependent genes, such as those encoding enzymes for host glycan utilisation or certain membrane transporters, and many genes commonly distributed at the species level, as was previously reported in studies with limited strains. As B. longum and B. breve are human-residential bifidobacteria (HRB), whereas B. animalis is a non-HRB species, several of the differences in these species' gene distributions might be the result of their adaptations to the nutrient environment. This information may aid both in selecting probiotic candidates and in understanding their potential function as probiotics.Strains of  Bifidobacterium has been classified into 48 taxa, including 39 species and nine subspecies [Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis) are commonly applied as bifidobacterial probiotics in the food industry. Many studies have been performed on the functionality of probiotic bifidobacteria in the host, and the properties and functionality of probiotics have generally been thought to be strain dependent [The genusbspecies , three oependent , 3. Howeependent , and antependent , 7 as weRecent work has provided genomic information for all bifidobacterial species/subspecies , 10. How B. longum, B. breve, and B. animalis, which are commonly applied as probiotics in the food industry. The use of a large set of genome sequences based on a relatively large number of strains of each species allowed the identification of pan-genome structures and the elucidation of differences in core genome structures among these species. In the present report, given that bifidobacteria have been reported to utilise many unique metabolic pathways for sugar fermentation [To further elucidate bifidobacterial genetic diversity and to evaluate species-level differences, we performed comparative genomic analyses of 49 strains belonging to three bifidobacterial species, orentation –19, addiThe bacterial strains used in this study and general information about these strains are listed in μL of the modified MRS, with a final concentration of 1%. Growth of each bifidobacterial strain was measured by absorbance at OD600 after cultivation under anaerobic conditions at 37°C for 24 h and 48 h. Experiments were performed in triplicate.Modified MRS was prepared by removing glucose from the original components and was supplemented with LNT  at a final concentration of 2%. An aliquot of seed culture from each bifidobacterial strain was then inoculated into 200  de novo assembly of the raw paired-end reads were performed using the CLC Genomics Workbench (v 6.0) software package  with default settings, except for contig length . Seventeen genomes that were sequenced de novo in this study were automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 2.0 and were manually checked as part of the process of genome submission to GenBank. Our sequencing efforts resulted in multiple contigs  according to the manufacturer's protocol. The preparation of genomic libraries was performed with 1 ng of genomic DNA using the Nextera XT DNA Sample Preparation Kit  according to the manufacturer's instructions. After PCR amplification and cleanup, the fragment size distribution of the tagmented DNA was analysed using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Analysis Kit . The libraries were sequenced using a MiSeq Personal Sequencing System and the MiSeq Reagent Kit v2 (500 cycles) (Illumina Inc.). Quality trimming and contigs . The datE-value applied in BLAST were 50 and 1e-8. Pan-genome profile analysis, genetic variation analysis of functional genes, and function enrichment analysis of gene clusters were then performed. KEGG Orthology (KO) numbers were assigned by the KEGG Automatic Annotation Server (KAAS) using the bidirectional best-hit method [For all of the 49 bifidobacterial strains included in this study, a pan-genome calculation was performed using the PGAP . The ORFt method .Pan-genome-based phylogenetic trees were generated according to a gene distance matrix, which was calculated based on genes that were absent or present in each strain , using U de novo sequence and annotation data reported herein have been deposited in GenBank under the accession numbers AVQA00000000-AVQE00000000 and AWFK00000000-AWFV00000000.The Gardnerella vaginalis ATCC 14018 as an outgroup species. Each phylogenetic tree was divided into two clusters. However, the classification of subspecies of certain strains based on the genotypes of the strains was contradictory to that previously defined based on phenotypes (http://dx.doi.org/10.1155/2015/567809). B. animalis subsp. animalis ATCC 27536 and ATCC 27674 fell into the clade with the type strain of B. animalis subsp. lactis, whereas B. longum subsp. longum JDM 301 and B. longum subsp. infantis 157F fell into the clades with the type strains of B. longum subsp. infantis and B. longum subsp. longum, respectively. In this study, we adopted subspecies of these strains based on the phylogenetic trees for further analyses.Based on the pan-genome profiles and 16S rRNA gene sequences, phylogenetic trees were constructed for each species, using B. longum, B. breve, and B. animalis (Supplementary file 4). These numbers are similar to those reported previously [ B. longum compared with a previous report [ B. longum has an open pan-genome; that is, B. longum may exhibit a robust ability to import new genes, allowing it to adapt to each ecological niche over its long history of evolution. In the current study, to reveal differences among species, a total of 90,442 genes from the 49 strains were jointly clustered. These genes were divided into 8,818 homologous clusters, and there were 584 common clusters among all of the bifidobacterial strains  were found in all strains except for B. animalis subsp. lactis AD011. Two of four gene homologues encoding glycoside hydrolases  were found in strains of B. breve as well as B. longum subsp. infantis. Fucosidase (Blon_2336) and sialidase (Blon_2348) homologues were found only in B. longum subsp. infantis. However, certain strains of B. breve possessed other gene clusters related to fucose and sialic acid incorporation (Seven homologous genes encoding extracellular solute-binding proteins predicted to bind oligosaccharides (SBP family 1) were found in only two strains ofporation . B. bifidum, B. longum subsp. longum, B. longum subsp. infantis, and B. breve have been reported to act as LNT consumers [β-1,3-galactosidase (Blon_2016) [ B. longum subsp. longum, B. longum subsp. infantis, and B. breve, but not B. animalis subsp. animalis and B. animalis subsp. lactis, in the utilisation of LNT (Supplementary file 5). Unexpectedly, Blon_2016 homologues were also present in B. animalis strains. Based on phylogenetic analysis, Yoshida et al. [ B. breve, B. longum subsp. infantis, and B. longum subsp. longum), whereas the gene in B. animalis was observed to be distant from this clade. These results imply the existence of different substrate specificity for LNT β-1,3-galactosidase, although further investigations are necessary to examine this issue. B. bifidum and certain strains of B. longum subsp. longum produce a secretory enzyme, LNBase, that hydrolyses LNT into lacto-N-biose (LNB) and lactose [ B. longum subsp. longum. The LNBase of B. longum subsp. longum has been reported to be able to hydrolyse the GlcNAcβ1-3Gal linkage in LNT, LNFP I, and sialyllacto-N-tetraose. These results imply the existence of different mechanisms for the utilisation of LNT decorated by fucose and sialic acid. B. bifidum might digest LNT after releasing its own fucosidase and sialidase, whereas B. longum subsp. longum lacks these enzymes but can directly degrade decorated LNT.Regarding the core structure of HMOs, three of the four predominant HMOs (lacto-N-fucopentaose I (LNFP I), lacto-N-difucohexaose I (LNDFH I), and lacto-N-tetraose (LNT)) exhibit the LNT structure. Therefore, the enzyme required for the utilisation of LNT is crucial in the utilisation of HMOs and the colonisation of the infant intestine. Certain strains belonging toonsumers , 30. Theon_2016) . In our a et al.  discover lactose , 33. In  B. longum and B. breve and in three strains of B. animalis subsp. animalis. This distribution is in complete accord with results regarding in vitro LNB utilisation [Subsequently, the liberated LNB is imported into the cells via the galacto-N-biose (GNB)/LNB transporter for further degradation . The dislisation . A previlisation .α-N-acetylgalactosaminidase, acting on core-3-type O-glycan (NagBb) [ B. longum subsp. infantis, 11 strains of B. longum subsp. longum, and 13 strains of B. breve in the present study. In contrast, a gene encoding endo-α-N-acetylgalactosaminidase, which is predicted to release GNB-containing glycans from core-1-type O-glycan mucin (BLD_1258) [ B. longum subsp. longum. In addition, the terminal ends of glycoconjugates in the suckling gut have been reported to predominantly consist of sialic acid, whereas in the adult, they predominantly consist of fucose [ B. longum subsp. infantis and B. breve contained a gene encoding sialidase, which might be useful for this species to colonise the infant intestine.Another substrate for this metabolic pathway is GNB, which is a core structure of gastrointestinal mucin. Mucins are extensively O-glycosylated proteins and are thought to serve as a potential carbon source for gut microbiota. Studies have revealed that the main core structures of gastric/duodenal and intestinal mucins are core-1, 2-type and core-3-type O-glycans, respectively , 38. A h (NagBb) , was obsLD_1258) , was obsf fucose , 42. In  B. longum and B. breve, but not B. animalis strains, were found to possess genes involved in the pathway for the utilisation of N-acetylglucosamine (GlcNAc) and GalNAc, which are the major constituents of host glycans. Furthermore, gene homologues encoding endo-beta-N-acetylglucosaminidase (BLD_0197) [Most strains ofLD_0197) , which r B. longum and B. breve, which are typical HRB, possess certain genes related to the pathway for the utilisation of LNT/LNB, which are core structures of type I oligosaccharides that are specific to human breast milk . All strains ofast milk . Among H samples , 45. In genomics , 27, 46.animalis . These r B. longum subsp. longum was shown to be genetically well equipped for the fermentation of plant-derived sugars , including taxon-specific transporters for GlcNAc, phosphonate, and urea, whereas B. animalis subsp. lactis possessed the lowest number of these homologues (33.7 ± 1.3). All three species can assimilate fructose; however, only species of B. longum exhibited a set of high-affinity fructose-specific ABC transporters , which have been reported to be involved in efficiently converting fructose to acetate under certain gut conditions [ B. longum subsp. longum lacked a homologous gene set for arabinogalactan transport. However, twelve strains of B. longum subsp. longum possessed a set of homologous genes encoding extracellular enzymes for the degradation of arabinogalactan (BLLJ_1840) [ B. breve also exhibited certain unique ABC transporters for lactose/L-arabinose, ribose, and sn-glycerol 3-phosphate.KO assignment indicated several differences in the distribution of carbohydrate transporters at the subspecies level. The subspecies that possessed the greatest number of transporter homologues wasantis 83. ± 2.1, iLJ_1840) . B. brev B. longum and B. breve possessed three homologues, encoding phosphoenolpyruvate-protein kinase, phosphocarrier protein, and beta-glucoside-PTS, whereas none of the B. animalis strains exhibited these genes. Additionally, nearly all strains of B. breve possessed homologues of N-acetylglucosamine, glucose, fructose, and ascorbate, which are involved in the PTS (We found distinctive differences between the three species in terms of the distribution of genes related to the phosphotransferase system (PTS), which is known to be a multicomponent system specific for sugar uptake that operates in global carbon regulation in many bacteria . Strains the PTS .Certain strains belonging to the three bifidobacterial species targeted in this study have been reported to exert numerous beneficial effects on human health as probiotics. Probiotic functionality is generally thought to be strain dependent. In fact, our analysis confirmed that certain genes, such as those encoding membrane transporters and enzymes for host glycan utilisation, are strain dependent. However, our data also demonstrated that there are common characteristics in each species that may be important in light of the species' health-promoting effects on their hosts. Additionally, differences in characteristics might be a result of adaptation to the nutrient environments of each species (such as HRB versus non-HRB). Our results support previous observations based on the investigation of certain type strains of bifidobacterial species and enable the qualification of several of these characteristics as species common or strain specific , 50, 52.These files indicate the phylogenetic trees to show the contradictory results based on phenotype and genotype.


Objective. This study was designed to compare the effect of Helicobacter pylori (H. pylori) infection treatment on serum zinc, copper, and selenium levels. Patients and Methods. We measured the serum zinc, copper, and selenium levels in H. pylori-positive and H. pylori-negative patients. We also evaluated the serum levels of these trace elements after H. pylori eradication. These serum copper, zinc, and selenium levels were determined by inductively coupled plasma mass spectrometry. Results. Sixty-three H. pylori-positive patients and thirty H. pylori-negative patients were studied. Serum copper, zinc, and selenium levels had no significant difference between H. pylori-positive and H. pylori-negative groups. There were 49 patients with successful H. pylori eradication. The serum selenium levels were lower after successful H. pylori eradication, but not significantly (P = 0.06). There were 14 patients with failed H. pylori eradication. In this failed group, the serum selenium level after H. pylori eradication therapy was significantly lower than that before H. pylori eradication therapy (P < 0.05). The serum zinc and copper levels had no significant difference between before and after H. pylori eradication therapies. Conclusion. H pylori eradication regimen appears to influence the serum selenium concentration (IRB number: KMUH-IRB-20120327). Helicobacter pylori (H. pylori), a spiral-shaped pathogenic bacterium found on the human gastric mucosa, was first isolated by Warren and Marshall in 1982. It is one of the most common worldwide human infections [ H. pylori plays an important role in the development of chronic gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Established indications for H. pylori eradication include gastric ulcer, duodenal ulcer, gastric mucosa-associated lymphoid tissue lymphoma, atrophic gastritis, after gastric cancer resection, uninvestigated dyspepsia, patients who are first degree relatives of patients with gastric cancer, and patient's wishes [ H. pylori infection.s wishes . TherefoCopper is an essential nutrient in human physiology and is an essential constituent of numerous enzymes. Copper deficiency may result in microcytic anemia, leukopenia, osteoporosis, new subperiosteal bone formation, and fibrosis of epiphysis . Zinc isHelicobacter pylori infection leads to chronic inflammation of gastric mucosa and peptic ulcer disease. It may influence the absorption of essential trace elements. The association between trace elements and H. pylori infection has been reported [ H. pylori eradication on the essential trace elements of zinc, copper, and selenium.reported . The aim H. pylori eradication; (c) patients with allergy history to PPI, or antibiotics; (d) patients who had ever taken PPI, antibiotics, and bismuth in the recent 4 weeks; (e) patients with severe systemic diseases ; (f) patients with previous gastric surgery; and (g) pregnant women. All of the clinical diagnoses were documented by endoscopic examination. The patients with H. pylori infection were classified as the study group, and the patients without H. pylori infection were the control group.Patients, who were older than 18 years and complained of dyspepsia or epigastralgia, were evaluated in our gastroenterology outpatient clinic. The exclusion criteria included (a) age less than 18 years; (b) patients who had had previous H. pylori infection was diagnosed by histology, culture, and rapid urease test. Patients who were infected with H. pylori were treated with lansoprazole 30 mg bid, amoxicillin 1 g bid, and clarithromycin 500 mg bid or levofloxacin 500 mg qd for 7–10 days. After 6–12 weeks of triple combination therapy, repeated gastroendoscopy with rapid urease test, histology, and culture was performed. If the patient refused repeated gastroendoscopy, 13C-urea breath tests were conducted to assess the status of H. pylori infection.The status ofThis study conformed to the Helsinki Declaration and was approved by the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (IRB number: KMUH-IRB-20120327). The written informed consents were collected from all participating patients. H. pylori was considered positive if one or more colonies showed gram negativity, oxidase (+), catalase (+), urease (+), and spiral or curved rods in morphology.Biopsy specimens were rubbed on the surface of a Columbia blood agar plate and then incubated at 35°C under microaerobic conditions for 4-5 days. Culture ofThe biopsy specimens were fixed with formalin, embedded in paraffin, and stained with hematoxylin and eosin. The result for the gram's stain was considered positive when curvy, gram-negative bacteria were found on the slide. They were interpreted and reported by the same pathologist.The results of the rapid urease test  were interpreted as positive if the color of the gel became pink or red after 6 hours at room temperature.13C-urea was manufactured by the Institute of Nuclear Energy Research, Taiwan. One hundred milliliters of fresh whole milk was used as the test meal. This detailed procedure was reported in our previous study [The us study . H. pylori infection was established if the culture was positive or both CLO test and histology were positive.For patients who received endoscopy,Blood samples were drawn from the median cubital vein in individuals and collected in vacutainer plain tubes. The centrifuge was set at a speed of 3000 rpm for 15 minutes under room temperature. After centrifugation, the supernatant was pipetted into an unused vial for −20°C preservation without any additives. The serum copper, zinc, and selenium levels were determined by inductively coupled plasma mass spectrometry (ICP-MS). At present, ICP-MS is the most sensitive analytical technique for the determination of trace elements in various matrices.t-test. The difference between the two groups was tested with paired t-test. A P value of <0.05 was considered statistically significant.All data were analyzed using Statistical Package for Social Sciences Version 14.0 software . Statistical differences between mean values were tested with Student's  H. pylori in the study group and 30 patients without H. pylori infection in the control group. The mean age was 53.87 ± 13.18 years in the study group and 48.00 ± 12.22 years in the control group. The endoscopic diagnosis in the study group was 11 patients with gastric ulcers, 28 patients with duodenal ulcers, 3 patients with gastritis, and 21 patients with reflux esophagitis. The control group had 30 patients with primary endoscopic finding of gastritis . Forty-nine patients had successful H. pylori eradication with a rate of 77.78% (49/63). After successful H. pylori eradication, the serum levels of copper, zinc, and selenium had no significant difference compared with before treatment . Among the patients with failure of H. pylori eradication, the serum copper and zinc levels were not significantly different between before and after treatments . However, the serum selenium level was significantly lower after failure of H. pylori eradication therapy (P < 0.05).H. pylori infection may result in stomach inflammation. H. pylori, when having altered gastric secretion coupled with tissue injury, leads to peptic ulcer disease and gastritis, and maybe progresses to atrophy, intestinal metaplasia, and eventually gastric carcinoma. H. pylori also leads to hypochlorhydria in H. pylori-related gastritis [astritis . The cha H. pylori infection [ H. pyloric infection and noninfection [ H. pylori- positive and H. pylori-negative patients . The definite mechanism responsible for decreasing serum selenium level after H. pylori eradication therapy is not clearly defined. However, the changes of microbiota in small intestine may play an important role in determining serum selenium level because it is mainly absorbed in the duodenum and cecum by active transport through a sodium pump [ H. pylori eradication regimen, may have a long-term impact on the native microbiota in the intestine for up to 4 years posttreatment [ H. pylori eradication regimen also results in bacterial and fungal overgrowth in small intestine [ H. pylori eradication therapy on decreasing serum selenium level, but the detailed mechanism needs to be further elucidated.Selenium has long been noted as an integral component of glutathione peroxidase, which is an important antioxidant against oxidative damage in human body . It is asubjects . Howeverdication . Furtherogenesis . This ise groups . Howevery Tables  and 4, eium pump . Previoureatment . Besidesntestine . The cha H. pylori infection, although the relationship is not significant in statistics. However, the serum selenium level decreases after H. pylori eradication therapy in both successful and failed eradication groups, and it achieves significantly lower levels of selenium after eradication therapy in the failed group. Our study shows that the H. pylori eradication regimen may influence the serum selenium level.Our study aims to explore the association of serum copper and zinc levels with


S. aureus infections in non-surgical healthcare settings.A protective effect of mupirocin has been seen among surgical, nonsurgical and dialysis patients. Our aim is to summarize evidence for mupirocin decolonization for prevention of S. aureus infections using meta-analytic methods.To identify the optimal setting and patient population to implement mupirocin decolonization for prevention of 2I statistics.We conducted systematic searches in PubMed, Cochrane Library Databases, Scopus, Web of Science, and ClinicalTrials.gov to identify papers published until 2013 on effectiveness of mupirocin in healthcare settings. Two investigators independently abstracted data with a pilot-tested form. Risk of bias was assessed using the Cochrane tool. The crude odds ratios were pooled (cpOR) using a random-effects model. Heterogeneity was evaluated using the Woolf’s test for homogeneity and S. aureus infections by 70%  and 60%  in both dialysis and non-dialysis settings, respectively. Nevertheless, there was highly significant (p=0.0009) and moderate heterogeneity (2I=46%) among studies. Studies were homogeneous (p>0.1) when stratified analyses were performed by specific clinical settings. Among the 6 studies that took place in adult intensive care units (ICUs), mupirocin decolonization was associated with a 56% reduction in the odds of S. aureus infection . There was also a protective effect of mupirocin against S. aureus exit site infections among patients undergoing peritoneal dialysis  and against bacteremia among hemodialysis patients .Of the 12,644 studies identified, 8 randomized controlled trials and 19 quasi-experimental studies met the study inclusion criteria. Mupirocin was observed to reduce the odds of S. aureus infections among both dialysis and adult ICU patient populations. Future studies should target other patient settings such as long-term care facilities.Mupirocin decolonization is protective against None declared.


Cochliobolus heterostrophus is a dothideomycete that causes Southern Corn Leaf Blight disease. There are two races, race O and race T that differ by the absence (race O) and presence (race T) of ~ 1.2-Mb of DNA encoding genes responsible for the production of T-toxin, which makes race T much more virulent than race O. The presence of repetitive elements in fungal genomes is considered to be an important source of genetic variability between different species.In silico evidence of recent activity was found for many of the transposons and analyses of expressed sequence tags (ESTs) demonstrated that these elements were actively transcribed. Various potentially active TEs were found near coding regions and may modify the expression and structure of these genes by acting as ectopic recombination sites. Transposons were found on scaffolds carrying polyketide synthase encoding genes, responsible for production of T-toxin in race T. Strong evidence of ectopic recombination was found, demonstrating that TEs can play an important role in the modulation of genome architecture of this species. The Repeat Induced Point mutation (RIP) silencing mechanism was shown to have high specificity in C. heterostrophus, acting only on transposons near coding regions.A detailed analysis of class I and II TEs identified in the near complete genome sequence of race O was performed. In total in race O, 12 new families of transposons were identified. C. heterostrophus, the RIP silencing mechanism is efficient and selective. The co-localization of effector genes and TEs, therefore, exposes those genes to high rates of point mutations. This may accelerate the rate of evolution of these genes, providing a potential advantage for the host. Additionally, it was shown that ectopic recombination promoted by TEs appears to be the major event in the genome reorganization of this species and that a large number of elements are still potentially active. So, this study provides information about the potential impact of TEs on the evolution of C. heterostrophus.New families of transposons were identified. In The online version of this article (doi:10.1186/1471-2164-15-536) contains supplementary material, which is available to authorized users. Cochliobolus heterostrophus  is the causative agent of Southern Corn Leaf Blight (SCLB) . An ORF similar to the gag region is sometimes found in a position upstream of the pol region, but its role remains unclear. At the 3’ end of the LINEs, a poly(A) tail, tandem repeats or an A-rich region may be present -transposase, which contains a characteristic motif of three acidic residues, two of which are aspartic acids and the last is a glutamic acid or, in some cases, a third aspartic acid [Tc1-Mariner transposons known as MITEs were also identified. These sequences lack one or more genes essential for transposition. However, in various species, a small number of Tc1-Mariner elements can be responsible for the origin and activation of a large population of non-autonomous elements [Copies of elements related to the tic acid , 40. Allelements .C. heterostrophus genome against the EST cluster, with the results demonstrating the presence of transcripts related to the major superfamilies of elements identified herein. “In silico” evidence of potential activity has also been reported by Martin et al. [Laccaria bicolor. The activation of TEs under stress conditions has been demonstrated in Aspergillus oryzae[Ophiostoma ulmi[A total of 147 elements, approximately 65% of the complete TEs found, are potentially active. This activity was demonstrated through alignment of the sequences of TEs identified in the n et al.  in Laccas oryzae and Ophioma ulmi. The effoma ulmi, 44, 45.C. heterostrophus race O. Therefore, recombination between retrotransposon sequences may have been or is a contributing factor in the reorganization of the C. heterostrophus genome. Complex DNA recombination events and the cultivation of monocultures of susceptible maize germplasm containing Tcms are considered to be causes of the Southern Corn Leaf Blight epidemic that occurred in the 1970s with the emergence of the C. heterostrophus race T [The evolution of chromosomal structure in Dothideomycetes, to a first approximation, appears to be the result of chromosomal rearrangements . In thiss race T .C. heterostrophus race O. Similarly, the analysis of scaffolds carrying genes responsible for the production of T-toxin in C. heterostrophus race T revealed the presence of various solo-LTRs, suggesting a high recombination rate in this region. The Tox1A and Tox1B loci, related to the production of T-toxin, are associated with a translocation involving race O chromosomes 6 and 12 [The presence of non-autonomous elements and solo-LTRs corroborates the notion of the high degree of ectopic recombination between sequences of TEs. These sequences generally result from recombination between sequences of TEs in the genome of 6 and 12 . The TEsN. crassa[, was identified in the C. heterostrophus genome. Another interesting result was that, of all the sequences analyzed, only families of transposons that contain at least one aligned copy near a coding region had RIPed sequences. Elements belonging to the families Gypsy-1_CH, Gypsy-5_CH, and Copia-1_CH did not show strong evidence of RIP activity. No coding sequences were found near any of these transposons, indicating that these TEs are most likely in heterochromatic regions. Thus, RIP in C. heterostrophus appears to be a highly selective mechanism, acting only on TE copies inserted near coding regions. A difference in the intensity with which RIP acts between the different transposable elements has been reported in other fungi; in Stagnospora nodorum, the Molly and Elsa transposons are more clearly affected by RIP [Aspergillus niger, RIP is considered a severe event and only two sequences of AniTa1 elements, of the 15 analyzed, exhibit evidence of RIP. Interestingly, these two sequences are found inserted into ORFs [C. heterostrophus has been shown by Ohm et al. [The adaptability of the host may be negatively affected by TEs that can cause gene deletions and duplications, chromosomal rearrangements and alterations in the expression of essential genes. However, some fungi have genetic silencing mechanisms known as RIP mechanisms to control repetitive DNA sequences such as the transposons. Despite evidence of RIP previously reported by Clutterbuck  and Ohm . crassa, was idd by RIP , while into ORFs . AnotherMycosphaerella fijiensis, where RIP has been shown to be very severe, several MFS and ABC transporters were identified near TEs [Cochliobolus carbonum, host-specific toxin genes are situated in transposon-rich regions of the genome [Aspergillus nidulans there is evidence of the involvement of transposons in the regulation of gene clusters related to secondary metabolism [Pyrenophora tritici-repentis, another member of the order Pleosporales, the analysis of TEs has suggested the involvement of TEs in the creation of new genes, diversification, horizontal gene transfer and trans-duplication [P. tritici-repentis isolates showed that pathogenicity in this species emerged through an influx of TEs, which created a genetically flexible situation that enabled an easy response to environmental changes [All genes up to 5,000 bp from the identified TEs were mapped. In addition to the possibility of suffering RIP when at a distance of less than 2,000 bp from the TEs . Sequencnear TEs . Genes rnear TEs , 52. Anoe genome . Althougtabolism . In Pyrelication . Additio changes .C. heterostrophus race O was performed. Twelve new families of transposons were identified, demonstrating the possible role of these elements in the genomic regulation and evolution of C. heterostrophus. In C. heterostrophus, the RIP silencing mechanism is efficient and selective, allowing movement of elements in heterochromatic regions, but silencing copies that may be inserted into coding regions. The major coding regions influenced by RIP mechanism were also characterized. Additionally, it was shown that ectopic recombination promoted by TEs appears to be the major event in the genome reorganization of this species and that a large number of elements are still potentially active.In this study, a complete characterization of the major TEs present in the genome of C. heterostrophus race O strain C5 v2.0 was obtained from the Joint Genome Institute (JGI) database (http://www.jgi.doe.gov/genome-projects/). Identification and classification of TE sequences in the genome of C. heterostrophus were performed using the RepeatMasker program . This program identifies copies of TEs by comparing genome sequences with sequences present in a previously described library of TEs (RepBase 16.12: http://www.girinst.org/repbase/update/index.html) [http://tlife.fudan.edu.cn/ltr_finder/) [http://expasy.org/) and Orf-finder (http://www.ncbi.nlm.nih.gov/projects/gorf/) programs. TE insertion sites, or TSRs (Target Site Repeat), were characterized by direct search of the sequences that flanked each TE.The genome of ex.html) . In thisfinder/)  and the finder/) . Class Ifinder/) . CompletSequences identified in this way were classified as complete elements, potentially active elements or degenerate sequences. Complete elements were those sequences with similarity to proteins related to the transposition machinery, conserved terminal repeats and target site duplications (TSDs), but lacking intact ORFs. Potentially active elements were complete elements that exhibited intact protein domains and ORFs characteristic of the superfamily of transposons. Finally, degenerate sequences were those sequences that displayed identity with consensus sequences of the major characterized TEs (RepBase). However, degenerate sequences lacked structural characteristics or sequences encoding transposition-related proteins.Copia and Gypsy retrotransposons because according to Wicker et al. [http://www.girinst.org/repbase/update/index.html) [http://www.girinst.org/repbase/update/browse.php with the following identifiers: Copia-1_CH and Copia-2_CH (Superfamily Copia); Gypsy-1_CH, Gypsy-2_CH, Gypsy-3_CH, Gypsy-4_CH, Gypsy-5_CH, Gypsy-6_CH, Gypsy-7_CH, and Gypsy-8_CH (Superfamily Gypsy); I-1_CH and I-2_CH (Superfamily I).To define families, the classification system proposed by Wicker et al.  was usedr et al.  the termex.html) . Finallyex.html) , and rephttp://www.ncbi.nlm.nih.gov/BLAST) against the RefSeq_protein database (Reference Sequence Protein) to determine the existence of protein-coding sequences near the TEs. The threshold used for protein identification was E-value < 10-20 and identity > 50%.After searching for complete TEs, approximately 5,000 bp upstream and downstream of each TE were analyzed by BLASTX , pol region (Gypsy), transposase (Tc1-Mariner) and ORF2 (I) were aligned using the Mega 4 program [DNA sequences associated with the synthesis of transposition proteins were analyzed for dinucleotide frequency and RIP index. Regions comprising  program . Only al program  to calcu program .C. heterostrophus genome database using the keyword “methyltransferase”. Alignments of different DMTs were performed to demonstrate the presence of conserved domains. GenBank accession numbers for C5-cytosine methyltransferase genes are as follows: Ajellomyces capsulatus (XP_001539629), Phaeosphaeria nodorum (XP_001797905), Penicillium chrysogenum (CAP86663), Pyrenophora tritici-repentis (XP_001935966), Emericella nidulans (AF428247), Coccidioides immitis RS (XP_001239116), Ascobolus immersus (AF025475), Aspergillus oryzae (BAE61916), Neurospora crassa (AAM27408), Cochliobolus heterostrophus , Aspergillus fumigatus (XP_747703), Gibberella zeae PH-1 (XP_388824), Aspergillus terreus (XP_001209776), and Magnaporthe oryzae (XP_366719).A search for the RID protein (DNA methyltransferase – DMT) was performed in the JGI C. heterostrophus genome was analyzed using RDP (recombination Detection Program) [Evidence of ectopic recombination in the transposon sequences found in the Program) , GeneconProgram) , BootscaProgram) , MaximumProgram) , ChimaerProgram) , Sister Program)  and 3SeqProgram)  implemenProgram) . TEs belProgram) , and seqC. heterostrophus C5 race O genome annotation. The library was obtained from the JGI genome database (http://www.jgi.doe.gov/). Sequences of complete elements and potentially active elements identified were aligned by BLASTN against a total of 88,751 expressed sequence tags (ESTs) [-5) in relation to the elements identified here were considered.In order to assess potential TE transcriptional activity, the EST-cluster database  was inspC. heterostrophus were downloaded from the Joint Genome Institute (JGI) database (http://www.jgi.doe.gov/genome-projects/). TE sequences from novel families are deposited at http://www.girinst.org/repbase/update/browse.php with the following identifiers: Copia-1_CH and Copia-2_CH (Superfamily Copia); Gypsy-1_CH, Gypsy-2_CH, Gypsy-3_CH, Gypsy-4_CH, Gypsy-5_CH, Gypsy-6_CH, Gypsy-7_CH, and Gypsy-8_CH (Superfamily Gypsy); I-1_CH and I-2_CH (Superfamily I). Supporting data are included as additional files.The genomes and EST-cluster of Additional file 1: Table S1: Sequences coding proteins downstream and upstream of full copies of the transposable elements. The table contains an analysis of the regions approximately 5,000 bp upstream and downstream of each transposable element. (DOCX 41 KB)C. heterostrophus race T. The figure contains the Scaffold 4 LU with transposons and Tox sequences. (DOCX 29 KB)Additional file 2: Figure S1: Transposon fragment containing an intact LTR found upstream from the LAM1 gene in the genome of


Feifei Feng, Zhuowei Li, Erin N. Potts-Kant, Yiming Wu, W. Michael Foster, Kristi L. Williams, and John W. Hollingsworthhttp://dx.doi.org/10.1289/ehp.1205188RETEnviron Health Perspect: Online: 1 July 2015Retraction of: Environ Health Perspect 120:1692–1698; doi:10.1289/ehp.1205188The authors request retraction of “Hyaluronan Activation of the Nlrp3 Inflammasome Contributes to the Development of Airway Hyperresponsiveness.” Due to concerns about the initial data from the animal physiology laboratory at Duke, the authors re-exported source data from the Flexivent machine and compared these data with raw data originally received from the animal pulmonary physiology laboratory. Results from these initial comparisons suggested potential inconsistencies in the data. Therefore, the authors proceeded to replicate experiments of animal airway physiology presented in Figure 2A. These replicate experiments failed to validate the originally reported role of Nlrp3, ASC, and caspase 1 in airway hyperresponsiveness after exposure to ozone. These observed inconsistencies significantly impact the overall conclusions of the manuscript. Therefore, all of the authors have agreed to retract this paper in an effort to correct the scientific record. The authors sincerely apologize for any inconvenience to the scientific community.


Background: Relapsing fever borreliae are vector-borne bacteria responsible for febrile infection in humans in North America, Africa, Asia, and in the Iberian Peninsula in Europe. Relapsing fever borreliae are phylogenetically closely related, yet they differ in pathogenicity and vectors. Their long-term taxonomy, based on geography and vector grouping, needs to be re-apprised in a genomic context. We therefore embarked into genomic analyses of relapsing fever borreliae, focusing on species found in Africa.Results: Genome-wide phylogenetic analyses group Old World Borrelia crocidurae, Borrelia hispanica, B. duttonii, and B. recurrentis in one clade, and New World Borrelia turicatae and Borrelia hermsii in a second clade. Accordingly, average nucleotide identity is 99% among B. duttonii, B. recurrentis, and B. crocidurae and 96% between latter borreliae and B. hispanica while the similarity is 86% between Old World and New World borreliae. Comparative genomics indicates that the Old World relapsing fever B. duttonii, B. recurrentis, B. crocidurae, and B. hispanica have a 2,514-gene pan genome and a 933-gene core genome that includes 788 chromosomal and 145 plasmidic genes. Analyzing the role that natural selection has played in the evolution of Old World borreliae species revealed that 55 loci were under positive diversifying selection, including loci coding for membrane, flagellar, and chemotaxis proteins, three categories associated with adaption to specific niches.Conclusion: Genomic analyses led to a reappraisal of the taxonomy of relapsing fever borreliae in Africa. These analyses suggest that B. crocidurae, B. duttonii, and B. recurrentis are ecotypes of a unique genomospecies, while B. hispanica is a distinct species. Borrelia includes the Lyme disease group of borreliae in North America and Europe and the relapsing fever group of borreliae in North America, Africa, Asia, and in the Iberian Peninsula in Europe    in TanzaB105117) .B. crocidurae, B. duttonii, B. recurrentis, B. hispanica  provides such an opportunity.Therefore, revisiting the long-term taxonomy of relapsing fever borreliae in Africa is needed. The recent availability of genome sequences for ispanica –16, B. tHere, we performed comparative genomics and genome-wide phylogenetic analyses to address the taxonomy of relapsing fever borreliae in a phylogenetic context, focusing on borreliae circulating in Africa.B. crocidurae (GenBank accession number CP003426), B. duttonii (CP000976), B. recurrentis (CP000993), B. hispanica (AYOU00000000), Borrelia miyamotoi (CP006647), Borrelia persica (AYOT00000000), Borrelia hermsii (CP000048), Borrelia turicatae (CP000049), Borrelia bissettii (CP002746), Borrelia garinii (CP003151), Borrelia alzelii (CP002933), and Borrelia burgdorferi (CP00222801). A bidirectional best hit (BBH) approach with an expect value cut-off of 10–3 was used to identify orthologous chromosomal genes; only genes having a similarity >30% and length coverage >70% were kept.All chromosomal and plasmidic sequences were retrieved from GenBank for genomes: To compare the genomes of the relapsing fever borreliae, each set of orthologous chromosomal gene sequences was first aligned using MAFFT . BayesiaThe Bayesian concordance analysis was carried out with BUCKy 1.4.0  using th2 distribution with one (M8a/M8) or two (M1a/M2 and M7/M8) degrees of freedom , with 371 bacterial genomes consisting of one chromosome and one plasmid and 364 bacterial genomes including 3–11 DNA fragments (chromosome and plasmid). The remaining bacterial genomes were efaciens , 24. Theelomeres . PhylogeBorrelia hermsii and B. turicatae chromosomes are collinear, whereas Old World borreliae genomes collinearity is disrupted by 1,134-bp insertion sequences (IS) IS605 OrfB  or 414-bp transposase IS200 (only in B. crocidurae). These IS are also carried by the plasmids of the three species and have high sequence identity [97–98%; Ref.  that are unique to B. hermsii, one hypothetical protein (AAX16910) missing from relapsing fever borreliae but present in the Lyme group borreliae, and the glutathione peroxidase-like BsaA gene (AAX17028) that is absent in non-B. hermsii borreliae but that has an ortholog in Geobacillus thermoglucosidasius and Bacillus species. A similar analysis using B. turicatae as a reference revealed one hypothetical protein (AAX17575) absent in non-B. turicatae borreliae, a second hypothetical protein (AAX18156) absent from relapsing fever borreliae but present in Lyme group borreliae, and the crossover junction endodeoxyribonuclease RuvC (AAX17369) gene that is absent in non-B. turicatae borreliae but has an ortholog in Clostridium acidurici. Comparison of New World and Old World relapsing borreliae genomes identified two genes missing from the New World relapsing fever borreliae: RNA polymerase sigma-54 factor (ACH93394) and antigen P35-like protein (ACH92961). Conversely, one gene encoding the DNA repair protein RadA (AAX17754) is missing from the Old World relapsing fever borreliae genome.Comparing the gene content of Borrelia pan genome includes 2,514 genes, and the number of protein coding genes (CDS) ranges from 990 in B. recurrentis to 2,140 in B. crocidurae. Of these, 933 (37.1%) represent the core genome and encode 788 core chromosome proteins and 145 core plasmid proteins. Interestingly, while the core genes represent <50% of the pan genome, they represent more than 81% of the genes classified into the cluster of orthologous group (COG) categories  and glycosyl transferase family GT28 and GT51 .The and GT51  previousand GT51 , suggestand GT51 . Among sB. duttonii, B. recurrentis, and B. crocidurae genomes, we used the 788 core chromosome genes and compared the phylogenetic signal obtained for each individual gene using a concordance analysis. This analysis reveals that the five genomes form two major clades: one containing B. duttonii, B. recurrentis, and B. crocidurae and second containing B. hermsii and B. turicatae; all 788 genes investigated support this clustering (concordance factor = 1).In order to further investigate the relationships between the B. duttonii, B. recurrentis, and B. crocidurae clade, with the first two forming a group and the third one being an outgroup (concordance factor = 0.94). Consistent with this, only 16 of the 788 genes supported an alternative grouping for these three genomes : 10 supported a B. recurrentis–B. crocidurae group and 6 a B. duttonii–B. crocidurae group. The tree topology was also conserved after including other relapsing and Lyme species  as well as proteins involved in cell motility (10%); the remaining conserved proteins represent a large part (80%) of the COG functions.The concordance analysis also reveals a second well-supported split in the Consistent with their high nucleotide identity (86–99%), there was no significant difference in codon usage between the five relapsing fever borreliae, with the most frequently used codons in the chromosome (as measured by the relative synonymous codon usage or RSCU) being AGA, which encodes arginine , and TTA, which encodes leucine . Similarly, the least used codons are CGG and CGC that encode arginine and CTG, which encodes leucine.To investigate how natural selection contributed to the evolution of the five relapsing fever borreliae genomes, we performed a genome-wide analysis of positive selection: significant evidence of positive diversifying selection was observed for 55 of the 788 core chromosome genes .Together with variation in plasmid content, positive selection represents one of the main molecular mechanisms contributing to vector–host adaptation. Our study revealed that 55 genes were the targets of positive diversifying selection during the evolution of the recurrent fever borreliae. Although these genes are randomly distributed in the chromosome, they code for proteins involved in translation, ribosomal structure and biogenesis, replication, recombination and repair, cell membrane biogenesis, cell motility, and signal transduction: these mechanisms are related to host interaction and might thus be important factors to restrict the habitats of B. hispanica, B. recurrentis, B. crocidurae, and B. duttonii and a clade B that includes New World B. turicatae and B. hermsii. This division is supported by the 788 genes used. The concordance analysis also strongly suggests that B. duttonii and B. recurrentis are closely related and form a clade that has B. crocidurae as an outgroup, strengthening a previous hypothesis that B. recurrentis is a subset of B. duttonii , and 91% between B. turicatae and B. hermsii, while the ANI between the two groups is at 86%. It was suggested that average nucleotide identity values of ~94% correspond to the cut-off for bacterial species definition (B. duttonii, B. crocidurae, and B. recurrentis would be well above that level and the three Old World species would thus be ecotypes of the same genomospecies. These data suggest updating the long-term taxonomy of these borreliae, in delineating a new species Borrelia africana, comprising of three subspecies crocidurae, duttonii, and recurrentis. Also, data here presented allow refining molecular tests for the accurate detection and identification of relapsing fever borreliae, in a point-of-care format (Current taxonomy of the relapsing fever borreliae derives from geographical and tick-specific relationships comforted by analyses of single genes or of several concatenated genes . The coeduttonii . Accordifinition , so thate format .Pierre Pontarotti and Michel Drancourt designed the study. Haitham Elbir, Laurent Abi-Rached, and Niyaz Yoosuf performed analyses. Haitham Elbir, Laurent Abi-Rached, Pierre Pontarotti, and Michel Drancourt analyzed the data and prepared the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose. To report a rare presentation of culture positive Mycobacterium chelonae (M. chelonae) corneal ulcer and its management. Case Report. We report a rare case with history of chronic pain and blurriness of vision. Examination revealed chronic nonhealing paracentral corneal ulcer inferiorly at 5 to 7 o'clock meridian with anterior chamber cells 1+ unresponsive to routine antibiotic and antifungal medications with Mantoux test (MT) positivity in a middle aged nondiabetic patient with no prior obvious history of trauma, ocular surgery, and contact lens usage. Discussion. Ziehl Neelsen (ZN) staining in nonhealing ulcer revealed acid fast bacilli typical of M. chelonae with subsequent culture positivity in Lowenstein Jensen (LJ) medium. Subsequent treatment with topical fortified amikacin and tobramycin resulted in rapid healing of corneal ulcer. Conclusion. M. chelonae presenting as a chronic nonhealing corneal ulcer spontaneously occurring in a healthy young adult with no predisposing factor draws the need to have a good index of suspicion by performing ZN stain and culture and its subsequent successful management with topical fortified amikacin and tobramycin. Mycobacterium chelonae (M. chelonae) is an omnipresent saprophyte present in soil, water, and air [ M. chelonae and M. fortuitum and scrofuloderma have been identified as uncommon infective causes of chronic nonhealing corneal ulcers [ M. chelonae being rapid growers lead to significant blinding complications [ Bacillus occurring in the absence of any predisposing factor in a healthy young adult has not been reported till date. Herein we report a very rare case of nonhealing corneal ulcer with Ziehl Neelsen (ZN) stain and culture positivity for M. chelonae and describe its management.The nontuberculous and air . In the  and air , 3. Risk and air , 3. Brea and air . Nonheall ulcers . A histol ulcers . Local il ulcers . Due to ications . Spontan∗ 3 mm horizontally oval corneal ulcer from 5 to 7 o'clock meridian inferiorly 2 mm away from limbus with underlying stromal edema . Iron reuptake test was negative. Identification to species level was achieved on the basis of the growth characteristics, including growth in less than 7 days, growth at 37°C, failure of growth in presence of sodium chloride 5%, pigment production, and positive catalase test. Antibiotic sensitivity with Kirby-Bauer disc diffusion method showed positive sensitivity to amikacin. Patient was started on topical fortified amikacin (50 mg/mL) and topical tobramycin (14 mg/mL) with rapid resolution of symptoms and healing of ulcer in 3 weeks  and Hepatitis B and Hepatitis C virus. TB gold test was negative. Upon presentation, best corrected visual acuity (BCVA) was 0.48 Log MAR in the right eye and 0.00 Log MAR in the left eye, respectively. Slit lamp biomicroscopy in the right eye revealed a paracentral 3 mm al edema . Corneal 3 weeks  leaving  M. chelonae infection presenting as a nonhealing corneal ulcer in a healthy young adult with no predisposing factor has not been reported till date. Nontuberculous mycobacteria (NTM) are aerobic, non-spore-forming, nonmotile acid fast bacilli. Runyon proposed a classification of nontuberculous mycobacteria, under which group IV is the M. fortuitum complex, consisting of M. fortuitum, M. chelonae, M. smegmatis, and M. vaccae [ M. fortuitum and M. chelonae are responsible for a growing number of skin and soft tissue diseases. NTM as a cause of nonhealing corneal ulcers is also well established. Girgis et al. in their retrospective study of ocular infections caused by nontuberculous mycobacteria reported 36.6% incidence of keratitis most commonly caused by M. abscessus/chelonae with identifiable risk factors being presence of biomaterials (63.1%), ocular surgery (24.1%), and steroid exposure (77%) [ M. chelonae keratitis and canaliculitis in a patient with a SmartPLUG [ M. chelonae keratitis following bilateral simultaneous LASIK with successful treatment with topical amikacin, azithromycin, and ciprofloxacin in most cases. Surgical debridement and flap removal were required in some. Yamaguchi et al. reported 39 eyes of 30 patients developing Mycobacterium keratitis following LASIK [ M. chelonae keratitis associated with soft contact lens usage [ M. chelonae and M. fortuitum. Typical “cracked windshield” appearance of cornea is considered diagnostic [ M. chelonae is mostly sensitive to tobramycin, whereas M. abscessus is not and is more sensitive to fluoroquinolones. We report on a case of spontaneously developed ZN smear and culture positive active NTM ocular infection with focus on cornea presenting as a nonhealing corneal ulcer with no known predisposing factor. Early recognition and prompt institution of topical amikacin and tobramycin prevented the ocular morbidity. Till date, M. chelonae keratitis has been reported consequently to some trauma or iatrogenic intervention or in immunosuppressed states. However, the possibility of incidental environmental inoculation cannot be ruled out. The present case stresses the importance of considering M. chelonae in the differential diagnosis of nonhealing corneal ulcer even in nonpredisposed healthy adults with no obvious history of ocular trauma and the role of subsequent early institution of appropriate sensitive drugs in preventing the consequent visual morbidity and the need of surgical intervention.Spontaneously occurring. vaccae . Nontube. vaccae . Both M.re (77%) . Feder emartPLUG  and FreimartPLUG  reportedng LASIK . Lalithang LASIK . Huang eng LASIK . They fons usage . Siong a surgery 4.1%, andagnostic . Most ofagnostic . Early r