Staphylococcus aureus. RNases in prokaryotes have been mainly studied in the two model organisms Escherichia coli and Bacillus subtilis. Based on identified RNases in these two models, putative orthologs have been identified in S. aureus. The main staphylococcal RNases involved in the processing and degradation of the bulk RNA are (i) endonucleases RNase III and RNase Y and (ii) exonucleases RNase J1/J2 and PNPase, having 5′ to 3′ and 3′ to 5′ activities, respectively. The diversity and potential roles of each RNase and of Hfq and RppH are discussed in the context of recent studies, some of which are based on next-generation sequencing technology.The regulation of RNA decay is now widely recognized as having a central role in bacterial adaption to environmental stress. Here we present an overview on the diversity of ribonucleases (RNases) and their impact at the posttranscriptional level in the human pathogen  Staphylococcus aureus is a main source of hospital-acquired infections causing pneumonia, endocarditis, osteomyelitis, soft-tissue, and skin infections [ S. aureus also causes serious nail infections (paronychia) and is a common cause of food poisoning due to the production of enterotoxin [ S. aureus infections is its ability to become resistant to multiple antibiotics including β-lactams (MRSA) and glycopeptides and also to more recent molecules such as linezolid and daptomycin [ S. aureus pathogenicity relate to the coordinated expression of numerous virulence factors. The combined risks of disease and diminishing efficacy of antibiotic treatments have incited the scientific community to investigate staphylococcal transcriptional and posttranscriptional regulation in detail.fections . S. aureerotoxin . A main ptomycin , 3, 4. Iptomycin . The und Escherichia coli, exonuclease activity is solely 3′ to 5′ while in Bacillus subtilis, 5′ to 3′ exonuclease activity was uncovered. Extensive studies on RNA processing and degradation reveal that while several RNases are present in other species of their respective taxons   activities contribute to RNA processing or degradation. RNA processing is a cleavage leading to functional transcripts, while RNA degradation results in RNAs transformed into oligonucleotides and nucleotides. Transcriptional and post-transcriptional regulatory pathways control protein production and contribute to homeostasis and adaptation to environmental stress. In bacteria, the first step of RNA decay is generally thought to involve removal of the RNA 5′-end pyrophosphate. It is followed by an endonucleolytic cleavage allowing exonucleolytic degradation. InRNase Z) , RNase E E. coli and the Firmicutes B. subtilis, results obtained for these species will be presented to discuss the recent knowledge on RNA decay in S. aureus.Since RNases were primarily studied in the Proteobacteria E. coli extracts more than forty years ago [ in vitro by metal chelators [ B. subtilis Mini-III RNase  to the large Homo sapiens Dicer1 protein . However, all family members possess a common RIIID-like domain that includes a nine-residue signature motif [RNase III is a double-strand (ds) specific RNase discovered inears ago . Its acthelators . RNase Ire motif .λ. RNase III cleaves a stem-loop in the 5′UTR region of the N gene transcript, thus releasing the Shine Dalgarno (SD) sequence and permitting recruitment of ribosomes [ Streptococcus pyogenes show that RNase III acts in concert with the CRISPR Csn1 protein to mature CRISPR RNAs (crRNA), resulting in prophage silencing [Through its ds-RNA specificity, RNase III is a key player in various cell processes. These roles include the maturation of ribosomal RNAs (rRNA) by cleaving stem-loops inside the primary rRNAs –10 and mibosomes . Due to ibosomes , 14. Recilencing . B. subtilis, amounts of 470 transcripts, representing 11% of total transcripts, were shown to be altered by RNase III depletion [ txpA/ratA and yonT/as-yonT TAs was sufficient to suppress the RNase III essentiality [Inepletion . Howeverntiality . S. aureus RNase; its role was mainly determined through the characterization of virulence genes regulated by the agr system [ agr system effector  rot mRNA (encoding a regulator of toxins) through imperfect base pairings involving two loop-loop interactions and of (ii) coa mRNA  via the binding of two distant regions of coa mRNA  strains [ S. aureus genome was identified. In the absence of RNase III, an accumulation of antisense transcripts and a decrease of short transcripts were observed, suggesting that RNase III likely eliminates a basal level of pervasive transcription [ B. subtilis, Enterococcus faecalis, Listeria monocytogenes, and Salmonella enterica. A correlation between the absence of RNase III and an increase of short transcripts was observed in all the tested Gram-positive bacteria, but not in the sole Gram-negative species tested (S. enterica) [ via RNase III, which consequently could control cellular protein levels. Second, RNase III could also eliminate transcriptional noise.A second study focusing on the role of RNase III at a genome scale was performed using a comparative transcriptomic analysis of wild-type and RNase III deficient (Δ strains . The autcription . To assenterica) . It will B. subtilis), has been described in low GC content Gram-positive bacteria. Mini-III plays a role in the maturation of 23S rRNA in B. subtilis [ S. aureus but to date has not been characterized  was identified as an RNase that cleaves single-stranded A- or AU-rich sequences [ yitJ riboswitch, but only in the presence of SAM, which contributes to forming a terminator structure. The initial rate of 5′ monophosphorylated RNA degradation is faster than for 5′ triphosphorylated RNAs. However, after prolonged in vitro incubation, the same amount of yitJ cleaved product was observed even for 5′ end triphosphorylation [The endonuclease RNase Y ofequences . It clearylation . These rrylation . Moreove infC-rpmI-rplT mRNAs encoding the elongation factor IF3 and ribosomal proteins L35 and L20 [ via its processing activity is also involved in regulation of the gapA operon and bsrG/SR4 type I toxin/antitoxin system [RNase Y is involved in the decay of polycistronic and L20 . This op and L20 . The pren system , 34. B. subtilis [ sinR mRNA resulting in the SinR repressor accumulation), modifications in folate and amino acid biosynthesis, extracellular polysaccharide synthesis, and an increase in penicillin-binding protein 2A mRNA stability [ B. subtilis physiology and metabolism.RNase Y depletion increases the half-life of bulk RNA levels insubtilis . Accordisubtilis  or 550 asubtilis . The protability . Overall S. aureus, the rny ortholog (aka cvfA) was discovered as a regulator of virulence genes using silkworm and mouse infection models [ rny impaired virulence notably by diminishing haemolysin production [ rny-dependent phenotypes [ B. subtilis, rny is not essential in S. aureus, as tested in the NCTC8325 and Newman strains [rny mutant and its corresponding isogenic wild-type strain [ saePQRS operon and expression of the two-component system SaeS/SaeR [ saePQRS operon, which differ in stability [ saeRS translation [Inn models , 37. Disoduction . RNase Yenotypes . As is t strains , 39. A re strain . Many ofaeS/SaeR . Differetability . RNase Y B. subtilis expressed in E. coli is processed by RNase E, suggesting that an RNase E functional equivalent exists in B. subtilis [ B. subtilis genome. Therefore, enzymes having RNase E-like activities  were searched in B. subtilis leading to the discovery of RNase J1 and RNase J2 (formerly YkqC and YmfA) encoded by rnjA and rnjB, respectively [ E. coli but comparable to what was observed for PNPase deletion in B. subtilis (see the following). Thirty percent of total transcripts are targeted by RNase J1 revealing a wide action of this RNase [ B. subtilis, the paradigm for RNA decay is an endonucleolytic cleavage by RNase Y, giving access to RNases J for 5′ to 3′ exonucleolytic activity, whereas PNPase performs a 3′ to 5′ exonucleolytic activity [ rnjA and rnjB genes in B. subtilis [ rnjA mutant is viable with a long doubling time (76 min instead of 26 min) with defects in sporulation, competence, and cell morphology, while the rnjB mutant has a growth rate similar to that of the wild-type strain [The threonyl-tRNA synthetase leader region fromsubtilis . Howeverectively , 45. As ectively . These eectively . The exoectively , 48. Theis RNase . In B. sactivity . As studsubtilis . The rnje strain . rnjA and rnjB genes were essential in S. aureus [ rnjA and rnjB. Each mutant exhibits poor growth at 42°C [ Sinorhizobium meliloti and B. subtilis [ S. aureus, the maturation of acpP (acyl-carrier protein) mRNA exemplifies the role of RNase J1 and J2. In the absence of RNase J1, this essential mRNA is cleaved by RNase J2, thus demonstrating the activity of this enzyme in S. aureus.Results from a saturated transposon mutagenesis suggested that. aureus . However at 42°C . The fac at 42°C . Overexp at 42°C . Specifisubtilis , 53. In  in vivo. RNase J2 has a narrower role than RNase J1; some functions of RNase J2 were observed in the RNase J1 mutant . However, the precise role of RNase J2 is not perfectly understood yet.RNase J is a complex enzyme made of two partners (J1 and J2) forming heteromers (either dimers or possibly tetramers)The polynucleotide phosphorylase (PNPase) is a 3′ to 5′ exonuclease with a peculiar mechanism for RNA decay; whereas “classical” RNases cleave RNA molecules by hydrolysis, the PNPase cleaves RNAs by phosphorolysis involving an inorganic phosphate and releasing a nucleotide diphosphate. This contributes to the dual role of PNPase which also acts as a polymerase when the concentration of inorganic phosphate is lower than those of nucleotide diphosphate .S. aureus encodes three 3′ to 5′ exonuclease orthologs, PNPase, RNase R, and YhaM. The global role of PNPase in RNA decay was determined at a genome scale, comparing wild-type and ΔpnpA isogenic strains [pnpA strain [ S. aureus. PNPase may be important for the degradation of the large amount of csp (cold shock protein) transcripts after a cold shock induction as demonstrated in E. coli [ S. aureus PNPase also interacts with RNase Y to degrade transcripts [ agr expression is decreased by the absence of RNase Y and this effect is suppressed in a pnp mutant. This phenotype is reversed when PNPase is expressed from an ectopic gene suggesting a direct role of PNPase in this process [ strains . While iA strain . PNPase  E. coli . A recennscripts . As an e process . This st E. coli by two independent teams [ E. coli, the major components of this complex are (i) RNase E, (ii) PNPase, (iii) RNA helicase B (RhlB), and (iv) the glycolytic enzyme enolase [ E. coli. Thus, RNase E is an endonuclease sensing 5′ monophosphate ends [The RNA degradosome was discovered during purification of RNase E fromnt teams , 59. Cop enolase . The funate ends  and PNPaate ends . RhlB unate ends . This roate ends . The rolate ends . B. subtilis was proposed with RNase Y as the central partner instead of RNase E. Protein-protein interactions demonstrated by a bacterial two-hybrid system led to the identification of RNases J1/J2, PNPase, a RNA helicase CshA, enolase, and phosphofructokinase, another glycolytic enzyme, as RNase Y partners [The existence of an RNA degradosome inpartners . Phospho S. aureus degradosome [ S. aureus and B. subtilis degradosome components are similar; however, the interactions between the different partners seem to be simpler in S. aureus [ B. subtilis, each partner interacts with at least three other partners whereas, in S. aureus, each partner seems to interact with two only other partners.A similar approach was used to identify theradosome  and led . aureus . In B. s S. aureus, and putative roles for the others are predicted based on assignments from other organisms. Among them, RNase P, a nucleoprotein complex shared by all kingdoms of life, removes 5′ extra-nucleotides from tRNA precursors [ rnpB and rnpA genes, respectively [ S. aureus sharing only 24% amino acid identity with E. coli RnpA; all conserved amino acids proved to be essential , which act together to scavenge and recycle nucleotides for new RNA transcripts [ nrnA orthologous gene in S. aureus, while to date, no study concerning this RNase family has been performed.Inease Orn . B. subtnscripts . Genome  S. aureus species. However, several acquired RNases have been described. These enzymes are, so far, part of toxin/antitoxin (TA) systems. TA systems can be divided in five groups according to the antitoxin function [ E. coli, MazF recognizes the 5′ end of ACA and cleaves just before the cytosine (A∧CA with “∧” represents the cleavage site), whereas in S. aureus, SaMazF cleaves inside a five-base sequence U∧ACAU [ S. aureus, SaMazE/F, SaPemI/K, and YefM-YoeB [RNases mainly belong to species core genomes. For instance, RNase III, RNase J1, RNase J2, and RNase Y are found in all isolates of thefunction . The twofunction . Severalfunction . The Mazfunction . In E. ce U∧ACAU , 87. Up efM-YoeB –91. E. coli, expression of MazF causes global mRNA degradation leading to reprogramming and growth arrest; cell death is rescued by MazE [ E. coli, MazF is involved in the cleavage of (i) mRNAs at ACA sequences in the vicinity of the AUG start codon and (ii) 16S rRNA within the 30S subunit [ S. aureus, MazF cleaves at U∧ACAU which is a relatively abundant sequence, for instance, inside the sraP gene, coding for a protein involved in the cell adhesion and thus virulence [In by MazE . However by MazE . The quo subunit . Modifie subunit . In S. airulence .Recently, another role of ribonuclease-encoding TA system has been described for SaPemI/K . This pl in vitro by cleaving free mRNA consistent with that previously observed in E. coli [The last system, YefM-YoeB, has a ribosome-dependent RNase activity. The toxin binds the A site of the 50S ribosomal subunit and then cleaves the mRNA three base pairs after the start codon . In addi E. coli .RNases encoded by TA systems may have a global impact on staphylococcal posttranscriptional regulation. Global scale experiments of these systems need to be performed. E. coli, RppH is responsible for the acceleration of the decay of hundreds of transcripts, demonstrating its importance in RNA stability [ E. coli RppH protein did not present any substrate specificity, at least in terms of the 5′-end nucleotide [Bs, the B. subtilis RppH ortholog [Bs drives pyrophosphate hydrolysis of a synthetic RNA when (i) at least two and preferably three or more nucleotides are unpaired at the 5′ end and (ii) if the second nucleotide is a guanosine and the third nucleotide is preferentially a purine [Bs crystallographic study revealing a binding pocket that fits a guanosine in the second position of substrates [ Bdellovibrio bacteriovorus RppH leads to a different interpretation, RppH recognizes the first nucleotide of the sequence. These results prompted us to inactivate and identify possible substrate specificity in S. aureus . Based on protein sequence identity (38% amino acid identity with RppHBs) and conserved synteny, we identified SAOUHSC_01913 as the gene expressing the S aureus RppH ortholog (RppHSa). The rppH gene of S. aureus strain HG003 [ S. aureus HG003 and HG003 ΔrppH in exponential phase were extracted, sequenced by RNA-seq and transcriptomes of these strains were compared using DeSeq tools [ rppH mutant (Sa in the tested condition possibly due to the presence of a second RppH-like enzyme that could compensate for the absence of RppHSa (SAOUHSC_01913).Enzymes without RNase activities, such as RppH and Hfq, can be involved in the decay of bulk RNA. RppH, for RNA pyrophosphate hydrolase, triggers RNA degradation by removing the 5′ pyrophosphate of mRNA . The remtability . The purcleotide . Howeverortholog , 97. Rppa purine . This obin HG003  was delein HG003 . Total Req tools . UnexpecH mutant . None of E. coli, Hfq was identified as an essential host factor for bacteriophage Qβ [ S. aureus (HfqSa) [ S. aureus remains unknown. The hfqSa deletion showed no phenotype when tested on over 1,500 tested growth conditions [ S. aureus as well as in B. subtilis has no impact on sRNA-mediated regulation, reviewed in [hfqSa is poorly or not expressed in the studied strains. In one report, hfqSa deletion in strains where Hfq was detected resulted in decreased toxicity and virulence, and over a hundred genes showed differential expression in an hfq-mutant compared to a wild-type strain using microarrays [ S. aureus NCTC8325. Surprisingly, the hfqSa gene fails to substitute for the hfq of Salmonella in sRNA-mediated regulation [Sa does not play a central role in posttranscriptional regulation. Further investigation will be necessary to understand the exact role of Hfq in S. aureus.A key non-RNase player in RNA processing and decay is the RNA-binding protein Hfq. Discovered more than forty years ago inphage Qβ . In manyphage Qβ . While t (HfqSa) , its funnditions . In contiewed in . It is croarrays . The obsgulation . Altoget S. aureus indicate that the scheme for RNA decay is similar to that in the low G+C content Firmicutes model B. subtilis. For instance, the absence of RNase E, replaced by RNase Js and Y, is also observed in S. aureus. RNases are key players of posttranscriptional regulation and therefore are involved in virulence factor regulation. As an example, RNase III controls the expression of factors involved in cell adhesions or factor involved in immunity escape via the degradation of sRNA/mRNA duplexes.Recent studies on the RNase functions in S. aureus and their potential effects on virulence gene regulation.Up to now, the impacts of only three RNases, that is, RNase III, Y, and Js, have been studied on the genome scale. Further studies will be needed to elucidate the precise roles of the other RNases present in


Arthroderma benhamiae produces the hydroxamate siderophores ferricrocin and ferrichrome C. Additionally, we show that the iron regulator HapX is crucial for the adaptation to iron starvation and iron excess, but is dispensable for virulence of A. benhamiae. Deletion of hapX caused downregulation of siderophore biosynthesis genes leading to a decreased production of siderophores during iron starvation. Furthermore, HapX was required for transcriptional repression of genes involved in iron-dependent pathways during iron-depleted conditions. Additionally, the ΔhapX mutant of A. benhamiae was sensitive to high-iron concentrations indicating that HapX also contributes to iron detoxification. In contrast to other pathogenic fungi, HapX of A. benhamiae was redundant for virulence and a ΔhapX mutant was still able to infect keratinized host tissues in vitro. Our findings underline the highly conserved role of the transcription factor HapX for maintaining iron homeostasis in ascomycetous fungi but, unlike in many other human and plant pathogenic fungi, HapX of A. benhamiae is not a virulence determinant.For many pathogenic fungi, siderophore-mediated iron acquisition is essential for virulence. The process of siderophore production and further mechanisms to adapt to iron limitation are strictly controlled in fungi to maintain iron homeostasis. Here we demonstrate that the human pathogenic dermatophyte  Arthroderma benhamiae belongs to a group of fungi known as dermatophytes, which exclusively infect keratinized structures such as hair, skin (stratum corneum) and nails of humans and animals  (SAB) and transformants of A. benhamiae LAU2354-2 were grown on SAB supplemented with 200 μg/ml hygromycin  or G418 , according to the selectable marker used. Additionally, all fungal strains used in this study were stored as frozen glycerol stocks at -80°C  KH2PO4; Carl Roth, Karlsruhe, Germany] if not otherwise stated. Further experiments were carried out at 30°C in Aspergillus minimal medium (AMM) containing 1% (w/v) glucose as the carbon source and 20 mM glutamine as the nitrogen source  fragment from pJetGFPACT1T1  x 100. The experiments were run in three biological replicates.For determination of the siderophore activity in culture supernatants of escribed . A volumNeilands . As a re4) and high iron conditions  for 5 d at 30°C and 200 rpm. For short-term iron stress the mycelium was grown for 3 d at 30°C and 200 rpm and shifted from -Fe to +Fe for 1 h (sFe). The frozen mycelium was ground with mortar and pestle and subsequently the RNeasy Plant Mini Kit  was used for total RNA isolation according to the manufacturer’s instructions. The quality and quantity of RNA was determined with a NanoDrop 1000 Spectrophotometer . For complete digestion of DNA 1000 ng RNA were treated with the TURBO DNA-free™ Kit  and the purified RNA was used for first strand cDNA synthesis with oligo d(t)23 VN primer  and RevertAid Reverse Transcriptase  according to the manufacturer’s instructions. The qRT-PCR experiments were performed with the StepOnePlus Real-Time PCR System . Gene-specific primers (A. benhamiae (ARB_04092) was chosen as internal control for normalization. Quantitative RT-PCR products were obtained using MyTaq HS Mix 2x  and EvaGreen  as fluorescent dye. PCR conditions were 95°C for 2 min followed by 40 cycles with 15 s at 95°C, 20 s at 60°C, 15 s at 72°C and a final step at 95°C for additional 15 s. For each primer pair a standard curve with serial dilutions of genomic DNA of A. benhamiae in technical triplicates was determined and the primer efficiency (100% ±10) was used for the calculation of transcript levels by the method described by Pfaffl et al. [A. benhamiae wild type during iron-replete conditions. The experiments were run in three biological replicates with technical duplicates.For RNA isolation fungal mycelium was harvested after cultivation in AMM during iron-replete conditions (+Fe), iron starvation  were used for the analysis of fungal growth on keratin substrates. Human scalp hair from a child and finger nails from a healthy female donor were cut from the donors themselves or the next of kin in their domestic home. The provided hair and clipped finger nail samples were autoclaved. Hair, nails and keratin powder were placed on water agar plates and inoculated with 3 plugs of fresh fungal mycelium from SAB agar plates. After incubation at 22°C for 25 d (keratin powder), 30 d (nails) and 40 d (hair) in the dark, mycelia formation on the keratin substrates was documented.The study did not include any diagnostic procedure or therapeutic method. Furthermore, the sample collection was non-invasive  and did not intrude into the privacy of the donor. Based on the regulations of the ethics commission at the Jena University Hospital, Jena (Germany), an approval of this study was not necessary in this case. Only human material  from voluntary donors were used. Additionally, the donors or the next of kin have provided written consent for the use of the samples for research and publication. The study does not contain any patient data. Only the first author had access to any potentially identifying donor information. Identifying details are omitted in the manuscript.A. benhamiae which has significant similarity to HapX of A. nidulans (49% identity) and A. fumigatus (48% identity). A. benhamiae hapX is encoded by an open reading frame of 1425 bp with a deduced protein of 474 amino acids. Furthermore, HapX of A. benhamiae displays all the typical characteristics which are common to this class of transcription factors, including the basic region leucine zipper (bZIP) and coiled-coil domains mediating DNA-binding, an N-terminal CCAAT-binding complex (CBC) domain, which is essential for interaction with the CBC subunit HapE [A. fumigatus, two of the four CRR are known to be involved in detoxification of iron excess [BLASTP search revealed a single HapX homologue in the genome of nit HapE  and fourn excess .hapX mutants were generated in the wild-type strain A. benhamiae LAU2354-2 by targeted hapX gene deletion with the hph resistance cassette  and the reconstituted strain AbenHAPXK1A (hapXC) were used for further analysis.To assess the functional role of HapX, Δcassette . To ensuapX gene . SoutherapX gene . The delhapX mutant during iron starvation, but not during iron-replete conditions  and m/z 769 (M-H−) were found by MS analyses. MS/MS analyses and dereplication with commercial databases suggested a potential identity of the compounds with the hydroxamate-type siderophores ferrichrome C and ferricrocin, respectively. To unequivocally prove the structures, their UV spectra, HRESIMS-spectra, MS/MS fragmentation pattern as well as HPLC retention times were compared to authentic standards  and sidA  of A. benhamiae are clustered , cycA (cytochrome C) and lysF (homoaconitase) are involved in heme biosynthesis, respiration and lysine biosynthesis, respectively [cccA, hemA, cycA and lysF were significantly increased in the ΔhapX mutant during iron starvation, but not during iron-replete conditions in comparison to the wild type  in A. benhamiae wild type but not in the ΔhapX mutant (cccA in A. benhamiae wild type and the ΔhapX mutant were observed during growth in medium supplemented with 3 mM FeSO4 (hFe)  .in vitro growth of A. benhamiae wild type, ΔhapX mutant and hapXC reconstituted strain on human hair and nails as well as on keratin powder derived from hooves and horns was analyzed. No growth differences were observed between A. benhamiae wild type, ΔhapX mutant and hapXC reconstituted strain during infection of all tested keratin substrates . Although SITs are quite conserved in siderophore producing and non-producing fungal species only a few SITs have been identified and functionally characterized, so far . In derF. oxysporum [A. benhamiae are clustered which is typical for genes encoding components of common pathways in filamentous fungi. Similar to U. maydis, S. pombe, F. graminearum and F. oxysporum the genes sidA and sidC of A. benhamiae are probably bidirectionally transcribed from a common promoter region [sidA and sidC of A. nidulans and A. fumigatus are located on different chromosomes [A. fumigatus [A. benhamiae  which are interconnected by a negative feed-back loop [A. benhamiae. SreA represses the expression of hapX and the siderophore system during iron sufficient conditions by an iron-sensing mechanism, while HapX represses sreA and activates the siderophore system during iron-limiting conditions resulting in efficient iron uptake and inhibition of iron-consuming pathways. As described above, deletion of hapX in A. benhamiae, A. fumigatus and A. nidulans resulted to some extent in a decreased siderophore production [A. fumgatus [Transcriptional regulation of iron homeostasis and regulation of the siderophore system in ack loop , 61, 62.oduction , 19. At fumgatus . Furtherfumgatus –67.A. fumigatus. The intermediate mevalonate produced by the HMG-CoA reductase serves as precursor for the biosynthesis of fusarinine C and TAFC [A. fumigatus, the transcript level of the gene encoding the HMG-CoA reductase, hmg1, is highly increased during iron starvation [hmg1 in A. benhamiae. This is in line with data from the non-siderophore producing fungi S. cerevisiae, C. neoformans and C. albicans [hmg1 in U. maydis [U. maydis [The main precursor for fungal siderophores is the non proteinogenic amino acid ornithine . Additioand TAFC . In A. farvation . In contalbicans –70. Simi. maydis  despite . maydis .A. benhamiae resulted in decreased growth, conidiation and complete deregulation of genes from iron-dependent pathways such as vacuolar iron storage, amino acid metabolism, respiration and heme biosynthesis during iron limitation. The transcript level of cccA (vacuolar iron importer), lysF (homoaconitase), cycA (cytochrome C) and hemA (5-amino-levulinic acid synthase) was highly upregulated in the ΔhapX mutant during iron starvation. Similar results have been obtained for loss of HapX homologues in A. nidulans, A. fumigatus, F. oxysporum, C. neoformans, C. albicans and S. pombe [hapX mutant of A. benhamiae causing a reddish pigmentation of the mycelium. The same has been previously reported from A. nidulans, A. fumigatus and F. oxysporum [Besides altered regulation of siderophore biosynthesis, deletion of HapX in S. pombe , 61, 62.xysporum , 19, 29.hapX inhibited growth of A. fumigatus, A. nidulans, F. oxysporum and A. benhamiae in the presence of excess iron . Similasistance . Additiomitation . CharactCAAT box . A similenhamiae .A. fumigatus, C. albicans, F. oxysporum and to a lesser extent in C. neoformans in murine infection models [hapX mutant of A. benhamiae did not show a virulence defect during in vitro infection of hair and nails. A major difference between the human pathogenic fungi A. fumigatus, C. albicans, C. neoformans and A. benhamiae is the infectious life cycle. In contrast to A. fumigatus, C. albicans and C. neoformans, which are able to grow invasively in immunocompromised individuals, A. benhamiae is restricted to superficial growth on keratinized host structures such as skin (stratum corneum), hair and nails of humans and animals. We hypothesized that skin, hair and nails constitute a highly iron-restricted environment, but the ability of the ΔhapX mutant of A. benhamiae to grow on these keratinized structures might result from sufficient iron acquisition. In support of this idea, it has been described that iron is excreted by skin through desquamation of iron-loaded epithelial cells [hapX mutant of A. benhamiae on keratin. Besides siderophores, the A. benhamiae genome encodes proteins which represent homologues of iron permeases and oxidoreductases known to be involved in reductive iron assimilation (RIA)  . These pnidulans , 78, 79.nidulans –82 whichinocytes . This rein vivo, but is redundant in A. benhamiae during in vitro infection of keratinized host tissues, which might reflect the different iron supply or requirements of fungi during their respective infectious life cycle.This study underlines the highly conserved role of the fungal-specific transcription factor HapX for adaptation to iron starvation and especially, its relevance for the downregulation of iron-consuming pathways and the activation of siderophore biosynthesis during iron deficiency in ascomycetous fungi. Furthermore, HapX is a virulence factor in many plant and human pathogenic fungi S1 FigThe N-terminal CBC binding domain is indicated by bold letters, the bZIP domain is shaded in black, the coiled-coil domain is highlighted in grey and the four conserved cysteine-rich regions are indicated by black lines.(TIF)Click here for additional data file.S2 Figm/z 771. (B) Ferrichrome C standard m/z 755. (C) Mycelial extract of A. benhamiae wild type m/z 771. (D) Mycelial extract of A. benhamiae wild type m/z 755.(A) Ferricrocin standard (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Epiphyas postvittana), was also quantified. Questionnaires were used to evaluate winegrowers’ perceptions of the value of and problems associated with such endemic plant species in their vineyards. Growth and survival rates of the 14 plant species, in eight families, were evaluated, with Leptinella dioica (Asteraceae) and Acaena inermis ‘purpurea’ (Rosaceae) having the highest growth rates in terms of area covered and the highest survival rate after 12 months. All 14 plant species suppressed weeds, with Leptinella squalida, Geranium sessiliforum (Geraniaceae), Hebe chathamica (Plantaginaceae), Scleranthus uniflorus (Caryophyllaceae) and L. dioica, each reducing weed cover by >95%. Plant species also differed in the diversity of arthropods that they supported, with the Shannon Wiener diversity index (H′) for these taxa ranging from 0 to 1.3. G. sessiliforum and Muehlenbeckia axillaris (Polygonaceae) had the highest invertebrate diversity. Density of spiders was correlated with arthropod diversity and G. sessiliflorum and H. chathamica had the highest densities of these arthropods. Several plant species associated with higher soil moisture content than in control plots. The best performing species in this context were A. inermis ‘purpurea’ and Lobelia angulata (Lobeliaceae). Soil beneath all plant species had a higher microbial activity than in control plots, with L. dioica being highest in this respect. Survival proportion to the adult stage of the moth pest, E. postvittana, on all plant species was poor (<0.3). When judged by a ranking combining multiple criteria, the most promising plant species were (in decreasing order) G. sessiliflorum, A. inermis ‘purpurea’, H. chathamica, M. axillaris, L. dioica, L. angulata, L. squalida and S. uniflorus. Winegrowers surveyed said that they probably would deploy endemic plants around their vines. This research demonstrates that enhancing plant diversity in vineyards can deliver SPUs, harbour ESPs and therefore deliver ES. The data also shows that growers are willing to follow these protocols, with appropriate advice founded on sound research.Vineyards worldwide occupy over 7 million hectares and are typically virtual monocultures, with high and costly inputs of water and agro-chemicals. Understanding and enhancing ecosystem services can reduce inputs and their costs and help satisfy market demands for evidence of more sustainable practices. In this New Zealand work, low-growing, endemic plant species were evaluated for their potential benefits as Service Providing Units (SPUs) or Ecosystem Service Providers (ESPs). The services provided were weed suppression, conservation of beneficial invertebrates, soil moisture retention and microbial activity. The potential Ecosystem Dis-services (EDS) from the selected plant species by hosting the larvae of a key vine moth pest, the light-brown apple moth ( Fagopyrum esculentum Moench. and the predators and parasites which it supports can deliver multiple ES, including enhanced biological control of insect pests  can be quantified and enhanced , 2016. Ect pests , 2008.In situ plant conservation continues to have a key role  between the rows and sometimes with a few other spontaneous weed species  every two weeks. The whole experiment utilised an area of the vineyard which was allocated by the company. No further space was available so plot size had to be restricted to the area around a single vine. Although this has implications for invertebrates moving between treatments, the latter were separated by two vines within a row and by an inter-row distance of 2.3 m, the latter comprising dense turf of perennial ryegrass. The trial was located in the Waipara region, North Canterbury, New Zealand  within the rows of grapevines . Mean annual rainfall at the site was 684 mm, mean January (summer) temperature was 23 °C and soil type was Glasnevin silty loam , 2002. TMuehlenbeckia axillaris (Hook.f.) Endl.  were New Zealand endemic species and all were perennial. Successful growth and survival of the plants were seen as prerequisites for their ability to provide sustainable benefits to the vineyard operation. Consequently, these parameters were assessed 6, 12 and 24 months after planting.Plant species were selected based on their growth habit (1–15 cm in height) to minimise interference with vine management. Species were further selected based on their shallow roots, floral characteristics and tolerance to frost, exposure, sun, drought and disturbance as well as practicalities such as cost and availability. All selected plants apart from Disphyma australe  Aiton, Muehlenbeckia ephedroides Hook.f. and Raoulia subsericea Hook.f. were not assessed due to their poor condition, growth and survival. Data were statistically analysed using a randomised block analysis of variance (ANOVA), followed by the unprotected Least Significant Difference (LSD) procedure at P = 0.05  in the five selected blocks. Percentage cover of the study plants and weeds was recorded. P = 0.05 , 1990.L. perenne) adjacent to the experimental plots in each of the ten blocks. The sampler was set on maximum power for 10 s, within which time an area of 0.04 m2 was sampled at each location. Collected invertebrates were stored in 70% ethanol before being brought to the laboratory for sorting and identification. Due to gaps in formal taxonomic definitions, individuals were assigned to RTUs (recognisable taxonomic unit) for statistical analysis of diversity and abundance. For the second and third sampling dates, R. subsericea, M. ephedroides and D. australe were not sampled nor analysed because of their poor growth and survival. The Shannon Wiener diversity index (H′) was used because it takes into account evenness and species richness  under-vine treatments were assessed for invertebrate diversity and abundance using a suction sampler , 1994. Irichness , 1988. Srichness , 2007, tGeranium sessiliforum Simpson et Thomson, Hebe chathamica Cockayne et allan, Leptinella dioica Hook.f., M. axillaris and Lobelia angulata G. Forst. Control plots (bare earth) were also assessed.The effect of plant species on soil moisture and microbial activity was assessed. Due to resource constraints, only six plant species  were assessed. These were Soil microbial activity was assessed by the TCC method see , 1995. TE. postvittana on the vegetative parts of the plant species was recorded in a laboratory bioassay. Species supporting high larval development rates could potentially exacerbate pest problems in the vineyard by acting as a suitable host. However, there is also the possibility that these species could act as trap plants  first-instar larvae were placed in each of six Petri dishes (15 × 120 mm) in each of ten treatments. Treatments comprised freshly cut plant material with shoots inserted into an Eppendorf tube filled with water. Each tube was placed in a Petri dish which was sealed with plastic food wrap to prevent larval escape. After seven days, plant material was examined and water changed or the plant replaced as necessary. The artificial diet treatment consisted of cut squares of the diet substrate on which first instar-larvae were placed. There were six replicates of each treatment , arranged in a randomised block design under a 16:8 L/D photoperiod at 20 °C ±3. The number of larvae surviving to each development stage  was recorded. A generalised linear model with a binomial distribution was used to determine the effect of treatment and development stage on E. postvittana survival.The larval development of p plants , 2008. Tp plants , 1965 weNOT to use endemic plants in or around your vineyard in the above ways?” (see ‘Winegrower questionnaires’). This information was used to ensure that recommendations to growers were feasible and to identify future research directions.Experimental work on ecosystem services enhancement in agriculture is of limited practical value unless agriculturalists are provided with ESPs , 2005 orL. dioica and A. inermis ‘purpurea’ showed greatest growth after 12 months while Anaphalioides bellidioides Glenny, M. ephedroides, R. subsericea and D. australe had little or no growth. After 24 months, survival remained high (≥90% ) for M. axillaris, L. dioica, Raoulia hookeri Allan var. hookeri, A. inermis ‘purpurea’ and G. sessiliflorum while that of other plants had begun to declineSignificant differences in coverage  between plant treatments were found after 6 and 12 months . L. dioiP < 0.05)  but also included Poaceae, Malvaceae and Asteraceae families.There was significantly more weed growth in the control compared to all plant treatments ( < 0.05) . L. squapression . Weeds cAt all sampling dates there was a significant effect of treatment on invertebrate diversity and there was greater overall abundance in the summer (January and March) than in winter (August) . A totalM. axillaris, G. sessiliflorum, A. bellidioides, L. dioica, L. squalida, L. angulata, A. inermis and R. hookeri had significantly higher diversity than either of the controls (P < 0.05) , while these and A. inermis, H. chathamica, A. bellidioides, L. squalida and L. dioica had significantly higher diversity indices than the bare earth control (P < 0.05)  (P < 0.05) (During early summer (January 2009),  < 0.05) . In late < 0.05) . In wint < 0.05) .A significant effect of treatment on spider density was found for all sampling dates, with highest spider abundance in March 2009 . Spider G. sessiliflorum and H. chathamica consistently had the highest densities of spiders. A. inermis ‘purpurea,’ A. bellidioides, L. angulata and M. axillaris also had significantly higher spider densities than did the bare earth control treatment on at least one of the sampling dates.Spider families included web-building spiders in the Theridiidae , Linyphiidae , Agelenidae (Koch) and Amaurobiidae (Thorell) families. Wandering/hunting spider families included Oxyopidae (Thorell), Salticidae , Gnaphosidae (Pocock), Clubionidae (Wagner) and Pisauridae (Simon).L. dioica treatment. In November 2009, it was significantly higher below L. angulata and A. inermis ‘purpurea’ compared to all other treatments (P < 0.05)  .L. dioica compared to that under the other plant treatments (P < 0.05)  . AlthougP < 0.001) and the larval instar reached (P < 0.001) on survival of the pest E. postvittana, but there was no significant interaction between treatment and instar (P = 0.99)  . SurvivaIn L. dioica ranked first for growth, survival and microbial activity, but ranked 10 out of 11 for spider density, and 7 out of 11 for invertebrate diversity. By comparison, G. sessiliflorum ranked first for weed suppression, invertebrate diversity and spider density, but ranked 8 out of 9 for leafroller (pest) survival.Some plant species were not evaluated for all characteristics, often because they had already been judged unsuitable. Only six species were assessed in all respects . None ofG. sessiliflorum, A. inermis ‘purpurea,’ H. chathamica, M. axillaris, L. dioica and L. angulata, with average ranks ranging from 3.8 to 5.0, respectively  ectively . None ofThe survey response rate was 30 out of 56 growers . The majGrowers were also asked to indicate whether certain factors had led them not to deploy endemic plants for the uses listed above . These, G. sessiliflorum, A. inermis ‘purpurea,’ H. chathamica, M. axillaris, L. dioica and L. angulata.Findings here suggest the selected endemic plants deployed beneath vines have the potential to improve pathways to ES provision  ultimately improving value to growers. Overall, certain endemic plant species may preserve biodiversity, enhance biological control of vineyard pests, provide weed suppression and improve soil health. Clearly further research is required, such as repeating the trial in different regions. In the first trial described in this paper, however, the most promising plant species were Management of weeds is a major concern of vineyard managers as these plants can compete with the vines’ surface ‘feeder’ roots for resources and can act as refuges for pests , scales (Hemiptera: Coccidae) and mites (Acari: Eriophyidae)  theory and practice , 2012. Iphyidae) , 2007. Tphyidae) , 2010. This pest , 2014.For all plant species, the estimated soil moisture was always similar to or higher than the control on all three sample dates. It is well established that competition for water between the crop and added plant biodiversity can be a major factor in farmers’ agronomic decision making , 2007. HL. dioica, A. inermis ‘purpurea’ and M. axillaris) to be the most promising for vineyard deployment by their growth and floral resource, L. dioica and M. axillaris supported the lowest mean larval survival and, along with the other plants tested  which have value beyond the farm , 2013. CA. inermis is endemic to New Zealand, it is now available commercially in the UK and USA and as seeds in New Zealand (http://www.nzseeds.co.nz/contact-us). The work presented here addresses a key current challenge, which is to maintain or enhance productivity of agro-ecosystems in a sustainable way and to reduce external costs by increasing the role that ES can play on farmland, while at the same time maintaining ecological integrity in the cultural landscape. This is critical to not only fulfilling international agreements on biodiversity protection, but also for the commercial benefit of an authentic ‘clean green’ brand. Meeting these challenges has been called ‘sustainable intensification’ (www.ipbes.net).Endemic New Zealand plants beneath grapevines can provide multiple potential ecosystem services, including weed suppression, biodiversity conservation, soil improvement and conservation biological control. In some cases in the current work, the plants constituted RTUs and harboured ESPs. For example, the added plant populations were SPUs for services such as biological control, they enhanced ESPs such as spiders and provided ES in the form of weed suppression and enhanced soil quality, expressed as higher moisture and microbial activity. Winegrowers are likely to establish endemic plants within vineyards if perceived and real barriers to such action are overcome. These include growers’ lack of knowledge, initial investment, risk and disruption to normal practices. Also, farmers learn about and adopt new practices in a range of ways, and social learning , 2007 isication’ , 2014 an10.7717/peerj.2042/supp-1Data S1Click here for additional data file.


Acinetobacter genus are present mainly in the external environment  and in animals, while in humans they may comprise physiological flora. The main pathogenic species is Acinetobacter baumannii complex, which constitutes a common cause of nosocomial infections, particularly in patients with underlying diseases and risk factors ; however, infections of the eye caused by strains of Acinetobacter spp. are very rare. We report a unique case of community-acquired corneal ulcer caused by Acinetobacter non-baumannii (possibly A. junii), in a patient with no risk factors identified. The case highlights the need for obtaining a sample from the cornea for bacteriological culture in the case of suspected ophthalmic infection as identification of the pathogen, and assessment of its susceptibility profile enables proper antibiotic therapy, improves the outcome and may constitute an eyesight-saving management.Rods of the  Acinetobacter genus are Gram-negative coccobacilli. They are present mainly in the external environment  and in animals, while in humans they may comprise physiological flora, particularly in hot and humid climate countries [Acinetobacter baumannii complex, which constitutes a common cause of nosocomial infections, particularly in patients with underlying diseases and risk factors  [Acinetobacter spp. are uncommon. We present a unique case of community-acquired Acinetobacter non-baumannii (possibly A. junii) corneal ulcer in a patient without recognised risk factors.Rods of the ountries . The maiilation) , 2. HoweA. junii in a 51-year-old woman with no significant past medical history. She was admitted to the E.R. with a diagnosis of corneal ulcer (ulcerative keratitis) and anterior uveitis of the right eye. For the previous 2 days she complained of flu-like symptoms and pain of the affected eyeball. She was generally in good health, with no history of ophthalmological diseases or treatment. Eight years earlier she was diagnosed with unexplained anaemia, but results of the recent blood tests were within the normal range.We report a community-acquired corneal ulcer caused by On examination the eyelid and conjunctiva were swollen, with deep and superficial conjunctival injection. There was a large , white and deep inflammatory infiltrate. A 2-mm hypopyon (accumulation of pus in the anterior chamber) was noticed. Best corrected visual acuity (BCVA) was 0.2.A. junii was isolated in pure culture and identified susceptible to all tested antimicrobials: aminoglycosides , tetracycline, chloramphenicol, norfloxacin and sulphonamides. The species identification and susceptibility testing was performed in a clinical microbiology laboratory using an automated system VITEK® 2 Compact (bioMerieux). It should be noted, however, that according to the present data the identification of A. junii by this automated system is uncertain, in contrast to A. baumannii complex [Corneal scrapings were taken at presentation for microbiological examination. A strain of  complex .Empiric antibiotic therapy was started on admission and comprised systemic ciprofloxacin (0.2 g b.d.) and topical chloramphenicol ointment (q.i.d.). After obtaining the culture result and an antibiogram of the isolate, this treatment was continued as the strain was susceptible to the antibiotics used in empiric therapy. In the meantime (whilst awaiting the culture results) there was a clinical improvement of the condition of the patient’s eye.Four days later the corneal ulcer continued to diminish in size and depth, with gradual epithelisation noted in the periphery of the affected area. Descemet’s membrane folds decreased substantially and hypopyon virtually resolved, while fine dust-like deposits were detected on the corneal endothelium. The infection resolved within 2 weeks, with only slight opacity, thinning and single pigment granules still present in the lower nasal quadrant of the cornea. The patient was discharged with BCVA 0.7.Acinetobacter species are opportunistic pathogens causing nosocomial infections [Acinetobacter spp. are community acquired, reported primarily from countries with tropical or subtropical climate, and mainly affected patients with some form of comorbidity or associated with heavy smoking and excess alcohol consumption [Acinetobacter infections include mainly the respiratory tract, bloodstream infections, peritoneum, urinary tract infection, surgical wounds, meningitis, skin and soft tissue infections, while eye infections are rare [fections . Relativsumption , 5. Clinare rare .Acinetobacter spp. comprises acute or chronic conjunctivitis, corneal ulcers and dacryocystitis [Acinetobacter [Acinetobacter have been implicated in the aetiology of endophthalmitis and preseptal cellulitis usually preceded by bacteraemia [Acinetobacter spp. is rare and may result from trauma, contact lens wear or ocular surgery, e.g. penetrating keratoplasty—PKP or cataract surgery [Acinetobacter spp. comprised 2 % of strains cultured from clinical specimens, being second most common pathogen (after Pseudomonas) among Gram-negative bacteria [Acinetobacter may precede its appearance in sputum or blood cultures 1–2 days later [Acinetobacter keratitis has also been reported in a patient with chronic lymphocytic leukaemia [The spectrum of ocular infections caused by cystitis , 7. Expotobacter . Rods ofteraemia , 8–10. K surgery –13. In abacteria . Interesys later . Acinetoeukaemia .Acinetobacter spp. may be particularly important in sedated intensive care unit patients [Ocular surface colonisation by patients . Incomplpatients .Acinetobacter spp. are endogenous, without any obvious predisposing factors [A. baumannii endophthalmitis has been reported, which resulted from intravitreal ranibizumab injection [A. baumannii as well as other species such as A. anitratus or A. lwoffii [Acinetobacter species, e.g. A. gyllenbergii-like isolate, have been implicated in ocular infections as well [In some cases infections caused by  factors , 9. Clin factors , 13. Recnjection . Eye inf lwoffii , 8, 11.  as well .Acinetobacter junii rarely causes infections in humans [A. junii constitute 3.6–4.8 % of all Acinetobacter spp. isolates—12/331 and 9/186, respectively [A. junii infections 80 % were hospital-acquired; four patients died (11.4 %) despite adequate antibiotic therapy in three cases [Acinetobacter junii has been associated mainly with bacteremia and sepsis in neonates and paediatric oncologic patients [A. junii in an adult oncologic patient [n humans . In largectively , 21. It ectively . Out of ee cases . Only onpatients . Linde e patient .A. junii-johnsonii. Corneal perforation due to A. junii has been reported very rarely [Henao-Martinez et al.  describey rarely , 24.A. non-baumannii species, including A. junii, may cause a community-acquired corneal ulceration in an otherwise healthy patient. Ophthalmologists should obtain a sample from the cornea for bacteriological culture in the case of suspected infection as identification of the unique causative pathogen, and its susceptibility profile enables administration of proper antibiotic therapy, improves the outcome and may constitute an eyesight-saving management.In conclusion it should be noted that


Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in southern China. The biosynthetic and metabolic pathways for a core secondary metabolite in L. macranthoides, chlorogenic acid (CGA), have been elucidated in many species. However, the mechanisms of CGA biosynthesis and the related gene regulatory network in L. macranthoides are still not well understood. In this study, CGA content was quantified by high performance liquid chromatography (HPLC), and CGA levels differed significantly among three tissues; specifically, the CGA content in young leaves (YL) was greater than that in young stems (YS), which was greater than that in mature flowers (MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads (average length 90 bp) and 76,453 unigenes (average length 703 bp). A total of 3,767 unigenes were involved in biosynthesis pathways of secondary metabolites. Of these unigenes, 80 were possibly related to CGA biosynthesis. Furthermore, differentially expressed genes (DEGs) were screened in different tissues including YL, MF and YS. In these tissues, 24 DEGs were found to be associated with CGA biosynthesis, including six phenylalanine ammonia lyase (PAL) genes, six 4-coumarate coenzyme A ligase (4CL) genes, four cinnamate 4-Hydroxylase (C4H) genes, seven hydroxycinnamoyl transferase/hydroxycinnamoyl-CoA quinate transferase HCT/HQT genes and one coumarate 3-hydroxylase (C3H) gene.These results further the understanding of CGA biosynthesis and the related regulatory network in L. macranthoides. Lonicera. macranthoides (L. macranthoides), a plant distributed widely in south China, belongs to Caprifoliaceae and is often used in traditional Chinese medicine. In the 2010 Chinese Pharmacopoeia, L. macranthoides is registered as “mountain honeysuckle” [L. macranthoides in the severe acute respiratory syndrome  and H1N1 (2009) outbreaks in China, the demand for L. macranthoides has dramatically increased. The entire plant of L. macranthoides, and particularly the buds and leaves, is used as medicine. Phenolic acids, flavonoids, volatile oils and saponins are major chemical components of L. macranthoides [L. macranthoides, which include antioxidant [L. macranthoides can be effectively applied in the treatment of H1N1 respiratory syndrome and hand-foot-and-mouth disease [L. macranthoides.SARS, 200 and H1N1ioxidant ,5, detoxioxidant , anti-inioxidant , anti-caioxidant ,9,10,11,ioxidant . Moreove disease . ChlorogAsteraceae, Solanaceae and Rubiaceae [Lonicera [CGA is a potent phenolic acids that is considered to have many important biological activities ,15,16,17ubiaceae . CGA accLonicera ,23.PAL, C4H and 4CL are key enzymes involved in the first three steps of CGA biosynthesis; however, the specific details of the roles of these enzymes in this process have not yet been fully elucidated [HQT) is a key enzyme acting downstream of the CGA metabolic pathway [HQT transgenic plants in tomato [HQT gene may be indispensable in CGA synthesis in L. japonica [CGA is a phenylpropanoid generated from the shikimic acid pathway of plant aerobic respiration. ucidated . Hydroxy pathway . The rat pathway  has beenn tomato . Moreovejaponica . Three djaponica . Second,japonica . Third, japonica . We hypoLonicera plants. Peng et al. (2010) cloned the HQT gene in L. japonica and demonstrated a correlation between HQT expression and CGA content in different tissues [PAL, 4CL, C4H and HQT using a transcriptome analysis in L. japonica, L. hypoglauca Miq. and L. macranthoides [L. japonica in recent years. Genes involved in the biosynthesis of the active ingredients in L. japonica were screened by transcriptome analysis of the flower and leaf tissues using 454 pyrosequencing [et al. (2012) identified genes associated with CGA and Luteoloside biosynthesis from transcriptome data in L. japonica [L. macranthoides, and the molecular mechanisms underlying the biosynthesis of important active components remain a mystery.There have been reports on the molecular mechanisms of CGA biosynthesis in  tissues . Yuan etnthoides . Severalquencing . Yuan etjaponica . HoweverL. macranthoides was performed using the Illumina HiSeq™ 2000 platform. To explore the correlation between key genes associated with the biosynthesis and metabolism of CGA and CGA contents, digital gene expression (DGE) analysis was performed on different tissues including flower, stem and leaf. RNA-Seq analysis identified differentially expressed genes (DEGs) that are potentially important for secondary metabolism and CGA biosynthesis in L. macranthoides. These results are practical genomic resources for future investigations into the biosynthesis of CGA and for potential genetic improvement of L. macranthoides.In the present study, CGA in different tissues were determined using high performance liquid chromatography (HPLC), and transcriptome-wide sequencing in L. macranthoides test site in Chongqing China . All of the tissues were cut into small pieces, frozen in liquid nitrogen and stored at -80°C until further use.Plant materials including mature flowers , young leaves  and young stem  were collected for transcriptome analysis and CGA content measurement. These samples were planted in the Tissue samples were processed in a series of steps, including lyophilization, grinding and passage through a 40 mesh sieve before CGA extraction. Then, 50 mL of ethanol  was mixed with 0.5 g of the processed samples, and the mixture was subjected to ultrasonic extraction for 30 min. It was then centrifuged at 4000 rpm for 10 min. The supernatant was filtered through 0.45 μM microfiltration membrane for CGA analysis.HPLC was performed on a Shimadzu LC-20A HPLC analyzer , equipped with a LC-20AT pump, SIL-20A autosampler, CBM-20A system controller, SPD-M20A diode array detector and CTO-20A column oven. Separation was carried out using a Shimadzu Shim-PackVP-ODS C18 column  and 2% formic acid  as the mobile phase at a 1.0 mL/min flow rate. The column and the detector were operated at 35°C. A volume of 20 μL was injected and the HPLC chromatogram was monitored at 320 nm. CGA standard (≥ 98%) was purchased from SIGMA . The CGA contents were analyzed in triplicate and calculated based on peak area measurements. Statistical significance was performed with SPSS using Duncan's new multiple range test.Total RNA was extracted from the tissues, including flower, leaf and stem, using TRIzol reagent . Equal amounts of RNA from each of the three, tissues were mixed for transcriptome sequencing. Poly A mRNA was enriched using oligo (dT) magnetic beads and fragmented. The short fragments were served as templates to synthesize first strand cDNA using random primers, and then the second cDNA strand was synthesized. Subsequently, cDNAs were end-repaired, A-tailed and ligated with sequencing adaptors. The cDNAs were size selected and PCR amplified to generate the final library for sequencing using the Illumina HiSeq™ 2000 platform.de novo transcriptome assembly.The raw reads were generated via image analysis and base calling procedures. Then, the raw reads were filtered before assembly and mapping by removing adaptors, reads with an N ratio greater than 5% and low-quality reads, which resulted in clean reads. Trinity software  was emplFirst, overlapping reads of a certain length were assembled into contigs. Then, the paired-end reads were mapped to contigs, and the different contigs in the same transcript were identified, as well as the distances between them. Subsequently, contigs were assembled using Trinity to generate a unigene sequence that could not be extended at either of the two ends. The final set of unigenes was obtained using homologous clustering of the assembled transcripts and by removing redundant unigenes using TGICL (TGI clustering tools) .After homologous clustering, the unigenes were divided into two categories, clusters and singletons. In the same cluster, there were several unigenes with relatively high similarity (greater than 70%), which are noted as ‘CL’ followed by the gene family ID. The singletons represent single unigenes and are noted as ‘Unigene’ followed by the gene ID. Finally, the unigenes were aligned to protein sequence database entries including those in the NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups of proteins (COG) databases using BLASTx . The best alignment result was used to determine the direction of the unigene sequence. If the above four databases were not available, ESTScan software was employed to determine the direction of the sequence .To obtain functional annotation of a given unigene, the sequence was aligned against protein sequence database entries including those in Nr, Swiss-Prot, KEGG and COG using BLASTx, and aligned against nucleotide sequence database using BLASTn with a significance threshold of e-value < 0.00001. Based on Nr annotation, the GO categories were obtained using Blast2GO software . Then, GTotal RNA was extracted from different tissues including YL, YS and MF. Three libraries were established for RNA-Seq analysis, which were then sequenced using the Illumina HiSeq™ 2000 platform.The raw image data obtained from the sequencing were transformed into raw reads with base calling. Poor quality reads were removed to obtain clean reads for subsequent analyses. Short read alignment software SOAP aligner/soap2  was usedGene expression was calculated using the number of reads aligned to a single gene and the total number of reads aligned to reference sequences in the Reads per Kb per Million reads (RPKM) method , and theDEGs were identified by comparing data obtained from different samples as described by Audic et al. . In multhttp://www.geneontology.org/), and the number of genes mapped to each term was noted. Next, hypergeometric test were used to determine GO terms that were significantly enriched in the DEGs compared with the entire genome background. For pathway enrichment analysis, all DEGs were mapped to the KEGG database, and pathways with Q value ≤ 0.05 were considered significantly enriched in DEGs.With Cluster software , Euclidehttps://www.genscript.com/ssl-bin/app/primer)   and a meThe raw transcriptome reads reported here have been deposited in the NCBI Short Read Archive under accession Nos. SRX1003341, and the raw DGE data were deposited under accession Nos. SRX1003347 (MF), SRX1003358 (YL) and SRX1003352 (YS).Experiment was carried out in the field own by College of Forestry and Life Science, and no specific permissions were required for these locations/activities. No endangered or protected species or locations were involved in this study.L. macranthoides were measured using HPLC (P < 0.05), YL  > YS (48.7857 ± 0.0523) > MF (37.4027 ± 0.2437).The CGA contents in the MF, YL and YS of ing HPLC . The CGAL. macranthoides were not available, transcriptome analysis of mixed tissues was performed to obtain genome data. A total of 60,287,590 raw reads were obtained, corresponding to 53,533,014 clean reads with an average length of 90 bp. These reads were further assembled into 145,214 sequences (contigs), with an average length of 342 bp  were annotated using BLASTx, with a threshold of 10L. macranthoides unigenes were assigned three GO categories, molecular function, cellular component and biological process. Based on sequence homology, 38,533 sequences were classified into 47 functional groups of the three ontologies , biosynthesis of secondary metabolites (3194) and plant-pathogen interaction (1633). These annotations provide valuable information for further investigations of the special metabolic processes, functions and pathways in L. macranthoides. Interestingly, 3767 unigenes were mapped to 33 pathways associated with the biosynthesis of secondary metabolites  and the percentage of clean reads in each library was more than 99.4% , 3,122,293 (86.72%) and 3,240,456 (87.10%) reads mapped to reference genes, respectively. Only reads aligning entirely inside exonic regions could be matched, whereas those from exon-exon junction regions could not be matched). For all the total mapped reads, the unique match and multi-position match in MF, YL and YS were 2,361,848 (66.53%) and 710,030 (20.00%), 2,438,931 (67.74%) and 683,362 (18.98%), and 2,495,081 (67.07%) and 745,375 (20.04%), respectively .Gene coverage is the percentage of a gene covered by the reads. This value is equal to the ratio of the base number in a gene covered by unique mapping reads to the total bases number of that gene. The distribution of each tissue in the reference genome's coverage and sequencing depth are listed in are listed in The differences in gene expression patterns were then analyzed for the pairs MF and YL, MF and YS, YS and YL . A totalL. macranthoides is a relative of L. japonica, and the CGA content in L. macranthoides was two-fold higher than of the content in L. japonica [L. macranthoides was greater than the number in L. japonica  was down-regulated in YS and MF compared with YL, and three genes  were up-regulated. Moreover, the expression levels of CL3556.contig2 and 4 were up- and down-regulated respectively and down-regulated in MF respectively as compared to YL, whereas CL3556.contig2 expression was down-regulated in YS with respect to YL. The six 4CL genes identified by RNA-Seq showed up-regulation in MF in comparison to YL. Compared to YL, except for CL3820.contig2, all 4CL genes were up-regulated in YS. Four C4H genes were differentially expressed in the three tissues. With respect to YL, except for CL6280.contig1 showing up-regulation, no obvious change was observed in the other three genes in YS. Furthermore, all four C4H genes were down-regulated in MF. Seven HCT/HQT genes were differentially expressed in different tissues. The other six genes showed significantly lower expression in MF than in YL excepted for Unigene13576. Similarly, the genes including CL7436.contig1, Unigene13202, Unigene29578 and Unigene34376 were dramatically down-regulated in YS compared with YL. The expression of one C3H gene (Unigene23258) was down-regulated in MF compared with YL, while it was up-regulated in YS. These DEGs, especially those functioning downstream in the metabolic pathway, may provide valuable clues for elucidating the CGA biosynthesis pathway.RNA-Seq showed that some of the genes predicted to be involved in CGA synthesis were differentially expressed among YL, YS and MF in  pathway . These gHCT and HQT are key genes involved in CGA biosynthesis in plants [HCT/HQT genes between L. macranthoides and other species, a phylogenetic tree was generated based on seven differentially expressed HCT/HQT genes and 18 sequences obtained from NCBI. Unigene32558 was found to share a relatively high sequence homology with the HCT genes in Cynara cardunculus , Platycodon grandiflorus (JN392753.1), Coffea canephora  and Coffea Arabica (EF143341.1) . It is noteworthy that the CL3069.contig1 and Unigene13202 share relatively high sequence homology with the HCT sequences in Pyrus bretschneideri (JQ280304.1) and Camellia sinensis (JQ619537.1), whereas Unigene13576, Unigene29578 and Unigene34376 did not show high sequence homology with HCT/HQT genes in other species.Many studies have shown that n plants ,25,26. T43341.1) . SimilarPAL: CL3556.contig4; C4H: CL11374.Contig2; HQT/HCT: CL7436.Contig1, Unigene13202_LmYL, and Unigene34376_LmYL) and six genes randomly selected from the expression profile data . All of these genes were differentially expressed among the three different tissues, and the data were consistent with the RNA-Seq data  [L. macranthoides remains unclear. Several studies have focused on the transcriptome and expression profile of genes related to CGA synthesis in L. japonica [L. macranthoides [L. macranthoides. We used the Illumina HiSeq™ 2000 platform to investigate gene expression profiles and the key pathways for CGA biosynthesis in L. macranthoides. CGA content differs significantly in different tissues of L. macranthoides. The RNA from MF, YS and YL samples of L. macranthoides was mixed in equal amounts for transcriptome sequencing. A total of 76,453 unigenes were obtained. Based on these data, RNA-Seq analysis was performed on the three different tissues and DEGs were identified.aponica) . Howeverjaponica ,29,30. Hnthoides ,26. MoreLonicera plants is determined by multiple factors including developmental stage and growth environment [L. macranthoides, which was highest in YL and lowest in MF  corresponded well with CGA content in different tissues (PAL1 (79% identity) in Coffea canephora was identified to be in high expression at the small green stage, which correlated with the high production of CGA during the SG stage [PAL in CGA accumulation. We also found that the transcripts of three C4H genes  were positively correlated with the CGA content, indicating their important role in CGA biosynthesis. On the contrary, a previous study in L. japonica displayed that LjC4H2 is critical gene that regulate CGA content within the organs, not LjC4H1 which showed highest identity to the C4H genes (CL11374.contig1 and CL11374.contig2) in L. macranthoides identified in our study [PAL, C4H and 4CL genes in different tissues were very weak Click here for additional data file.S2 FigAfter filtering the only adaptor sequences, containing N sequences and low quality sequences, the three RNA-Seq libraries still generated over 3.5 million clean reads in each library and the percentage of clean reads among raw tags in each library over 99.4%.(JPG)Click here for additional data file.S1 FileA, Classfication of raw reads. B, Distribution analysis of the RNA-Seq tags in the three libraries.(JPG)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Lactobacillus spp. potentially contribute to health by modulating bacterial biofilm formation, but their effects on the overall oral microbiota remain unclear.Lactobacillus reuteri DSM 17938 and PTA 5289 consumption. Forty-four adults were assigned to a test group (n = 22) that received lactobacilli lozenges (108 CFU of each strain/lozenge) or a control group that received placebo (n = 22). Presence of L. reuteri was confirmed by cultivation and species specific PCR. Tooth biofilm samples from 16 adults before, during, and after exposure were analyzed by pyrosequencing. A total of 1,310,292 sequences were quality filtered. After removing single reads, 257 species or phylotypes were identified at 98.5% identity in the Human Oral Microbiome Database. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria were the most abundant phyla. Streptococcus was the most common genus and the S. oralis/S. mitis/S. mitis bv2/S. infantis group comprised the dominant species. The number of observed species was unaffected by L. reuteri exposure. However, subjects who had consumed L. reuteri were clustered in a principal coordinates analysis relative to scattering at baseline, and multivariate modeling of pyrosequencing microbiota, and culture and PCR detected L. reuteri separated baseline from 12-week samples in test subjects. L. reuteri intake correlated with increased S. oralis/S. mitis/S. mitis bv2/S. infantis group and Campylobacter concisus, Granulicatella adiacens, Bergeyella sp. HOT322, Neisseria subflava, and SR1 [G-1] sp. HOT874 detection and reduced S. mutans, S. anginosus, N. mucosa, Fusobacterium periodicum, F. nucleatum ss vincentii, and Prevotella maculosa detection. This effect had disappeared 1 month after exposure was terminated.Oral microbiota was characterized via 454-pyrosequencing of the 16S rDNA hypervariable region V3-V4 after 12 weeks of daily L. reuteri consumption did not affect species richness but induced a shift in the oral microbiota composition. The biological relevance of this remains to be elucidated.NCT02311218ClinicalTrials.gov  Differences between groups were tested with parametric or non-parametric tests depending on the data measurement and distribution levels. P < 0.05 was considered statistically significant.http://www.homd.org)  sp. HOT874, and the S. oralis/S. mitis/S. mitis bv2/S. infantis group were associated with consumption of the L. reuteri-containing lozenges for 12 weeks . The PCoA projection of beta diversity in the baseline samples versus the 1-month follow-up samples showed no sample clustering, such as that observed in the 12-week treatment samples . FurtherL. reuteri strains DMS 17938 and PTA 5289 for 12 weeks would modify the microbiota composition and species richness in tooth biofilms. The main findings were as follows: (i) the composition shifted but species richness remained unaffected, (ii) the shift normalized within 1 month after terminating exposure, and (iii) the test strains could not be detected in approximately 30% of the participants despite receiving daily booster doses.The present study evaluated if consuming the probiotic S. mutans and S. sobrinus) [L. reuteri DSM 17938 and PTA 5289 ingestion have been previously associated with reduced oral levels of mutans streptococci [S. mutans among total streptococci was lower in the test group after a 12-week period during which L. reuteri strains DSM 17938 and PTA 5289 were ingested. This finding partly reflected a reduction in mutans streptococci in some subjects  and partly reflected the increased numbers of other streptococci . However, this result should be interpreted with caution as mutans streptococcal colonization was not an inclusion criterion in the present study, in contrast to the referred studies [S. mutans (none were colonized with S. sobrinus). A potentially beneficial effect of L. reuteri exposure was the reduced prevalence of F. nucleatum ss vincentii and F. periodicum, which may be linked to reduced biofilm formation and gingivitis development during the ingestion of probiotic bacteria [The ecology of the gastrointestinal microbiota, including that of the mouth, is virtually stable once the period of variation in early childhood has passed . Previouobrinus) –27. L. rptococci ,25. Howeptococci ,48, the ptococci –51. In t studies ,25 and fbacteria .L. reuteri for a longer period (12 weeks) and mapped the microbiota by sequencing an approximately 400-base pair section of the 16S rRNA gene, revealed a transient reduction in diversity in the tooth biofilm samples, although this shift normalized within 1 month after intake was discontinued. This finding is in accordance with studies showing transient fluctuations in gut bacterial profiles after introducing probiotic products or diet changes along with a rapid reversion of the microbial community to its previous stable state [Two previous studies evaluated oral microbiota after the short-term use (3 or 4 weeks) of probiotic bacteria ,53. One le state .Streptococcus genus. For example, the relative prevalence rates of the 4 species in the most prevalent group  cannot be distinguished. Therefore, the species-level sequence abundance is partly due to the restrictive identification criteria and partly to the overall limitations of using 16S rRNA gene variation for species identification [The final number of species/phylotypes detected in the present study was somewhat lower than that reported in other studies; however, the phylum, genus, and species representation follow the patterns reported in other studies that analyzed comparable samples and populations , 55. Thefication , 57.L. reuteri, on the composition of tooth biofilm microbiota using 16S rRNA pyrosequencing. Multiplex sequencing is cost-effective for the following reasons: (i) thousands of sequences can be simultaneously obtained from a single sample [ii) open-ended view of the microbiome is provided, and (iii) answers regarding microbial richness and ecological stability and shift are facilitated [i) limitations on taxonomic resolution due to the short-length sequence limit [(ii), loss of an unknown amount of taxa because of the rather high detection limit [(iii) distortion of the true microbiota profile at various steps, i.e. contaminations in DNA extraction and purification, and PCR and sequencing errors and data analyses and interpretation [et al. [6, 104, and 102 CFU/mL, respectively. Those authors also reported that only 35% of bacterial phylotypes were detected in fecal samples by pyrosequencing. The fact that no L. reuteri or other lactobacilli (except two single sequences that were discarded according to the filtering criteria) was detected in 12-week samples from the test group, despite culture and PCR results demonstrating that the species and specifically the test strains were present in 6 of 7 test subjects, illustrates the underestimated number of detected species in the present study. Accordingly, metagenomic studies may need to consider this limitation as well as additional approaches for identifying less abundant, condition-associated genera/species. In contrast, the strengths of the study include the RCT design and high compliance and a double-blinded, randomized design in addition to the application of a new metagenomic approach combined with strain-specific PCR and cultivation.To the best of our knowledge, this is the first study to evaluate the effects of a probiotic bacterium, e sample , (ii) opilitated . Drawbacce limit  (ii), loon limit , and  have been made for decades . The facnosus GG ,34,64,65nosus GG ,66–68.L. reuteri consumption induces a shift in oral microbiota composition but does not affect the total species richness. The biological relevance of this finding as well as the individuality of this effect remains to be elucidated.The conclusion from the present study is that S1 CONSORT Checklist(PDF)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.S1 TableL. reuteri or placebo lozenge are listed.Mean abundances  for test and control subjects at baseline and after 12 weeks exposure to an (DOCX)Click here for additional data file.S2 TableL. reuteri or placebo lozenge are listed.Mean abundances  for test and control subjects at baseline and after 12 weeks exposure to an (DOCX)Click here for additional data file.


AbstractNeoperla, N. nigromarginatasp. n. and N. similiflavescenssp. n., are described from Dabie Mountains of Central China in the Liankangshan National Nature Reserve. The new species are compared with related congeners.Two new species of the stonefly genus  Neoperla is the most species-rich stonefly genus in the family Perlidae in China , Xinxiang, and the Entomological Museum of China Agricultural University (CAU), Beijing. They were examined with the aid of a Motic SMZ 168 microscope and the color illustrations were captured using digitalized Motic Images Advanced 3.2 software. All specimens were kept in 80% ethanol. Aedeagi were everted using the cold maceration technique of Taxon classificationAnimaliaPlecopteraPerlidaeLi & Zhangsp. n.http://zoobank.org/14EAFB99-1450-4DF2-8ACF-E7FDB99620A331°64,39'N, 114°87,95'E, light trap, 17 June 2014, W.H. Li. Paratypes: 4 females (2 in CAU), the same locality and data as holotype.Holotype. male (HIST), China: Henan Province, Xinyang City, Xin County, Liankangshan National Nature Reserve, Laomiao Protection station, Distance between ocelli slightly wider than diameter of the ocellus. Head pale yellow to brownish with black areas, slightly wider than pronotum, with a black rectangular area covering ocelli, a large black anterior spot and two small lateral spots present on frons Fig. ; compounMale. Forewing length 11.8 mm. Tergum 7 with an anteromedian pair of sclerotized, upraised, nipple-shaped processes and a distal subquadrate process on posterior margin, covered with small sensilla basiconica Fig. . Tergum Forewing length 13.4–14.0 mm. General color pattern similar to male. Sternum 7 slightly sclerotized except the posterior margin slightly produced forming sclerotized subgenital fig. Subgenital fig a small dark brown tab with slightly emarginate tip Fig. . SternumThe specific epithet refers to the dark lateral margins of pronotum.China (Henan Province).Neoperla montivaga species group as defined by Neoperla flagellata Li & Murányi, 2012 and Neoperla tuberculata Wu, 1937 . However, Neoperla nigromarginata is easily distinguishable from the latter two species by the distinctively pigmented pronotal lateral margins, short aedeagal sac (nearly as long as tube) with only dorsal spine patch at base and absence of a sac loop  forming a loop with lateral spine patches at base of aedeagal sac , China: Henan Province, Xinyang City, Xin County, Liankangshan National Nature Reserve, Laomiao Protection station, Forewing length 13.9 mm. Distance between ocelli barely as wide as diameter of the ocellus. Head slightly wider than pronotum, mostly yellow brown, lateral margins and occiput behind compound eyes pale, a triangular dark area covering ocelli, a dark spot in front of M-line and U-shaped brownish spot between M-line and ocellus, M-line pale; antennae brown to dark brown, scape darker; compound eyes black; mouthparts brown Fig. . PronotuTerminalia. Tergum 7 with trapezoidal median process at posterior margin, covered by numerous tiny sensilla basiconica, anterior and lateral margins forming elevated figau but with posteromedian concativity in which process of tergum 8 lies. Sclerotized process of tergum 8 recurved backward and triangular in shape. Tergum 9 with two submedial patches of long hairs. Hemitergal processes of tergum 10 strongly sclerotized and straight .PageBreakPageBreakPageBreakNeoperla montivaga species group as defined by Neoperla flavescens Chu originally known from Zhejiang Province, and recently also found from several provinces  of China (Neoperla flavescens Chu by the shape of ventral projection of aedeagal tube. The projection in Neoperla similiflavescens is generally triangular in lateral view and tapers to a triangular tip (Fig. Neoperla flavescens (Figs 4d & 5 in Neoperla similiflavescens is generally brownish in color and spots on frons are well defined and small (Fig. Neoperla flavescens, the general body color is brown and darker, the spots on frons are larger and obscure (Fig. The new species may be assigned to the tip Fig.  whereas all Fig.  while inure Fig. .Neoperla finding. Previous study on the stoneflies from the Dabie Mountains only includes the description of Neoperla jigongshana Li & Li, 2014 (Neoperla species are known from the Dabie Mountains presently.The present study is based on the insect collection of two years surveys to the Liankangshan National Nature Reserve organized by administrative bureau of the Reserve. In the year 2013 only several stonefly nymphs were collected before the present Li, 2014  from Mou


Hafnia alvei, a gram-negative facultative anaerobic, rod-shaped bacterium, is a rare cause of infection in humans. We report on a renal transplant patient who developed H. alvei pyelonephritis and urosepsis. The source of infection remains enigmatic but is most likely the intestinal tract. Appropriate antibiotic therapy with cefepime followed by oral ciprofloxacin brought about rapid resolution of symptoms and complete recovery. H. alvei may cause severe infection in transplant patients without predisposing factors such as hospitalization, invasive procedures, or antibiotic treatment. Hafnia alvei is a facultative anaerobic gram-negative bacterium belonging to the Enterobacteriaceae family. Although this genus was first described in 1954, very little is known about its role in human infectious diseases. H. alvei can be recovered from soil, water, and many animal species, especially mammals, where it colonises primarily the gastrointestinal tract [ H. alvei seems to be a member of the normal gut microbiome as a nonpathogenic inhabitant, although some studies conclude that these bacteria cause significant clinical gastroenteritis [ H. alvei is a microorganism with limited pathogenicity in humans that may cause clinically significant infections only in immunocompromised individuals, for example, those with malignancy or after organ transplantation [ H. alvei urosepsis.nteritis , especianteritis , but alsantation . We hereThe 56-year-old woman developed end-stage renal disease due to IgA nephropathy. In August 2012 she underwent preemptive kidney transplantation, receiving a kidney allograft from her husband. Her posttransplant course was stable with a serum creatinine of 0.90 mg/dL (eGFR 75 mL/min), and medical therapy consisted of tacrolimus (blood levels between 5 and 7 ng/mL), azathioprine, simvastatin, and pantoprazole. H. alvei identified by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF) . No coinfection with other bacteria could be identified. The results of antimicrobial susceptibility testing using the VITEK 2 system (bioMerieux) are shown in Two days before admission in November 2014 she experienced headache, shivering, nausea, repeated vomiting, and diarrhea. At admission she had fever of 38°C. Blood tests showed a mild leukocytosis of 10.5 G/L (neutrophils 84%) and CRP of 20 mg/dL and her serum creatinine had increased to 1.30 mg/dL. An ultrasound of the transplant was unremarkable. The urinary sediment contained multiple leukocytes and bacteria. A diagnosis of transplant pyelonephritis was made and cefepime 2 g b.i.d. was commenced. Cultures taken from urine and blood samples taken on admission grewUnder antibiotic therapy the patient's symptoms and fever subsided and serum creatinine decreased to 0.90 mg/dL within two days. After five days antibiotic therapy was switched to ciprofloxacin 500 mg b.i.d. and the patient was discharged. At an outpatient control one week later she was in excellent clinical condition, laboratory parameters had normalised, and antibiotic therapy could be discontinued. H. alvei in human clinical infection, some common themes do emerge. First of all, H. alvei is rarely found in human specimens, mostly from the respiratory and gastrointestinal tract, urine, blood, wounds, and abscesses [ H. alvei can cause acute gastroenteritis is still a matter of debate [ H. alvei in 80 specimens but considered it to be pathogenetic in only three patients (two with septicemia and one with peritonitis). Secondly, most patients seem to have underlying conditions which predispose them to infection. For example, in the series studied by Günthard and Pennekamp, 93% of the patients had an underlying disease, mainly malignancy. Thirdly, in most cases infection with H. alvei is nosocomial. Rodríguez-Guardado in a collective of 36 patients with extraintestinal infection due to H. alvei over an eleven-year period found that 25 were nosocomial and eleven community-acquired [ Staphylococcus aureus was seen [Although relatively little is known about the role ofbscesses , 10. In f debate . Güntharacquired . And las H. alvei isolates have been detected over the last ten years. Most positive specimens were obtained from respiratory tract secretions in intubated patients or from infections after abdominal surgery. Only three positive blood cultures were found and two of these patients had an underlying malignancy.At our institution 40 H. alvei infection have been described in stem cell and particularly solid organ transplant recipients. Intestinal colonization and possible infection with H. alvei have been reported in a 9-year-old girl after hematopoietic stem cell transplantation for Fanconi's anemia. Two patients, one 61-year-old woman and a 2-year-old boy, developed hepatic abscess after liver transplantation. The first patient had coinfection with Enterococcus faecalis and Candida albicans and the second with Enterococcus faecalis. Immunosuppressive and antimicrobial treatment are specified in Several cases of H. alvei infection has also been reported in two renal transplant recipients.Clinically significantThe first patient was a 69-year-old female who developed sepsis and pneumonia nine years after renal transplantation. Basal immunosuppression consisted of cyclosporine, azathioprine, and steroids. The patient also suffered from hepatitis C infection. She developed hypotension, oliguria, and coagulopathy and recovered after hemodynamic support and antibiotic therapy with imipenem and clarithromycin. H. alvei pyelonephritis developed after ureteral stent implantation for hydroureteronephrosis. The patient recovered after treatment with ceftriaxone, cefixime, and removal of the stent.The second case was a 45-year-old female suffering from ESRD due to diabetic nephropathy. Her course after transplantation was complicated by acute humoral rejection and chronic rejection. H. alvei in large amounts [ H. alvei is part of the normal gut microbiota, we believe that the gastrointestinal tract, as common in urinary tract infections, was the most likely source of infection. None of these kidney transplant patients had a coinfection with other more aggressive bacteria. The two other patients, however, had additional comorbidities, namely, diabetes in one and hepatitis C infection in the other patient. In one patient H. alvei infection developed after an invasive procedure. The other two patients had triple immunosuppressive therapy including prednisolone, whereas our patient only had double therapy without steroids. All three patients ultimately recovered after appropriate antibiotic therapy.Our case is similar to these two reports. We also were unable to identify the source of the infection. Certain foods, especially meat and fish, may harbour amounts . As H. a H. alvei can cause severe infection such as urosepsis, pyelonephritis, and pneumonia in renal allograft recipients. Antimicrobial therapy with carbapenems, quinolones, or fourth-generation cephalosporins is necessary to cure the infection.In conclusion,


Methotrexate (MTX) is a cornerstone and safe disease-modifying anti-rheumatic drug (DMARD) in the treatment of juvenile idiopathic arthritis (JIA). Despite its safety, MTX intolerance occurs frequently, leading to non-compliance, thus hampering efficacy and potentially leading to early discontinuation.The aim of this study was to construct a risk model to predict MTX intolerance.In a large, prospective JIA cohort, potential predictors  were determined at the time of MTX start. The Methotrexate Intolerance Severity Score was employed to measure MTX intolerance in the first year of treatment. MTX intolerance was most prevalent at 6 or 12 months after MTX start. This was defined as the outcome for the prediction model. The model was developed in 152 patients using multivariate logistic regression analysis. It was subsequently internally validated using bootstrapping.The prediction model included the following predictors: JIA subtype, antinuclear antibody, parent/patient assessment of pain, Juvenile Arthritis Disease Activity Score-27, thrombocytes, alanine aminotransferase and creatinine. The model classified 77.5% of patients correctly, changing to 66.7% after internal validation by bootstrapping. The prediction model was transformed into a risk score (range 0-17). At an optimal cut-off of ≥6, sensitivity was 82.0%, specificity 56.1%, positive predictive value was 58.7% and negative predictive value 80.4%.The prediction model combined routine clinical variables and showed good predictive power to detect MTX intolerance. This easy-to-use tool could assist clinicians in identifying patients at risk to develop MTX intolerance. Consequently, clinicians can monitor them closely and intervene timely before MTX intolerance hampers MTX efficacy.None declared.