P. patens). With the increase of SSRs repeat number the percentage of A/T in C. reinhardtii had no significant change, while the percentage of A/T in terrestrial plants species gradually declined. Third, in dinucleotide SSRs, the percentage of AT/TA increased along with the evolution of plant kingdom and the repeat number increased in terrestrial plants species. This trend was more obvious in dicotyledon than monocotyledon. The percentage of CG/GC showed the opposite pattern to the AT/TA. Forth, in trinucleotide SSRs, the percentages of combinations including two or three A/T were in a rising trend along with the evolution of plant kingdom; meanwhile with the increase of SSRs repeat number in plants species, different species chose different combinations as dominant SSRs. SSRs in C. reinhardtii, P. patens, Z. mays and A. thaliana showed their specific patterns related to evolutionary position or specific changes of genome sequences. The results showed that, SSRs not only had the general pattern in the evolution of plant kingdom, but also were associated with the evolution of the specific genome sequence. The study of the evolutionary regularities of SSRs provided new insights for the analysis of the plant genome evolution.Simple sequence repeats (SSRs) are widespread units on genome sequences, and play many important roles in plants. In order to reveal the evolution of plant genomes, we investigated the evolutionary regularities of SSRs during the evolution of plant species and the plant kingdom by analysis of twelve sequenced plant genome sequences. First, in the twelve studied plant genomes, the main SSRs were those which contain repeats of 1–3 nucleotides combination. Second, in mononucleotide SSRs, the A/T percentage gradually increased along with the evolution of plants (except for  Plant genomes are filled with low-complexity repetitive sequences. One of the most frequent low complexity sequences is simple sequence repeats  –4. SSRs Brassicales did so again in the Cenozoic [P. patens is a paleopolyploid as well. The genome duplication to have occurred between 30 and 60 million years ago [SSRs in plant genome sequences evolve along with the plant gene and genome evolution. Gene and genome duplications are major driving forces of gene diversification and evolution . AngiospCenozoic , 18. Mosears ago . Interesears ago –22. It hears ago .At the same time SSRs themselves are variations. One striking feature of SSRs is its high mutation rate . It is eThe distributions and characteristics of SSRs in plant genomes and their relation with the annotated genome components, mainly as genes sequences (including introns and exons), promoters and transposable elements, have been investigated –38. HoweArabidopsis thaliana Col-0 , Glycine max (G. max), Vitis vinifera (V. vinifera), Solanum lycopersicum (S. lycopersicum)), four monocotyledon species (Brachypodium distachyon (B. distachyon), Oryza sativa Japonica Group (O. sativa), Sorghum bicolor (S. bicolor), Zea mays (Z. mays)), one ferm species (Selaginella moellendorffii (S. moellendorffii)), one moss species (Physcomitrella patens (P. patens)), and two algae species (Chlamydomonas reinhardtii (C. reinhardtii), Volvox carteri (V. carteri)) were selected for analysis. To analyze whether the SSR distribution pattern is occurring randomly, the other two ecotypes of A. thaliana and Drosophila melanogaster (D. melanogaster) were selected for calculation. The genome sequences of A. thaliana (Col-0), B. distachyon, G. max, O. sativa (Japonica Group), S. lycopersicum, V. vinifera and D. melanogaster were downloaded from the National Center for Biotechnology Information (NCBI) genome database (ftp://ftp.ncbi.nlm.nih.gov/genomes/). The genome sequences of C. reinhardtii, P. patens, S. moellendorffii, S. bicolor and Z. mays were downloaded from the Ensembl plant database (http://plants.ensembl.org/). The genome sequence of V. carteri was downloaded from the PlantGDB dababase (http://www.plantgdb.org/). The genome sequences of A. thaliana ecotypes Ler-0 and Ws-0 were downloaded from http://mus.well.ox.ac.uk/19genomes/fasta/MASKED/. Details showed in In this study, four dicotyledon species  in a species.In the percentage analysis of SSRs section, we classified nucleotide combinations according to the principle of complementary base and sequence of nucleotide combination and analyzed the data according to different nucleotide combination groups. In mononucleotide SSRs, we classified adenine (A) repeat SSRs and thymine (T) repeat SSRs as a group; cytosine (C) repeat SSRs and guanine (G) repeat SSRs as another group. In dinucleotide SSRs, twelve nucleotide combinations were classified into four groups, named In the percentage analysis of SSRs based on repeat number section, we chose SSRs containing the same repeat number and having more than 1000 total SSRs number to analyze. We chose SSRs groups with the same repeat number and the same nucleotide number SSRs units as a whole (100%) in a species.The symmetrized Kullback–Leibler divergence analysis , a quantC. reinhardtii , V. carteri  and A. thaliana  (referring to the ecotype Col-0 hereinafter if not labeled) were smaller than the others, and the Z. mays genome  was the largest (C. reinhardtii (63.87%) and V. carteri (56.11%) genomes were GC-rich, and the other genomes were AT-rich (S. moellendorffii (45.23%) and monocotyledon (43.55%~46.89%) were approximately equal and the GC content in moss P. patens (33.60%) was close to that of dicotyledon (33.95%~36.03%)  .P. patens and dicotyledon were significantly higher than other plants. The SSRs densities from trinucleotide to hexanucleotide in C. reinhardtii and V. carteri were higher than those of other plants, which was consistent with the results of zhao et al. [S. moellendorffii were lower than other plants.We analyzed the SSRs number and SSRs density (SSRs number / mega bases) in the plant genome sequences . The deno et al. . While tS. moellendorffii (86.15%) and monocotyledon (71.96%~89.17%). While the percentages of A/T in moss P. patens (97.30%) and dicotyledon (96.01%~98.76%) were approximately equal. There was a special case that Z. mays had significantly lower A/T (71.96%) than other monocotyledon (85.85%~89.17%). The algae C. reinhardtii and V. carteri had the higher C/G content (74.55%~91.07%)  , which wP. patens was a special case which showed the same trend as dicotyledon  was obviously higher than that of the A/T repeats in algae  of C/Gs .B. distachyon). Meanwhile, AT/TA combination was dominant in dicotyledon and moss P. patens. In monocotyledon (except for S. bicolor) and fern S. moellendorffii, AG/CT/GA/TC combination was dominant and the percentage increased along with the increase of repeat number. However the percentage of AG/CT/GA/TC combination declined along with the increase of repeat number in dicotyledon and moss P. patens. The percentages of CG/GC combination decreased along with the increase of repeat number in the twelve plants. Dicotyledon and moss were significantly lower in percentage of CG/GC combination than other plants , the combinations of CCG/CGG/CGC/GCG/GCC/GGC were dominant SSRs. In moss P. patens and dicotyledon , AAT/ATT/ATA/TAT/TAA/TTA combinations were dominant SSRs, and the percentages increased along with the increase of repeat number. The percentage of SSRs with trinucleotide combinations in A. thaliana and Z. mays differed from other plants along with the increase of repeat number   preferentially chose CCG/CGG/CGC/GCG/GCC/GGC as dominant SSRs, moss P. patens and dicotyledon  chose AAT/ATT/ATA/TAT/TAA/TTA as dominant SSRs  . This redon Figs  and 8A. don Figs . In the don Figs , the tredon Figs . The perdon Figs . Howeverdon Figs . Forth, don Figs . Meanwhiant SSRs . It is w animals –44. Our Toth et al.  thought C. reinhardtii is a unicellular green algae whose lineage diverged from terrestrial plants over one billion years ago. Many C. reinhardtii and angiosperm genes are derived from ancestral green plant genes [C. reinhardtii and animals are derived from the last plant-animal common ancestor and many of these have been lost in angiosperms [C. reinhardtii also displays extensive metabolic flexibility under the control of regulatory genes that allow it to inhabit distinct environmental niches and to survive fluctuations in nutrient availability [C. reinhardtii and terrestrial plant genome sequences.nt genes . Genes siosperms . C. reinlability . This maP. patens is a paleopolyploid. The genome sequences and construction of linearized phylogenetic trees suggest that a large-scale duplication, possibly involving the whole genome, has occurred between 30 and 60 million years ago [P. patens revealed different biases of gene retention compared with seed plants [P. patens genome sequences were obviously different from C. reinhardtii , pentanucleotide and hexanucleotide SSRs  were lower than other monocotyledon plants in this paper    over theAG/CT/GA/TC and AAG/CTT/AGA/TCT/GAA/TTC combinations in A. thaliana were higher than other studied dicotyledons , Landsberg erecta (Ler-0) and Wassilewskija (Ws-0)). We found there were different SSRs regularities among three ecotypes. But the differences within the three ecotypes are smaller than that between species  consist of C/G, were gradually declining. And the percentages of SSRs, which (mainly) consist of A/T, were gradually increased. At the same time, for a particular species, SSRs composition and percentage were changed accompanied by the genome/genes varies . Thus the regularities of SSRs in the twelve plant genome sequences can provide clues for revealing the evolution of plant genomes.Given the current of sequenced plant genome restrictions, fern and moss chose only one species, in the paper we cannot large sample analysis of SSRs feature in different evolutionary position plants.S1 Fig(TIF)Click here for additional data file.S1 File(RAR)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


Salmonella enterica is a zoonotic foodborne pathogen that causes acute gastroenteritis in humans. We assessed the virulence potential of one-hundred and six Salmonella strains isolated from food animals and products. A high through-put virulence genes microarray demonstrated Salmonella Pathogenicity Islands (SPI) and adherence genes were highly conserved, while prophages and virulence plasmid genes were variably present. Isolates were grouped by serotype, and virulence plasmids separated S. Typhimurium in two clusters. Atypical microarray results lead to whole genome sequencing (WGS) of S. Infantis Sal147, which identified deletion of thirty-eight SPI-1 genes. Sal147 was unable to invade HeLa cells and showed reduced mortality in Galleria mellonella infection model, in comparison to a SPI-1 harbouring S. Infantis. Microarray and WGS of S. Typhimurium Sal199, established for the first time in S. Typhimurium presence of cdtB and other Typhi-related genes. Characterization of Sal199 showed cdtB genes were upstream of transposase IS911, and co-expressed with other Typhi-related genes. Cell cycle arrest, cytoplasmic distension, and nuclear enlargement were detected in HeLa cells infected by Sal199, but not with S. Typhimurium LT2. Increased mortality of Galleria was detected on infection with Sal199 compared to LT2. Thus, Salmonella isolates were rapidly characterized using a high through-put microarray; helping to identify unusual virulence features which were corroborated by further characterisation. This work demonstrates that the use of suitable screening methods for Salmonella virulence can help assess the potential risk associated with certain Salmonella to humans. Incorporation of such methodology into surveillance could help reduce the risk of emergence of epidemic Salmonella strains. Salmonella (NTS) represent an important public health problem and economic burden worldwide [Salmonella serovars associated with gastroenteritis transmit through the faecal-oral route, either directly or via contaminated food or water [Salmonella serotypes can cause gastroenteritis, a few serotypes such as S. Typhi can cause invasive infection.Foodborne diseases caused by non-typhoidal orldwide . In Euroorldwide . Salmoneor water . Gastroeor water , a serioor water . The incor water . While aSalmonella serotypes depend upon the virulence potential of the microorganism and the susceptibility of the host. Bacterial virulence factors are necessary to adhere, invade and replicate inside host cells. These are encoded by genes present on a wide range of genetic elements, including the bacterial chromosome, plasmids, prophages and Salmonella Pathogenicity Islands (SPIs). Some SPIs are conserved throughout the genus, e.g. SPI-1, which encodes a Type 3 Secretion System 1 (T3SS-1) which is important for infection of the host cell and SPI-2, which encodes T3SS-2 that facilitates intracellular survival and replication [e.g. SPI-7 in S. Typhi [Salmonella genome comprises DNA of bacteriophage origin which encodes a variety of virulence factors [Salmonella serotypes associated with infections of humans and animals, including the Salmonella virulence plasmid. In addition to the risk presented by NTS serotypes as a potential foodborne hazard, there has been a rapid emergence in recent years of multidrug-resistant isolates of Salmonella [Salmonella disease of food origin to help assess and understand the risk posed by strains from different sources. High through-put DNA microarrays have been developed which enable detection of large numbers of virulence genes, allowing many isolates to be studied simultaneously [Salmonella high through-put chips [Salmonella glass-slide microarray chips which have been used to look at virulence profile previously [The differences in pathogenicity of lication . Other SS. Typhi , and inclmonella , 9. So, aneously –13. Thisut chips ; 12, whieviously ; 14; 15.S. enterica in order to better understand the potential risk that they might present. A customised Salmonella virulence gene microarray was constructed targeting Salmonella pathogenicity islands, virulence regulators, prophages, toxin, and virulence plasmids genes. The array screening informed the selection of atypical isolates for further in vitro and in vivo virulence characterisation using whole genome sequencing, adhesion/invasion assays, Galleria mellonella infection model, and flow cytometry.The aim of the present study was to assess the diversity of virulence genes in Salmonella strains were selected from a previous epidemiological study [A total of 106 al study . StrainsSalmonella virulence determinants . An Unweighted Pair Group Method with Arithmetic mean (UPGMA) dendrogram was used to cluster isolates based on their virulence determinants.http://frodo.wi.mit.edu/) using the reference sequences given in Salmonella strain was identified for each virulence gene (PCR primers for each virulence gene were designed using the Primer3Web software (version 0.4.0) (nce gene  and presS. Typhimurium LT2 (BWA software) [S. Typhimurium Sal199, S. Infantis Sal147, and S. Infantis Sal280 are deposited in the European Nucleotide Archive (ENA) under the study accession number PRJEB9808, which is available from http://www.ebi.ac.uk/ena/data/view/PRJEB9808.The genomic DNA from selected isolates showing atypical microarray results was pair-end sequenced using an Illumina Miseq Sequencer. Raw sequences were filtered and trimmed to minimized sequencing errors (Trimmomatic software) . The rawoftware)  and SAMToftware) . BetweenS. Typhimurium Sal199 and S. Typhimurium LT2 strains grown on Mueller-Hinton broth for 2, 16 and 24 h at 37°C by using Tri reagent  as described before [envF, pagC, pagD, pltA, pltB, cdtB and three typhoid-related virulence genes taiA, tcfA and hlyE  agarose gels.Total RNA was isolated from d before . Reverseand hlyE . GenomicS. Typhimurium Sal199 strain were performed as described previously by Spanó et al. [S. Typhimurium LT2 as negative control. Briefly, HeLa cells were seeded into 6-well tissue culture plates at a density of 1×105 cells per well. The cells were then incubated for 72 h at 37°C with 5% CO2. After the incubation, cells were washed three times with Phosphate-Buffered Saline 0.1M, pH 7.2 (PBS), fixed with 3% paraformaldehyde, and observed under light microscope Axiovert 40 CFL , for demonstration of morphological changes in the cells. To measure the cell cycle arrest flow cytometry was followed as described by Mezal et al. [-1, and 100 μg of RNase A mL-1) and incubated at room temperature in the dark for 15 min. Approximately, 1.0×104 cells were examined using a Fluorescence-activated cell sorting (FACS) analysis with the excitation set at 488 nm and the emission set at 630 nm. The experiment was conducted in triplicate.Cytolethal toxin assays for ó et al.  with minl et al. . The celS. Infantis isolates, Sal147 missing 14 SPI-1 genes, and Sal280 with all SPI-1 genes were selected. Adhesion and invasion assays were conducted using HT-29 colon human cells in presence of gentamicin as previously described [10 transformed to equalise the variances for each strain regardless of the mean. The resulting four means for adhesion and four means for invasion were subjected to a two-ways analysis of variance (ANOVA) using the statistical package GraphPad Prism 5 . A p-value of < 0.05 was taken to indicate statistical significance.Two escribed . Mean ofG. mellonella was used as an in vivo virulence model, as previously described [in vivo virulence of Sal199, Sal147, and Sal280, in comparison with to S. Typhimurium LT2. Briefly, Salmonella grown in Mueller-Hinton broth for 2, 16 and 24h at 37°C, was harvested to give a final concentration of 108 bacteria in 1 mL 0.1 M PBS (pH 7.2). Ten larvae were injected with a 10 μL 104 CFU mL-1 in the right fore pro-leg, using a Hamilton syringe . All larvae were incubated at 37°C for 24 h before determining rates of survival and macroscopic appearance. Non-injected and 0.1 M PBS-injected animals were included in each experiment as controls. The exact dose was determined after plating the inoculum on LB agar. Representative results were obtained from three independent experiments. Data are expressed as percentage of survival, and analysed using the statistical package GraphPad Prism 5 . A p-value of < 0.05 was taken to indicate statistical significance.escribed  to compaSalmonella isolates. The microarray results were transformed to a binary form i.e. present or absent  isolated from a food product of swine origin was deficient in many SPI-1 genes. Sixteen isolates of non-common serotypes, lacked rhuM , and seven isolates lacked sugR  from SPI-3. Virulence determinants of SPI-4 and SPI-5 were conserved in all isolates. Some islet genes were present in all serotypes  or effector proteins .The results indicated that virulence determinants located in SPIs 1–6 were highly conserved . In mosterotypes . The islgogB were present in half of S. Typhimurium and S. Infantis isolates. The Gifsy-2-associated gene sodCI was detected in 100% of S. Enteritidis and 96.6% strains from S. Typhimurium and the non-common serotypes. The Gifsy-3-associated gene sspH2 was reported in 89.8% strains of S. Typhimurium, and 90.5% of S. Enteritidis.The prophage genes showed the greatest variation in their occurrence . Gifsy-1spvC and spvR are located on the Salmonella virulence plasmid. They were present in all isolates of S. Enteritidis and in 33.9% of S. Typhimurium. Gene rck was detected in all S. Enteritidis, and in 32.2% S. Typhimurium isolates.The genes agfA, bcfC, bcfG, stdB, stiC, STM4595 were present in all isolates. The sefA/sefR was found in 90.5% S. Enteritidis. All S. Enteritidis isolates had the plasmid-located genes . Typhi colonization factor (tcf) operon was found in 18.9% of isolates from serotypes Typhimurium, Mbandaka, Havana, Virchow, and Infantis. However, the presence of pilus-fimbrial pilV gene was only found in three isolates of non-determined serotype.The fimbrial genes cdtB gene by array and Sal147 which showed absence of SPI-1 genes included on the array. The WGS data was analysed by mapping contigs to the S. Typhimurium LT2 genome, which was used as reference. In S. Typhimurium Sal199, the insertion of cdtB toxin together with pltA and pltB, was positioned upstream of the transposase IS911. Analysis of S. Infantis Sal147 genome, corroborated arrays results showing deletion of a large number of SPI-1 genes, namely avrA, sprB, hilC, orgC, orgB, orgA, prgK, prgJ, prgI, prgH, hilD, hilA, iagB, sptP, sicP, iacP, sipA, sipD, sipC, sipB, sicA, spaS, spaR, spaQ, spaP, spaO, invJ, invI, invC, invB, invA, invE, invG, invF. Overall, both genomes were very elastic with many phages, and mobile elements being inserted or deleted, with respect to LT2.Whole genome sequencing (WGS) was performed on two isolates which showed atypical microarray results; Sal199 which showed presence of the cdtB gene on S. Typhimurium Sal199. The cdtB gene is encoded on SPI-11, together with pltA and pltB; when these genes are expressed together they form a tripartite toxin, which was originally described in S. Typhi [taiA, tcfA, and hlyE which are other typhoid associated genes, RT-PCR was performed. The RT-PCR experiments showed the expression of SPI-11 genes, as well as the expression of typhoid-related virulence genes , with a large accumulation of cells in the G0 / G1. In contrast, HeLa cells infected with Sal199 become arrested in G2 / M phase after 72 h in comparison to un-infected cells . When comparing the two S. Infantis strains no statistically significant difference in adhesion was observed, but S. Infantis Sal147, was unable to invade HT-29 cells, unlike Sal280  than S. Typhimurium Sal199 that presented a mean survival of 10% (range 0–20%). S. Typhimurium LT2 and S. Infantis Sal280 presented a survival rate of 16.7% (range 10–20%)  . TherefoS. enterica is one of the most important causes of food-borne diseases worldwide. In this study we have developed and tested a high through-put microarray chip for Salmonella virulence genes using a collection of Salmonella isolates recovered from farm animals, slaughterhouses and retail meat, in order to assess the ability of this array to help identify the potential risk associated with Salmonella harbouring different virulence determinants entering the food chain and causing disease in man.Infection with S. Infantis, while prophages and virulence plasmid were generally variable among the strains and serotypes, as reported previously [sodCI (encoding a periplasmatic superoxide dismutase) and sspH2 (encoding an ubiquitin ligase protein), suggesting its stable maintenance. A core set of fimbrial genes were detected in all strains which could contribute to the colonization in a broad host range; while presence of particular fimbrial genes in S. Enteritidis, probably contribute to host specificity [S. Infantis strains were swine-associated and most showed presence of SPI-3 sugR gene, contrasting with previous reports [Salmonella. S. Typhimurium strains harbouring the spv and rck genes, which are required for bacterial multiplication in the reticulo-endothelial system are more able to produce systemic disease [pef fimbrial operon and rck gene that are plasmid borne and play a role in stages of the infection process, were present in all S. Enteritidis and in some S. Typhimurium isolates allowing their separation into two groups which may correspond to a difference in the potential for these S. Typhimurium strains to cause infection and pathogenesis. It also highlights the virulence plasticity of S. enterica, and its ability to adapt to others hosts. Therefore having a more extensive knowledge of virulence genes will help understands the evolution in different serotypes and to infer their relative virulence. Genome variability is common among S. Typhimurium isolates [S. Typhimurium strains harboured one or more antibiotic resistance determinant, including to Extended Spectrum β-Lactamases (ESBL), with more than 37.2% showing a multidrug resistance (MDR) phenotype [This study showed that adhesion and SPI-located genes were highly conserved in all strains except one eviously ; 14; 27.cificity ; 14. All disease . These pisolates  and may henotype .cdtB gene, in association with pltA and pltB genes in S. Typhimurium Sal199 strain isolated from raw poultry meat. CdtB forms a tripartite toxin with PltA and PltB, causing cell-cycle arrest in S. Typhi, thereby increasing its virulence potential [cdtB gene has been described in only six NTS serotypes from human clinical and poultry origin [S. Typhimurium. This study shows that the HeLa cells infected with S. Typhimurium Sal199 became distended with prominent cell cycle arrest compared with the control (un infected cells and cells infected with S. Typhimurium LT2). Presence of cdtB, pltA and pltB upstream of an IS911 transposase might have resulted in horizontal transfer of these genes from a S. Typhi to Sal199, a S. Typhimurium strain. To the best of our knowledge, this is the first report detecting the presence of this typhoid-related toxin in S. Typhimurium, which is one of the most common serotypes associated with human gastroenteritis. Furthermore, the increased virulence of Sal199 is corroborated by our in vivo findings which showed increased mortality of G. mellonella by S. Typhimurium Sal199 with respect to S. Typhimurium LT2, which might be associated with expression of the CdtB toxin.Moreover, using the virulence arrays we detected a otential . Our finotential ; 24. They origin –31, but S. Infantis Sal147. Adhesion/invasion assays showed that this strain was able to adhere but not invade a human colon cell line in comparison to controls. These results indicate a possible reduction in the virulence capacity for Sal147 in comparison to another S. Infantis isolate and LT2, which was confirmed using the G. mellonella in vivo virulence model and suggests a reduced risk to humans.Another interesting finding from using the virulence arrays, and confirmed by WGS, was the absence of thirty-three SPI-1 genes in the MDR Salmonella strains isolated from farm animals, slaughter houses and food products which can enter the food chain and cause infection in humans. Identification of strains with high virulence potential and infectivity in the food animal breeding section at an early stage may help facilitate interventions that could reduce the risk of dissemination of epidemic strains such as S. Enteritidis PT4, S. Typhimurium DT104 and monophasic S. Typhimurium DT193, which have all entered the food chain to cause human infections. In addition to providing details of the virulence profile of isolates this chip also helped group isolates by serotype, which indicates that isolates of the same serotype usually harbour the same virulence profile, and so this chip can be used as a surrogate for serotyping in future. In addition, it has the benefit of spotting isolates from a serotype with atypical virulence profile which may pose either increased or decreased risk to humans. Currently, these arrays are a cheaper and more accessible option than some of the other methods explored in this study including whole genome sequencing, but this may change in future. Also, they can be used by any laboratory worldwide, as a rapid and useful epidemiological surveillance tool to detect and assess the risk posed to humans by Salmonella isolated from the food chain and to help investigate outbreaks.Therefore, in this study we have clearly shown the benefits of using a virulence genes microarray to assess the risk of infection associated with S1 Table(XLS)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Staphylococcus genus. The newest probiotics based on representatives of the normal commensal microflora – non-pathogenic lactic acid bacteria with antibacterial and immunomodulatory properties may become alternative treatment methods for patients with uncomplicated urinary tract infections and vaginosis.It is known that uncomplicated infections of urinary tract and vaginosis are often caused by opportunistic commensal bacteria of the In vivo experimental studies were performed on six-week-old female BALB/c mice. Staphylococcosis was modeled through intravaginal administration of the S. aureus strain 8325-4 to mice, in doses of 5 x 107 cells per animal. Twenty-four hours after infection, mice were given an intravaginal injection of a probiotic bacteria suspension at a dose of 1 × 106 cells per animal, once per day for 7 days.L. casei IMV B-7280, L. acidophilus IMV B-7279, L. delbrueckii subsp. bulgaricus IMB B-728, B. animalis VKL and B. animalis VKB in vitro had different antagonistic effect on museums and clinical strains of pathogenic and opportunistic microorganisms. L. acidophilus IMV B-7279, L. delbrueckii subsp. bulgaricus IMB B-7280 and B. animalis VKB showed efficient antagonistic activity. Staphylococcus genus bacteria were sensitive to all these probiotic strains. Biocompatibility of lacto- and bifidobacteria was shown, that indicates the possibility of different strain’s compositions creation.It was found that identified and characterized probiotic strains Staphylococcus infected mice resulted in a significant increase in lacto- and bifidobacteria number in the vagina under physiological norm and in cases of staphylococcal infection. This indicates that all of these probiotic strains temporarily colonized mice vagina. Antistaphylococcal activity in vivo was confirmed by the significant reduction of S. aureus 8325-4 colonies, which were seeded out from infected mice vagina. L. casei IMV B-7280 - B. animalis VKL - B. animalis VKB composition’s antistaphylococcal action was the most effective and more active than in each of strains alone or in different compositions of two or three bacteria.It was shown that intravaginal probiotic strains administration to intact or Immunomodulatory properties of studied strains in monoculture or in different compositions in cases of experimental intravaginal staphylococcosis in mice caused phagocytic cells functional activity increasing, immune cell activation and interferon-γ and IL-12 production normalization in different periods of observation. However, decreased production of IL-4 indicated the ability of these probiotic strains to balance the immune response towards increasing the level of Th1-type cytokines that guide cell type development of the immune response.L. casei IMV B-7280, L. acidophilus IMV B-7279, B. animalis VKL and B. animalis VKB in monoculture or in different compositions are promising to create a highly effective probiotic with antistaphylococcal and immunomodulating activity for farm animals’ treatment from infectious and inflammatory diseases of the urogenital tract, in particular, induced by staphylococci.Probiotic strains of


Hirsutella minnesotensis [Ophiocordycipitaceae ] is a dominant endoparasitic fungus by using conidia that adhere to and penetrate the secondary stage juveniles of soybean cyst nematode. Its genome was de novo sequenced and compared with five entomopathogenic fungi in the Hypocreales and three nematode-trapping fungi in the Orbiliales (Ascomycota). The genome of H. minnesotensis is 51.4 Mb and encodes 12,702 genes enriched with transposable elements up to 32%. Phylogenomic analysis revealed that H. minnesotensis was diverged from entomopathogenic fungi in Hypocreales. Genome of H. minnesotensis is similar to those of entomopathogenic fungi to have fewer genes encoding lectins for adhesion and glycoside hydrolases for cellulose degradation, but is different from those of nematode-trapping fungi to possess more genes for protein degradation, signal transduction, and secondary metabolism. Those results indicate that H. minnesotensis has evolved different mechanism for nematode endoparasitism compared with nematode-trapping fungi. Transcriptomics analyses for the time-scale parasitism revealed the upregulations of lectins, secreted proteases and the genes for biosynthesis of secondary metabolites that could be putatively involved in host surface adhesion, cuticle degradation, and host manipulation. Genome and transcriptome analyses provided comprehensive understanding of the evolution and lifestyle of nematode endoparasitism. Hirsutella minnesotensis  is the representative of nematode endoparasitic fungi. This species mainly invades the secondary stage juveniles of cyst nematodes by producing conidia that adhere to, penetrate cuticle, and eventually kill the nematode  Heterodera glycines in the United States and China  for medicinal application  in Minnesota, USA , adapter contamination (15-bp overlap between adapter and reads), and duplication contamination were removed to obtain clean reads before assembly. The Kmer value of 51 was used for the genome assembly. The genome was sequenced to 128.3-fold coverage and assembled using SOAPdenovo . SignalP 3.0  and TargetP (http://www.cbs.dtu.dk/services/TargetP/) were used to predict potential secreted proteins.To maximize accuracy, EvidenceModeler algorithms  were useudogenes . All prehttp://www.repeatmasker.org/). The REPET pipeline  against the RepeatMasker library  was used to construct gene families for the H. minnesotensis and 14 additional genomes . Sequences with an e value cutoff of 1e-5 and at least 40% identity over 60% coverage were considered orthologs (http://loco.biosci.arizona.edu/r8s/) calibrated against the origin of Ascomycota at 500–600 Ma (OrthoMCL (rthologs . To inferthologs  for eachrthologs . The divrthologs  with r8srthologs  version 0–600 Ma .E < 1 × 10−4) (http://merops.sanger.ac.uk/). False positives were eliminated following unsuccessful searches against peptidase units and peptidase domains of MEROPS (E < 1 × 10−4) and the Pfam databases, respectively. Prediction of putative secreted proteases was then identified using a combination of the SignalP 3.0 and TargetP 1.1 servers.The families of proteases were classified by full-length BLASTp against the MEROPS peptidase database (http://antismash.secondarymetabolites.org/) with Hidden Markov Model (HMM) signatures was used to identify and annotate putative polyketide synthase (PKS), nonribosomal peptide synthetase (NRPS), and terpene synthase (TPS) genes and gene clusters, and to predict the PKS and NRPS domain architecture in all 16 genomes. Sequences were aligned with MUSCLE. RAxML (H. minnesotensis and other fungi was considered significantly different (The AntiSMASH pipeline (E. RAxML  was usedifferent .http://www.cazy.org/). Whole genome blast searches were conducted against protein sequences in the pathogen–host interaction database (http://www.phi-base.org/) (E < 1 ×10−5). Transporters were classified based on the Transport Classification Database (http://www.tcdb.org/tcdb/). The cytochrome P450s were identified according to Dr Nelson’s P450 database (http://drnelson.utmem.edu/CytochromeP450.html). Lipases were classified according to BLASTp (E < 1 ×10−5) results obtained against the Lipase Engineering Database (http://www.led.uni-stuttgart.de/). Additionally, G-protein-coupled receptors (GPCRs), protein kinases, and transcription factors were classified by BLASTp against GPCDB sequences containing seven transmembrane helices (http://www.cbs.dtu.dk/services/TMHMM/), KinBase (http://kinase.com/), and Fungal Transcription Factor Database (http://ftfd.snu.ac.kr/), respectively. CAFÉ  were treated with different lectins at the concentration of 100 μg/ml, including mannose binding lectin, concanavalin A lectin, Ricin B lectin, and lectin from he fungi . Three rH. minnesotensis strain 3608 was generated using an Illumina Hiseq 2000 sequencer with 128.3-fold coverage and assembled using SOAPdenovo , which is the result of the expansion of certain families. Overall 17,154 occurrences of TEs belonging to two classes were identified. Retrotransposons are dominated by Gypsy (7.18% of the genome) and Copia (4.60%) superfamilies of the long terminal repeat retrotransposons and the I superfamily (4.50%) of the long interspersed elements . The terminal inverted repeats are the dominant superfamily belonging to Class II DNA transposons (8.10% of the genome).The richness of TEs is the most remarkable genome feature of this nematode endoparasitic fungus. The massive proliferation of TEs accounts for nearly 32% of the genome . The abundant retrotransposons are enriched in the regions in which the gene order is not conserved , contributing appreciably to the relative expansion of gene-poor regions. In contrast, DNA transposons are relatively evenly distributed across the genomes . To determine whether the proliferation of TEs in H. minnesotensis genome was related to the expansion of specific gene families, GO and Pfam domain enrichment tests were performed for 50-kb genomic windows containing more than 60% TEs. Significant enrichment of genes involved in secondary metabolism and several families of transcription factors was observed in TE-rich regions . Additionally, large numbers of species-specific genes are also overrepresented in the TE-rich region .Like other fungal genomes enriched in repeat sequences , TEs in H. minnesotensis genome by the high value of TpA/ApT (1.10) and low ratio of (CpA+TpG)/(ApC+GpT) (0.99) . One homolog (HIM_04934) of DNA methyltransferase RID gene in Neurospora crassa (NCU02034) required for RIP was identified in H. minnesotensis genome. This supports the active RIP defense mechanism in H. minnesotensis.RIP mutation is a genome defense mechanism in fungi during which duplicated sequences are mutated from CpA to TpA . A signiFusarium spp. . Protein-coding genes involved in secondary metabolism, transcription factors, and members of expanded species-specific multigene families that are overrepresented in the regions flanking TEs have undergone more RIP than other genes.TEs are frequently a target of RIP mutations, which also preferentially occur in the flanking regions harboring the TEs. Similarly to plant pathogens such as ium spp.  and Dothes fungi , based oH. minnesotensis are shared, whereas 2,132 genes are completely unique for this fungus , indicating their unique gene functions. Of the remaining with Pfam domains, the major functional categories included TEs, secondary metabolism, and transcription factors , which were also identified as significantly enriched domains in the fungus .Compared with 14 other sequenced Ascomycota genomes, most orthologous genes  in s fungus . DespiteH. minnesotensis clusters with insect pathogens and is closely related to the caterpillar fungus O. sinensis .A phylogenomic tree generated based on 898 single-copy orthologs revealed that the nematode endoparasitic fungus sinensis . HirsuteH. minnesotensis has fewer (40) lectin genes than trapping fungi (average 69) . Concanavalin A-like lectin binding to α-d-glucose and α-d-mannose is the most abundant family in nematophagous fungi genomes. Compared with trapping fungi, several families including H-type lectin and fucose-specific lectin which bind to N-acetyl-d-galactosamine and fucose, respectively, are absent in H. minnesotensis genome. In addition, trapping fungi encode a bulb-type lectin which may be used as a defense against nematodes rather than to adhere to them (The adhesion of nematophagous fungi to their host might be mediated by the interaction between lectins on the surface of trapping devices or adhesive spores and carbohydrate ligands on the nematode cuticle . Lectins to them . TherefoH. minnesotensis on lectin-treated nematodes significantly decreased compared with the control without lectin treatment (P < 0.05), which supports the hypothesis that lectins might involve in the process of recognition and adhesion to the host .Following the sugar inhibition experiment procedure , we pretH. minnesotensis is rich of protein, and proteases such as serine proteases have been demonstrated to be involved in the infection of nematodes by fungi , but representing fewer genes than those found in three insect pathogens Me. robertsii (401), B. bassiana (359), and C. militaris (341) . Approximately 18% of these proteases identified in H. minnesotensis contain a signal peptide, which is more likely to be involved in pathogen–host interactions  to assist their adaptation on more complex insect cuticles compared with nematode cuticles.The nematode cuticle as the primary barrier  encounteby fungi , 2009. Tractions , such asractions , whereasH. minnesotensis and other reference fungi are the serine proteases, which comprise approximately 50% of all secreted proteases . Serine proteases, especially the subtilases (affiliated into two families: The subtilisin-like protease S8 and the serine-carboxyl protease S53), are involved in lethal activity and the infection process of nematodes (H. minnesotensis compared with the insect pathogen Me. robertsii and trapping fungi A. oligospora and Mo. haptotylum. A phylogenetic tree based on the conserved enzymatic S8/S53-subtilisin/kexin/sedolisin domains (PF00082) revealed that subtilase genes in the endoparasitic fungus diverged from those of insect fungi, supporting the origin of endoparasitic species from insect fungi and their similar functions in protein degradation. Compared with insect fungi, S53 serine proteases, which adapt to the specific insect hosts coupled with S8 proteases , which resembles insect pathogens (average 155) but is significantly less than plant pathogens  . Similar to insect pathogens, several cellulase families including GH6, GH7, GH12, GH45, and GH61 are contracted or even absent in the H. minnesotensis genome. Hirsutella minnesotensis as well as insect pathogens also lack several hemicellulase families like GH10, GH11, GH26, GH29, and GH67. With fewer cellulases and hemicellulases, carbohydrate-binding module 1 protein domains enhancing the localization of CAZy enzymes on the surface of crystalline cellulose  genome. In comparison with plant pathogens, H. minnesotensis has much fewer putative cutinases (2 vs. average 10) and pectin lyases (2 vs. average 10) (H. minnesotensis encodes more GH18 chitinases (23) involved in cuticle degradation than nematode-trapping fungi (average 11) and plant pathogens (16) .In plant pathogens, carbohydrate-active enzymes are required to degrade complex plant cell wall composed of cellulose, hemicelluloses, and/or pectin . Hirsuteellulose  are abserage 10)  that arerage 10) . Chitinarage 10) . SimilarH. minnesotensis resemble the rice-blast fungus Magnaporthe oryzae Pth11-like proteins which are required for sensing the host surface stimuli to produce appressorium in plant pathogens  than most insect pathogens (average 22) . Besides, one homolog of Saccharomyces cerevisiae GPR-1, which is activated in yeast during nitrogen starvation  lack this GPR1-like GPCR .Fungal GPCRs are key components of signaling pathways for pheromone/nutrient sensing and host recognition . Most GPathogens . Similararvation , is alsoH. minnesotensis encodes more protein kinases (139) than the trapping fungi (average 115) . Especially, serine/arginine-rich protein kinases involved in the modulation of salt tolerance, membrane potential, and potassium transporter in S. cerevisiae (H. minnesotensis genomes (6) compared with those of trapping fungi (average 1) .Reversible protein phosphorylation by protein kinase plays critical roles in the regulation of fungal growth, pathogenesis, stress responses, toxin production, and sexual reproduction . The nemrevisiae  are expaHirsutella minnesotensis encodes large number of transcription factors (979) more than nematode-trapping fungi (average 374) and insect pathogens (average 422) . The most of H. minnesotensis gene expansion involves in Zn2/Cys6, centromere protein B, homeodomain-like, CCHC zinc finger, and bZIP transcription factors. These families especially the Zn2/Cys6 type play important role in regulating protein and polysaccharide degradation . Totally, 101 core genes were identified, compared with 10–19 in trapping fungi and 28–70 in insect fungi . The numbers of Type I PKS biosynthetic genes possessed by H. minnesotensis (26) are significantly more than trapping fungi  and insect pathogens . Hirsutella minnesotensis also encodes abundant of putative NRPS genes (23) in comparison with trapping fungi (average 2) and insect pathogens (average 12). Phylogenetic analyses indicate the PKS genes of nematode endoparasitic fungus evolved from those of insect pathogens . Three, six, five, and ten PKS genes are clustered with other genes involved in the biosynthesis of pigments, mycotoxins, lovastatin, and alternapyrone, respectively , indicating that H. minnesotensis might produce secondary metabolites with similar structures. Six NRPS and two terpene genes are unique to the nematode endoparasitic fungus, suggesting lineage-specific expansion of these families in H. minnesotensis genome.Genes encoding secondary metabolic enzymes such as PKS, the NRPS, and the TPS are significantly expanded in H. minnesotensis genome. Although most PKS and NRPS genes are orthologous across the nematophagous fungi and insect fungi, the accessory genes in the secondary metabolite gene clusters are more dynamic. Only five secondary metabolism gene clusters are shared between H. minnesotensis and other insect pathogens with limited synteny (A). This cluster diversity seems to have resulted from gene duplication or loss related to TEs associated with the secondary metabolism gene clusters (B). Thirty-two of 94 gene clusters in H. minnesotensis are located in TE-rich regions (>60% TE).The core genes for secondary metabolite production in fungal genomes are typically located in clusters with other accessory genes, such as those encoding dehydrogenases, methyltransferases, acetyl transferases, prenyltransferases, oxidoreductases, and cytochrome P450s . In tota synteny A. This cclusters B. ThirtyHirsutella minnesotensis was investigated about the infection process on nematodes by the RNA-seq (RNA sequencing) approach. The relative expression of all H. minnesotensis genes was determined by Illumina sequencing across four infection stages, including i) H. minnesotensis cultures on CMA medium free of nematodes to produce large amount of spores (0 h), ii) spore adhesive stage after 8 h of culture with nematode challenge (8 h), iii) infection peg formation stage after 16 h of nematode challenge (16 h), and iv) stage of fungal mycelium growth in the body of nematode after 36 h (36 h). In total, 9,277 of 12,702 predicted genes were expressed during the infection process, representing 73.0% of the H. minnesotensis transcriptome. After infection on nematodes for 8, 16 and 36 h, 1,433, 1,448 and 973 putative protein-coding genes were significantly up- or downregulated (P < 0.05), respectively, compared with that of the culture on CMA without nematodes. Many of these differentially expressed genes encode known proteins (approximately 70%), a significantly greater proportion than for nondifferentially expressed genes.A, clusters 1–8) accounted for about 93% of the differentially expressed genes in the four stages. Most of the blue bins showed enrichment for particular clusters of gene expression (fig. 5B). Genes that encode proteins involved in translation, cytoplasm, respiration activity, hydrolase activity, proteasome, transmembrane transport, lipid metabolism, and organic substance transport are greatly enriched in clusters 1 and 2, representing genes that are expressed at the highest levels at the spore adhesion stage. High expression of genes responsible for the proteasome and hydrolase activity indicated their important role in cuticle degradation. Genes that showed peak expression during the period when fungal mycelium growth within the nematode body included those that are required for phospholipid metabolism, macromolecular metabolism, amino acid metabolism, protein degradation, serine-type peptidase activity, and secondary metabolism, suggesting that the fungal transcriptome undergoes detoxification in host body, degrades nematode protein substrates, and involves nutrient uptake.GO terms and Pfam domains were used to assign genes to functional categories and group the genes by parasitic dynamics using the K-Means clustering algorithm. Eight main clusters . In addition, H. minnesotensis encodes 16 adhesive proteins containing a CFEM  that are used by pathogenic fungi to recognize and attach to their hosts (P < 0.05) at the adhesive stage (8 h) .Recognition and adhesion are the first two steps of fungal invasion of nematodes. In the genome of ir hosts , and fouP < 0.05)  during the penetration stage and may be involved in degrading the nematode cuticle. One gene involved in cell wall assembly , one in cell wall beta-glucan synthesis , and three genes containing cell wall galactomannoprotein  were highly expressed during the infection peg formation. Swelling at the peg tip indicates the stress response to reactive oxygen, nitrogen, and chlorine species generated by the defense action of the host. Four genes  encoding thioredoxin (PF00085) acting as antioxidants were activated during infection stage.Secreted enzymes, including one pepsin-like aspartic peptidases (HIM_11269) and five subtilisin serine peptidases , were significantly upregulated .After the interaction of the fungus and the nematode during the peg stage, the fungus conquers the nematode defense and consumes the nematode body content for growth. Both the endopeptidases M4 and M43, and exopeptidases M14 and M28  were highly expressed at this stage (36 h). In addition, one general substrate transporter (HIM_03796), two oligopeptide transporters , and one amino acid transporter (HIM_07163) were also strongly activated to facilitate the nutrient transportation. The transcriptome profile of H. minnesotensis. The PKC1 (HIM_00683), Bck1 , and Mkk1  genes were relatively highly expressed at 16 h compared with 8 h . Adaptation to stress  mainly occurs through the high osmolarity glycerol (HOG) pathway . The significant upregulation of protein kinase A (PKA) during the last stage revealed the importance of the cyclic AMP-protein kinase A pathway for filamentation . Meanwhile, two genes (HIM_01527 and HIM_07761) resembling the STM1-like GPCR responsible for triggering adaptation to nitrogen starvation were upregulated during the whole infection process. Overall, the nematode endoparasitic fungus is able to respond to diverse host-derived stimuli using distinct or shared signal pathways involving mitogen-activated protein kinase and cAMP-PKA to carry out their infection cycle on the nematode hosts .Like the infection structures of appressoria for entomopathogenic fungi  and the  pathway . The MAPHirsutella minnesotensis is one of the most important endoparasites of SCNs. Different from other animal pathogens such as nematode-trapping fungi and entomopathogenic fungi, H. minnesotensis produces adhesive spores to adhere to, penetrate, and eventually kill their nematode host. The analysis of H. minnesotensis genome compared with genomes of other insect pathogens in Hypocreales and trapping fungi in Orbiliales and the transcriptome of H. minnesotensis on C. elegans have provided insights into the evolution and life strategy of nematode endoparasites.H. minnesotensis genome compared with other animal pathogen genomes (except O. sinensis) is that H. minnesotensis contains a strikingly rich and diverse population of TEs, associated with its relatively larger genome size. An expanded TE-rich genome is also a feature of some biotrophic obligate plant pathogens, such as Blumeria graminis .The most significant characteristic of graminis  and Puccgraminis , the ectnosporum , and thesinensis  that aresinensis  increasesinensis . A posit regions . Overrepptations , the pron nature . Repetitium spp.  and Dothes fungi  in whiches fungi . The potHirsutella minnesotensis is a member of the order Hypocreales which is an important group of fungi that has various lifestyles, including plant pathogens, endophytes, hyperparasites, insect pathogens, and nematode parasites. The phylogenomic analyses demonstrated that H. minnesotensis is evolutionary close to the insect fungi T. inflatum and O. sinensis in the family Ophiocordycipitaceae and evolved into nematode endoparasite independently out of nematode-trapping fungi lineage  and serine-carboxyl proteinases (S53) involve in the penetration and colonization on hosts for nematophagous fungi and entomopathogenic fungi , including the wide-spectrum antibiotic phomalactone  actively against entomopathogenic fungi, plant pathogenic fungi, insects and plant . Hirsutella minnesotensis and trapping fungi also encode GPR1 nutrient sensors, which are absent in entomopathogenic fungi. These similar expansion of GPCRs associated with functions necessary for nutrient sense and host recognition reflects convergent evolution between nematode endoparasitic fungus and trapping fungi. However, H. minnesotensis encodes expanded protein kinase involved in regulation of fungal growth, pathogenesis, toxin production, and sexual reproduction compared with trapping fungi especially has a complex signal transduction cascade controlling stress response. Moreover, transcription factors belonging to Zn2/Cys6 fungal type, zinc finger, and bZIP which are expressed during pathogenesis .


Neoverruca are abundant near deep-sea hydrothermal vents of the northwestern Pacific Ocean, and are useful for understanding processes of population formation and maintenance of deep-sea vent faunas. Using next-generation sequencing, we isolated 12 polymorphic microsatellite loci from Neoverruca sp., collected in the Okinawa Trough. These microsatellite loci revealed 2–19 alleles per locus. The expected and observed heterozygosities ranged from 0.286 to 1.000 and 0.349 to 0.935, respectively. Cross-species amplification showed that 9 of the 12 loci were successfully amplified for Neoverruca brachylepadoformis in the Mariana Trough. A pairwise FST value calculated using nine loci showed significant genetic differentiation between the two species. Consequently, the microsatellite markers we developed will be useful for further population genetic studies to elucidate genetic diversity, differentiation, classification, and evolutionary processes in the genus Neoverruca.Barnacles of the genus  Neoverruca [Imbricaverruca [Neoverruca has no fossil record, the first appearance of close, but extinct ancestors is in the Mesozoic Era [Neoverruca is a very primitive barnacle. Presently, the genus Neoverruca contains only one described species, Neoverrucabrachylepadoformis. Based on morphology, individuals in the Okinawa Trough and Izu-Ogasawara Arc are distinct from N. brachylepadoformis in the Mariana Trough, located south of the Izu-Ogasawara Arc [et al. [Neoverruca sp., and investigated population structure, focusing on mitochondrial cytochrome oxidase c subunit I (COI). They discovered significant genetic differentiation between the two regions. Further population genetic analysis of this species using high-resolution genetic markers will be needed for detailed estimation of genetic diversity and structure. Therefore, microsatellite markers have been developed for further population genetic studies of Neoverruca sp. We isolated and characterized novel polymorphic microsatellite loci for Neoverruca sp. using next-generation sequencing and investigated cross-species amplification for N.brachylepadoformis collected from the Mariana Trough. Furthermore, we estimated the extent of genetic differentiation between Neoverruca sp. and N.brachylepadoformis using developed microsatellite loci, since Watanabe et al. [Larval dispersal in ecosystems such as deep-sea hydrothermal vents is essential for maintenance of benthic populations and for establishment of new colonies. For vent-endemic benthos, planktonic gametes or larvae provide the only means of migration between hydrothermal vents . Howeveroverruca  and Imbraverruca . The foraverruca ,5. Thougzoic Era ,6. Hencewara Arc ,5. WatanNeoverruca sp., and assembled each read pair. We used assembled 736,240 sequences longer than 100 bp  for simple sequence repeat identification. Sequence repeats were used for microsatellite detection and primer design. Ninety-six primer pairs were designed for screening amplifiable microsatellite loci . Twelve of these loci were successfully amplified and subsequently analyzed in Neoverruca sp., although Nsp_11 and Nsp_80 were not amplified in two and one individual(s), respectively. The number of alleles per locus ranged from 2 to 19 (p < 0.05).We obtained 291,118,462 bp  of raw sequence data from genomic DNA of  2 to 19 . Values Neoverruca sp., nine were successfully amplified in N. brachylepadoformis. Three loci, including Nsp_11 and Nsp_23, which showed high polymorphism in Neoverruca sp., were hardly amplified in N. brachylepadoformis. The number of individual N. brachylepadoformis that showed successful amplification at these nine loci was 14 to 19 (out of 19) .Of the 12 loci isolated and characterized from t of 19) . These nt of 19) . Values FST implied significant differentiation between Neoverruca sp. and N. brachylepadoformis. This differentiation is consistent with divergent patterns in molecular phylogenetic trees previously constructed for the COI haplotype [In this study, the number of useful loci and the significance of the pairwise aplotype . Using ni.e., between the Okinawa Trough and the Izu-Ogasawara Arc, which influences the formation and maintenance of populations within these species. Furthermore, these markers are expected to help elucidate genetic diversity, differentiation, classification, and evolutionary processes in genus Neoverruca. They may even be useful in clarifying the taxonomic diversification concerning this genus.Further population genetic studies using these markers will enable us to elucidate the level of present and historical dispersal and recruitment between these areas, Neoverruca sp. was collected at the Iheya Depression  in the Okinawa Trough during cruise of R/V “Kaiyo” (Cruise No. KY11-02 Leg. 2), using the remotely operated vehicle (ROV) “Hyper-Dolphin” (Dive No. HPD#1245). Specimens were preserved in ethanol, and genomic DNA was isolated using proteinase K and phenol-chloroform extraction. DNA was further purified using ethanol precipitation and a QIAquick PCR purification kit . Extracted DNA was sequenced as 300 bp paired end reads using a MiSeq sequencer , according to manufacturer’s instructions. Sequencing adapters were trimmed with fastq-mcf in ea-utils version 1.1.2-537 [For isolation of whole genomic DNA, Neoverruca sp. collected at the Iheya North field  with aforementioned procedures and conditions. These specimens were collected at the Alice Springs field  were calculated with GenAlEx version 6.5 [N. brachylepadoformis. In addition, the number of alleles, expected and observed heterozygosity, and deviation index from Hardy-Weinberg equilibrium were also calculated in N. brachylepadoformis. To estimate the level of genetic differentiation between populations of Neoverruca sp. and N. brachylepadoformis, pairwise FST was calculated using GenAlEx focusing cross-species amplifiable loci. The test for statistical significance was based on 999 random permutations.For successfully amplified microsatellite loci, the number of alleles, expected and observed heterozygosity, and deviation index from Hardy-Weinberg equilibrium (sion 6.5 . The sofsion 6.5  performesion 6.5 ,14,15 unNeoverruca sp. to estimate the processes of population formation and maintenance. In addition, nine of the primers can amplify microsatellites in N. brachylepadoformis. These markers are expected to help elucidate genetic diversity, differentiation, classification, and evolutionary processes in the genus Neoverruca.Twelve microsatellite loci developed in this study were successfully amplified and should have utility for further population genetics of


Salmonella spp. during 2005–2013. A total of 2120 (1.7%) Salmonella spp. were isolated and the prevalence of Salmonella spp. decreased significantly over time . Among the typhoidal Salmonella (TS) serogroups, S. Typhi was predominant  followed by S. Paratyphi B  and S. Paratyphi A . Of the nontyphoidal Salmonella (NTS) isolates, the serogroup C1  was predominant followed by B , C2 , E , and D . Most of the resistance was found towards nalidixic acid (40%), ampicillin (36%), cotrimoxazole (20%), chloramphenicol (13%), ciprofloxacin (4%), and ceftriaxone (4%). Interestingly, 32% of the isolates showed reduced susceptibility to Cip. Multiantibiotic resistance  was more common among TS than NTS strains (P < 0.001). Among the representative ceftriaxone-resistant isolates, blaTEM gene was detected among 88% (7/8) of the strains, whereas only one strain of S. Typhi was positive for both blaTEM and blaCTX-M genes. The study reflects higher prevalence of MAR Salmonella spp. and is the first to report the blaTEM gene mediated ESBL production among Salmonellae in Bangladesh. Emergence of MAR Salmonella spp. in particular ESBL strains should be considered a public health concern.Salmonellosis, an acute invasive enteric infection, is endemic in Bangladesh. We analyzed 128,312 stool samples of diarrheal patients to identify  Salmonella (NTS) infection is a global public health concern, particularly in Salmonella endemic low and middle income countries (LMICs). Treatment is critical for persons with severe disease, particularly children and immune compromised people. Treatment with an appropriate antibiotic can shorten the duration of symptoms, significantly reduce severity of disease and the risk of transmission, and prevent potentially lethal complications. Emergence of resistance to first-line therapy like ampicillin, chloramphenicol, and cotrimoxazole including ciprofloxacin among Salmonella spp. during the last decades has complicated the situation [ Salmonella infection resistant to these drugs, extended-spectrum cephalosporins (ESCs) are considered as an alternative therapeutic choice. With the increased use of β-lactam antibiotics to treat enteric infection, Salmonella spp. had acquired resistant to third generation cephalosporin antibiotics in different parts of the world and had been associated with clinical treatment failure [Salmonellosis due to nontyphoidalituation , 2. For  failure , 4.Extended-spectrum beta-lactamases (ESBL) are usually encoded by large plasmids (≥100 kb) that are transferable from strain to strain and between bacterial species –7. ResisblaCTX-M and blaTEM type ESBL producer S. Typhi in one-year-old child with recurrent high-grade fever [β-lactam antibiotics among Salmonella species [blaTEM gene mediated ESBL production among Salmonella spp. isolated from stool specimen of patients with diarrhea in an urban setting of Bangladesh.Earlier we have reported the emergence of de fever . In rece species . Here, w Salmonella spp. Antibiotic susceptibility test was done by Kirby-Bauer disk diffusion test. The putative extended-spectrum beta-lactamase (ESBL) producing strains were tested by the double-disc synergy method and detection of β-lactamase genes  was performed by PCR as describe elsewhere [As part of a microbiological analysis of stool sample received at Dhaka Treatment Centre of International Centre for Diarrheal Diseases Research, Bangladesh  during 2005–2013, we have analyzed 128,312 fecal specimen following standard microbiological method to identifylsewhere . Salmonella infection showed a decreasing trend and the proportion came down significantly from 2% in 2005 to 1.0% in 2013 (P < 0.001). Of the total Salmonella spp. , nontyphoidal Salmonella (NTS) isolates were more frequently isolated than typhoidal Salmonella (TS) . Demographic information showed that Salmonellae were isolated from patients of all age groups with a maiden age of 4.04 years. The male/female ratio of patients was 1.05. Of the total Salmonella positive patients, 51.2% (n = 1086) were children aged less than five years. Among the TS serogroups, S. Typhi was predominant  followed by S. Paratyphi B  and S. Paratyphi A . Of the NTS isolates, serogroups C1 strains were more frequently isolated , followed by B , C2 , E , D , G , nontypeable Salmonella , S. Typhimurium , and A  serogroup. The yearly distribution of Salmonella spp. showed distinct seasonality with higher isolation during May to October in each year. Temporal shift was noted in the prevalence of serogroups, seasonality, gender distribution, and resistance pattern between TS and NTS isolates.Overall, the annual incidence of Salmonella isolates showed resistance to nalidixic acid (Na) followed by 36% to ampicillin (Amp), 20% to cotrimoxazole (Sxt), 4% to ciprofloxacin (Cip), 13% to chloramphenicol (C), and 4% to ceftriaxone (Cro). Interestingly, 32% of the isolates were with reduced susceptibility to ciprofloxacin. Resistance to ≥3 antibiotic classes was more common among TS strains than NTS counterpart . Among the representative Salmonella isolates with unusual resistant phenotype (AmpCCipCroNaSxt)R, first identified in 2011, blaTEM gene was detected in 88% (7/8) of the strains ( S. Typhi was positive for both blaTEM and blaCTX-M genes which is a very rare phenomenon. In our earlier report, we noticed that this same phenomenon was observed in one S. Typhi isolated from blood of a typhoid patient [ Salmonella isolated from stool specimen of diarrheal patients in urban Dhaka, Bangladesh. Detailed molecular characterization including sequencing is necessary for further geno- and subtyping of these strains.Around 40% of the strains . Interes patient . These f Salmonella spp. among children aged <5 years and blaTEM gene mediated ESBL production among Salmonella spp. isolated from stool sample of diarrheal patient in urban Bangladesh. Therefore, it is important to establish a surveillance program to understand actual disease burden due to Salmonella as well as promote specific and actual line of therapy for Salmonellosis.The study finding reflects the higher prevalence of MAR


Rickettsia felis is an emerging insect-borne rickettsial pathogen and the causative agent of flea-borne spotted fever. First described as a human pathogen from the USA in 1991, R. felis is now identified throughout the world and considered a common cause of fever in Africa. The cosmopolitan distribution of this pathogen is credited to the equally widespread occurrence of cat fleas , the primary vector and reservoir of R. felis. Although R. felis is a relatively new member of the pathogenic Rickettsia, limited knowledge of basic R. felis biology continues to hinder research progression of this unique bacterium. This is a comprehensive review examining what is known and unknown relative to R. felis transmission biology, epidemiology of the disease, and genetics, with an insight into areas of needed investigation. Rickettsia felis, has progressed from a sporadic disease in the USA to a common cause of fever in Africa . Giviewed in ). This tt system •. Recentted Fig.  26•]. A•. AR. feR. felis infection was misdiagnosed as flea-borne endemic typhus (Rickettsia typhi) because the available serological reagents were unable to distinguish between the two rickettsial species [R. felis among endemic typhus patients was initiated because field surveys revealed the presence of this agent within suspected vectors and mammalian hosts of R. typhi in the USA [Didelphis virginians) as respective vectors and hosts of R. felis in suburban regions of California and Texas [R. typhi with rat fleas and commensal rats (Rattus sp.) [R. felis among Oriental rat fleas and Norwegian rats (Rattus norvegicus) than R. typhi in endemic typhus areas of Los Angeles [R. felis-infected fleas. The vulnerability of human populations to flea-borne rickettsiae is of particular concern in developed countries where aggressive pest management programs may not control for ectoparasites, which can result in the relocation of arthropods to new hosts  following rodent extermination. Given the indiscriminate feeding habits of cat fleas [R. felis is essentially a household rickettsiosis in human populations where peri-domestic animals  are in close contact.Flea-borne spotted fever is considered an emergent global threat to human health, with cases likely underestimated due to similarities in clinical signs with other febrile illnesses  and limited access to appropriate laboratory tests  , 10, 15. species . A retro the USA –29. Compnd Texas , 27, 29.tus sp.) . Interes Angeles . It is uR. felis has been conducted almost exclusively in Africa due to the considerable frequency of flea-borne spotted fever in hospitalized febrile patients. In sub-Saharan Africa, R. felis is described as a common (3–15 %) cause of illness among patients with “fever of unknown origin” in malaria-endemic regions [R. felis infections was higher than that of malaria in two of the studied villages of Senegal [R. felis infections reported within the last 5 years is in stark contrast to the total number of infections (∼100 human cases) documented worldwide [R. felis is classified as an emerging pathogen, it is unclear whether this increased incidence in Africa reflects an overall trend or represents an endemic state previously unknown for this disease. Commonalities  were observed between the epidemiology of R. felis and Plasmodium falciparum infections in Africa, which were initially hypothesized to coincide because of a proposed common vector, Anopheles mosquitoes [Anopheles in the transmission of R. felis was ambiguous; however, the transmission potential of R. felis by A. gambiae  was recently demonstrated in a simulated model [R. felis in Africa include numerous species of fleas, mosquitoes, and mites, as well as an individual bed bug [R. felis in Africa is unknown, but molecular evidence for the presence of R. felis in African apes  was derived from PCR-positive stool samples [R. felis in Africa might be primates, including humans [R. felis infections were PCR-positive for rickettsial DNA [Much of the latest work concerning the epidemiology of  regions , 32, 33.orldwide . Again, ed model . Other a samples . It was g humans . As suchsial DNA . Converssial DNA .Rickettsia (Rickettsiaceae) was designated as typhus group (TG) or spotted fever group (SFG) rickettsiae; however, R. felis displayed phenotypic oddities that confounded its categorization as either TG or SFG, e.g., association with an insect, hemolytic activity, actin-based motility, transovarial maintenance in the vector host, and serological cross-reactivity [R. felis revealed a large genome size relative to other rickettsiae, and the presence of plasmids [Rickettsia, including a sister clade of the SFG now known as the transitional group (TRG) and a non-pathogenic clade, thought to be basal to all other groups, called the ancestral group (AG) [R. felis is a member of the TRG rickettsiae, which may explain certain anomalies  as this bacterium continues to undergo major life history transitions.Historically, the genus activity . Additioplasmids . Combineoup (AG) . R. feliR. felis have been isolated from colonized and wild-caught arthropods [Liposcelis bostrychophila (Insecta: Psocoptera) [R. felis is an obligate mutualist required for the early development of the oocyte and is maintained 100 % transovarially [R. felis employ different transmission routes for sustained infection within unique arthropod populations [R. felis host specialization, the sequenced genomes of two strains, R. felis (str. LSU-Lb) isolated from a booklice colony and R. felis (str. LSU) isolated from a cat flea colony, were compared to the flea-derived R. felis reference strain  [R. felis, suggesting that spatial isolation  and potential host specialization (flea vs. booklouse) have resulted from genetic divergence [R. felis str. LSU-Lb assembly provides evidence for host-specific strain variation [R. felis strains, with some strains having no plasmids and others having two [R. felis str. LSU-Lb by a colony of cat fleas, as well as subsequent prevalence and infection load dynamics . Thus, the selective forces operating on R. felis genomes from strains associated with different arthropod vectors remain unknown and require further examination.Several strains of thropods , 40, inccoptera) . In the varially , 43. Givulations . In an eRWXCal2) . Sequencvergence . Specifiariation . This diving two , 46. Towics Fig. . SurprisR. felis-like organisms (RFLOs) in different arthropods, including cat fleas  in the sequences of the 16S rRNA (rrs) (≥99.8 %) gene and four protein-coding genes, the gltA (≥99.9 %), ompA (≥98.8 %), and ompB (≥99.2 %) genes and gene D (≥99.3 %) to existing Rickettsia species [rrs gene is the borderline for separation of 2 Rickettsia species, whereas 1 % divergence is known to mark the borders of naturally occurring bacterial species [Rickettsia species isolated from cat fleas, Candidatus Rickettsia asemboensis and Candidatus Rickettsia senegalensis, showed 99.5 and 99.65 % similarity to the rrs gene in validated species of R. felis, respectively [R. felis isolates due to spatial isolation, a more suitable approach to justify the separation of RFLOs into species may be to seek ecological, genomic, or phenotypic differences among the major clusters resolved by MLST [Candidatus Rickettsia asemboensis was sequenced [Within the last decade, numerous reports have identified as Table , throughes level . As such species . The con species . For exaectively •, 50. Gi by MLST . Recentlequenced , and futR. felis infections in endemic-disease foci remains unclear  and flea-borne endemic typhus (R. typhi) [R. felis-infected cat fleas generate feces with detectable levels of rickettsial transcript [Bartonella henselae, achieves successful transmission from adult fleas to their progeny via vertical non-transovarial transmission [Bartonella species was demonstrated, but a previous study showed the absence of transovarial transmission of B. henselae within flea colonies [Bartonella-positive adult flea feces, then the larvae acquired an infection that was maintained through adulthood [R. felis should be tested within cat flea colonies as an additional route of pathogen maintenance in vector populations  , 52. Theanscript , the trasmission . Verticacolonies ; howeverdulthood . Thus, vons Fig. .R. felis throughout the world. Doubts have been raised about whether R. felis transmission from mammal to arthropod occurs given the efficiency of pathogen transfer between co-feeding fleas without a systemic vertebrate infection [R. felis infections in endemic disease foci. Transmission of R. felis within cat flea colonies has proved variable and adaptable, with decreased colony prevalence signaling to increase infection burdens in individual fleas [R. felis in nature. The latest reports from urban environments have emphasized the potential of domestic cats and dogs as mammalian reservoirs of R. felis infections [R. felis where components of this enzootic cycle are present, e.g., free-ranging cats and dogs, commensal rodents and opossums, and associated flea species. Future studies should address Koch’s postulates to identify R. felis as the causative agent of vertebrate infection, specifically isolation of R. felis for culture from these proposed reservoir hosts.The lack of a description of a definitive vertebrate host impedes epidemiological studies of nfection •; howeveal fleas . Thus, ofections –61, whilfections , 62. AccR. felis infections in febrile and afebrile patients were diagnosed by PCR detection in human blood samples [R. felis, as they are for another insect-borne rickettsial species (R. prowazekii). The transmission cycle for R. prowazekii is louse to human to louse, with lice ingesting bacteria by blood-feeding on infected humans and subsequently transferring the bacterium to humans by excretion of infectious feces at the bite site [R. prowazekii is Brill-Zinsser disease, or recrudescent typhus, in which mild symptoms reappear after a latent period [R. felis DNA was detected in the blood of a patient at multiple time points over a 1.5-month interval. While this initial observation suggests episodic rickettsial infection (relapse or reinfection) in humans, these samples were taken from a child in the absence of antimicrobial therapy [R. felis should be investigated further with adult patients administered antibiotic treatment. Additional studies reported that not all patients diagnosed as PCR-positive for R. felis infection generated anti-rickettsial antibodies, which researchers proposed supports the notion of a recurrent infection [R. felis infection based on PCR-positive blood samples. For example, R. felis DNA was detected in skin swabs from healthy individuals in a Senegal village where roughly 7 % of the villagers possess an R. felis infection [R. felis in blood samples from asymptomatic persons challenges existing paradigms about pathogenic rickettsiae. Such as, the magnitude of rickettsial growth required for PCR detection in the bloodstream of patients is typically fatal, yet these afebrile individuals had no adverse symptoms [R. felis has not been isolated from a vertebrate host, even in severe human cases. Thus, a human isolate must be obtained before conclusions are drawn on the role of people in R. felis epidemiology.Recently,  samples , 63; thuite site . A delayt period . Humans t period . Similar therapy . The occnfection , 64; hownfection , 66. Thinfection . Furthersymptoms . RickettR. felis genotype appears to be vast, with different isolates shown to consist of unique individual qualities. Whether RFLOs warrant species designation is unclear, but there are disparities among this genogroup that may lead to a microbial-dependent influence on R. felis prevalence. For example, interspecific competition of rickettsiae in ticks is well documented, with a primary infection responsible for the interference or blocking of a secondary infection [R. felis infections appear low or absent may be due to an interference event followed by perpetuation of the primary infection within a closed arthropod population. The transmission biology as well as the pathogenicity of RFLOs is unknown, but these organisms are detected in arthropods known to bite humans. Future work with RFLOs should identify, if any, phenotypic characteristics associated with genotypic diversity and focus on acquisition, dissemination, and transmission of these organisms by their respective arthropod hosts (Fig. The genetic diversity within the nfection –70. Thussts Fig. .R. felis from additional countries, and the influx of RFLOs may result in a similar trend. Active surveillance of R. felis infections among hospitalized febrile patients will determine when an endemic state has been reached by this emerging pathogen, as well as indicate spread to populations outside of endemic disease foci. Advance genetic analyses of Rickettsia species should include criteria for ecological, genomic and phenotypic differences in addition to sequence homology. In order to determine the specific roles of both the vertebrate and arthropod host in the transmission cycle of R. felis, it is critical to continue the development and implementation of molecular tools and bioassays necessary for more accurate risk assessment and efficacious control measures.Every year, there are new reports of arthropod, animal, and human cases of


Nonomuraea sp. TP-A0861 produces the nonribosomal peptide myxochelin A, which is known as a microbial siderophore. Here, we report its draft genome sequence. The genome contains at least three nonribosomal peptide synthetase gene clusters, one of which is proposed to be responsible for the biosynthesis of myxochelin A. Nonomuraea sp. TP-A0861 isolated from a soybean root was found to produce myxochelin A . The whole genome of the Nonomuraea sp. TP-A0861 monoisolate was read by using a combined strategy of shotgun sequencing with GS FLX+ (Roche)  and paired-end sequencing with MiSeq (Illumina) . These reads were assembled using Newbler version 2.8 software, and subsequently finished using GenoFinisher software  contains eleven NRPS modules and the other (ORF 888 to ORF 891) two modules (An NRPS gene cluster in scaffold 03 encodes orthologues (ORF 606 to ORF 611) of a Sg a15 . This in modules , suggestNonomuraea candida NRRL B-24552 (JOAG00000000.1), Nonomuraea coxensis DSM 45129 (ARBV00000000.1), and Nonomuraea kuesteri NRRL B-24325 (JOAM00000000.1) were released to the public. However, no scientific papers regarding these strains have been published yet. Therefore, this is the first report of the genome sequence of the genus Nonomuraea. The genome sequence of Nonomuraea sp. TP-A0861 will provide valuable information to elucidate the potential of Nonomuraea strains as a source of new bioactive compounds.Recently, draft genome sequences of Nonomuraea sp. TP-A0861 has been deposited in the DDBJ/ENA/GenBank database under the accession number BBZG00000000. The version described in this paper is the first version, BBZG01000000.The draft genome sequence of


H. pylori infection and there is growing evidence that H. pylori co-infection is associated with parameters of HIV disease progression. The objective of this study was to investigate the prevalence of H. pylori infection, and the association with clinical, immunological and virological parameters in a large cohort of HIV-infected individuals and uninfected controls in a West African country.Worldwide, there is a high co-endemicity of HIV and H. pylori status was determined using stool antigen testing. HIV-related, clinical and socio-demographic parameters were recorded and analyzed according to H. pylori status.HIV-patients  and HIV-negative individuals (n = 107) were recruited at a university hospital in Ghana. H. pylori infection was significantly lower in HIV-positive compared to HIV-negative individuals . In HIV patients, H. pylori prevalence decreased in parallel with CD4+ T cell counts. In ART-naïve HIV-infected individuals, but not in those taking ART, H. pylori infection was associated with higher CD4 cell counts  and lower HIV-1 viral loads . The findings could not be explained by socio-demographic confounders or reported use of antibiotics. Having no access to tap water and higher CD4+ T cell counts were identified as risk factors for H. pylori infection.The prevalence of H. pylori prevalence was inversely correlated with the degree of immunosuppression. In ART-naïve individuals, H. pylori infection is associated with favorable immunological and virological parameters. The underlying mechanisms for this association are unclear and warrant investigation. Helicobacter pylori (H. pylori) infection has attracted attention. A number of studies have reported lower H. pylori prevalence rates in HIV-infected compared to HIV-negative individuals [H. pylori infection is considered a disease of poverty, and poor socioeconomic status has been associated with rather disadvantageous outcomes of HIV infection [H.pylori status is sorely lacking. As a consequence, interpretation and comparison of results are difficult and data published to date is partly inconsistent [Recently, the interplay between the Human immunodeficiency virus (HIV) and ividuals ,2. This nfection . The undH. pylori infection, the investigation of possible interplay is of interest. Over the past few years it has become clear, that the gastrointestinal tract (GIT) plays an important role in the pathophysiology of HIV/AIDS. Chronic immune activation, associated with intestinal barrier dysfunction, has been identified as central pathomechanism in HIV disease [H. pylori colonize the gastric and duodenal mucosae and induce a specific local and also systemic immune response, involving, among others, CD4+ T cells, dendritic cells, regulatory T cells (Treg) and Th17 cells, with all of these also playing a role in HIV pathogenesis [Considering the significant epidemiological and pathophysiological overlap of HIV and  disease . H. pyloogenesis –8.H. pylori co-infection has not been systematically studied in sub-Saharan Africa, where more than two thirds of HIV-infected individuals live, and where, at the same time, the vast majority of the population gets infected with H. pylori during childhood [H. pylori infection, and its association with clinical, immunological and virological parameters in a large cohort of HIV-infected individuals and uninfected controls in a West African country.The association of HIV and hildhood –11. The This cross-sectional study was conducted at the Komfo Anokye Teaching Hospital, a tertiary referral hospital in the Ashanti Region of Ghana. Between November 2011 and November 2012, consecutive adult HIV-infected patients presenting to the HIV outpatient clinic, and HIV-negative blood donors presenting to the blood bank of the hospital, were offered participation in the study. All participants gave a written informed consent prior to enrolment. The study was conducted in conformity with the Helsinki declaration, and was approved by the appropriate ethics committees of the Kwame Nkrumah University of Science and Technology (Ghana) and of the medical association in Hamburg (Germany).H. pylori using the RidaScreen® FemtoLab H. pylori stool antigen test . The sensitivity and specificity of this test has been described to be 98% and 96.7% in pediatric patients and 93% and 90% in adult patients [Demographic, socioeconomic, and clinical data, as well as a detailed medical history were recorded using standardized questionnaires, which were completed by trained study personnel. In particular, time since diagnosis of HIV infection, duration and kind of antiretroviral therapy (ART), co-medications, and clinical parameters were documented. Routine laboratory parameters were extracted from patient’s folders. EDTA blood samples were obtained for the analysis of CD4/CD8 T cell counts, using a FACSCalibur® flow cytometer . HIV-1 and 2 antibody testing was done using the First Response® HIV-1/2 test  and the Genscreen® ULTRA HIV Ag-Ab Assay . EDTA plasma and native stool samples were freshly frozen at -80°C and transported to Germany on dry ice. Stool was tested for patients ,13. HIV-H. pylori infection and other demographic, clinical and laboratory parameters, using only parameters with a significance level of ≤0.05 in bivariate analysis and a correlation coefficient of ≤0.10 in the multivariate regression model. Missing data were excluded from analysis. Statistical analyses were conducted with SPSS version 19 software .Parametric variables were compared using the Student’s t-test, non-parametric variables were compared using the Mann-Whitney U-test. Categorical data were analyzed using Chi-squared or Fisher’s exact test. A multivariable logistic regression model was used to analyze the association between H. pylori testing were available for 952 HIV-positive (86.9%) and 100 HIV-negative individuals (93.5%). HIV-positive, compared to HIV-negative individuals, were more often female, significantly older, had a lower BMI, lower socioeconomic status, lower CD4 and higher CD8 T cell counts . In HIV-positive individuals, H. pylori prevalence declined in parallel with CD4+ T cell counts, from 64.8% in patients with more than 800 CD4 T cells/μL, to 41.4% in patients with less than 200 CD4 T cells/μL. The same trend was observed in HIV-negative individuals, without reaching statistical significance  and less likely to have attained a tertiary level education . There were no significant differences in other demographic variables assessed, or in WHO clinical HIV disease stages , higher CD4/CD8 ratios  and lower HIV-1 viral loads  compared to those without H. pylori co-infection. H. pylori positive patients in this group also had higher mean hemoglobin levels , and lower platelet counts , as shown in H. pylori positive and negative individuals.Among ART-naïve HIV patients, those with H. pylori infection was also not associated to increased frequencies of gastrointestinal symptoms in H. pylori positive, compared to negative patients, with weight loss , epigastric discomfort , and diarrhea  being the most common symptoms. In the HIV-infected, ART-exposed group, no significant associations between H. pylori status and CD4+ T cell count, HIV-1 viral load, or the proportion of patients with undetectable viral load were observed. However, significantly lower CD8+ T cell counts , and a trend towards higher CD4/CD8 ratios , as possible indicator of decreased immune activation, were noted among those patients with H. pylori co-infection [nfection –17.H. pylori infection. A weak trend towards higher CD4/CD8 ratios was also observed in those HIV-negative individuals with H. pylori infection, compared to those without H. pylori infection .Among HIV-negative controls, no differences in baseline characteristics, symptoms, or socio-demographic parameters were observed between individuals with and without H. pylori infection  for H. pylori infection was 0.82 for those patients having access to tap water and 1.37 for those with >200 CD4 T cells/μl within the group of HIV-positive patients. No risk factors were identified to be associated with H. pylori infection in the HIV-negative group (data not shown).Using a logistic multivariable regression model including parameters with p≤0.05 in the univariate analysis and a correlation coefficient of ≤0.1 in the regression model, only CD4+ T cell count  and having access to tap water  were associated with nfection . SignifiH. pylori and HIV infection in sub-Saharan Africa, where both infections are highly co-endemic. We assessed the prevalence of H. pylori co-infection in a large cohort of unselected adult HIV-infected individuals and HIV-negative controls, and its association with clinical, immunological and virological parameters. We found a graded decrease in H. pylori prevalence in relation to the level of immune competence, being 88% in HIV-negative and 51.5% in HIV-positive individuals. Among HIV positive individuals, H. pylori prevalence declined in tandem with CD4+ T cell counts. A similar trend was observed in HIV-negative individuals, although statistical significance was not attained.This is the first and largest study to systematically investigate the interplay between H. pylori prevalence in HIV-positive compared to HIV-negative individuals, and also among patients with AIDS compared to matched HIV-infected patients without AIDS [H. pylori status, often lacking in previous studies have been explored in the present study. Furthermore, studies including asymptomatic patients used serological tests to determine H. pylori status, which have been shown to be problematic especially in HIV-infected individuals [H. pylori stool antigen tests, as employed in the present study, are non-invasive and have a proven high sensitivity and specificity, making them suitable tools for epidemiologic studies including HIV-infected individuals [Our results are in accordance with previous epidemiologic studies, indicating a lower out AIDS ,2. Howevividuals . H. pyloividuals ,13.H. pylori is generally considered a disease of poverty and known to be associated with poor hygienic conditions, HIV-negative participants in our study, having a clearly higher H. pylori prevalence, ironically had indicators of a higher socioeconomic status. This suggests that the significant differences in H. pylori prevalence observed between the HIV positive and HIV negative participants may not be explained wholly by socioeconomic disparities. Indeed, the HIV negative participants had more frequent access to tap water compared to HIV-positive individuals, and having no access to tap water was independently associated with the H. pylori infection in our study. Besides indicating poor sanitary conditions, the lack of access to tap water might also directly promote H. pylori acquisition by consumption of contaminated drinking water, e.g. from wells. An association between H. pylori and the consumption of water from wells has previously been reported from India, [H. pylori has also been identified in drinking water samples from Pakistan by PCR [Although m India,  and H. pn by PCR .H. pylori prevalence with the progression of immunodeficiency in HIV-positive individuals, with the same trend being observed in HIV-negative individuals, but without reaching statistical significance. The underlying mechanisms responsible for this association between immune competence and H. pylori prevalence are still unclear, although several hypotheses have been offered [H. pylori eradication [H. pylori status and reported intake of antibiotics in the past six months before recruitment. Furthermore, only few patients reported taking antibiotics in this period of time, making it unlikely that the observed differences in H. pylori prevalence are explained by unintended eradication in our study population.We also found a significant graded decrease in  offered . The mosdication . We founH. pylori eradication [H. pylori eradication rate of 19% for monotherapy regimens has been reported [H. pylori status in univariate, but not in multivariate logistic regression analysis. Co-trimoxazole has not been reported to have activity against H. pylori, and a culture medium containing trimethoprim and sulfamethoxazole has been developed to selectively isolate H. pylori from animal samples [H. pylori [H. pylori replication by concurrent tuberculosis treatment, or even clearance of the infection, is thus conceivable. However, it is to be noted that HIV patients with advanced disease are often prescribed Co-trimoxazole prophylaxis against opportunistic infections and are also more likely to receive anti-tubercular therapy for tuberculosis hence the observed lack of significant association in multivariate analyses between use of these antibiotics and risk of H. pylori co-infection. These findings suggest that progressive HIV disease rather than antibiotic usage may account for the diminution in frequency of H. pylori co-infection.Antibiotic monotherapy has been reported to have only minor efficacy in dication . Using areported . In our  samples . In cont. pylori . A tempoH. pylori infection requires an intact mucosal cellular immunity, and that the loss of the CD4+ T cell population in the gastric mucosa may prevent H. pylori persistence [H. pylori prevalence with CD4+ T cell count would be consistent with this theory, although there is no evidence that impaired T cell immunity itself might cause a loss of H. pylori infection. CD4+ T cells have been shown to be increased in H. pylori gastritis, but gastric inflammation has been shown to correlate with lower H. pylori bacterial load, and pro-inflammatory genetic profiles are associated to lower H. pylori seroprevalence [H. pylori infection, regulatory T cells have the ability to override this T cell driven immunity [H. pylori persistence [H. pylori persistence in the context of HIV infection.Another proposed hypothesis is that the maintenance of sistence ,25,26. Hevalence –29. Whilimmunity . Althougsistence . FurtherH. pylori infection is linked to a number of adverse clinical effects, such as iron deficiency anemia, childhood growth faltering, other gastrointestinal infections and chronic diarrhea[H. pylori infection was not associated to the presence of diarrhea, anemia, malnutrition or parasitic diseases (data not shown). Indeed a paradoxical protective effect of H. pylori infection against tuberculosis has been reported [H. pylori infection is associated with enhanced Th1-type immune responses to TB antigens [H. pylori infection is associated with decreased markers of immune activation in ART-naïve HIV infected patients [H. pylori infection may influence susceptibility to HIV infection or the natural course of HIV disease. A large proportion of HIV-infected individuals worldwide are co-infected with H. pylori, hence such interaction could be relevant for the understanding of HIV immunopathology, and could also have public health implications, especially considering the ongoing efforts to develop an H. pylori vaccine [ diarrhea–35. In oreported . Furtherantigens . We havepatients . Considepatients –17, it i vaccine .H. pylori within the group of HIV patients. Since we included unselected HIV patients, the group was heterogenous, among others, in terms of ART status and clinical stage of HIV disease. The group of patients taking ART in particular was heterogeneous, and we did not record details on the history and efficacy of ART in terms of CD4+ T cell recovery and virological suppression, limiting the informative value of the analysis in this subgroup. Importantly, the cross sectional study design did not allow for the investigation of causal relationships concerning the described associations.There are some limitations of our study to be mentioned. The sample size of our HIV-negative group was smaller than that of HIV-positive individuals, and differed in terms of age and gender distribution. However, the main focus of this study was to analyze the effect of H. pylori infection is associated with higher CD4+ T cell counts and lower HIV-1 viral loads in ART-naïve patients. Our findings could not be explained by typical confounders as socioeconomic factors, time since diagnosis of HIV infection or unintended H. pylori eradication by antibiotic use for other infectious conditions. Considering the pathophysiological overlap of both chronic infections, the effects of H. pylori infection on the systemic immune response, and subsequently on the natural course of HIV disease, warrants further investigation employing prospective studies.In conclusion, we have shown that S1 Table(DOCX)Click here for additional data file.