Draft Genome Sequences of 12 Monophasic Salmonella enterica subsp. enterica Serotype Typhimurium 1,4,[5], 12:i:- Strains Isolated from Wild Griffon Vultures in Eastern Spain

[EN] Monophasic Salmonella enterica subsp. enterica serovar Typhimurium is one of the most common zoonotic pathogens. Salmonella species reside in a wide variety of hosts, including wild animals. Thus, we report here the genome sequences of 12 monophasic S. Typhimurium strains isolated from healthy wild vultures to gain better insight into their epidemiology and host-pathogen interactions.This work was funded by Generalitat Valenciana (Government of Valencia) and by CEU-UCH (Consolidacion de Indicadores INDI15/16, INDI16/20, and INDI17/25).Marín, C.; D'auria, G.; Martínez-Priego, L.; Marco-Jiménez, F. (2019). Draft Genome Sequences of 12 Monophasic Salmonella enterica subsp. enterica Serotype Typhimurium 1,4,[5], 12:i:- Strains Isolated from Wild Griffon Vultures in Eastern Spain. Microbiology Resource Announcements. 8(42):1-3. https://doi.org/10.1128/MRA.00570-19S13842Blanco, G. (2018). Supplementary feeding as a source of multiresistantSalmonellain endangered Egyptian vultures. Transboundary and Emerging Diseases, 65(3), 806-816. doi:10.1111/tbed.12806Krawiec, M., Kuczkowski, M., Kruszewicz, A., & Wieliczko, A. (2015). Prevalence and genetic characteristics of Salmonella in free-living birds in Poland. BMC Veterinary Research, 11(1), 15. doi:10.1186/s12917-015-0332-xMolina-López, R. A., Vidal, A., Obón, E., Martín, M., & Darwich, L. (2015). Multidrug-resistantSalmonella entericaSerovar Typhimurium Monophasic Variant 4,12:i:- Isolated from Asymptomatic Wildlife in a Catalonian Wildlife Rehabilitation Center, Spain. Journal of Wildlife Diseases, 51(3), 759-763. doi:10.7589/2015-01-019Marin, C., Torres, C., Marco-Jiménez, F., Cerdà-Cuéllar, M., Sevilla, S., Ayats, T., & Vega, S. (2018). Supplementary feeding stations for conservation of vultures could be an important source of monophasic Salmonella typhimurium 1,4,[5],12:i:-. Science of The Total Environment, 636, 449-455. doi:10.1016/j.scitotenv.2018.04.310Marin, C., Palomeque, M.-D., Marco-Jiménez, F., & Vega, S. (2014). Wild Griffon Vultures (Gyps fulvus) as a Source of Salmonella and Campylobacter in Eastern Spain. PLoS ONE, 9(4), e94191. doi:10.1371/journal.pone.0094191Schmieder, R., & Edwards, R. (2011). Quality control and preprocessing of metagenomic datasets. Bioinformatics, 27(6), 863-864. doi:10.1093/bioinformatics/btr026Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114-2120. doi:10.1093/bioinformatics/btu170Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., … Homer, N. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics, 25(16), 2078-2079. doi:10.1093/bioinformatics/btp352Quinlan, A. R., & Hall, I. M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6), 841-842. doi:10.1093/bioinformatics/btq033Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. Bioinformatics, 30(14), 2068-2069. doi:10.1093/bioinformatics/btu153Lima, T., Auchincloss, A. H., Coudert, E., Keller, G., Michoud, K., Rivoire, C., … Bairoch, A. (2009). HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot. Nucleic Acids Research, 37(Database), D471-D478. doi:10.1093/nar/gkn661Finn, R. D., Coggill, P., Eberhardt, R. Y., Eddy, S. R., Mistry, J., Mitchell, A. L., … Bateman, A. (2015). The Pfam protein families database: towards a more sustainable future. Nucleic Acids Research, 44(D1), D279-D285. doi:10.1093/nar/gkv1344Seribelli, A. A., Frazão, M. R., Gonzales, J. C., Cao, G., Leon, M. S., Kich, J. D., … Falcão, J. P. (2018). Draft Genome Sequences of 20 Salmonella enterica subsp. enterica Serovar Typhimurium Strains Isolated from Swine in Santa Catarina, Brazil. Genome Announcements, 6(16), e00232-18. doi:10.1128/genomea.00232-1

Starkeya nomas sp. nov., a prosthecate and budding bacterium isolated from an immunocompromized patient

Strain HF14-78462T is an environmental bacterium found in clinical samples from an immunocompromized patient in 2014 at Hospital Universitari i Politècnic La Fe (Valencia, Spain). Phenotypically, strain HF14-78462T cells were Gram-stain-negative, aerobic, non-spore forming and non-motile small rods which formed mucous and whitish-translucent colonies when incubated at 20-36 °C. Phylogenetic analyses based on the 16S rRNA genes and the whole genomes of closest sequenced relatives confirmed that strain HF14-78462T is affiliated with the genus Starkeya. The strain was oxidase, catalase and urease positive; but indole, lysine decarboxylase, ornithine decarboxylase and DNase negative, did not produce H2S and was able to utilize a wide variety of carbon sources including acetamide, adonitol, amygdalin, l-arabinose, citric acid, glucose, mannitol and melibiose. Unlike Starkeya novella and Starkeya koreensis, strain HF14-78462T failed to grow in thiosulphate-oxidizing media and had a narrower temperature growth range. Its genome was characterized by a size of 4.83 Mbp and a C+G content of 67.75 mol%. Major fatty acids were C18:1 ω7c, cyclo C19 : 0 and C16 : 0, its polar acids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and an aminophospholipid; while the ubiquinones were Q9 (1.8 %) and Q10 (98.2 %). Digital DNA-DNA hybridization values were 41 and 41.4 against S. novella and S. koreensis, respectively, while average nucleotide identity values were around 84 %. Phenotypic, average nucleotide identity and phylogenomic comparative studies suggest that strain HF14-78462T is a new representative of the genus Starkeya and the name Starkeya nomas sp. nov. is proposed. The type strain is HF14-78462T (=CECT 30124T=LMG 31874T).Financial support was obtained by the IIS project 2013/0437.S

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate

The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.This work was funded by grant CP09/00049 Miguel Servet, Instituto de Salud Carlos III, Spain to GD; by projects SAF2009-13032-C02-01 and SAF 2012-31187 (AM), BFU2009-12895-CO2-01 and SAF2010-16240 (FC) from the Spanish Ministry for Science and Innovation (MCINN), FU2008-04501-E from Spanish Ministry for Science and Innovation(MCINN) in the frame of ERA-Net PathoGenoMics and Prometeo/2009/092 from Conselleria D’Educació Generalitat Valenciana,Spain, to AM. MD is recipient of a fellowship from Spanish Ministry of Education FPU2010. MGG was supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation (Grant number BES-2008-006029

Tobacco rattle virus 16K silencing suppressor binds AGO4 and inhibits formation of RNA silencing complexes

35 p.-5 fig.1 tab. supl.The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression.This work was supported by grants BIO2006-13107 and BIO2009-12004 from the Spanish Ministry of Science and Innovation (MICINN-FEDER funding program) (CL), grants OTKA NN 107787 and NN 111024 from the Hungarian Scientific Research Fund and (LL), and grant AGL2008-03482 from MICINN (TC). LFC was the recipient of a JAE-Doc Contract from CSIC. IGB and IG were supported by graduate fellowships from MICINN. LL was supported from TÁMOP-4.2.2.A11/1/KONV-2012-0035.Peer reviewe

Clustering analysis of the k-mer abundance distribution.

<p>Comparison of the relative abundances of 6-mer in the different datasets using hierarchical clustering. As observed, the most likely conformation shows aggregation of <i>E. coli</i> with DS methodology, while <i>B. subtilis</i> is associated with MDA.</p

Results of <i>E. coli</i> genome mapping and blast to NCBI database.

<p>Proportions (in %) of Mbp mapped by SSAHA2 to <i>E. coli</i> genome are shown for MDA and DS sequences, separately for each sequencing run. It can be observed that the percentage of mapped DS reads were significantly higher than the MDA reads. The reads that were not mapped to <i>E. coli</i> were analyzed by blast in “nr” database. However, most reads remained unidentified, especially in the case of MDA.</p

Flowchart of the minimal library preparation protocol.

<p>Panel A: The experimental work started with cell sorting, where 20,000 cells were separated in two replicates to confirm the whole experiment. The DNA from 20,000 cells was extracted and split into halves, where one half was amplified with GenomiPhi (MDA) and a second half was processed without whole genome amplification (DS). The shotgun libraries were prepared with the same alternative protocol for the both MDA and DSsamples. Library quality control points were the test PCR with emPCR primers to prove the removal of self-ligated adaptors and the library concentration checking with qPCR. The MDAsample and DSsample with different MIDs in two repetitions were combined into two sequencing runs as sho! wn in the scheme. Panel B: DNA amount requirements in the standard Rapid Library Preparation Method Manual GS FLX+ Series – XL+ (May 2011) compared with the amounts actually needed for sequencing on a selected PTP region. The minimal amount of prepared library required for proceeding to emPCR step in the standard 454 protocol may lose 99% of input DNA during the library preparation step. Then, this amount is diluted to a working stock of 10–7 molecules, defined as the best starting point to perform the emPCR titration step. However, if the exact number of molecules is quantified with qPCR, the emPCR titration step can be omitted, so actually only 0.13 pg of prepared library are needed for sequencing on 1/8 region of PTP (equivalent to 340,000 ssDNA molecules). This allows to use an alternative shotgun protocol where the DNA losses are reduced.</p

Profiling the Bladder Microbiota in Patients With Bladder Cancer

Evidence suggests that microbiota may contribute to the pathogenesis of several diseases, including cancer. In the case of bladder cancer, preliminary studies have found alterations in the urinary microbiota of patients with urothelial carcinoma compared with healthy individuals. Conversely, the urinary microbiota differ between men and women, and it has been hypothesized that these differences are associated with the lower incidence of bladder cancers in women. The objective of this study was to characterize the bladder microbiota in paired samples of tumor and non-tumor mucosa of patients with malignant bladder neoplasia using next-generation sequencing. In addition, we aimed to study potential differences in microbial composition in tumor samples according to clinical and pathological variables, and to determine possible microbial profiles. We found significant differences in microbial richness at the genus level, with a higher richness observed in the non-tumor compared with the tumor mucosa. It was also shown that Actinobacteria were significantly more enriched in the non-tumor compared with the tumor mucosa (P = 0.014). In the multivariate analysis, we found significant differences in microbial composition according to tumor grade (P = 0.03 and 0.04 at the phylum and genus levels, respectively). Moreover, we detected a higher microbial richness in non-tumor vs. tumor tissues which agrees with the global assumption that microbial richness is an indicator of health. The greater abundance of members of the Actinobacteria phylum in the non-neoplastic bladder mucosa samples supports the hypothesis that a higher abundance of Actinomycetes is associated with a lower rate of bladder cancer in women and suggests a protective role for these microbiota. We detected a microbial profile that was enriched for Enterococcus in low-grade tumors. Finally, we identified the presence of two clusters in the microbial composition of the tumor mucosa samples, significantly enriched for the genera Barnesiella, Parabacteroides, Prevotella, Alistipes, and Lachnospiracea_incertae_sedis (Cluster 1), or Staphylococcus (Cluster 2). Further longitudinal studies are needed to assess the role of the bladder microbiota in carcinogenesis.This work was supported by the Conselleria de Educación, Cultura y Deporte de la Generalitat Valenciana, Spain (GV/2016/175).Peer reviewe

Distribution of coverage throughout the <i>E. coli</i> genome.

<p>The comparison of the genome coverage obtained by MDA and DS methods. The genome coverage of MDA reads was characterized by unequal distribution with many gaps and several areas with extremely high coverage (up to 121 x), while the highest coverage obtained by DS was only 15 x and it was better distributed throughout the whole genome.</p