Rouxiella badensis sp. nov. and Rouxiella silvae sp. nov. isolated from peat bog soil and emendation description of the genus Rouxiella.

Four bacterial strains isolated from peat bog soil or swampy meadow in Baden-Württemberg (Germany) and found to have rrs sequences close to that of Rouxiella chamberiensis were compared to this species by using multi-locus sequence analysis and phenotypic tests. The four strains constituted two discrete groups (referred to as the Baden and the Silva groups) belonging to the genus Rouxiella. These groups differed in their ability to grow at 37 °C, reduce nitrate into nitrite, and to produce acid from several carbohydrates. Two novel species are, therefore, proposed: Rouxiella badensis sp. nov. for the Baden group (type strain, 323T=CIP 111153T=DSM 100043T) and Rouxiella silvae for the Silva group (type strain, 213T=CIP 111154T=DSM 103735T). The definition of the genus Rouxiellahas also been emended in order to take these two novel species into account.

Genome-wide mosaicism within Mycobacterium abscessus: evolutionary and epidemiological implications.

BACKGROUND: In mycobacteria, conjugation differs from the canonical Hfr model, but is still poorly understood. Here, we quantified this evolutionary processe in a natural mycobacterial population, taking advantage of a large clinical strain collection of the emerging pathogen Mycobacterium abscessus (MAB). RESULTS: Multilocus sequence typing confirmed the existence of three M. abscessus subspecies, and unravelled extensive allelic exchange between them. Furthermore, an asymmetrical gene flow occurring between these main lineages was detected, resulting in highly admixed strains. Intriguingly, these mosaic strains were significantly associated with cystic fibrosis patients with lung infections or chronic colonization. Genome sequencing of those hybrid strains confirmed that half of their genomic content was remodelled in large genomic blocks, leading to original tri-modal 'patchwork' architecture. One of these hybrid strains acquired a locus conferring inducible macrolide resistance, and a large genomic insertion from a slowly growing pathogenic mycobacteria, suggesting an adaptive gene transfer. This atypical genomic architecture of the highly recombinogenic strains is consistent with the distributive conjugal transfer (DCT) observed in M. smegmatis. Intriguingly, no known DCT function was found in M. abscessus chromosome, however, a p-RAW-like genetic element was detected in one of the highly admixed strains. CONCLUSION: Taken together, our results strongly suggest that MAB evolution is sporadically punctuated by dramatic genome wide remodelling events. These findings might have far reaching epidemiological consequences for emerging mycobacterial pathogens survey in the context of increasing numbers of rapidly growing mycobacteria and M. tuberculosis co-infections.

Pantoea coffeiphila sp. nov., cause of the 'potato taste' of Arabica coffee from the African Great Lakes region.

Six isolates recovered from coffee seeds giving off a potato-like flavour were studied. Gene sequencing (rrs and rpoB) showed they belong to the genus Pantoea. By DNA-DNA hybridization, the isolates constituted a genomic species with less than 17% relatedness to 96 strains representing enterobacterial species. Multilocus sequence analysis (gyrB, rpoB, atpD and infB genes) showed the isolates to represent a discrete species of the genus Pantoea. Nutritional versatility of the novel species was poor. The novel species is proposed as Pantoea coffeiphila sp.nov. and its type strain is Ca04(T) ( =CIP 110718(T) =DSM 28482(T)).

Rouxiella chamberiensis gen. nov., sp. nov., a member of the family Enterobacteriaceae isolated from parenteral nutrition bags.

Parenteral nutrition bags for newborns were found contaminated by a previously undescribed member of the family Enterobacteriaceae. The six isolates studied by rrs gene (encoding 16S rRNA) sequence analysis and multi-locus sequence analysis (MLSA) formed a discrete branch close to the genera Ewingella, Rahnella, Yersinia,Hafnia and Serratia. Phenotypically, the new taxon was distinct from these five genera. The new taxon gave positive results in Voges-Proskauer, Simmons citrate and o-nitrophenyl-ß-galactoside hydrolysis tests; fermented d-glucose, d-mannitol, l-rhamnose, melibiose, l-arabinose and d-xylose; hydrolysed aesculin; and did not ferment maltose, trehalose, raffinose, d-sorbitol, sucrose or cellobiose. Tests for motility, gas production, urease, gelatinase and nitrate reduction were also negative. All isolates failed to grow at 37 °C. The DNA G+C content of strain 130333T was 53 mol%. On the basis of data obtained in this study, the six isolates represent a novel species of a new genus in the family Enterobacteriaceae, named Rouxiella chamberiensis gen. nov., sp. nov. The type strain of the type species is 130333T ( = CIP 110714T = DSM 28324T).

A Six Years (2010-2016) Longitudinal Survey of the Four Serotypes of Dengue Viruses in Lao PDR.

Dengue fever is the most prevalent arthropod-borne viral infection of humans in tropical and subtropical countries. Since 1979, dengue has been reported to be endemic in the Lao People's Democratic Republic (PDR), as in many countries in Southeast Asia, with a complex circulation of the four dengue viruses' serotypes (DENV-1 to DENV-4). By sequencing the complete envelope protein, we explored a panel of samples from five Lao Provinces (Vientiane capital, Luangprabang, Bolikhamxay, Saravane, Attapeu) to enrich knowledge about the co-circulation of DENVs in Lao PDR between 2010 and 2016. Phylogenetic analyses highlighted the specific circulation of DENV-1 genotype I, DENV-2 genotype Asian I, DENV-4 genotype I and the co-circulation of DENV-3 genotype II and III. The continuous co-circulation of the four serotypes was underlined, with genotype or cluster shifts among DENV-3 and DENV-1. These data suggested the emergence or re-emergence of DENV strains associated with epidemic events, potentially linked to the exchanges within the territory and with neighboring countries. Indeed, the increasing local or regional connections favored the dissemination of new isolates or new clusters around the country. Since 2012, the surveillance and alert system created in Vientiane capital by the Institut Pasteur du Laos appears to be a strategic tool for monitoring the circulation of the four serotypes, especially in this endemic country, and allows for improving dengue epidemiological knowledge to anticipate epidemic events better.

Evolution of synchronous female bilateral breast cancers and response to treatment.

Synchronous bilateral breast cancer (sBBC) occurs after both breasts have been affected by the same germline genetics and environmental exposures. Little evidence exists regarding immune infiltration and response to treatment in sBBCs. Here we show that the impact of the subtype of breast cancer on levels of tumor infiltrating lymphocytes (TILs, n = 277) and on pathologic complete response (pCR) rates (n = 140) differed according to the concordant or discordant subtype of breast cancer of the contralateral tumor: luminal breast tumors with a discordant contralateral tumor had higher TIL levels and higher pCR rates than those with a concordant contralateral tumor. Tumor sequencing revealed that left and right tumors (n = 20) were independent regarding somatic mutations, copy number alterations and clonal phylogeny, whereas primary tumor and residual disease were closely related both from the somatic mutation and from the transcriptomic point of view. Our study indicates that tumor-intrinsic characteristics may have a role in the association of tumor immunity and pCR and demonstrates that the characteristics of the contralateral tumor are also associated with immune infiltration and response to treatment.

Automated phosphopeptide identification using multiple MS/MS fragmentation modes.

Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, respectively), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana . We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.

Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the Central African Republic.

Monkeypox is an emerging and neglected zoonotic disease whose number of reported cases has been gradually increasing in Central Africa since 1980. This disease is caused by the monkeypox virus (MPXV), which belongs to the genus Orthopoxvirus in the family Poxviridae. Obtaining molecular data is particularly useful for establishing the relationships between the viral strains involved in outbreaks in countries affected by this disease. In this study, we evaluated the use of the MinION real-time sequencer as well as different polishing tools on MinION-sequenced genome for sequencing the MPXV genome originating from a pustular lesion in the context of an epidemic in a remote area of the Central African Republic. The reads corresponding to the MPXV genome were identified using two taxonomic classifiers, Kraken2 and Kaiju. Assembly of these reads led to a complete sequence of 196,956 bases, which is 6322 bases longer than the sequence previously obtained with Illumina sequencing from the same sample. The comparison of the two sequences showed mainly indels at the homopolymeric regions. However, the combined use of Canu with specific polishing tools such as Medaka and Homopolish was the best combination that reduced their numbers without adding mismatches. Although MinION sequencing is known to introduce a number of characteristic errors compared to Illumina sequencing, the new polishing tools allow a better-quality MinION-sequenced genome, thus to be used to help determine strain origin through phylogenetic analysis.

New insights into Human Pegivirus-1 (HPgV-1) genotypes diversity in sub-Saharan Africa.

In the framework of a viral discovery research program using metagenomics, Human Pegivirus-1 reads (HPgV-1, formerly known as GBV-C) were detected in plasma pools of healthy blood donors from seven sub-Saharan African countries. For five of these countries, Mauritania, Mali, Niger, Burundi and Madagascar, no data about HPgV-1 genotypes was reported to date. To confirm our metagenomic findings and further investigate the genotype diversity and distribution of HPgV-1 in Africa, 400 blood donations from these five localities as well as from Cameroon, the Democratic Republic of Congo (DRC) and the Burkina Faso were screened with a RT-nested PCR targeting the viral 5'NCR region. Amplified products were sequenced, and the virus was genotyped by phylogenetic analysis. Out of the 400 plasma samples tested, 65 were positive for HPgV-1 RNA and 61 were successfully genotyped. Among these, 54 strains (88.5%) clustered with genotype 1, six (9.8%) with genotype 2 and one (1.6%) with genotype 5. Genotype 1 was observed in all countries studied, except in Madagascar, genotype 2 was detected in Mauritania and Madagascar, and genotype 5 in DRC. Overall, our results extend the geographic distribution of HPgV-1 in Africa and provide six additional nearly complete genomes. Considering that some HPgV-1 genotypes have been reported as potential predictive indicators of lower disease progression in HIV-1 infected subjects, further investigations should be conducted to better understand the positive impact, if any, of this virus.

Molecular characterization of a new highly divergent Mobala related arenavirus isolated from Praomys sp. rodents.

Arenaviruses represent a family of viruses that are naturally present in rodents belonging to subfamily Murinae, Neotominae or Sigmodontinae. Except for Lassa virus, little information is available on other Old-World arenaviruses. Here, we describe strain AnRB3214, a virus isolated from a presumed Praomys sp. rodent in the Central African Republic in 1981 and assigned to Ippy virus based on antigenic similarity. The strain was simultaneously sequenced on Illumina NovaSeq 6000 and MinION Mk1B devices and analysed with various bioinformatics tools. We show that the best genome coverage and depth were obtained with the Kaiju and Minimap2 classification and identification tools, on either the MinION or the Illumina reads. The genetic analysis of AnRB3214 fragments showed 68% to 79% similarity with the Mobala and Gairo mammarenaviruses at the nucleic acid level. Strain AnRB3214 had a truncated nucleoprotein smaller than that of other Old World arenaviruses. Molecular clock analysis suggests that this strain diverged from Mobala virus at least 400 years ago. Finally, this study illustrates the importance of genomics in the identification of archived viruses and expands on the diversity of African arenaviruses, because strain AnRB3214 is either a variant or a close relative of Mobala virus, and not Ippy virus.

No evidence for a pathogen associated with pulmonary MALT lymphoma: a metagenomics investigation.

Mucosa-associated lymphoid tissue (MALT) lymphoma is generally associated with chronic antigen stimulation: auto-antigens or of microbial origin. Only one study suggested association between Achromobacter xylosoxidans and pulmonary MALT lymphoma. We aimed to investigate the presence of virus or any infectious agents in pulmonary MALT lymphoma by using metagenomic next-generation sequencing (mNGS).All lung samples were centrally reviewed. The t(11;18) (q21;q21) was evaluated by FISH analysis. The snap frozen large lung biopsies were analyzed by mNGS. After lung biopsies homogenization total nucleic acids (RNA and DNA) were extracted, amplified and classified according to their taxonomic assignment, after exclusion of host DNA.We included 13 samples from pulmonary MALT lymphoma (mean age: 60.3 years, 7 women, 3 with auto-immune background) and 10 controls. The diagnosis of MALT lymphoma was confirmed for the 13 samples, 3 showed API2-MALT1 translocation (23%). No evidence of the presence of a specific pathogen was clearly identified in the group of patients with pulmonary MALT lymphoma. We identifiedA. xylosoxidans sequence in 4/13 patients and in 4/10 controls.This study did not find evidence for a DNA or RNA virus, a fungi, a parasite or a bacteria associated with pulmonary MALT lymphoma either in the stroma or in tumor cells.

Deep sequencing applied to the analysis of viromes in patients with beta-thalassemia.

To date, blood banks apply routine diagnosis to a specific spectrum of transfusion-transmitted viruses. Even though this measure is considered highly efficient to control their transmission, the threat imposed by emerging viruses is increasing globally, which can impact transfusion safety, especially in the light of the accelerated viral discovery by novel sequencing technologies. One of the most important groups of patients, who may indicate the presence of emerging viruses in the field of blood transfusion, is the group of individuals who receive multiple transfusions due to hereditary hemoglobinopathies. It is possible that they harbor unknown or unsuspected parenterally-transmitted viruses. In order to elucidate this, nucleic acids from 30 patients with beta-thalassemia were analyzed by Illumina next-generation sequencing and bioinformatics analysis. Three major viral families: Anelloviridae, Flaviviridae and Hepadnaviridae were identified. Among them, anelloviruses were the most representative, being detected with high number of reads in all tested samples. Human Pegivirus 1 (HPgV-1, or GBV-C), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) were also identified. HBV and HCV detection was expected due to the high seroprevalence in patients with beta thalassemia. Our results do not confirm the presence of emerging or unsuspected viruses threatening the transfusion safety at present, but can be used to actively search for viruses that threaten blood transfusion safety. We believe that the application of viral metagenomics in multiple-transfused patients is highly useful to monitor possible viral transfusion threats and for the annotation of their virome composition.

Alignment of LC-MS images, with applications to biomarker discovery and protein identification.

LC-MS-based approaches have gained considerable interest for the analysis of complex peptide or protein mixtures, due to their potential for full automation and high sampling rates. Advances in resolution and accuracy of modern mass spectrometers allow new analytical LC-MS-based applications, such as biomarker discovery and cross-sample protein identification. Many of these applications compare multiple LC-MS experiments, each of which can be represented as a 2-D image. In this article, we survey current approaches to LC-MS image alignment. LC-MS image alignment corrects for experimental variations in the chromatography and represents a computational key technology for the comparison of LC-MS experiments. It is a required processing step for its two major applications: biomarker discovery and protein identification. Along with descriptions of the computational analysis approaches, we discuss their relative merits and potential pitfalls.

Assessing computational tools for the discovery of transcription factor binding sites.

The prediction of regulatory elements is a problem where computational methods offer great hope. Over the past few years, numerous tools have become available for this task. The purpose of the current assessment is twofold: to provide some guidance to users regarding the accuracy of currently available tools in various settings, and to provide a benchmark of data sets for assessing future tools.

Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples.

Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.

SARS-CoV related Betacoronavirus and diverse Alphacoronavirus members found in western old-world.

The emergence of SARS-CoV and MERS-CoV, triggered the discovery of a high diversity of coronaviruses in bats. Studies from Europe have shown that coronaviruses circulate in bats in France but this reflects only a fraction of the whole diversity. In the current study the diversity of coronaviruses circulating in western Europe was extensively explored. Ten alphacoronaviruses in eleven bat species belonging to the Miniopteridae, Vespertilionidae and Rhinolophidae families and, a SARS-CoV-related Betacoronavirus in Rhinolophus ferrumequinum were identified. The diversity and prevalence of bat coronaviruses presently reported from western Europe is much higher than previously described and includes a SARS-CoV sister group. This diversity demonstrates the dynamic evolution and circulation of coronaviruses in this species. That said, the identified coronaviruses were consistently associated with a particular bat species or genus, and these relationships were maintained no matter the geographic location. The observed phylogenetic grouping of coronaviruses from the same species in Europe and Asia, emphasizes the role of host/pathogen coevolution in this group.

High prevalence of cyclovirus Vietnam (CyCV-VN) in plasma samples from Madagascan healthy blood donors.

Cycloviruses, small ssDNA viruses belonging to the Circoviridae family, have been suggested as possible causes of enteric, respiratory and neurological disorders in human patients. One of these species, cyclovirus-Vietnam (CyCV-VN), initially isolated from cerebrospinal fluid samples of patients with unexplained neurological disorders, has since been reported in serum samples from chronically patients infected with HBV, HCV or HIV, in Italy. On the other hand, CyCV-VN was not detected in serum samples from healthy individuals. Here, we report on a high prevalence of 43.4% (40/92) of CyCV-VN in plasma samples from asymptomatic blood donors from Madagascar. Interestingly, this virus was not detected by metagenomics and PCR in six other African countries, suggesting regional differences in CyCV-VN prevalence across Africa. Phylogenetic analysis based on the complete genomes showed that CyCV-VN sequences isolated from blood were most closely related to sequences previously reported from human stool in Madagascar. Further investigations using larger cohorts are required to determine the global epidemiology, the natural history and the pathological significance, if any, of CyCV-VN infection in humans.

Comparison of statistical significance criteria.

We study and compare two classes of statistical criteria to assess the significance of exceptional words. Indeed, the Z-score-like criteria, or the normal approximation that is a strict equivalent, suffer from several drawbacks in terms of sensitivity and specificity. Thanks to the combinatorial structure of words, a computation of the exact P-value has been made possible by recent mathematical results. We study here the drawbacks of the Z-score, the choice of the threshold and the tightness to the P-value. A major conclusion is that the normal approximation is always very poor and overestimates statistical significance.

Genomic epidemiology and global diversity of the emerging bacterial pathogen Elizabethkingia anophelis.

Elizabethkingia anophelis is an emerging pathogen involved in human infections and outbreaks in distinct world regions. We investigated the phylogenetic relationships and pathogenesis-associated genomic features of two neonatal meningitis isolates isolated 5 years apart from one hospital in Central African Republic and compared them with Elizabethkingia from other regions and sources. Average nucleotide identity firmly confirmed that E. anophelis, E. meningoseptica and E. miricola represent demarcated genomic species. A core genome multilocus sequence typing scheme, broadly applicable to Elizabethkingia species, was developed and made publicly available (http://bigsdb.pasteur.fr/elizabethkingia). Phylogenetic analysis revealed distinct E. anophelis sublineages and demonstrated high genetic relatedness between the African isolates, compatible with persistence of the strain in the hospital environment. CRISPR spacer variation between the African isolates was mirrored by the presence of a large mobile genetic element. The pan-genome of E. anophelis comprised 6,880 gene families, underlining genomic heterogeneity of this species. African isolates carried unique resistance genes acquired by horizontal transfer. We demonstrated the presence of extensive variation of the capsular polysaccharide synthesis gene cluster in E. anophelis. Our results demonstrate the dynamic evolution of this emerging pathogen and the power of genomic approaches for Elizabethkingia identification, population biology and epidemiology.

Mycobacterial Pan-Genome Analysis Suggests Important Role of Plasmids in the Radiation of Type VII Secretion Systems.

In mycobacteria, various type VII secretion systems corresponding to different ESX (ESAT-6 secretory) types, are contributing to pathogenicity, iron acquisition, and/or conjugation. In addition to the known chromosomal ESX loci, the existence of plasmid-encoded ESX systems was recently reported. To investigate the potential role of ESX-encoding plasmids on mycobacterial evolution, we analyzed a large representative collection of mycobacterial genomes, including both chromosomal and plasmid-borne sequences. Data obtained for chromosomal ESX loci confirmed the previous five classical ESX types and identified a novel mycobacterial ESX-4-like type, termed ESX-4-bis. Moreover, analysis of the plasmid-encoded ESX loci showed extensive diversification, with at least seven new ESX profiles, identified. Three of them (ESX-P clusters 1-3) were found in multiple plasmids, while four corresponded to singletons. Our phylogenetic and gene-order-analyses revealed two main groups of ESX types: 1) ancestral types, including ESX-4 and ESX-4-like systems from mycobacterial and non-mycobacterial actinobacteria and 2) mycobacteria-specific ESX systems, including ESX-1-2-3-5 systems and the plasmid-encoded ESX types. Synteny analysis revealed that ESX-P systems are part of phylogenetic groups that derived from a common ancestor, which diversified and resulted in the different ESX types through extensive gene rearrangements. A converging body of evidence, derived from composition bias-, phylogenetic-, and synteny analyses points to a scenario in which ESX-encoding plasmids have been a major driving force for acquisition and diversification of type VII systems in mycobacteria, which likely played (and possibly still play) important roles in the adaptation to new environments and hosts during evolution of mycobacterial pathogenesis.