Environmental Metagenomic Assemblies Reveal Seven New Highly Divergent Chlamydial Lineages and Hallmarks of a Conserved Intracellular Lifestyle.

The javax.xml.bind.JAXBElement@c89e628 phylum exclusively encompasses bacteria sharing a similar obligate intracellular life cycle. Existing 16S rDNA data support a high diversity within the phylum, however genomic data remain scarce owing to the difficulty in isolating strains using culture systems with eukaryotic cells. Yet, javax.xml.bind.JAXBElement@5026e79c genome data extracted from large scale metagenomic studies might help fill this gap. This work compares 33 cultured and 27 environmental, uncultured chlamydial genomes, in order to clarify the phylogenetic relatedness of the new chlamydial clades and to investigate the genetic diversity of the javax.xml.bind.JAXBElement@50520457 phylum. The analysis of published chlamydial genomes from metagenomics bins and single cell sequencing allowed the identification of seven new deeply branching chlamydial clades sharing genetic hallmarks of parasitic javax.xml.bind.JAXBElement@4f9dc5d2 . Comparative genomics suggests important biological differences between those clades, including loss of many proteins involved in cell division in the genus javax.xml.bind.JAXBElement@ce6c2af , and loss of respiratory chain and tricarboxylic acid cycle in several species. Comparative analyses of chlamydial genomes with two proteobacterial orders, the javax.xml.bind.JAXBElement@6b1bdea8 and the javax.xml.bind.JAXBElement@2b2b0f20 showed that genomes of different javax.xml.bind.JAXBElement@ab9fcc2 families are much more similar than genomes of different javax.xml.bind.JAXBElement@3070a480 families. On the other hand, the chlamydial 16S rRNAs exhibit a higher sequence conservation than their javax.xml.bind.JAXBElement@5abc2b8 counterparts, while chlamydial proteins exhibit increased sequence divergence. Studying the diversity and genome plasticity of the entire javax.xml.bind.JAXBElement@760a8f22 phylum is of major interest to better understand the emergence and evolution of this ubiquitous and ancient clade of obligate intracellular bacteria

Taxogenomics of the order Chlamydiales.

Bacterial classification is a long-standing problem for taxonomists and species definition itself is constantly debated among specialists. The classification of strict intracellular bacteria such as members of the order Chlamydiales mainly relies on DNA- or protein-based phylogenetic reconstructions because these organisms exhibit few phenotypic differences and are difficult to culture. The availability of full genome sequences allows the comparison of the performance of conserved protein sequences to reconstruct Chlamydiales phylogeny. This approach permits the identification of markers that maximize the phylogenetic signal and the robustness of the inferred tree. In this study, a set of 424 core proteins was identified and concatenated to reconstruct a reference species tree. Although individual protein trees present variable topologies, we detected only few cases of incongruence with the reference species tree, which were due to horizontal gene transfers. Detailed analysis of the phylogenetic information of individual protein sequences (i) showed that phylogenies based on single randomly chosen core proteins are not reliable and (ii) led to the identification of twenty taxonomically highly reliable proteins, allowing the reconstruction of a robust tree close to the reference species tree. We recommend using these protein sequences to precisely classify newly discovered isolates at the family, genus and species levels

Génomique et métagénomique bactériennes: applications cliniques et importance médicale [Bacterial genomics and metagenomics: clinical applications and medical relevance]

New sequencing technologies provide in a short time and at low cost high amount of genomic sequences useful for applications such as: a) development of diagnostic PCRs and/or serological tests; b) detection of virulence factors (virulome) or genes/SNPs associated with resistance to antibiotics (resistome) and c) investigation of transmission and dissemination of bacterial pathogens. Thus, bacterial genomics of medical importance is useful to clinical microbiologists, to infectious diseases specialists as well as to epidemiologists. Determining the microbial composition of a sample by metagenomics is another application of new sequencing technologies, useful to understand the impact of bacteria on various non-infectious diseases such as obesity, asthma, or diabetes. Genomics and metagenomics will likely become a specialized diagnostic analysis

One Year Genome Evolution of Lausannevirus in Allopatric versus Sympatric Conditions.

Amoeba-resisting microorganisms raised a great interest during the last decade. Among them, some large DNA viruses present huge genomes up to 2.5 Mb long, exceeding the size of small bacterial genomes. The rate of genome evolution in terms of mutation, deletion, and gene acquisition in these genomes is yet unknown. Given the suspected high plasticity of viral genomes, the microevolution of the 346 kb genome of Lausannevirus, a member of Megavirales, was studied. Hence, Lausannevirus was co-cultured within the amoeba Acanthamoeba castellanii over one year. Despite a low number of mutations, the virus showed a genome reduction of 3.7% after 12 months. Lausannevirus genome evolution in sympatric conditions was investigated by its co-culture with Estrella lausannensis, an obligate intracellular bacterium, in the amoeba A. castellanii during one year. Cultures were split every 3 months. Genome sequencing revealed that in these conditions both, Lausannevirus and E. lausannensis, show stable genome, presenting no major rearrangement. In fact, after one year they acquired from 2 to 7 and from 4 to 10 mutations per culture for Lausannevirus and E. lausannensis, respectively. Interestingly, different mutations in the endonuclease encoding genes of Lausannevirus were observed in different subcultures, highlighting the importance of this gene product in the replication of Lausannevirus. Conversely, mutations in E. lausannensis were mainly located in a gene encoding for a phosphoenolpyruvate-protein phosphotransferase (PtsI), implicated in sugar metabolism. Moreover, in our conditions and with our analyses we detected no horizontal gene transfer during one year of co-culture

Pathogenic Determinants of the Mycobacterium kansasii Complex: An Unsuspected Role for Distributive Conjugal Transfer.

The Mycobacterium kansasii species comprises six subtypes that were recently classified into six closely related species; Mycobacterium kansasii (formerly M. kansasii subtype 1), Mycobacterium persicum (subtype 2), Mycobacterium pseudokansasii (subtype 3), Mycobacterium ostraviense (subtype 4), Mycobacterium innocens (subtype 5) and Mycobacterium attenuatum (subtype 6). Together with Mycobacterium gastri, they form the M. kansasii complex. M. kansasii is the most frequent and most pathogenic species of the complex. M. persicum is classically associated with diseases in immunosuppressed patients, and the other species are mostly colonizers, and are only very rarely reported in ill patients. Comparative genomics was used to assess the genetic determinants leading to the pathogenicity of members of the M. kansasii complex. The genomes of 51 isolates collected from patients with and without disease were sequenced and compared with 24 publicly available genomes. The pathogenicity of each isolate was determined based on the clinical records or public metadata. A comparative genomic analysis showed that all M. persicum, M. ostraviense, M innocens and M. gastri isolates lacked the ESX-1-associated EspACD locus that is thought to play a crucial role in the pathogenicity of M. tuberculosis and other non-tuberculous mycobacteria. Furthermore, M. kansasii was the only species exhibiting a 25-Kb-large genomic island encoding for 17 type-VII secretion system-associated proteins. Finally, a genome-wide association analysis revealed that two consecutive genes encoding a hemerythrin-like protein and a nitroreductase-like protein were significantly associated with pathogenicity. These two genes may be involved in the resistance to reactive oxygen and nitrogen species, a required mechanism for the intracellular survival of bacteria. Three non-pathogenic M. kansasii lacked these genes likely due to two distinct distributive conjugal transfers (DCTs) between M. attenuatum and M. kansasii, and one DCT between M. persicum and M. kansasii. To our knowledge, this is the first study linking DCT to reduced pathogenicity

Evaluation of the BD Phoenix™ CPO Detect Test for the detection of carbapenemase producers.

Becton-Dickinson recently developed the Phoenix™ CPO (carbapenemase-producing organism) Detect Test, a growth-based test embedded in Gram-negative (GN) panels for the detection and confirmation of bacteria producing class A, B and D carbapenemases. This study aimed to (a) determine the performance of the CPO test, and (b) assess its added value in routine diagnostic workflows. The performance of the BD Phoenix CPO test was analysed retrospectively on a collection of 185 molecularly characterized strains, including 92 CPOs, and prospectively on 135 and 160 routine isolates with and without CPO suspicion, respectively. In the retrospective study the CPO test exhibited 92.4% accuracy (95%CI 87.6-95.8), 97.8% sensitivity (95%CI 92.4-99.7) and 87.1% specificity (95%CI 78.6-93.2) for carbapenemase detection. The CPO test provided a classification to class A, B, and D for 81.3% of detected carbapenemases with 94.6% accuracy (95%CI 86.7-98.5). In the prospective study the CPO test detection performance showed 77.8% accuracy (95%CI 68.8-84.5), 100% sensitivity (95%CI 91.2-100) and 67.8% specificity (95%CI 57.3-77.1) with 135 CPO-suspicious isolates and 98.8% accuracy and specificity (95%CI 95.6-99.9) with 160 non-CPO-suspicious isolates. Compared to routine testing, the implementation of the CPO test allowed a mean reduction of 21.3 h (95%CI 17.6-25) in turnaround time, 16.8 min (95%CI 13.4-20.2) in hands-on time, and 20.6 CHF (95%CI 16.5-24.8) in costs. The CPO test is reliable for the detection of CPO with a high sensitivity. However, the relatively low detection specificity required the use of additional confirmatory methods. The carbapenemase classification accuracy is robust in providing preliminary results before molecular characterization. Finally, the implementation of the test in routine workflows allowed a significant reduction in turnaround time, hands-on time and cost compared to the conventional approach

Cedratvirus lausannensis - digging into Pithoviridae diversity.

Amoeba-infecting viruses have raised scientists' interest due to their novel particle morphologies, their large genome size and their genomic content challenging previously established dogma. We report here the discovery and the characterization of Cedratvirus lausannensis, a novel member of the Megavirales, with a 0.75-1 µm long amphora-shaped particle closed by two striped plugs. Among numerous host cell types tested, the virus replicates only in Acanthamoeba castellanii leading to host cell lysis within 24 h. C. lausannensis was resistant to ethanol, hydrogen peroxide and heating treatments. Like 30 000-year-old Pithovirus sibericum, C. lausannensis enters by phagocytosis, releases its genetic content by fusion of the internal membrane with the inclusion membrane and replicates in intracytoplasmic viral factories. The genome encodes 643 proteins that confirmed the grouping of C. lausannensis with Cedratvirus A11 as phylogenetically distant members of the family Pithoviridae. The 575,161 bp AT-rich genome is essentially devoid of the numerous repeats harbored by Pithovirus, suggesting that these non-coding repetitions might be due to a selfish element rather than particular characteristics of the Pithoviridae family. The discovery of C. lausannensis confirms the contemporary worldwide distribution of Pithoviridae members and the characterization of its genome paves the way to better understand their evolution

A predation assay using amoebae to screen for virulence factors unearthed the first W. chondrophila inclusion membrane protein.

Waddlia chondrophila is an intracellular bacterium phylogenetically related to the well-studied human and animal pathogens of the Chlamydiaceae family. In the last decade, W. chondrophila was convincingly demonstrated to be associated with adverse pregnancy outcomes in humans and abortions in animals. All members of the phylum Chlamydiae possess a Type Three Secretion System that they use for delivering virulence proteins into the host cell cytosol to modulate their environment and create optimal conditions to complete their life cycle. To identify W. chondrophila virulence proteins, we used an original screening approach that combines a cosmid library with an assay monitoring resistance to predation by phagocytic amoebae. This technique combined with bioinformatic data allowed the identification of 28 candidate virulence proteins, including Wimp1, the first identified inclusion membrane protein of W. chondrophila

Comparative genomics of Neisseria meningitidis strains: new targets for molecular diagnostics.

In 2010, Jaton et al. (False-negative PCR result due to gene polymorphism: the example of Neisseria meningitidis. J Clin Microbiol 2010;48:4590-2) reported an isolate of Neisseria meningitidis serogroup B that was not detected by the ctrA quantitative real-time PCR (qRT-PCR) used in our diagnostic laboratory. Sequence analysis of ctrA revealed several single nucleotide polymorphisms responsible for the negative qRT-PCR. Therefore, we sequenced the genome of this isolate and performed comparative genomics to propose new gene targets for the specific detection of N. meningitidis from clinical specimens. We identified 11 genes as specific to N. meningitidis genomes and common to at least 177 (97%) of the 183 genomes available. Among them, three genes (metA, tauE and shlA) were selected to develop new qRT-PCRs for the detection of N. meningitidis DNA. The three qRT-PCRs were highly sensitive and specific, and they exhibited a good reproducibility when tested on plasmidic positive controls and genomic DNA extracted from strains of N. meningitidis and other relevant bacterial species. The clinical sensitivity and specificity of metA and tauE qRT-PCRs were both 100% based on a testing of cerebrospinal fluid samples positive for N. meningitidis or other clinically relevant bacteria. Despite a 100% specificity, the sensitivity of the shlA qRT-PCR was only 70%. We thus recommend using the metA and/or tauE qRT-PCRs developed here. To prevent PCR failure in the presence of new polymorphic strains, the detection of dual targets by duplex qRT-PCR would be more accurate and suitable for the diagnosis of N. meningitidis from clinical specimens

Judges' perceptions of expert reports: The effect of neuroscience evidence.

This article explores the impact of neuroscience evidence on how expert reports are perceived and their effects on the decisions made by trial judges. Experimental psychology has demonstrated a number of cognitive effects arising from exposure to neuroimaging data which may bias judgments and lead to (mis)interpretations that can affect decisions. We conducted a study on a sample of 62 Swiss and French judges in order to determine whether their perceptions of the credibility, quality and scientific basis of a psychiatric evaluation of a criminal defendant vary according to whether or not the evaluation includes neuroscientific data. Quantitative analyses were conducted in order to evaluate significant differences between the two conditions (one-way analyses of variance) and moderation and conditional analyses to examine whether the participants' sex and length of professional experience moderated the effect of the conditions. Terminological and thematic analyses were carried out on open questions. Quantitative and qualitative results suggest that the presence of neuroscience data in an expert report affects judges' perceptions of the quality, credibility, and scientificity (reliability, objectivity, scientific basis) of the report, and the persuasiveness of the evidence it provided. Moreover, this phenomenon was stronger in more experienced judges than in less experienced judges