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April 21, 2020

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Phenotypic and Genomic Analyses of Burkholderia stabilis Clinical Contamination, Switzerland.

A recent hospital outbreak related to premoistened gloves used to wash patients exposed the difficulties of defining Burkholderia species in clinical settings. The outbreak strain displayed key B. stabilis phenotypes, including the inability to grow at 42°C; we used whole-genome sequencing to confirm the pathogen was B. stabilis. The outbreak strain genome comprises 3 chromosomes and a plasmid, sharing an average nucleotide identity of 98.4% with B. stabilis ATCC27515 BAA-67, but with 13% novel coding sequences. The genome lacks identifiable virulence factors and has no apparent increase in encoded antimicrobial drug resistance, few insertion sequences, and few pseudogenes, suggesting this outbreak was an opportunistic infection by an environmental strain not adapted to human pathogenicity. The diversity among outbreak isolates (22 from patients and 16 from washing gloves) is only 6 single-nucleotide polymorphisms, although the genome remains plastic, with large elements stochastically lost from outbreak isolates.

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April 21, 2020

Insights into the evolution and drug susceptibility of Babesia duncani from the sequence of its mitochondrial and apicoplast genomes.

Babesia microti and Babesia duncani are the main causative agents of human babesiosis in the United States. While significant knowledge about B. microti has been gained over the past few years, nothing is known about B. duncani biology, pathogenesis, mode of transmission or sensitivity to currently recommended therapies. Studies in immunocompetent wild type mice and hamsters have shown that unlike B. microti, infection with B. duncani results in severe pathology and ultimately death. The parasite factors involved in B. duncani virulence remain unknown. Here we report the first known completed sequence and annotation of the apicoplast and mitochondrial genomes of B. duncani. We found that the apicoplast genome of this parasite consists of a 34?kb monocistronic circular molecule encoding functions that are important for apicoplast gene transcription as well as translation and maturation of the organelle’s proteins. The mitochondrial genome of B. duncani consists of a 5.9?kb monocistronic linear molecule with two inverted repeats of 48?bp at both ends. Using the conserved cytochrome b (Cytb) and cytochrome c oxidase subunit I (coxI) proteins encoded by the mitochondrial genome, phylogenetic analysis revealed that B. duncani defines a new lineage among apicomplexan parasites distinct from B. microti, Babesia bovis, Theileria spp. and Plasmodium spp. Annotation of the apicoplast and mitochondrial genomes of B. duncani identified targets for development of effective therapies. Our studies set the stage for evaluation of the efficacy of these drugs alone or in combination against B. duncani in culture as well as in animal models.Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

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April 21, 2020

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

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April 21, 2020

Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

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April 21, 2020

Reduced frequency of HIV superinfection in a high-risk cohort in Zambia.

Rates of HIV-1 superinfection, re-infection with a genetically distinct virus despite HIV-1 specific immune responses, vary in different risk populations. We previously found the rates of superinfection were similar to primary HIV infection (PHI) in a Zambian heterosexual transmission cohort. Here, we conduct a similar analysis of 47 HIV-positive Zambians from an acute infection cohort with more frequent follow-up, all infected by non-spousal partners. We identified only one case of superinfection in the first two years, significantly fewer than in our previous study, which was likely due to increased counseling during acute infection and an overall population-wide decline in factors associated with HIV transmission. The predominant virus detected after superinfection was a recombinant of the transmitted founder (TF) and the superinfecting strain. The superinfected individual mounted a neutralizing antibody response to the primary TF virus, which remained TF-specific over time and even after superinfection, did not neutralize the superinfecting variant.Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

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April 21, 2020

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

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April 21, 2020

Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes.

Symbiosis is a major force of evolutionary change, influencing virtually all aspects of biology, from population ecology and evolution to genomics and molecular/biochemical mechanisms of development and reproduction. A remarkable example is Wolbachia endobacteria, present in some parasitic nematodes and many arthropod species. Acquisition of genomic data from diverse Wolbachia clades will aid in the elucidation of the different symbiotic mechanisms(s). However, challenges of de novo assembly of Wolbachia genomes include the presence in the sample of host DNA: nematode/vertebrate or insect. We designed biotinylated probes to capture large fragments of Wolbachia DNA for sequencing using PacBio technology (LEFT-SEQ: Large Enriched Fragment Targeted Sequencing). LEFT-SEQ was used to capture and sequence four Wolbachia genomes: the filarial nematode Brugia malayi, wBm, (21-fold enrichment), Drosophila mauritiana flies (2 isolates), wMau (11-fold enrichment), and Aedes albopictus mosquitoes, wAlbB (200-fold enrichment). LEFT-SEQ resulted in complete genomes for wBm and for wMau. For wBm, 18 single-nucleotide polymorphisms (SNPs), relative to the wBm reference, were identified and confirmed by PCR. A limit of LEFT-SEQ is illustrated by the wAlbB genome, characterized by a very high level of insertion sequences elements (ISs) and DNA repeats, for which only a 20-contig draft assembly was achieved.

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April 21, 2020

High quality reference genomes for toxigenic and non-toxigenic Vibrio cholerae serogroup O139.

Toxigenic Vibrio cholerae of the O139 serogroup have been responsible for several large cholera epidemics in South Asia, and continue to be of clinical and historical significance today. This serogroup was initially feared to represent a new, emerging V. cholerae clone that would lead to an eighth cholera pandemic. However, these concerns were ultimately unfounded. The majority of clinically relevant V. cholerae O139 isolates are closely related to serogroup O1, biotype El Tor V. cholerae, and comprise a single sublineage of the seventh pandemic El Tor lineage. Although related, these V. cholerae serogroups differ in several fundamental ways, in terms of their O-antigen, capsulation phenotype, and the genomic islands found on their chromosomes. Here, we present four complete, high-quality genomes for V. cholerae O139, obtained using long-read sequencing. Three of these sequences are from toxigenic V. cholerae, and one is from a bacterium which, although classified serologically as V. cholerae O139, lacks the CTXf bacteriophage and the ability to produce cholera toxin. We highlight fundamental genomic differences between these isolates, the V. cholerae O1 reference strain N16961, and the prototypical O139 strain MO10. These sequences are an important resource for the scientific community, and will improve greatly our ability to perform genomic analyses of non-O1 V. cholerae in the future. These genomes also offer new insights into the biology of a V. cholerae serogroup that, from a genomic perspective, is poorly understood.

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April 21, 2020

An integrated whole genome analysis of Mycobacterium tuberculosis reveals insights into relationship between its genome, transcriptome and methylome.

Human tuberculosis disease (TB), caused by Mycobacterium tuberculosis (Mtb), is a complex disease, with a spectrum of outcomes. Genomic, transcriptomic and methylation studies have revealed differences between Mtb lineages, likely to impact on transmission, virulence and drug resistance. However, so far no studies have integrated sequence-based genomic, transcriptomic and methylation characterisation across a common set of samples, which is critical to understand how DNA sequence and methylation affect RNA expression and, ultimately, Mtb pathogenesis. Here we perform such an integrated analysis across 22?M. tuberculosis clinical isolates, representing ancient (lineage 1) and modern (lineages 2 and 4) strains. The results confirm the presence of lineage-specific differential gene expression, linked to specific SNP-based expression quantitative trait loci: with 10 eQTLs involving SNPs in promoter regions or transcriptional start sites; and 12 involving potential functional impairment of transcriptional regulators. Methylation status was also found to have a role in transcription, with evidence of differential expression in 50 genes across lineage 4 samples. Lack of methylation was associated with three novel variants in mamA, likely to cause loss of function of this enzyme. Overall, our work shows the relationship of DNA sequence and methylation to RNA expression, and differences between ancient and modern lineages. Further studies are needed to verify the functional consequences of the identified mechanisms of gene expression regulation.

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April 21, 2020

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

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April 21, 2020

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

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April 21, 2020

Meiotic sex in Chagas disease parasite Trypanosoma cruzi.

Genetic exchange enables parasites to rapidly transform disease phenotypes and exploit new host populations. Trypanosoma cruzi, the parasitic agent of Chagas disease and a public health concern throughout Latin America, has for decades been presumed to exchange genetic material rarely and without classic meiotic sex. We present compelling evidence from 45 genomes sequenced from southern Ecuador that T. cruzi in fact maintains truly sexual, panmictic groups that can occur alongside others that remain highly clonal after past hybridization events. These groups with divergent reproductive strategies appear genetically isolated despite possible co-occurrence in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes. Our study reinvigorates the hunt for the site of genetic exchange in the T. cruzi life cycle, provides tools to define the genetic determinants of parasite virulence, and reforms longstanding theory on clonality in trypanosomatid parasites.

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April 21, 2020

FDA-ARGOS is a database with public quality-controlled reference genomes for diagnostic use and regulatory science.

FDA proactively invests in tools to support innovation of emerging technologies, such as infectious disease next generation sequencing (ID-NGS). Here, we introduce FDA-ARGOS quality-controlled reference genomes as a public database for diagnostic purposes and demonstrate its utility on the example of two use cases. We provide quality control metrics for the FDA-ARGOS genomic database resource and outline the need for genome quality gap filling in the public domain. In the first use case, we show more accurate microbial identification of Enterococcus avium from metagenomic samples with FDA-ARGOS reference genomes compared to non-curated GenBank genomes. In the second use case, we demonstrate the utility of FDA-ARGOS reference genomes for Ebola virus target sequence comparison as part of a composite validation strategy for ID-NGS diagnostic tests. The use of FDA-ARGOS as an in silico target sequence comparator tool combined with representative clinical testing could reduce the burden for completing ID-NGS clinical trials.

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