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April 21, 2020

Enrichment of oral microbiota in early cystic precursors to invasive pancreatic cancer.

Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic cysts that can progress to invasive pancreatic cancer. Associations between oncogenesis and oral microbiome alterations have been reported. This study aims to investigate a potential intracystic pancreatic microbiome in a pancreatic cystic neoplasm (PCN) surgery patient cohort.Paired cyst fluid and plasma were collected at pancreatic surgery from patients with suspected PCN (n=105). Quantitative and qualitative assessment of bacterial DNA by qPCR, PacBio sequencing (n=35), and interleukin (IL)-1ß quantification was performed. The data were correlated to diagnosis, lesion severity and clinical and laboratory profile, including proton-pump inhibitor (PPI) usage and history of invasive endoscopy procedures.Intracystic bacterial 16S DNA copy number and IL-1ß protein quantity were significantly higher in IPMN with high-grade dysplasia and IPMN with cancer compared with non-IPMN PCNs. Despite high interpersonal variation of intracystic microbiota composition, bacterial network and linear discriminant analysis effect size analyses demonstrated co-occurrence and enrichment of oral bacterial taxa including Fusobacterium nucleatum and Granulicatella adiacens in cyst fluid from IPMN with high-grade dysplasia. The elevated intracystic bacterial DNA is associated with, but not limited to, prior exposure to invasive endoscopic procedures, and is independent from use of PPI and antibiotics.Collectively, these findings warrant further investigation into the role of oral bacteria in cystic precursors to pancreatic cancer and have added values on the aetiopathology as well as the management of pancreatic cysts. © Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

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April 21, 2020

Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100

The range of naturally occurring variation in the ability of Bdellovibrio strains to attack and kill Gram-negative bacteria is not well understood. Defining phenotypic and associated genotypic variation among Bdellovibrio may further our understanding of how this genus impacts microbial communities. In addition, comparisons of the predatory phenotypes of divergent strains may inform the development of Bdellovibrio as biocontrol agents to combat bacterial infections. We isolated Bdellovibrio sp. NC01 from soil and compared its genome and predatory phenotypes to B. bacteriovorus type strain HD100. Based on analysis of 16S rRNA gene sequences and average amino acid identity, NC01 belongs to a different species than HD100. Genome-wide comparisons and individual gene analyses indicated that eight NC01 genome regions were likely acquired by horizontal gene transfer (HGT), further supporting an important role for HGT in Bdellovibrio genome evolution. Within these regions, multiple protein-coding sequences were assigned predicted functions related to transcriptional regulation and transport; however, most were annotated as hypothetical proteins. Compared to HD100, NC01 has a limited prey range and kills E. coli ML35 less efficiently. Whereas HD100 drastically reduces the ML35 population and then maintains low prey population density, NC01 causes a smaller reduction in ML35, after which the prey population recovers, accompanied by a decrease in NC01. In addition, NC01 forms turbid plaques on lawns of E. coli ML35, in contrast to clear plaques formed by HD100. Characterizing variation in interactions between Bdellovibrio and Gram-negative bacteria, such as observed with NC01 and HD100, is valuable for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.

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April 21, 2020

A novel blaSIM-1-carrying megaplasmid pSIM-1-BJ01 isolated from clinical Klebsiella pneumonia

A rare carbapenem-resistant gene blaSIM-1 was found in a 316-kb megaplasmid designated pSIM-1-BJ01 isolated from a clinical strain Klebsiella pneumonia 13624. The plasmid pSIM-1-BJ01 was fully sequenced and analyzed. Its length is 316,557 bp and it has 342 putative open reading frames with two multidrug-resistant regions and a total of 19 resistant genes. Its backbone was highly homologous to the newly reported plasmid pRJA166a, which was isolated from a clinical third-generation cephalosporin-resistant hypervirulen strain K. pneumonia ST23. The plasmid pSIM-1-BJ01 was verified to be able to transfer to Escherichia coli. The emergency of the transferable blaSIM-1-carrying multidrug-resistant plasmid pSIM-1-BJ01 suggests the spread of blaSIM among Enterobacteriaceae is possible. Therefore, the data presented herein provided insights into the genomic diversity and evolution of blaSIM-carrying plasmids, as well as the dissemination and epidemiology of blaSIM among Enterobacteriaceae in public health system.

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April 21, 2020

Complete genome screening of clinical MRSA isolates identifies lineage diversity and provides full resolution of transmission and outbreak events

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices, but most applications are focused on conserved core genomic regions due to limitations of short-read technologies. In this study we established a long-read technology-based WGS screening program of all first-episode MRSA blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads revealed widespread gain/loss of accessory mobile genetic elements among established hospital- and community-associated lineages impacting >10% of each genome, and frequent megabase-scale inversions between endogenous prophages. We also characterized an outbreak of a CC5/ST105/USA100 clone among 3 adults and 18 infants in a neonatal intensive care unit (NICU) lasting 7 months. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its origins and progression, which was characterized by multiple sub-transmissions and likely precipitated by equipment sharing. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance and persistence. This included a DNA-recognition domain recombination in the hsdS gene of a Type-I restriction-modification system that altered DNA methylation. RNA-Seq profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall our findings demonstrate that long-read sequencing substantially improves our ability to characterize accessory genomic elements that impact MRSA virulence and persistence, and provides valuable information for infection control efforts.

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April 21, 2020

The landscape of SNCA transcripts across synucleinopathies: New insights from long reads sequencing analysis

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3textquoteright UTR lengths, as well as novel 5textquoteright starts and 3textquoteright ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114kb SNCA region, with the longest phased block excedding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.

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April 21, 2020

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

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April 21, 2020

Morphological and genomic characterisation of the hybrid schistosome infecting humans in Europe reveals a complex admixture between Schistosoma haematobium and Schistosoma bovis parasites

Schistosomes cause schistosomiasis, the worldtextquoterights second most important parasitic disease after malaria. A peculiar feature of schistosomes is their ability to produce viable and fertile hybrids. Originally only present in the tropics, schistosomiasis is now also endemic in Europe. Based on two genetic markers the European species had been identified as a hybrid between the ruminant-infective Schistosoma bovis and the human-infective Schistosoma haematobium.Here we describe for the first time the genomic composition of the European schistosome hybrid (77% of S. haematobium and 23% of S. bovis origins), its morphometric parameters and its compatibility with the European vector snail and intermediate host Compatibility is a key parameter for the parasites life cycle progression. We also show that egg morphology (a classical diagnostic parameter) does not allow for differential diagnosis while genetic tests do so. Additionally, we performed genome assembly improvement and annotation of S. bovis, the parental species for which no satisfactory genome assembly was available.For the first time since the discovery of hybrid schistosomes, these results reveal at the whole genomic level a complex admixture of parental genomes highlighting (i) the high permeability of schistosomes to other speciestextquoteright alleles, and (ii) the importance of hybrid formation for pushing species boundaries not only conceptionally but also geographically.

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April 21, 2020

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s.

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype. Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020

Evolution and global transmission of a multidrug-resistant, community-associated MRSA lineage from the Indian subcontinent

The evolution and global transmission of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here, we trace the recent origins and global spread of a multidrug resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data shows that the clone emerged on the Indian subcontinent in the early 1970s and disseminated rapidly in the 1990s. Short-term outbreaks in community and healthcare settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the divergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological transmission of community-associated MRSA. Our study demonstrates the importance of whole genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.Importance The Bengal Bay clone (ST772) is a community-acquired and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we show that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally resulting in small-scale community and healthcare outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug-resistance of healthcare-associated S. aureus lineages. This study demonstrates the importance of whole genome sequencing for the surveillance of highly antibiotic resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.

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April 21, 2020

Early Sex-chromosome Evolution in the Diploid Dioecious Plant Mercurialis annua.

Suppressed recombination allows divergence between homologous sex chromosomes and the functionality of their genes. Here, we reveal patterns of the earliest stages of sex-chromosome evolution in the diploid dioecious herb Mercurialis annua on the basis of cytological analysis, de novo genome assembly and annotation, genetic mapping, exome resequencing of natural populations, and transcriptome analysis. The genome assembly contained 34,105 expressed genes, of which 10,076 were assigned to linkage groups. Genetic mapping and exome resequencing of individuals across the species range both identified the largest linkage group, LG1, as the sex chromosome. Although the sex chromosomes of M. annua are karyotypically homomorphic, we estimate that about a third of the Y chromosome has ceased recombining, containing 568 transcripts and spanning 22.3 cM in the corresponding female map. Nevertheless, we found limited evidence for Y-chromosome degeneration in terms of gene loss and pseudogenization, and most X- and Y-linked genes appear to have diverged in the period subsequent to speciation between M. annua and its sister species M. huetii which shares the same sex-determining region. Taken together, our results suggest that the M. annua Y chromosome has at least two evolutionary strata: a small old stratum shared with M. huetii, and a more recent larger stratum that is probably unique to M. annua and that stopped recombining about one million years ago. Patterns of gene expression within the non-recombining region are consistent with the idea that sexually antagonistic selection may have played a role in favoring suppressed recombination.Copyright © 2019, Genetics.

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April 21, 2020

A microbial factory for defensive kahalalides in a tripartite marine symbiosis.

Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont (“Candidatus Endobryopsis kahalalidefaciens”) uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. “Ca E. kahalalidefaciens” has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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April 21, 2020

The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4+ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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April 21, 2020

Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Salmonella Typhimurium ST313

Background: Salmonella Typhimurium ST313 exhibits signatures of adaptation to invasive human infection, including higher resistance to humoral immune responses than gastrointestinal isolates. Full resistance to antibody-mediated complement killing (serum resistance) among nontyphoidal Salmonellae is uncommon, but selection of highly resistant strains could compromise vaccine-induced antibody immunity. Here, we address the hypothesis that serum resistance is due to a distinct genotype or transcriptome response in S. Typhimurium ST313.

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