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Asset Tag: Infectious disease

April 21, 2020

Haplotype-phased genome assembly of virulent Phythophthora ramorum isolate ND886 facilitated by long-read sequencing reveals effector polymorphisms and copy number variation.

Phytophthora ramorum is a destructive pathogen that causes Sudden Oak Death. The genome sequence of P. ramorum isolate Pr102 was previously produced using Sanger reads, and contained 12 Mb of gaps. However, isolate Pr102 had shown reduced aggressiveness and genome abnormalities. In order to produce an improved genome assembly for P. ramorum, we performed long read sequencing of highly aggressive P. ramorum isolate CDFA1418886 (abbreviated as ND886). We generated a 60.5 Mb assembly of the ND886 genome using the Pacific Biosciences sequencing platform. The assembly includes 302 primary contigs (60.2 Mb) and 9 unplaced contigs (265 Kb). Additionally, we found a “Highly repetitive” component from the Pacbio unassembled unmapped reads containing tandem repeats that are not part of the 60.5 Mb genome. The overall repeat content in the primary assembly was much higher than the Pr102 Sanger version (48% vs. 29%) indicating that the long reads have captured repetitive regions effectively. The 302 primary contigs were phased into 345 haplotype blocks and 222,892 phased variants, of which the longest phased block was 1,513,201 bp with 7,265 phased variants. The improved phased assembly facilitated identification of 21 and 25 Crinkler effectors and 393 and 394 RXLR effector genes from two haplotypes. Of these, 24 and 25 RXLR effectors were newly predicted from Haplotype A and Haplotype B, respectively. In addition, 7 new paralogs of effector Avh207 were found in contig 54, not reported earlier. Comparison of the ND886 assembly with Pr102 V1 assembly suggests that several repeat-rich smaller scaffolds within the Pr102 V1 assembly were possibly misassembled; these regions are fully encompassed now in ND886 contigs. Our analysis further reveals that Pr102 is a heterokaryon with multiple nuclear types in the sequences corresponding to contig 10 of ND886 assembly.

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April 21, 2020

Insertion sequences drive the emergence of a highly adapted human pathogen.

Pseudomonas aeruginosa is a highly adaptive opportunistic pathogen that can have serious health consequences in patients with lung disorders. Taxonomic outliers of P. aeruginosa of environmental origin have recently emerged as infectious for humans. Here, we present the first genome-wide analysis of an isolate that caused fatal haemorrhagic pneumonia. In two clones, CLJ1 and CLJ3, sequentially recovered from a patient with chronic pulmonary disease, insertion of a mobile genetic element into the P. aeruginosa chromosome affected major virulence-associated phenotypes and led to increased resistance to the antibiotics used to combat the infection. Comparative genome, proteome and transcriptome analyses revealed that this ISL3-family insertion sequence disrupted the genes for flagellar components, type IV pili, O-specific antigens, translesion polymerase and enzymes producing hydrogen cyanide. Seven-fold more insertions were detected in the later isolate, CLJ3, than in CLJ1, some of which modified strain susceptibility to antibiotics by disrupting the genes for the outer-membrane porin OprD and the regulator of ß-lactamase expression AmpD. In the Galleria mellonella larvae model, the two strains displayed different levels of virulence, with CLJ1 being highly pathogenic. This study revealed insertion sequences to be major players in enhancing the pathogenic potential of a P. aeruginosa taxonomic outlier by modulating both its virulence and its resistance to antimicrobials, and explains how this bacterium adapts from the environment to a human host.

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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

Molecular Characterization of a Multidrug-Resistant Klebsiella pneumoniae Strain R46 Isolated from a Rabbit

To investigate the mechanisms of multiple resistance and the horizontal transfer of resistance genes in animal pathogens, we characterized the molecular structures of the resistance gene-related sequences in a multidrug-resistant Klebsiella pneumoniae strain R46 isolated from a rabbit. Molecular cloning was performed to clone the resistance genes, and minimum inhibitory concentrations (MICs) were measured to determine the resistance characteristics of the cloned genes and related strains. A conjugation experiment was conducted to assess the transferability of the resistance plasmids. Sequencing and comparative genomic methods were used to analyze the structures of the resistance gene-related sequences. The K. pneumoniae R46 genome consisted of a chromosome and three resistance plasmids named pR46-27, pR46-42, and pR46-270, respectively. The whole genome encoded 34 antibiotic resistance genes including a newly identified chromosome-encoded florfenicol resistance gene named mdfA2. pR46-270, besides encoding 26 antibiotic resistance genes, carried four clusters of heavy metal resistance genes and several virulence-related genes or gene clusters. The plasmid-encoded resistance genes were mostly associated with mobile genetic elements. The plasmid with the most similarity to the floR gene-harboring plasmid pR46-27 was pCTXM-2271, a plasmid from Escherichia coli. The results of this work demonstrated that the plasmids with multidrug resistance genes were present in animal-derived bacteria and more florfenicol resistance genes such as mdfA2 could be present in bacterial populations. The resistance genes encoded on the plasmids may spread between the bacteria of different species or genera and cause the resistance dissemination.

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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

Impact of antibiotic treatment and host innate immune pressure on enterococcal adaptation in the human bloodstream.

Multidrug-resistant enterococcal strains emerged in the early 1980s and are now among the leading causes of drug-resistant bacterial infection worldwide. We used functional genomics to study an early bacterial outbreak in patients in a Wisconsin hospital between 1984 and 1988 that was caused by multidrug-resistant Enterococcus faecalis The goal was to determine how a clonal lineage of E. faecalis became adapted to growth and survival in the human bloodstream. Genome sequence analysis revealed a progression of increasingly fixed mutations and repeated independent occurrences of mutations in a relatively small set of genes. Repeated independent mutations suggested selection within the host during the course of infection in response to pressures such as host immunity and antibiotic treatment. We observed repeated independent mutations in a small number of loci, including a little studied polysaccharide utilization pathway and the cydABDC locus. Functional studies showed that mutating these loci rendered E. faecalis better able to withstand antibiotic pressure and innate immune defenses in the human bloodstream. We also observed a shift in mutation pattern that corresponded to the introduction of carbapenem antibiotics in 1987. This work identifies pathways that allow enterococci to survive the transition from the human gut into the bloodstream, enabling them to cause severe bacteremia associated with high mortality. 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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April 21, 2020

Complete Genome Sequence of a Parabacteroides distasonis Strain (CavFT hAR46) Isolated from a Gut Wall-Cavitating Microlesion in a Patient with Severe Crohn’s Disease.

Crohn’s disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans. There is evidence that Parabacteroides distasonis could contribute to IBD. Here, we present the complete genome sequence of a strain designated CavFT-hAR46, which was isolated from a gut intramural cavernous fistulous tract (CavFT) microlesion in a CD patient.Copyright © 2019 Yang et al.

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April 21, 2020

Complete Genome Sequence of GD1108, a Moderate-Virulence Strain of Human-Associated ST398 Methicillin-Susceptible Staphylococcus aureus.

Staphylococcus aureus multilocus sequence type 398 (ST398) is responsible for an increasing number of severe infections in humans. There are no reports detailing if all ST398 strains are equally virulent. We present the genome sequence of the moderate-virulence ST398 methicillin-susceptible Staphylococcus aureus strain GD1108, determined in a Caenorhabditis elegans infection model, to reveal the ST398 sublineage virulence.Copyright © 2019 McClure et al.

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