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Asset Tag: Third generation sequencing

July 7, 2019

A murine herpesvirus closely related to ubiquitous human herpesviruses causes T-cell depletion.

The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4(+) T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Quantifying the importance of the rare biosphere for microbial community response to organic pollutants in a freshwater ecosystem.

A single liter of water contains hundreds, if not thousands, of bacterial and archaeal species, each of which typically makes up a very small fraction of the total microbial community (<0.1%), the so-called "rare biosphere." How often, and via what mechanisms, e.g., clonal amplification versus horizontal gene transfer, the rare taxa and genes contribute to microbial community response to environmental perturbations represent important unanswered questions toward better understanding the value and modeling of microbial diversity. We tested whether rare species frequently responded to changing environmental conditions by establishing 20-liter planktonic mesocosms with water from Lake Lanier (Georgia, USA) and perturbing them with organic compounds that are rarely detected in the lake, including 2,4-dichlorophenoxyacetic acid (2,4-D), 4-nitrophenol (4-NP), and caffeine. The populations of the degraders of these compounds were initially below the detection limit of quantitative PCR (qPCR) or metagenomic sequencing methods, but they increased substantially in abundance after perturbation. Sequencing of several degraders (isolates) and time-series metagenomic data sets revealed distinct cooccurring alleles of degradation genes, frequently carried on transmissible plasmids, especially for the 2,4-D mesocosms, and distinct species dominating the post-enrichment microbial communities from each replicated mesocosm. This diversity of species and genes also underlies distinct degradation profiles among replicated mesocosms. Collectively, these results supported the hypothesis that the rare biosphere can serve as a genetic reservoir, which can be frequently missed by metagenomics but enables community response to changing environmental conditions caused by organic pollutants, and they provided insights into the size of the pool of rare genes and species. IMPORTANCE A single liter of water or gram of soil contains hundreds of low-abundance bacterial and archaeal species, the so called rare biosphere. The value of this astonishing biodiversity for ecosystem functioning remains poorly understood, primarily due to the fact that microbial community analysis frequently focuses on abundant organisms. Using a combination of culture-dependent and culture-independent (metagenomics) techniques, we showed that rare taxa and genes commonly contribute to the microbial community response to organic pollutants. Our findings should have implications for future studies that aim to study the role of rare species in environmental processes, including environmental bioremediation efforts of oil spills or other contaminants. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

First report of blaOXA-499 as a carbapenemase gene from Acinetobacter pittii.

We identified the carbapenemase gene blaOXA-499, a variant of blaOXA-143 from a clinical isolate of Acinetobacter pittii for the first time. OXA-499 shared 93.1% amino acid identity with OXA-143 and the gene was located on the chromosome. By cloning the OXA-499 encoding gene into the pWH1266 vector and transforming into susceptible Acinetobacter spp, we were able to show that OXA-499 confers resistance to carbapenems. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Perturbations of phosphatidate cytidylyltransferase (CdsA) mediate daptomycin resistance in Streptococcus mitis by a novel mechanism.

Streptococcus mitis/oralis is an important pathogen, causing life-threatening infections such as endocarditis and severe sepsis in immunocompromised patients. The ß-lactam antibiotics are the usual therapy of choice for this organism, but their effectiveness is threatened by the frequent emergence of resistance. The lipopeptide daptomycin (DAP) has been suggested for therapy against such resistant S. mitis/oralis strains due to its in vitro bactericidal activity and demonstrated efficacy against other Gram-positive pathogens. Unlike other bacteria, however, S. mitis/oralis has the unique ability to rapidly develop stable, high-level resistance to DAP upon exposure to the drug both in vivo and in vitro Using isogenic DAP-susceptible and DAP-resistant S. mitis/oralis strain pairs, we describe a mechanism of resistance to both DAP and cationic antimicrobial peptides that involves loss-of-function mutations in cdsA (encoding a phosphatidate cytidylyltransferase). CdsA catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin. DAP-resistant S. mitis/oralis strains demonstrated a total disappearance of phosphatidylglycerol, cardiolipin, and anionic phospholipid microdomains from membranes. In addition, these strains exhibited cross-resistance to cationic antimicrobial peptides from human neutrophils (i.e., hNP-1). Interestingly, CdsA-mediated changes in phospholipid metabolism were associated with DAP hyperaccumulation in a small subset of the bacterial population, without any binding by the remaining larger population. Our results indicate that CdsA is the major mediator of high-level DAP resistance in S. mitis/oralis and suggest a novel mechanism of bacterial survival against attack by antimicrobial peptides of both innate and exogenous origins. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

The pathogenic potential of Proteus mirabilis is enhanced by other uropathogens during polymicrobial uirinary tract infection.

Urinary catheter use is prevalent in health care settings, and polymicrobial colonization by urease-positive organisms, such as Proteus mirabilis and Providencia stuartii, commonly occurs with long-term catheterization. We previously demonstrated that coinfection with P. mirabilis and P. stuartii increased overall urease activity in vitro and disease severity in a model of urinary tract infection (UTI). In this study, we expanded these findings to a murine model of catheter-associated UTI (CAUTI), delineated the contribution of enhanced urease activity to coinfection pathogenesis, and screened for enhanced urease activity with other common CAUTI pathogens. In the UTI model, mice coinfected with the two species exhibited higher urine pH values, urolithiasis, bacteremia, and more pronounced tissue damage and inflammation compared to the findings for mice infected with a single species, despite having a similar bacterial burden within the urinary tract. The presence of P. stuartii, regardless of urease production by this organism, was sufficient to enhance P. mirabilis urease activity and increase disease severity, and enhanced urease activity was the predominant factor driving tissue damage and the dissemination of both organisms to the bloodstream during coinfection. These findings were largely recapitulated in the CAUTI model. Other uropathogens also enhanced P. mirabilis urease activity in vitro, including recent clinical isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa We therefore conclude that the underlying mechanism of enhanced urease activity may represent a widespread target for limiting the detrimental consequences of polymicrobial catheter colonization, particularly by P. mirabilis and other urease-positive bacteria. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Surveillance of bat coronaviruses in Kenya identifies relatives of human coronaviruses NL63 and 229E and their recombination history.

Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5′ and 3′ ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination “hot spot” in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances.IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequence of a Staphylococcus epidermidis strain with exceptional antimicrobial activity.

Staphylococcus epidermidis is a Gram-positive bacterium that is prevalent on human skin. The species is associated with skin health, as well as with opportunistic infections. Here, we report the complete genome sequence of S. epidermidis 14.1.R1, isolated from human skin. In bacterial interference assays, the strain showed exceptional antimicrobial activity. Copyright © 2017 Lassen et al.

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July 7, 2019

Complete genome sequence of Staphylococcus succinus 14BME20 isolated from a traditional Korean fermented soybean food.

The complete genome sequence of Staphylococcus succinus 14BME20, isolated from a Korean fermented soybean food and selected as a possible starter culture candidate, was determined. Comparative genome analysis with S. succinus CSM-77 from a Triassic salt mine revealed the presence of strain-specific genes for lipid degradation in strain 14BME20. Copyright © 2017 Jeong and Lee.

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July 7, 2019

Hybrid sequencing and map finding (HySeMaFi): optional strategies for extensively deciphering gene splicing and expression in organisms without reference genome.

Using second-generation sequencing (SGS) RNA-Seq strategies, extensive alterative splicing prediction is impractical and high variability of isoforms expression quantification is inevitable in organisms without true reference dataset. we report the development of a novel analysis method, termed hybrid sequencing and map finding (HySeMaFi) which combines the specific strengths of third-generation sequencing (TGS) (PacBio SMRT sequencing) and SGS (Illumina Hi-Seq/MiSeq sequencing) to effectively decipher gene splicing and to reliably estimate the isoforms abundance. Error-corrected long reads from TGS are capable of capturing full length transcripts or as large partial transcript fragments. Both true and false isoforms, from a particular gene, as well as that containing all possible exons, could be generated by employing different assembly methods in SGS. We first develop an effective method which can establish the mapping relationship between the error-corrected long reads and the longest assembled contig in every corresponding gene. According to the mapping data, the true splicing pattern of the genes was reliably detected, and quantification of the isoforms was also effectively determined. HySeMaFi is also the optimal strategy by which to decipher the full exon expression of a specific gene when the longest mapped contigs were chosen as the reference set.

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July 7, 2019

Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of loci encoding increased protein quality control mechanisms.

Salmonella enterica subsp. enterica bacteria are important foodborne pathogens with major economic impact. Some isolates exhibit increased heat tolerance, a concern for food safety. Analysis of a finished-quality genome sequence of an isolate commonly used in heat resistance studies, S. enterica subsp. enterica serovar Senftenberg 775W (ATCC 43845), demonstrated an interesting observation that this strain contains not just one, but two horizontally acquired thermotolerance locus homologs. These two loci reside on a large 341.3-kbp plasmid that is similar to the well-studied IncHI2 R478 plasmid but lacks any antibiotic resistance genes found on R478 or other IncHI2 plasmids. As this historical Salmonella isolate has been in use since 1941, comparative analysis of the plasmid and of the thermotolerance loci contained on the plasmid will provide insight into the evolution of heat resistance loci as well as acquisition of resistance determinants in IncHI2 plasmids. IMPORTANCE Thermal interventions are commonly used in the food industry as a means of mitigating pathogen contamination in food products. Concern over heat-resistant food contaminants has recently increased, with the identification of a conserved locus shown to confer heat resistance in disparate lineages of Gram-negative bacteria. Complete sequence analysis of a historical isolate of Salmonella enterica serovar Senftenberg, used in numerous studies because of its novel heat resistance, revealed that this important strain possesses two distinct copies of this conserved thermotolerance locus, residing on a multireplicon IncHI2/IncHI2A plasmid. Phylogenetic analysis of these loci in comparison with homologs identified in various bacterial genera provides an opportunity to examine the evolution and distribution of loci conferring resistance to environmental stressors, such as heat and desiccation.

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July 7, 2019

Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses.

Candida auris, a multidrug-resistant yeast that causes invasive infections, was first described in 2009 in Japan and has since been reported from several countries.To understand the global emergence and epidemiology of C. auris, we obtained isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela during 2012-2015 and the type specimen from Japan. Patient information was available for 41 of the isolates. We conducted antifungal susceptibility testing and whole-genome sequencing (WGS).Available clinical information revealed that 41% of patients had diabetes mellitus, 51% had undergone recent surgery, 73% had a central venous catheter, and 41% were receiving systemic antifungal therapy when C. auris was isolated. The median time from admission to infection was 19 days (interquartile range, 9-36 days), 61% of patients had bloodstream infection, and 59% died. Using stringent break points, 93% of isolates were resistant to fluconazole, 35% to amphotericin B, and 7% to echinocandins; 41% were resistant to 2 antifungal classes and 4% were resistant to 3 classes. WGS demonstrated that isolates were grouped into unique clades by geographic region. Clades were separated by thousands of single-nucleotide polymorphisms, but within each clade isolates were clonal. Different mutations in ERG11 were associated with azole resistance in each geographic clade.C. auris is an emerging healthcare-associated pathogen associated with high mortality. Treatment options are limited, due to antifungal resistance. WGS analysis suggests nearly simultaneous, and recent, independent emergence of different clonal populations on 3 continents. Risk factors and transmission mechanisms need to be elucidated to guide control measures. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019

Single-Molecule sequencing of the Drosophila serrata genome.

Long-read sequencing technology promises to greatly enhance de novo assembly of genomes for nonmodel species. Although the error rates of long reads have been a stumbling block, sequencing at high coverage permits the self-correction of many errors. Here, we sequence and de novo assemble the genome of Drosophila serrata, a species from the montium subgroup that has been well-studied for latitudinal clines, sexual selection, and gene expression, but which lacks a reference genome. Using 11 PacBio single-molecule real-time (SMRT cells), we generated 12 Gbp of raw sequence data comprising ~65 × whole-genome coverage. Read lengths averaged 8940 bp (NRead50 12,200) with the longest read at 53 kbp. We self-corrected reads using the PBDagCon algorithm and assembled the genome using the MHAP algorithm within the PBcR assembler. Total genome length was 198 Mbp with an N50 just under 1 Mbp. Contigs displayed a high degree of chromosome arm-level conservation with the D. melanogaster genome and many could be sensibly placed on the D. serrata physical map. We also provide an initial annotation for this genome using in silico gene predictions that were supported by RNA-seq data. Copyright © 2017 Allen et al.

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