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Asset Tag: Whole Genome Sequencing,

September 22, 2019

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

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September 22, 2019

Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease.

Nontypeable Haemophilus influenzae (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

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September 22, 2019

Heterologous expression guides identification of the biosynthetic gene cluster of chuangxinmycin, an indole alkaloid antibiotic.

The indole alkaloid antibiotic chuangxinmycin, from Actinobacteria Actinoplanes tsinanensis, containing a unique thiopyrano[4,3,2- cd]indole scaffold, is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase. The chuangxinmycin biosynthetic gene cluster was identified by in silico analysis of the genome sequence, then verified by heterologous expression. Systemic gene inactivation and intermediate identification determined the minimum set of genes for unique thiopyrano[4,3,2- cd]indole formation and the concerted action of a radical S-adenosylmethionine protein plus an unknown protein for addition of the 3-methyl group. These findings set a solid foundation for comprehensively investigating the biosynthesis, optimizing yield, and generating new analogues of chuangxinmycin.

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September 22, 2019

Distinct evolutionary patterns of Neisseria meningitidis serogroup B disease outbreaks at two universities in the USA.

Neisseria meningitidis serogroup B (MnB) was responsible for two independent meningococcal disease outbreaks at universities in the USA during 2013. The first at University A in New Jersey included nine confirmed cases reported between March 2013 and March 2014. The second outbreak occurred at University B in California, with four confirmed cases during November 2013. The public health response to these outbreaks included the approval and deployment of a serogroup B meningococcal vaccine that was not yet licensed in the USA. This study investigated the use of whole-genome sequencing(WGS) to examine the genetic profile of the disease-causing outbreak isolates at each university. Comparative WGS revealed differences in evolutionary patterns between the two disease outbreaks. The University A outbreak isolates were very closely related, with differences primarily attributed to single nucleotide polymorphisms/insertion-deletion (SNP/indel) events. In contrast, the University B outbreak isolates segregated into two phylogenetic clades, differing in large part due to recombination events covering extensive regions (>30?kb) of the genome including virulence factors. This high-resolution comparison of two meningococcal disease outbreaks further demonstrates the genetic complexity of meningococcal bacteria as related to evolution and disease virulence.

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September 22, 2019

Case report of an extensively drug-resistant Klebsiella pneumoniae infection with genomic characterization of the strain and review of similar cases in the United States

Reports of extensively drug-resistant and pan-drug-resistant Klebsiella pneumoniae (XDR-KP and PDR-KP) cases are increasing worldwide. Here, we report a case of XDR-KP with an in-depth molecular characterization of resistance genes using whole-genome sequencing, and we review all cases of XDR-KP and PDR-KP reported in the United States to date.

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September 22, 2019

Virgibacillus phasianinus sp. nov., a halophilic bacterium isolated from faeces of a Swinhoe’s pheasant, Lophura swinhoii.

A rod-shaped, Gram-stain-positive, motile and aerobic bacterium, designated LM2416T, was isolated from faeces of Lophuras winhoii living in Seoul Grand Park, Gyeonggi-do, Republic of Korea. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain LM2416T belonged to the genus Virgibacillus, sharing high 16S rRNA gene sequence similarities to Virgibacillus necropolis LMG 19488T (99.0?%), Virgibacillus carmonensis LMG 20964T (98.4?%), Virgibacillus arcticus Hal 1T (98.3?%) and Virgibacillus flavescens S1-20T (97.9?%). The isolate grew at 10-30?°C, pH 6-7 and 0-20?% (w/v) NaCl. Optimal growth was observed at 30?°C, pH 6-7 and 10?% (w/v) NaCl. The major fatty acid was anteiso-C15?:?0. Polar lipids were composed of phosphatidylglycerol, diphosphatidylglycerol, three unknown phospholipids and two unknown aminophospholipids. The main menaquinone was MK-7. Strain LM2416T had alanine, lysine, glutamic acid, glycine and aspartic acid as cell-wall amino acids and ribose as a cell-wall sugar. The whole genome sequences of strain LM2416T and V. necropolis KCTC 3820T were sequenced by PacBio RS II sequencing. The genome sequence-based G+C?content of strain LM2416T was 39.5?mol%. The orthologous average nucleotide identity value, showing genetic relatedness between strain LM2416T and V. necropolis KCTC 3820T, was 78.3?%. Based on the phylogenetic, biochemical, chemotaxonomic and genotypic data presented in this study, strain LM2416T is considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus phasianinus is proposed. The type strain is LM2416T (=KCTC 33927T=JCM 32144T).

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September 22, 2019

Gene presence-absence polymorphism in castrating anther-smut fungi: Recent gene Gains and Phylogeographic Structure.

Gene presence-absence polymorphisms segregating within species are a significant source of genetic variation but have been little investigated to date in natural populations. In plant pathogens, the gain or loss of genes encoding proteins interacting directly with the host, such as secreted proteins, probably plays an important role in coevolution and local adaptation. We investigated gene presence-absence polymorphism in populations of two closely related species of castrating anther-smut fungi, Microbotryum lychnidis-dioicae (MvSl) and M. silenes-dioicae (MvSd), from across Europe, on the basis of Illumina genome sequencing data and high-quality genome references. We observed presence-absence polymorphism for 186 autosomal genes (2% of all genes) in MvSl, and only 51 autosomal genes in MvSd. Distinct genes displayed presence-absence polymorphism in the two species. Genes displaying presence-absence polymorphism were frequently located in subtelomeric and centromeric regions and close to repetitive elements, and comparison with outgroups indicated that most were present in a single species, being recently acquired through duplications in multiple-gene families. Gene presence-absence polymorphism in MvSl showed a phylogeographic structure corresponding to clusters detected based on SNPs. In addition, gene absence alleles were rare within species and skewed toward low-frequency variants. These findings are consistent with a deleterious or neutral effect for most gene presence-absence polymorphism. Some of the observed gene loss and gain events may however be adaptive, as suggested by the putative functions of the corresponding encoded proteins (e.g., secreted proteins) or their localization within previously identified selective sweeps. The adaptive roles in plant and anther-smut fungi interactions of candidate genes however need to be experimentally tested in future studies.

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September 22, 2019

Multi-omics approach identifies novel pathogen-derived prognostic biomarkers in patients with Pseudomonas aeruginosa bloodstream infection

Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.

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September 22, 2019

Comparative genomics of bdelloid rotifers: Insights from desiccating and nondesiccating species.

Bdelloid rotifers are a class of microscopic invertebrates that have existed for millions of years apparently without sex or meiosis. They inhabit a variety of temporary and permanent freshwater habitats globally, and many species are remarkably tolerant of desiccation. Bdelloids offer an opportunity to better understand the evolution of sex and recombination, but previous work has emphasised desiccation as the cause of several unusual genomic features in this group. Here, we present high-quality whole-genome sequences of 3 bdelloid species: Rotaria macrura and R. magnacalcarata, which are both desiccation intolerant, and Adineta ricciae, which is desiccation tolerant. In combination with the published assembly of A. vaga, which is also desiccation tolerant, we apply a comparative genomics approach to evaluate the potential effects of desiccation tolerance and asexuality on genome evolution in bdelloids. We find that ancestral tetraploidy is conserved among all 4 bdelloid species, but homologous divergence in obligately aquatic Rotaria genomes is unexpectedly low. This finding is contrary to current models regarding the role of desiccation in shaping bdelloid genomes. In addition, we find that homologous regions in A. ricciae are largely collinear and do not form palindromic repeats as observed in the published A. vaga assembly. Consequently, several features interpreted as genomic evidence for long-term ameiotic evolution are not general to all bdelloid species, even within the same genus. Finally, we substantiate previous findings of high levels of horizontally transferred nonmetazoan genes in both desiccating and nondesiccating bdelloid species and show that this unusual feature is not shared by other animal phyla, even those with desiccation-tolerant representatives. These comparisons call into question the proposed role of desiccation in mediating horizontal genetic transfer.

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September 22, 2019

Genome-wide comparison reveals a probiotic strain Lactococcus lactis WFLU12 isolated from the gastrointestinal tract of olive flounder (Paralichthys Olivaceus) harboring genes supporting probiotic action.

Our previous study has shown that dietary supplementation with Lactococcus lactis WFLU12 can enhance the growth of olive flounder and its resistance against streptococcal infection. The objective of the present study was to use comparative genomics tools to investigate genomic characteristics of strain WFLU12 and the presence of genes supporting its probiotic action using sequenced genomes of L. lactis strains. Dispensable and singleton genes of strain WFLU12 were found to be more enriched in genes associated with metabolism (e.g., energy production and conversion, and carbohydrate transport and metabolism) than pooled dispensable and singleton genes in other L. lactis strains, reflecting WFLU12 strain-specific ecosystem origin and its ability to metabolize different energy sources. Strain WFLU12 produced antimicrobial compounds that could inhibit several bacterial fish pathogens. It possessed the nisin gene cluster (nisZBTCIPRKFEG) and genes encoding lysozyme and colicin V. However, only three other strains (CV56, IO-1, and SO) harbor a complete nisin gene cluster. We also found that L. lactis WFLU12 possessed many other important functional genes involved in stress responses to the gastrointestinal tract environment, dietary energy extraction, and metabolism to support the probiotic action of this strain found in our previous study. This strongly indicates that not all L. lactis strains can be used as probiotics. This study highlights comparative genomics approaches as very useful and powerful tools to select probiotic candidates and predict their probiotic effects.

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September 22, 2019

Genomic analysis of a pan-resistant isolate of Klebsiella pneumoniae, United States 2016.

Antimicrobial resistance is a threat to public health globally and leads to an estimated 23,000 deaths annually in the United States alone. Here, we report the genomic characterization of an unusualKlebsiella pneumoniae, nonsusceptible to all 26 antibiotics tested, that was isolated from a U.S.The isolate harbored four known beta-lactamase genes, including plasmid-mediatedblaNDM-1andblaCMY-6, as well as chromosomalblaCTX-M-15andblaSHV-28, which accounted for resistance to all beta-lactams tested. In addition, sequence analysis identified mechanisms that could explain all other reported nonsusceptibility results, including nonsusceptibility to colistin, tigecycline, and chloramphenicol. Two plasmids, IncA/C2 and IncFIB, were closely related to mobile elements described previously and isolated from Gram-negative bacteria from China, Nepal, India, the United States, and Kenya, suggesting possible origins of the isolate and plasmids. This is one of the firstK. pneumoniaeisolates in the United States to have been reported to the Centers for Disease Control and Prevention (CDC) as nonsusceptible to all drugs tested, including all beta-lactams, colistin, and tigecycline. IMPORTANCE Antimicrobial resistance is a major public health threat worldwide. Bacteria that are nonsusceptible or resistant to all antimicrobials available are of major concern to patients and the public because of lack of treatment options and potential for spread. AKlebsiella pneumoniaestrain that was nonsusceptible to all tested antibiotics was isolated from a U.S.Mechanisms that could explain all observed phenotypic antimicrobial resistance phenotypes, including resistance to colistin and beta-lactams, were identified through whole-genome sequencing. The large variety of resistance determinants identified demonstrates the usefulness of whole-genome sequencing for detecting these genes in an outbreak response. Sequencing of isolates with rare and unusual phenotypes can provide information on how these extremely resistant isolates develop, including whether resistance is acquired on mobile elements or accumulated through chromosomal mutations. Moreover, this provides further insight into not only detecting these highly resistant organisms but also preventing their spread.

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September 22, 2019

Chinook salmon (Oncorhynchus tshawytscha) genome and transcriptome.

When unifying genomic resources among studies and comparing data between species, there is often no better resource than a genome sequence. Having a reference genome for the Chinook salmon (Oncorhynchus tshawytscha) will enable the extensive genomic resources available for Pacific salmon, Atlantic salmon, and rainbow trout to be leveraged when asking questions related to the Chinook salmon. The Chinook salmon’s wide distribution, long cultural impact, evolutionary history, substantial hatchery production, and recent wild-population decline make it an important research species. In this study, we sequenced and assembled the genome of a Chilliwack River Hatchery female Chinook salmon (gynogenetic and homozygous at all loci). With a reference genome sequence, new questions can be asked about the nature of this species, and its role in a rapidly changing world.

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September 22, 2019

Characterization of two novel bacteriophages infecting multidrug-resistant (MDR) Acinetobacter baumannii and evaluation of their therapeutic efficacy in vivo.

Acinetobacter baumannii is emerging as a challenging nosocomial pathogen due to its rapid evolution of antibiotic resistance. We report characterization of two novel bacteriophages, PBAB08 and PBAB25, infecting clinically isolated, multidrug-resistant (MDR) A. baumannii strains. Both phages belonged to Myoviridae of Caudovirales as their morphology observed under an electron microscope. Their genomes were double stranded linear DNAs of 42,312 base pairs and 40,260 base pairs, respectively. The two phages were distinct from known Acinetobacter phages when whole genome sequences were compared. PBAB08 showed a 99% similarity with 57% sequence coverage to phage AB1 and PBAB25 showed a 97% similarity with 78% sequence coverage to phage IME_AB3. BLASTN significant alignment coverage of all other known phages were <30%. Seventy six and seventy genes encoding putative phage proteins were found in the genomes of PBAB08 and PBAB25, respectively. Their genomic organizations and sequence similarities were consistent with the modular theory of phage evolution. Therapeutic efficacy of a phage cocktail containing the two and other phages were evaluated in a mice model with nasal infection of MDR A. baumannii. Mice treated with the phage cocktail showed a 2.3-fold higher survival rate than those untreated in 7 days post infection. In addition, 1/100 reduction of the number of A. baumannii in the lung of the mice treated with the phage cocktail was observed. Also, inflammatory responses of mice which were injected with the phage cocktail by intraperitoneal, intranasal, or oral route was investigated. Increase in serum cytokine was minimal regardless of the injection route. A 20% increase in IgE production was seen in intraperitoneal injection route, but not in other routes. Thus, the cocktail containing the two newly isolated phages could serve as a potential candidate for therapeutic interventions to treat A. baummannii infections.

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September 22, 2019

Inferring the minimal genome of Mesoplasma florum by comparative genomics and transposon mutagenesis.

The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain. On the basis of random transposon mutagenesis, we also estimated that ~290 out of 720 genes are essential for M. florum L1 in rich medium. We next evaluated different genome reduction scenarios for M. florum L1 by using gene conservation and essentiality data, as well as comparisons with the first working approximation of a minimal organism, Mycoplasma mycoides JCVI-syn3.0. Our results suggest that 409 of the 473 M. mycoides JCVI-syn3.0 genes have orthologs in M. florum L1. Conversely, 57 putatively essential M. florum L1 genes have no homolog in M. mycoides JCVI-syn3.0. This suggests differences in minimal genome compositions, even for these evolutionarily closely related bacteria. IMPORTANCE The last years have witnessed the development of whole-genome cloning and transplantation methods and the complete synthesis of entire chromosomes. Recently, the first minimal cell, Mycoplasma mycoides JCVI-syn3.0, was created. Despite these milestone achievements, several questions remain to be answered. For example, is the composition of minimal genomes virtually identical in phylogenetically related species? On the basis of comparative genomics and transposon mutagenesis, we investigated this question by using an alternative model, Mesoplasma florum, that is also amenable to genome reduction efforts. Our results suggest that the creation of additional minimal genomes could help reveal different gene compositions and strategies that can support life, even within closely related species.

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September 22, 2019

Genomic architecture of haddock (Melanogrammus aeglefinus) shows expansions of innate immune genes and short tandem repeats.

Increased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates, including teleosts. Sequencing of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss of the major histocompatibility complex (MHC) II genes 100 million years ago. An improved version of the Atlantic cod genome assembly shows an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous assemblies are therefore needed to further investigate the unusual immune system of the Gadiformes, and whether the high density of tandem repeats found in Atlantic cod is a shared trait in this group.Here, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) – a relative of Atlantic cod – using a combination of PacBio and Illumina reads. Comparative analyses reveal that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. A characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For the NOD-like receptors (NLRs), another gene family associated with the innate immune system, we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes.The generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod – but possibly a feature common to all, or most, codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization of these genes, i.e. the number of NLRs in different teleost species might be underestimates.

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