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July 7, 2019  |  

Whole-genome sequencing: opportunities and challenges for public health, food-borne outbreak investigations, and the global food supply.

Food-borne disease is burdensome, af- fecting 1 in 6 persons or an estimated 48 million ill, 128 000 hospitalized, and 3000 deaths in the United States annually. In addition, societal costs from lost lives, lost labor, lost wages, and even lost revenue in the food industry are substan- tial. Globally the burden is even higher, and multinational outbreaks due to the global movement of contaminated foods are being described increasingly. The glo- bal food supply links nations and econo- mies, emphasizing the need to view food safety with an integrated farm-to-fork lens. As predicted, advances in molecular techniques and information management have been transformative for food-borne disease investigation.


July 7, 2019  |  

High incidence of invasive group A Streptococcus disease caused by strains of uncommon emm types in Thunder Bay, Ontario, Canada.

An outbreak of type emm59 invasive group A Streptococcus (iGAS) disease was declared in 2008 in Thunder Bay District, Northwestern Ontario, two years after a country-wide emm59 epidemic was recognized in Canada. Despite a declining number of emm59 infections since 2010, numerous cases of iGAS disease continue to be reported in the area. We collected clinical information on all iGAS cases recorded in Thunder Bay District from 2008-2013. We also emm typed and sequenced the genomes of all available strains isolated in 2011-2013 from iGAS infections, and from severe cases of soft tissue infections. We used whole-genome data to investigate the population structure of GAS strains of the most frequently isolated emm types. We report increased incidence of iGAS in Thunder Bay compared to the metropolitan area of Toronto/Peel and the province of Ontario. Illicit drug use, alcohol abuse, homelessness and hepatitis C infection were underlying diseases or conditions that might have predisposed patients to iGAS disease. Most cases were caused by clonal strains of “skin” or “generalist” emm types (i.e. emm82, emm87, emm101, emm4, emm83, and emm114), uncommonly seen in other areas of the province. We observed rapid waxing and waning of emm types causing disease and their replacement by other emm types associated with the same tissue tropisms. Thus, iGAS disease in Thunder Bay District predominantly affects a select population of disadvantaged persons and is caused by clonally related strains of a few “skin” and “generalist” emm types less commonly associated with iGAS in other areas of Ontario. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Identification and resolution of microdiversity through metagenomic sequencing of parallel consortia.

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Clonal Complex 17 group B Streptococcus strains causing invasive disease in neonates and adults originate from the same genetic pool.

A significant proportion of group B Streptococcus (GBS) neonatal disease, particularly late-onset disease, is associated with strains of serotype III, clonal complex (CC) 17. CC17 strains also cause invasive infections in adults. Little is known about the phylogenetic relationships of isolates recovered from neonatal and adult CC17 invasive infections. We performed whole-genome-based phylogenetic analysis of 93 temporally and geographically matched CC17 strains isolated from both neonatal and adult invasive infections in the metropolitan region of Toronto/Peel, Canada. We also mined the whole-genome data to reveal mobile genetic elements carrying antimicrobial resistance genes. We discovered that CC17 GBS strains causing neonatal and adult invasive disease are interspersed and cluster tightly in a phylogenetic tree, signifying that they are derived from the same genetic pool. We identified limited variation due to recombination in the core CC17 genome. We describe that loss of Pilus Island 1 and acquisition of different mobile genetic elements carrying determinants of antimicrobial resistance contribute to CC17 genetic diversity. Acquisition of some of these mobile genetic elements appears to correlate with clonal expansion of the strains that possess them. Our results provide a genome-wide portrait of the population structure and evolution of a major disease-causing clone of an opportunistic pathogen.


July 7, 2019  |  

Genomic analyses reveal that partial sequence of an earlier pseudorabies virus in China is originated from a Bartha-vaccine-like strain.

Pseudorabies virus (PRV), the causative agent of Aujeszky?s disease, has gained increased attention in China in recent years as a result of the outbreak of emergent pseudorabies. Several genomic and partial sequences are available for Chinese emergent and European-American strains of PRV, but limited sequence data exist for the earlier Chinese strains. In this study, we determined the complete genomic sequence of one earlier Chinese strain SC and one emergent strain HLJ8. Compared with other known sequences, we demonstrated that PRV strains from distinct geographical regions displayed divergent evolution. Additionally, we report for the first time, a recombination event between PRV strains, and show that strain SC is a recombinant of an endemic Chinese strain and a Bartha-vaccine-like strain. These results contribute to our understanding of PRV evolution. Copyright © 2016 Elsevier Inc. All rights reserved.


July 7, 2019  |  

Complete closed genome sequences of Salmonella enterica subsp. enterica serotypes Anatum, Montevideo, Typhimurium, and Newport, isolated from beef, cattle, and humans.

Salmonella enterica spp. are a diverse group of bacteria with a wide range of virulence potential. To facilitate genome comparisons across this virulence spectrum, we present eight complete closed genome sequences of four S. enterica serotypes (Anatum, Montevideo, Typhimurium, and Newport), isolated from various cattle samples and from humans. Copyright © 2016 Harhay et al.


July 7, 2019  |  

Population structure and antimicrobial resistance profiles of Streptococcus suis serotype 2 sequence type 25 strains

Strains of serotype 2 Streptococcus suis are responsible for swine and human infections. Different serotype 2 genetic backgrounds have been defined using multilocus sequence typing (MLST). However, little is known about the genetic diversity within each MLST sequence type (ST). Here, we used whole-genome sequencing to test the hypothesis that S. suis serotype 2 strains of the ST25 lineage are genetically heterogeneous. We evaluated 51 serotype 2 ST25 S. suis strains isolated from diseased pigs and humans in Canada, the United States of America, and Thailand. Whole-genome sequencing revealed numerous large-scale rearrangements in the ST25 genome, compared to the genomes of ST1 and ST28 S. suis strains, which result, among other changes, in disruption of a pilus island locus. We report that recombination and lateral gene transfer contribute to ST25 genetic diversity. Phylogenetic analysis identified two main and distinct Thai and North American clades grouping most strains investigated. These clades also possessed distinct patterns of antimicrobial resistance genes, which correlated with acquisition of different integrative and conjugative elements (ICEs). Some of these ICEs were found to be integrated at a recombination hot spot, previously identified as the site of integration of the 89K pathogenicity island in serotype 2 ST7 S. suis strains. Our results highlight the limitations of MLST for phylogenetic analysis of S. suis, and the importance of lateral gene transfer and recombination as drivers of diversity in this swine pathogen and zoonotic agent.


July 7, 2019  |  

Evolutionary history of the global emergence of the Escherichia coli epidemic clone ST131.

Escherichia colisequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n= 215) of sequenced ST131E. coliisolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of ablaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration ofblaCTX-Mwithin subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, theblaCTX-M-14/14-likegroup. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages ofE. coli These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages.IMPORTANCEEscherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specificE. colilineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements. Copyright © 2016 Stoesser et al.


July 7, 2019  |  

PEPR: pipelines for evaluating prokaryotic references.

The rapid adoption of microbial whole genome sequencing in public health, clinical testing, and forensic laboratories requires the use of validated measurement processes. Well-characterized, homogeneous, and stable microbial genomic reference materials can be used to evaluate measurement processes, improving confidence in microbial whole genome sequencing results. We have developed a reproducible and transparent bioinformatics tool, PEPR, Pipelines for Evaluating Prokaryotic References, for characterizing the reference genome of prokaryotic genomic materials. PEPR evaluates the quality, purity, and homogeneity of the reference material genome, and purity of the genomic material. The quality of the genome is evaluated using high coverage paired-end sequence data; coverage, paired-end read size and direction, as well as soft-clipping rates, are used to identify mis-assemblies. The homogeneity and purity of the material relative to the reference genome are characterized by comparing base calls from replicate datasets generated using multiple sequencing technologies. Genomic purity of the material is assessed by checking for DNA contaminants. We demonstrate the tool and its output using sequencing data while developing a Staphylococcus aureus candidate genomic reference material. PEPR is open source and available at https://github.com/usnistgov/pepr .


July 7, 2019  |  

Genetic diversity of O-antigens in Hafnia alvei and the development of a suspension array for serotype detection.

Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.


July 7, 2019  |  

Whole genome DNA sequence analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS) to Salmonella subspecies enterica serotype Tennessee (S. Tennessee) to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana), which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP) analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs), suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks. Using WGS can delimit contamination sources for foodborne illnesses across multiple outbreaks and reveal otherwise undetected DNA sequence differences essential to the tracing of bacterial pathogens as they emerge.


July 7, 2019  |  

Evaluation of an optimal epidemiologic typing scheme for Legionella pneumophila with whole genome sequence data using validation guidelines.

Sequence-based typing (SBT), analogous to multi-locus sequence typing (MLST), is the current gold-standard typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila However, as common sequence types (STs) cause many infections, some investigations remain unresolved. Here, various whole genome sequencing (WGS)-based methods were evaluated according to published guidelines, including: i) single nucleotide polymorphism (SNP)-based; ii) extended multi-locus sequence typing (MLST) using different numbers of genes; iii) gene presence/absence, and iv) kmer-based. L. pneumophila serogroup 1 isolates (n=106) from the standard “typing panel”, previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI) were tested together with another 229 isolates.Over 98% isolates were considered typable using the mapping- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50-gene) to 86.8% (1455-gene) whilst only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP-based), and all values are higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ~50 genes provides optimal epidemiological concordance whilst substantially improving the discrimination offered by SBT, and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila. Copyright © 2016 David et al.


July 7, 2019  |  

1,135 genomes reveal the global pattern of polymorphism in Arabidopsis thaliana.

Arabidopsis thaliana serves as a model organism for the study of fundamental physiological, cellular, and molecular processes. It has also greatly advanced our understanding of intraspecific genome variation. We present a detailed map of variation in 1,135 high-quality re-sequenced natural inbred lines representing the native Eurasian and North African range and recently colonized North America. We identify relict populations that continue to inhabit ancestral habitats, primarily in the Iberian Peninsula. They have mixed with a lineage that has spread to northern latitudes from an unknown glacial refugium and is now found in a much broader spectrum of habitats. Insights into the history of the species and the fine-scale distribution of genetic diversity provide the basis for full exploitation of A. thaliana natural variation through integration of genomes and epigenomes with molecular and non-molecular phenotypes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.


July 7, 2019  |  

Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi.

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.


July 7, 2019  |  

Species- and strain-specific adaptation of the HSP70 super family in pathogenic trypanosomatids.

All eukaryotic genomes encode multiple members of the heat shock protein 70 (HSP70) family, which evolved distinctive structural and functional features in response to specific environmental constraints. Phylogenetic analysis of this protein family thus can inform on genetic and molecular mechanisms that drive species-specific environmental adaptation. Here we use the eukaryotic pathogen Leishmania spp. as a model system to investigate the evolution of the HSP70 protein family in an early-branching eukaryote that is prone to gene amplification and adapts to cytotoxic host environments by stress-induced and chaperone-dependent stage differentiation. Combining phylogenetic and comparative analyses of trypanosomatid genomes, draft genome of Paratrypanosoma and recently published genome sequences of 204 L. donovani field isolates, we gained unique insight into the evolutionary dynamics of the Leishmania HSP70 protein family. We provide evidence for (i) significant evolutionary expansion of this protein family in Leishmania through gene amplification and functional specialization of highly conserved canonical HSP70 members, (ii) evolution of trypanosomatid-specific, non-canonical family members that likely gained ATPase-independent functions, and (iii) loss of one atypical HSP70 member in the Trypanosoma genus. Finally, we reveal considerable copy number variation of canonical cytoplasmic HSP70 in highly related L. donovani field isolates, thus identifying this locus as a potential hot spot of environment-genotype interaction. Our data draw a complex picture of the genetic history of HSP70 in trypanosomatids that is driven by the remarkable plasticity of the Leishmania genome to undergo massive intra-chromosomal gene amplification to compensate for the absence of regulated transcriptional control in these parasites. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


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