Epidemic Clostridioides difficile Ribotype 027 Lineages: Comparisons of Texas Versus Worldwide Strains

Abstract Background The epidemic Clostridioides difficile ribotype 027 strain resulted from the dissemination of 2 separate fluoroquinolone-resistant lineages: FQR1 and FQR2. Both lineages were reported to originate in North America; however, confirmatory large-scale investigations of C difficile ribotype 027 epidemiology using whole genome sequencing has not been undertaken in the United States. Methods Whole genome sequencing and single-nucleotide polymorphism (SNP) analysis was performed on 76 clinical ribotype 027 isolates obtained from hospitalized patients in Texas with C difficile infection and compared with 32 previously sequenced worldwide strains. Maximum-likelihood phylogeny based on a set of core genome SNPs was used to construct phylogenetic trees investigating strain macro- and microevolution. Bayesian phylogenetic and phylogeographic analyses were used to incorporate temporal and geographic variables with the SNP strain analysis. Results Whole genome sequence analysis identified 2841 SNPs including 900 nonsynonymous mutations, 1404 synonymous substitutions, and 537 intergenic changes. Phylogenetic analysis separated the strains into 2 prominent groups, which grossly differed by 28 SNPs: the FQR1 and FQR2 lineages. Five isolates were identified as pre-epidemic strains. Phylogeny demonstrated unique clustering and resistance genes in Texas strains indicating that spatiotemporal bias has defined the microevolution of ribotype 027 genetics. Conclusions Clostridioides difficile ribotype 027 lineages emerged earlier than previously reported, coinciding with increased use of fluoroquinolones. Both FQR1 and FQR2 ribotype 027 epidemic lineages are present in Texas, but they have evolved geographically to represent region-specific public health threats.

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Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin-based serotyping of Pasteurella multocida

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638717732371 ◽

2017 ◽

Vol 30 (1) ◽

pp. 42-55 ◽

Cited By ~ 2

Author(s):

Karen J. LeCount ◽

Linda K. Schlater ◽

Tod Stuber ◽

Suelee Robbe Austerman ◽

Timothy S. Frana ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Restriction Endonuclease ◽

Genome Sequencing ◽

Pasteurella Multocida ◽

Restriction Endonuclease Analysis ◽

The United States ◽

Snp Analysis ◽

Whole Genome ◽

Gel Diffusion ◽

Endonuclease Analysis

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.

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SeqSero2: Rapid and Improved Salmonella Serotype Determination Using Whole-Genome Sequencing Data

Applied and Environmental Microbiology ◽

10.1128/aem.01746-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 25

Author(s):

Shaokang Zhang ◽

Hendrik C. den Bakker ◽

Shaoting Li ◽

Jessica Chen ◽

Blake A. Dinsmore ◽

...

Keyword(s):

Public Health ◽

United States ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Large Scale ◽

Draft Genome ◽

The United States ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Link Type

ABSTRACT SeqSero, launched in 2015, is a software tool for Salmonella serotype determination from whole-genome sequencing (WGS) data. Despite its routine use in public health and food safety laboratories in the United States and other countries, the original SeqSero pipeline is relatively slow (minutes per genome using sequencing reads), is not optimized for draft genome assemblies, and may assign multiple serotypes for a strain. Here, we present SeqSero2 (github.com/denglab/SeqSero2; denglab.info/SeqSero2), an algorithmic transformation and functional update of the original SeqSero. Major improvements include (i) additional sequence markers for identification of Salmonella species and subspecies and certain serotypes, (ii) a k-mer based algorithm for rapid serotype prediction from raw reads (seconds per genome) and improved serotype prediction from assemblies, and (iii) a targeted assembly approach for specific retrieval of serotype determinants from WGS for serotype prediction, new allele discovery, and prediction troubleshooting. Evaluated using 5,794 genomes representing 364 common U.S. serotypes, including 2,280 human isolates of 117 serotypes from the National Antimicrobial Resistance Monitoring System, SeqSero2 is up to 50 times faster than the original SeqSero while maintaining equivalent accuracy for raw reads and substantially improving accuracy for assemblies. SeqSero2 further suggested that 3% of the tested genomes contained reads from multiple serotypes, indicating a use for contamination detection. In addition to short reads, SeqSero2 demonstrated potential for accurate and rapid serotype prediction directly from long nanopore reads despite base call errors. Testing of 40 nanopore-sequenced genomes of 17 serotypes yielded a single H antigen misidentification. IMPORTANCE Serotyping is the basis of public health surveillance of Salmonella. It remains a first-line subtyping method even as surveillance continues to be transformed by whole-genome sequencing. SeqSero allows the integration of Salmonella serotyping into a whole-genome-sequencing-based laboratory workflow while maintaining continuity with the classic serotyping scheme. SeqSero2, informed by extensive testing and application of SeqSero in the United States and other countries, incorporates important improvements and updates that further strengthen its application in routine and large-scale surveillance of Salmonella by whole-genome sequencing.

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Genomic and phylogenetic analysis ofSalmonellaTyphimurium and its monophasic variants responsible for invasive endemic infections in Colombia

10.1101/588608 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yan Li ◽

Caisey V. Pulford ◽

Paula Díaz ◽

Blanca M. Perez-Sepulveda ◽

Carolina Duarte ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Nalidixic Acid ◽

Genome Sequencing ◽

Phylogenetic Trees ◽

Evolutionary History ◽

Large Scale ◽

Multidrug Resistant ◽

Human Infection ◽

Sequence Type ◽

Whole Genome

AbstractSalmonellosis is an endemic human infection, associated with both sporadic cases and outbreaks throughout Colombia. Typhimurium is the most common Colombian serovar ofSalmonella enterica, responsible for 32.5% of theSalmonellainfections. Whole genome sequencing (WGS) is being used increasingly in Europe and the USA to study the epidemiology ofSalmonella, but there has not yet been a WGS-based analysis ofSalmonellaassociated with bloodstream infection in Colombia. Here, we analysed 209 genome sequences of ColombianS. Typhimurium and monophasicS. 4,[5],12:i:-isolates from Colombia from 1999 to 2017. We used a core genome-based maximum likelihood tree to define seven distinct clusters which were predominantly Sequence Type (ST) 19 isolates. We also identified the first ST313 and monophasic ST34 isolates to be reported in Colombia. The history of each cluster was reconstructed with a Bayesian tree to reveal a timeline of evolution. Cluster 7 was closely related to European multidrug-resistant (MDR) DT104. Cluster 4 became the dominant variant ofSalmonellain 2016, and resistance to nalidixic acid was associated with a plasmid-encodedqnrB19gene. Our findings suggest multiple transfers ofS. Typhimurium between Europe and Colombia.Author summaryThe large-scale genome sequencing ofSalmonellaTyphimurium and monophasicSalmonella4,[5],12:i:-involved bloodstream isolates from Colombia. The two serovars were responsible for about 1/3 ofSalmonellainfections in Colombia in the past 20 years. To identify the population structure we used Whole Genome Sequencing, performedin silicosequence typing, obtained phylogenetic trees, inferred the evolutionary history, detected the plasmids and prophages, and associated the antibiotic resistance (AMR) genotype with phenotype. Different clusters showed temporal replacement. The Colombian sequence type 313 was distinct from African lineages due to the absence of a key virulence-related gene,bstA. One of the Colombian clusters is likely to belong to the global epidemic of DT104, according to the evolutionary history and the AMR profile. The most common cluster in recent years was resistant to nalidixic acid and carried a plasmid-mediated antibiotic resistant geneqnrB19. Our findings will inform the ongoing efforts to combat Salmonellosis by Colombian public health departments.

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Population Dynamics of Staphylococcus aureus in Cystic Fibrosis Patients To Determine Transmission Events by Use of Whole-Genome Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.00164-17 ◽

2017 ◽

Vol 55 (7) ◽

pp. 2143-2152 ◽

Cited By ~ 13

Author(s):

Andrea Ankrum ◽

Barry G. Hall

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Population Dynamics ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Phylogenetic Trees ◽

Hospital Environment ◽

Snp Analysis ◽

Whole Genome ◽

Content Type

ABSTRACT Strict infection control practices have been implemented for health care visits by cystic fibrosis (CF) patients in an attempt to prevent transmission of important pathogens. This study used whole-genome sequencing (WGS) to determine strain relatedness and assess population dynamics of Staphylococcus aureus isolates from a cohort of CF patients as assessed by strain relatedness. A total of 311 S. aureus isolates were collected from respiratory cultures of 115 CF patients during a 22-month study period. Whole-genome sequencing was performed, and using single nucleotide polymorphism (SNP) analysis, phylogenetic trees were assembled to determine relatedness between isolates. Methicillin-resistant Staphylococcus aureus (MRSA) phenotypes were predicted using PPFS2 and compared to the observed phenotype. The accumulation of SNPs in multiple isolates obtained over time from the same patient was examined to determine if a genomic molecular clock could be calculated. Pairs of isolates with ≤71 SNP differences were considered to be the “same” strain. All of the “same” strain isolates were either from the same patient or siblings pairs. There were 47 examples of patients being superinfected with an unrelated strain. The predicted MRSA phenotype was accurate in all but three isolates. Mutation rates were unable to be determined because the branching order in the phylogenetic tree was inconsistent with the order of isolation. The observation that transmissions were identified between sibling patients shows that WGS is an effective tool for determining transmission between patients. The observation that transmission only occurred between siblings suggests that Staphylococcus aureus acquisition in our CF population occurred outside the hospital environment and indicates that current infection prevention efforts appear effective.

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Whole genome sequencing of Clostridioides difficile PCR ribotype 046 suggests transmission between pigs and humans

PLoS ONE ◽

10.1371/journal.pone.0244227 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244227

Author(s):

Anders Werner ◽

Paula Mölling ◽

Anna Fagerström ◽

Fredrik Dyrkell ◽

Dimitrios Arnellos ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

General Pattern ◽

Snp Analysis ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Neonatal Pigs ◽

Hospital Outbreak ◽

Clostridioides Difficile

Background A zoonotic association has been suggested for several PCR ribotypes (RTs) of Clostridioides difficile. In central parts of Sweden, RT046 was found dominant in neonatal pigs at the same time as a RT046 hospital C. difficile infection (CDI) outbreak occurred in the southern parts of the country. Objective To detect possible transmission of RT046 between pig farms and human CDI cases in Sweden and investigate the diversity of RT046 in the pig population using whole genome sequencing (WGS). Methods WGS was performed on 47 C. difficile isolates from pigs (n = 22), the farm environment (n = 7) and human cases of CDI (n = 18). Two different core genome multilocus sequencing typing (cgMLST) schemes were used together with a single nucleotide polymorphisms (SNP) analysis and the results were related to time and location of isolation of the isolates. Results The pig isolates were closely related (≤6 cgMLST alleles differing in both cgMLST schemes) and conserved over time and were clearly separated from isolates from the human hospital outbreak (≥76 and ≥90 cgMLST alleles differing in the two cgMLST schemes). However, two human isolates were closely related to the pig isolates, suggesting possible transmission. The SNP analysis was not more discriminate than cgMLST. Conclusion No general pattern suggesting zoonotic transmission was apparent between pigs and humans, although contrasting results from two isolates still make transmission possible. Our results support the need for high resolution WGS typing when investigating hospital and environmental transmission of C. difficile.

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Rare Non-coding Variation Identified by Large Scale Whole Genome Sequencing Reveals Unexplained Heritability of Type 2 Diabetes

10.1101/2020.11.13.20221812 ◽

2020 ◽

Author(s):

Jennifer Wessel ◽

Timothy D Majarian ◽

Heather M Highland ◽

Sridharan Raghavan ◽

Mindy D Szeto ◽

...

Keyword(s):

Type 2 Diabetes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Large Scale ◽

Regulatory Elements ◽

Whole Genome Sequence ◽

European Ancestry ◽

Substantial Contribution ◽

Whole Genome

Type 2 diabetes is increasing in all ancestry groups1. Part of its genetic basis may reside among the rare (minor allele frequency <0.1%) variants that make up the vast majority of human genetic variation2. We analyzed high-coverage (mean depth 38.2x) whole genome sequencing from 9,639 individuals with T2D and 34,994 controls in the NHLBI’s Trans-Omics for Precision Medicine (TOPMed) program2 to show that rare, non-coding variants that are poorly captured by genotyping arrays or imputation panels contribute h2=53% (P=4.2×10−5) to the genetic component of risk in the largest (European) ancestry subset. We coupled sequence variation with islet epigenomic signatures3 to annotate and group rare variants with respect to gene expression4, chromatin state5 and three-dimensional chromatin architecture6, and show that pancreatic islet regulatory elements contribute to T2D genetic risk (h2=8%, P=2.4×10−3). We used islet annotation to create a non-coding framework for rare variant aggregation testing. This approach identified five loci containing rare alleles in islet regulatory elements that suggest novel biological mechanisms readily linked to hypotheses about variant-to-function. Large scale whole genome sequence analysis reveals the substantial contribution of rare, non-coding variation to the genetic architecture of T2D and highlights the value of tissue-specific regulatory annotation for variant-to-function discovery.

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Quality Control and Integration of Genotypes from Two Calling Pipelines for Whole Genome Sequence Data in the Alzheimer’s Disease Sequencing Project

10.1101/318857 ◽

2018 ◽

Cited By ~ 1

Author(s):

Adam C. Naj ◽

Honghuang Lin ◽

Badri N. Vardarajan ◽

Simon White ◽

Daniel Lancour ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Quality Control ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Large Scale ◽

Read Depth ◽

Whole Genome Sequence ◽

Whole Genome ◽

Sequencing Project

AbstractThe Alzheimer’s Disease Sequencing Project (ADSP) performed whole genome sequencing (WGS) of 584 subjects from 111 multiplex families at three sequencing centers. Genotype calling of single nucleotide variants (SNVs) and insertion-deletion variants (indels) was performed centrally using GATK-HaplotypeCaller and Atlas V2. The ADSP Quality Control (QC) Working Group applied QC protocols to project-level variant call format files (VCFs) from each pipeline, and developed and implemented a novel protocol, termed “consensus calling,” to combine genotype calls from both pipelines into a single high-quality set. QC was applied to autosomal bi-allelic SNVs and indels, and included pipeline-recommended QC filters, variant-level QC, and sample-level QC. Low-quality variants or genotypes were excluded, and sample outliers were noted. Quality was assessed by examining Mendelian inconsistencies (MIs) among 67 parent-offspring pairs, and MIs were used to establish additional genotype-specific filters for GATK calls. After QC, 578 subjects remained. Pipeline-specific QC excluded ~12.0% of GATK and 14.5% of Atlas SNVs. Between pipelines, ~91% of SNV genotypes across all QCed variants were concordant; 4.23% and 4.56% of genotypes were exclusive to Atlas or GATK, respectively; the remaining ~0.01% of discordant genotypes were excluded. For indels, variant-level QC excluded ~36.8% of GATK and 35.3% of Atlas indels. Between pipelines, ~55.6% of indel genotypes were concordant; while 10.3% and 28.3% were exclusive to Atlas or GATK, respectively; and ~0.29% of discordant genotypes were. The final WGS consensus dataset contains 27,896,774 SNVs and 3,133,926 indels and is publicly available.AbbreviationsAD, Alzheimer’s disease; QC, Quality Control; LSSAC, Large-Scale Sequencing and Analysis Center; Broad, Broad Institute Genomics Service; Baylor, Baylor College of Medicine Human Genome Sequencing Center; WashU, Washington University-St. Louis McDonnell Genome Institute; WGS, whole genome sequencing; WES, whole exome sequencing; indel, insertion-deletion variants; VCF, variant control format; MI, Mendelian inconsistency; MC, Mendelian consistency; GWAS, genome-wide association study; VR, referent allele read depth; DP, overall read depth; MS, mapping score; GQ, genotype quality score; Ti/Tv, Transition/Transversion; CS, concordance code

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Genomic Context of Azole Resistance Mutations in Aspergillus fumigatus Determined Using Whole-Genome Sequencing

mBio ◽

10.1128/mbio.00536-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 60

Author(s):

Alireza Abdolrasouli ◽

Johanna Rhodes ◽

Mathew A. Beale ◽

Ferry Hagen ◽

Thomas R. Rogers ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

The United States ◽

Azole Resistance ◽

Snp Analysis ◽

Genome Wide Association Studies ◽

Whole Genome ◽

Content Type ◽

Genome Wide

ABSTRACT A rapid and global emergence of azole resistance has been observed in the pathogenic fungus Aspergillus fumigatus over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves mutations in the cyp51A gene, which encodes a protein targeted by triazole antifungal drugs. Whole-genome sequencing (WGS) was performed for high-resolution single-nucleotide polymorphism (SNP) analysis of 24 A. fumigatus isolates, including azole-resistant and susceptible clinical and environmental strains obtained from India, the Netherlands, and the United Kingdom, in order to assess the utility of WGS for characterizing the alleles causing resistance. WGS analysis confirmed that TR34/L98H (a mutation comprising a tandem repeat [TR] of 34 bases in the promoter of the cyp51A gene and a leucine-to-histidine change at codon 98) is the sole mechanism of azole resistance among the isolates tested in this panel of isolates. We used population genomic analysis and showed that A. fumigatus was panmictic, with as much genetic diversity found within a country as is found between continents. A striking exception to this was shown in India, where isolates are highly related despite being isolated from both clinical and environmental sources across >1,000 km; this broad occurrence suggests a recent selective sweep of a highly fit genotype that is associated with the TR34/L98H allele. We found that these sequenced isolates are all recombining, showing that azole-resistant alleles are segregating into diverse genetic backgrounds. Our analysis delineates the fundamental population genetic parameters that are needed to enable the use of genome-wide association studies to identify the contribution of SNP diversity to the generation and spread of azole resistance in this medically important fungus. IMPORTANCE Resistance to azoles in the ubiquitous ascomycete fungus A. fumigatus was first reported from clinical isolates collected in the United States during the late 1980s. Over the last decade, an increasing number of A. fumigatus isolates from the clinic and from nature have been found to show resistance to azoles, suggesting that resistance is emerging through selection by the widespread usage of agricultural azole antifungal compounds. Aspergillosis is an emerging clinical problem, with high rates of treatment failures necessitating the development of new techniques for surveillance and for determining the genome-wide basis of azole resistance in A. fumigatus.

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