scholarly journals Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Katherine E. Bowden ◽  
Sandeep J. Joseph ◽  
John C. Cartee ◽  
Noa Ziklo ◽  
Damien Danavall ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Intracellular Bacterium ◽  
Genomic Diversity ◽  
Clinical Samples ◽  
Comparative Genomic ◽  
Sexual Networks ◽  
Whole Genome ◽  
Target Enrichment ◽  
Content Type ◽  
Sex With Men

ABSTRACT Chlamydia trachomatis, an obligately intracellular bacterium, is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. Numbers of U.S. infections of the urogenital tract and rectum have increased annually. Because C. trachomatis is not easily cultured, comparative genomic studies are limited, restricting our understanding of strain diversity and emergence among populations globally. While Agilent SureSelectXT target enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal samples, public access to these libraries is not available. We therefore designed an RNA bait library (34,795 120-mer probes based on 85 genomes, versus 33,619 probes using 74 genomes in a previous one) to augment organism sequencing from clinical samples that can be shared with the scientific community, enabling comparison studies. We describe the library and limit of detection for genome copy input, and we present results of 100% efficiency and high-resolution determination of recombination and identical genomes within vaginal-rectal specimen pairs in women. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, which limits our understanding of urogenital and rectal C. trachomatis transmission and impact on morbidity. To provide a publicly available workflow for whole-genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we developed and report on an RNA bait library to enrich the organism from clinical samples for sequencing. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis and identified recombinant and identical C. trachomatis genomes in paired vaginal-rectal samples from women. Our workflow provides a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital, and rectal infections and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

scholarly journals Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs

2020 ◽  
Author(s):  
Katherine E. Bowden ◽  
Sandeep J. Joseph ◽  
John Cartee ◽  
Noa Ziklo ◽  
Damien Danavall ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Intracellular Bacterium ◽  
Clinical Samples ◽  
Sexual Networks ◽  
Whole Genome ◽  
Target Enrichment ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Improve Efficiency ◽  
Sex With Men

AbstractChlamydia trachomatis is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. U.S. cases have been steadily increasing for more than a decade in both the urogenital tract and rectum. C. trachomatis is an obligate intracellular bacterium that is not easily cultured, limiting the capacity for genome studies to understand strain diversity and emergence among various patient populations globally. While Agilent SureSelectXT target-enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival and rectal samples, efficiencies are only 60-80% for ≥95-100% genome coverage. We therefore re-designed and expanded the RNA bait library to augment enrichment of the organism from clinical samples to improve efficiency. We describe the expanded library, the limit of detection for C. trachomatis genome copy input, and the 100% efficiency and high-resolution of generated genomes where genomic recombination among paired vaginal and rectal specimens from four patients was identified. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, among geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.ImportanceChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, and there is limited information on rectal C. trachomatis transmission and its impact on morbidity. To improve efficiency of previous studies involving whole genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we expanded the RNA bait library to augment enrichment of the organism from clinical samples. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis. We show the new system is sensitive for near identical genomes of C. trachomatis from two body sites in four women. Further, we provide a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital and rectal infections, and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

scholarly journals Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains

2021 ◽  
Vol 7 (6) ◽  
Author(s):  
Helena M. B. Seth-Smith ◽  
Angèle Bénard ◽  
Sylvia M. Bruisten ◽  
Bart Versteeg ◽  
Björn Herrmann ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Genome Sequencing ◽  
Lymphogranuloma Venereum ◽  
Genomic Diversity ◽  
Invasive Infection ◽  
Content Type ◽  
Genomic Changes ◽  
Variable Domains ◽  
Sex With Men ◽  
Sexually Transmissible Infection

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis , is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

scholarly journals Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Jean F. Challacombe ◽  
Jeannine M. Petersen ◽  
La Verne Gallegos-Graves ◽  
David Hodge ◽  
Segaran Pillai ◽  
...  
Keyword(s):  
New Species ◽  
16S Rrna ◽  
Response Times ◽  
Amino Acid Sequences ◽  
Clinical Samples ◽  
Whole Genome ◽  
Multiple Gene ◽  
Content Type ◽  
Environmental Surveys ◽  
Genome Comparisons

ABSTRACT Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

scholarly journals Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jin Sun No ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Seung-Ho Lee ◽  
Jeong-Ah Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Samples ◽  
Whole Genome ◽  
Target Enrichment ◽  
Next Generation ◽  
Apodemus Agrarius ◽  
Target Capture ◽  
Generation Sequencing

Abstract Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

scholarly journals Streptococcus gallolyticus subsp. gallolyticus from Human and Animal Origins: Genetic Diversity, Antimicrobial Susceptibility, and Characterization of a Vancomycin-Resistant Calf Isolate Carrying avanA-Tn1546-Like Element

2015 ◽  
Vol 59 (4) ◽  
pp. 2006-2015 ◽  
Author(s):  
Beatriz Romero-Hernández ◽  
Ana P. Tedim ◽  
José Francisco Sánchez-Herrero ◽  
Pablo Librado ◽  
Julio Rozas ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antibiotic Susceptibility ◽  
Resistant Isolate ◽  
Comparative Genomic ◽  
Gene Gain ◽  
Whole Genome ◽  
Content Type ◽  
Streptococcus Gallolyticus

ABSTRACTThe aim of this work was to characterize the antibiotic susceptibility and genetic diversity of 41Streptococcus gallolyticussubsp.gallolyticusisolates: 18 isolates obtained from animals and 23 human clinical isolates. Antibiotic susceptibility was determined by the semiautomatic Wider system and genetic diversity by pulsed-field gel electrophoresis (PFGE) with SmaI. Animal isolates grouped separately in the PFGE analysis, but no statistical differences in antimicrobial resistance were found between the two groups. The LMG 17956 sequence type 28 (ST28) strain recovered from the feces of a calf exhibited high levels of resistance to vancomycin and teicoplanin (MIC, ≥256 mg/liter). Its glycopeptide resistance mechanism was characterized by Southern blot hybridization and a primer-walking strategy, and finally its genome, determined by whole-genome sequencing, was compared with four closely relatedS. gallolyticussubsp.gallolyticusgenomes. Hybridization experiments demonstrated that a Tn1546-like element was integrated into the bacterial chromosome. In agreement with this finding, whole-genome sequencing confirmed a partial deletion of thevanY-vanZregion and partial duplication of thevanHgene. The comparative genomic analyses revealed that the LMG 17956 ST28 strain had acquired an unusually high number of transposable elements and had experienced extensive chromosomal rearrangements, as well as gene gain and loss events. In conclusion,S. gallolyticussubsp.gallolyticusisolates from animals seem to belong to lineages separate from those infecting humans. In addition, we report a glycopeptide-resistant isolate from a calf carrying a Tn1546-like element integrated into its chromosome.

scholarly journals Whole-Genome Sequences and Classification of Streptococcus agalactiae Strains Isolated from Laboratory-Reared Long-Evans Rats (Rattus norvegicus)

2017 ◽  
Vol 5 (1) ◽  
Author(s):  
C. Bodi Winn ◽  
J. Dzink-Fox ◽  
Y. Feng ◽  
Z. Shen ◽  
V. Bakthavatchalu ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Genomic Analysis ◽  
Opportunistic Pathogen ◽  
Comparative Genomic ◽  
Whole Genome ◽  
Genome Sequences ◽  
Content Type ◽  
Long Evans ◽  
Fatal Septicemia

ABSTRACT In collaboration with the CDC’s Streptococcus Laboratory, we report here the whole-genome sequences of seven Streptococcus agalactiae bacteria isolated from laboratory-reared Long-Evans rats. Four of the S. agalactiae isolates were associated with morbidity accompanied by endocarditis, metritis, and fatal septicemia, providing an opportunity for comparative genomic analysis of this opportunistic pathogen.

scholarly journals Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments

2014 ◽  
Vol 80 (15) ◽  
pp. 4616-4625 ◽  
Author(s):  
Thomas Denes ◽  
Kitiya Vongkamjan ◽  
Hans-Wolfgang Ackermann ◽  
Andrea I. Moreno Switt ◽  
Martin Wiedmann ◽  
...  
Keyword(s):  
New York ◽  
Dairy Farm ◽  
Morphological Diversity ◽  
Genomic Diversity ◽  
Comparative Genomic ◽  
The Novel ◽  
Important Food ◽  
Content Type ◽  
Small Genome ◽  
Food Borne

ABSTRACTThe genusListeriais ubiquitous in the environment and includes the globally important food-borne pathogenListeria monocytogenes. While the genomic diversity ofListeriahas been well studied, considerably less is known about the genomic and morphological diversity ofListeriabacteriophages. In this study, we sequenced and analyzed the genomes of 14Listeriaphages isolated mostly from New York dairy farm environments as well as one relatedEnterococcus faecalisphage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. TheseListeriaphages, based on gene orthology and morphology, together with previously sequencedListeriaphages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large-genome (∼135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (∼66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) ofE. faecalisphages, with medium-sized genomes (∼56 kb), was identified, which grouped together and shares morphological features with the novelListeriaphage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.

scholarly journals Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

2015 ◽  
Vol 53 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Louise J. Pankhurst ◽  
Luke W. Anson ◽  
Marcus R. Morgan ◽  
Deborah Gascoyne-Binzi ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Solid Phase ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

scholarly journals Deciphering the Origins and Tracking the Evolution of Cholera Epidemics with Whole-Genome-Based Molecular Epidemiology

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Yonatan H. Grad ◽  
Matthew K. Waldor
Keyword(s):  
Point Mutations ◽  
Comparative Genomic ◽  
Whole Genome ◽  
Cholera Outbreak ◽  
Outbreak Strain ◽  
Security Forces ◽  
Content Type ◽  
Cholera Epidemic ◽  
Genomic Analyses ◽  
The World

ABSTRACT The devastating Haitian cholera outbreak that began in October 2010 is the first known cholera epidemic in this island nation. Epidemiological and genomic data have provided strong evidence that United Nations security forces from Nepal introduced toxigenic Vibrio cholerae O1, the cause of epidemic cholera, to Haiti shortly before the outbreak arose. However, some have contended that indigenous V. cholerae contributed to the outbreak. In a recent paper (mBio 4:e00398-13, 2013), L. S. Katz et al. explored the nature and rate of changes in this ancient pathogen’s genome during an outbreak, based on whole-genome sequencing of 23 Haitian V. cholerae clinical isolates obtained over a 20-month period. Notably, they detected point mutations, deletions, and inversions but found no insertion of horizontally transmitted DNA, arguing strongly against the idea that autochthonous V. cholerae donated DNA to the outbreak strain. Furthermore, they found that Haitian epidemic V. cholerae isolates were virtually untransformable. Comparative genomic analyses revealed that the Haitian isolates were nearly identical to isolates from Nepal and that the Nepalese-Haitian isolates were distinguishable from isolates circulating elsewhere in the world. Reconstruction of the phylogeny of the Haitian isolates was consistent with a single introduction of V. cholerae to Haiti sometime between late July and late October 2010, dates remarkably concordant with epidemiological observations. In aggregate, this paper provides additional compelling evidence that the V. cholerae strain responsible for the Haitian cholera epidemic came from Nepal and illustrates the power of whole-genome-based analyses for epidemiology, pathogen evolution, and forensics.

scholarly journals The Pangenome Structure of Escherichia coli: Comparative Genomic Analysis of E. coli Commensal and Pathogenic Isolates

2008 ◽  
Vol 190 (20) ◽  
pp. 6881-6893 ◽  
Author(s):  
David A. Rasko ◽  
M. J. Rosovitz ◽  
Garry S. A. Myers ◽  
Emmanuel F. Mongodin ◽  
W. Florian Fricke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Sequencing ◽  
Genomic Analysis ◽  
Comparative Genomic Analysis ◽  
Genomic Diversity ◽  
Comparative Genomic ◽  
Whole Genome ◽  
E Coli ◽  
Important Group ◽  
Commensal Species

ABSTRACT Whole-genome sequencing has been skewed toward bacterial pathogens as a consequence of the prioritization of medical and veterinary diseases. However, it is becoming clear that in order to accurately measure genetic variation within and between pathogenic groups, multiple isolates, as well as commensal species, must be sequenced. This study examined the pangenomic content of Escherichia coli. Six distinct E. coli pathovars can be distinguished using molecular or phenotypic markers, but only two of the six pathovars have been subjected to any genome sequencing previously. Thus, this report provides a seminal description of the genomic contents and unique features of three unsequenced pathovars, enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. We also determined the first genome sequence of a human commensal E. coli isolate, E. coli HS, which will undoubtedly provide a new baseline from which workers can examine the evolution of pathogenic E. coli. Comparison of 17 E. coli genomes, 8 of which are new, resulted in identification of ∼2,200 genes conserved in all isolates. We were also able to identify genes that were isolate and pathovar specific. Fewer pathovar-specific genes were identified than anticipated, suggesting that each isolate may have independently developed virulence capabilities. Pangenome calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors. This comparative study of the species E. coli, while descriptive, should provide the basis for future functional work on this important group of pathogens.

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