scholarly journals 5-fluorocytosine resistance is associated with hypermutation and alterations in capsule biosynthesis in Cryptococcus

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
R. Blake Billmyre ◽  
Shelly Applen Clancey ◽  
Lucy X. Li ◽  
Tamara L. Doering ◽  
Joseph Heitman
Keyword(s):  
Whole Genome Sequencing ◽  
Mismatch Repair ◽  
Genome Sequencing ◽  
Resistance Genes ◽  
Fungal Pathogen ◽  
Glucuronic Acid ◽  
Nucleotide Metabolism ◽  
Whole Genome ◽  
Dna Mismatch

AbstractPatients infected with the fungal pathogen Cryptococcus are most effectively treated with a combination of 5-fluorocytosine (5FC) and amphotericin B. 5FC acts as a prodrug, which is converted into toxic 5-fluorouracil (5FU) upon uptake into fungal cells. However, the pathogen frequently develops resistance through unclear mechanisms. Here we show that resistance to 5FC in Cryptococcus deuterogattii is acquired more frequently in isolates with defects in DNA mismatch repair that confer an elevated mutation rate. We use whole genome sequencing of 16 independent isolates to identify mutations associated with 5FC resistance in vitro. We find mutations in known resistance genes (FUR1 and FCY2) and in a gene UXS1, previously shown to encode an enzyme that converts UDP-glucuronic acid to UDP-xylose for capsule biosynthesis, but not known to play a role in 5FC metabolism. Mutations in UXS1 lead to accumulation of UDP-glucuronic acid and alterations in nucleotide metabolism, which appear to suppress toxicity of both 5FC and its toxic derivative 5FU.

scholarly journals Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair DefectiveEscherichia coli

Genetics ◽  
2018 ◽  
Vol 209 (4) ◽  
pp. 1029-1042 ◽  
Author(s):  
Patricia L. Foster ◽  
Brittany A. Niccum ◽  
Ellen Popodi ◽  
Jesse P. Townes ◽  
Heewook Lee ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Mismatch Repair ◽  
Genome Sequencing ◽  
Base Pair ◽  
Dna Mismatch Repair ◽  
Whole Genome ◽  
Dna Mismatch ◽  
Base Pair Substitution

scholarly journals Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella

2016 ◽  
Vol 60 (9) ◽  
pp. 5515-5520 ◽  
Author(s):  
Patrick F. McDermott ◽  
Gregory H. Tyson ◽  
Claudine Kabera ◽  
Yuansha Chen ◽  
Cong Li ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Resistance Genes ◽  
The United States ◽  
Whole Genome ◽  
Content Type ◽  
Structural Resistance ◽  
Retail Meat

ABSTRACTLaboratory-basedin vitroantimicrobial susceptibility testing is the foundation for guiding anti-infective therapy and monitoring antimicrobial resistance trends. We used whole-genome sequencing (WGS) technology to identify known antimicrobial resistance determinants among strains of nontyphoidalSalmonellaand correlated these with susceptibility phenotypes to evaluate the utility of WGS for antimicrobial resistance surveillance. Six hundred fortySalmonellaof 43 different serotypes were selected from among retail meat and human clinical isolates that were tested for susceptibility to 14 antimicrobials using broth microdilution. The MIC for each drug was used to categorize isolates as susceptible or resistant based on Clinical and Laboratory Standards Institute clinical breakpoints or National Antimicrobial Resistance Monitoring System (NARMS) consensus interpretive criteria. Each isolate was subjected to whole-genome shotgun sequencing, and resistance genes were identified from assembled sequences. A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified. There were more unique resistance genes (n =59) in the 104 human isolates than in the 536 retail meat isolates (n =36). Overall, resistance genotypes and phenotypes correlated in 99.0% of cases. Correlations approached 100% for most classes of antibiotics but were lower for aminoglycosides and beta-lactams. We report the first finding of extended-spectrum β-lactamases (ESBLs) (blaCTX-M1andblaSHV2a) in retail meat isolates ofSalmonellain the United States. Whole-genome sequencing is an effective tool for predicting antibiotic resistance in nontyphoidalSalmonella, although the use of more appropriate surveillance breakpoints and increased knowledge of new resistance alleles will further improve correlations.

scholarly journals Determinants of base-pair substitution patterns revealed by whole-genome sequencing of DNA mismatch repair defectiveEscherichia coli

2018 ◽  
Author(s):  
Patricia L. Foster ◽  
Brittany A. Niccum ◽  
Ellen Popodi ◽  
Jesse P. Townes ◽  
Heewook Lee ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Mismatch Repair ◽  
Genome Sequencing ◽  
Base Pair ◽  
Whole Genome ◽  
Dna Mismatch ◽  
In The Wild ◽  
Leading Strand ◽  
Base Pair Substitution ◽  
Lagging Strand

ABSTRACTMismatch repair (MMR) is a major contributor to replication fidelity, but its impact varies with sequence context and the nature of the mismatch. Mutation accumulation experiments followed by whole-genome sequencing of MMR-defectiveE. colistrains yielded ≈30,000 base-pair substitutions, revealing mutational patterns across the entire chromosome. The base-pair substitution spectrum was dominated by A:T > G:C transitions, which occurred predominantly at the center base of 5′NAC3′+5′GTN3′ triplets. Surprisingly, growth on minimal medium or at low temperature attenuated these mutations. Mononucleotide runs were also hotspots for base-pair substitutions, and the rate at which these occurred increased with run length. Comparison with ≈2000 base-pair substitutions accumulated in MMR-proficient strains revealed that both kinds of hotspots appeared in the wild-type spectrum and so are likely to be sites of frequent replication errors. In MMR-defective strains transitions were strand biased, occurring twice as often when A and C rather than T and G were on the lagging-strand template. Loss of nucleotide diphosphate kinase increases the cellular concentration of dCTP, which resulted in increased rates of mutations due to misinsertion of C opposite A and T. In anmmr ndkdouble mutant strain, these mutations were more frequent when the template A and T were on the leading strand, suggesting that lagging-strand synthesis was more error-prone or less well corrected by proofreading than was leading strand synthesis.

scholarly journals Single-cell whole-genome sequencing reveals mutational landscapes of DNA mismatch repair deficiency in mouse primary fibroblasts

2019 ◽  
Author(s):  
Lei Zhang ◽  
Xiao Dong ◽  
Xiaoxiao Hao ◽  
Moonsook Lee ◽  
Zhongxuan Chi ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Single Cell ◽  
Mismatch Repair ◽  
Genome Sequencing ◽  
Somatic Mutations ◽  
Fold Increase ◽  
Whole Genome ◽  
Wild Type ◽  
Dna Mismatch ◽  
Mmr Deficiency

AbstractDNA Mismatch repair (MMR) deficiency is a major cause of hereditary non-polyposis colorectal cancer, and is also associated with increased risk of several other cancers. This is generally ascribed to the role of MMR in avoiding mutations by correcting DNA replication errors. In MMR knockout mice very high frequencies of somatic mutations, up until 100-fold of background, have been reported. However, these results have been obtained using bacterial reporter transgenes, which are not representative for the genome overall, and mutational patterns of MMR deficiency remain largely unknown. To fill this knowledge gap, we performed single-cell whole-genome sequencing of lung fibroblasts of Msh2−/− and wild-type mice. We observed a 4-fold increase of somatic single nucleotide variants (SNVs) in the fibroblasts of Msh2−/− mice compared to those of wild-type mice. The SNV signature of Msh2 deficiency was found to be driven by C>T and T>C transitions. By comparing it to human cancer signatures, we not only confirmed the inferred MMR-deficiency-related etiology of several cancer signatures but also suggested that MMR deficiency is likely the cause of a cancer signature with its etiology previously unknown. We also observed a 7-fold increase of somatic small insertions and deletions (INDELs) in the Msh2−/− mice. An elevated INDEL frequency has also been found in human MMR-related cancers. INDELs and SNVs distributed differently across genomic features in the Msh2−/− and control cells, with evidence of selection pressure and repair preference. These results provide insights into the landscape of somatic mutations in normal somatic cells caused by MMR deficiency.SignificanceOur results show that MMR deficiency in the mouse is associated with a much lower elevation of somatic mutation rates than previously reported and provides the first MMR whole-genome mutational landscapes in normal somatic cells in vivo.

scholarly journals SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weili Cai ◽  
Schyler Nunziata ◽  
John Rascoe ◽  
Michael J. Stulberg
Keyword(s):  
Whole Genome Sequencing ◽  
Molecular Characterization ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Coverage ◽  
Metagenomic Sample ◽  
Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

2021 ◽  
Author(s):  
◽  
Mariah Taylor ◽  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Animal Health ◽  
Functional Domains ◽  
Whole Genome ◽  
Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

scholarly journals Whole-Genome Sequencing Reveals Breast Cancers with Mismatch Repair Deficiency

2017 ◽  
Vol 77 (18) ◽  
pp. 4755-4762 ◽  
Author(s):  
Helen Davies ◽  
Sandro Morganella ◽  
Colin A. Purdie ◽  
Se Jin Jang ◽  
Elin Borgen ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Mismatch Repair ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Breast Cancers ◽  
Mismatch Repair Deficiency ◽  
Repair Deficiency

scholarly journals Whole-Genome Sequencing Reveals a Prolonged and Persistent Intrahospital Transmission of Corynebacterium striatum, an Emerging Multidrug-Resistant Pathogen

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
Xuebing Wang ◽  
Haijian Zhou ◽  
Dongke Chen ◽  
Pengcheng Du ◽  
Ruiting Lan ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Resistance Genes ◽  
Antimicrobial Agents ◽  
Chronically Ill ◽  
Multidrug Resistant ◽  
Resistance Rate ◽  
Genomic Diversity ◽  
Whole Genome ◽  
Corynebacterium Striatum

ABSTRACT Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen that occurs primarily among immunocompromised and chronically ill patients. However, little is known about the genomic diversity of C. striatum, which contributes to its long-term persistence and transmission in hospitals. In this study, a total of 192 C. striatum isolates obtained from 14 September 2017 to 29 March 2018 in a hospital in Beijing, China, were analyzed by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was conducted on 91 isolates. Nearly all isolates (96.3%, 183/190) were MDR. The highest resistance rate was observed for ciprofloxacin (99.0%, 190/192), followed by cefotaxime (90.6%, 174/192) and erythromycin (89.1%, 171/192). PFGE separated the 192 isolates into 79 pulsotypes, and differences in core genome single-nucleotide polymorphisms (SNPs) partitioned the 91 isolates sequenced into four clades. Isolates of the same pulsotype were identical or nearly identical at the genome level, with some exceptions. Two dominant subclones, clade 3a, and clade 4a, were responsible for the hospital-wide dissemination. Genomic analysis further revealed nine resistance genes mobilized by eight unique cassettes. PFGE and whole-genome sequencing revealed that the C. striatum isolates studied were the result mainly of predominant clones spreading in the hospital. C. striatum isolates in the hospital progressively acquired resistance to antimicrobial agents, demonstrating that isolates of C. striatum may adapt rapidly through the acquisition and accumulation of resistance genes and thus evolve into dominant and persistent clones. These insights will be useful for the prevention of C. striatum infection in hospitals.

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